Directory of Open Access Journals (Sweden)
M. Kristen Hall
2016-06-01
Full Text Available Glycosylation modulates growth, maintenance, and stress signaling processes. Consequently, altered N-glycosylation is associated with reduced fitness and disease. Therefore, expanding our understanding of N-glycans in altering biological processes is of utmost interest. Herein, clustered regularly interspaced short palindromic repeats/caspase9 (CRISPR/Cas9 technology was employed to engineer a glycosylation mutant Chinese Hamster Ovary (CHO cell line, K16, which expresses predominantly hybrid type N-glycans. This newly engineered cell line enabled us to compare N-glycan effects on cellular properties of hybrid type N-glycans, to the well-established Pro−5 and Lec1 cell lines, which express complex and oligomannose types of N-glycans, respectively. Lectin binding studies revealed the predominant N-glycan expressed in K16 is hybrid type. Cell dissociation and migration assays demonstrated the greatest strength of cell–cell adhesion and fastest migratory rates for oligomannose N-glycans, and these properties decreased as oligomannose type were converted to hybrid type, and further decreased upon conversion to complex type. Next, we examined the roles of three general types of N-glycans on ectopic expression of E-cadherin, a cell–cell adhesion protein. Microscopy revealed more functional E-cadherin at the cell–cell border when N-glycans were oligomannose and these levels decreased as the oligomannose N-glycans were processed to hybrid and then to complex. Thus, we provide evidence that all three general types of N-glycans impact plasma membrane architecture and cellular properties.
Biological significance of complex N-glycans in plants and their impact on plant physiology.
Strasser, Richard
2014-01-01
Asparagine (N)-linked protein glycosylation is a ubiquitous co- and post-translational modification which can alter the biological function of proteins and consequently affects the development, growth, and physiology of organisms. Despite an increasing knowledge of N-glycan biosynthesis and processing, we still understand very little about the biological function of individual N-glycan structures in plants. In particular, the N-glycan-processing steps mediated by Golgi-resident enzymes create a structurally diverse set of protein-linked carbohydrate structures. Some of these complex N-glycan modifications like the presence of β1,2-xylose, core α1,3-fucose or the Lewis a-epitope are characteristic for plants and are evolutionary highly conserved. In mammals, complex N-glycans are involved in different cellular processes including molecular recognition and signaling events. In contrast, the complex N-glycan function is still largely unknown in plants. Here, in this short review, I focus on important recent developments and discuss their implications for future research in plant glycobiology and plant biotechnology.
Structural characterization of complex O-linked glycans from insect-derived material.
Garenaux, Estelle; Maes, Emmanuel; Levêque, S; Brassart, Colette; Guerardel, Yann
2011-07-01
Although insects are among the most diverse groups of the animal kingdom and may be found in nearly all environments, one can observe an obvious lack of structural data on their glycosylation ability. Hymenoptera is the second largest of all insect orders with more than 110,000 identified species and includes the most famous examples of social insects' species such as wasps, bees and ants. In this report, the structural variety of O-glycans has been studied in two Hymenoptera species. In a previous study, we showed that major O-glycans from common wasp (Vespula germanica) salivary mucins correspond to T and Tn antigen, eventually substituted by phosphoethanolamine or phosphate groups. More detailed structural analysis performed by mass spectrometry revealed numerous minor O-glycan structures bearing Gal, GlcNAc, GalNAc and Fuc residues. Thus, in order to investigate glycosylation diversity in insects, we used common wasp nest (V. germanica) and hornet nest (Vespa cabro) as starting materials. These materials were submitted to reductive β-elimination and the released oligosaccharide-alditols further fractionated by multidimensional HPLC. Tandem mass spectrometry analyses combined with NMR data revealed the presence of various families of complex O-glycans differing accordingly to both core structures and external motifs. Glycans from wasp were characterized by the presence of core types 1 and 2, Lewis X and internal Gal-Gal motifs. We also observed unusual O-glycans containing a reducing GalNAc unit directly substituted by a fucose residue. In contrast, hornet O-glycans appeared as a rather homogeneous family of core 1 type O-glycans extended by galactose oligomers. Copyright © 2011 Elsevier Ltd. All rights reserved.
Simple Sugars to Complex Disease—Mucin-Type O-Glycans in Cancer
Kudelka, Matthew R.; Ju, Tongzhong; Heimburg-Molinaro, Jamie; Cummings, Richard D.
2017-01-01
Mucin-type O-glycans are a class of glycans initiated with N-acetylgalactosamine (GalNAc) α-linked primarily to Ser/Thr residues within glycoproteins and often extended or branched by sugars or saccharides. Most secretory and membrane-bound proteins receive this modification, which is important in regulating many biological processes. Alterations in mucin-type O-glycans have been described across tumor types and include expression of relatively small-sized, truncated O-glycans and altered terminal structures, both of which are associated with patient prognosis. New discoveries in the identity and expression of tumor-associated O-glycans are providing new avenues for tumor detection and treatment. This chapter describes mucin-type O-glycan biosynthesis, altered mucin-type O-glycans in primary tumors, including mechanisms for structural changes and contributions to the tumor phenotype, and clinical approaches to detect and target altered O-glycans for cancer treatment and management. PMID:25727146
Solid-phase glycan isolation for glycomics analysis
Yang, Shuang; Zhang, Hui
2012-01-01
Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. ...
Production of complex multiantennary N-glycans in Nicotiana benthamiana plants.
Nagels, Bieke; Van Damme, Els J M; Pabst, Martin; Callewaert, Nico; Weterings, Koen
2011-03-01
In recent years, plants have been developed as an alternative expression system to mammalian hosts for the production of therapeutic proteins. Many modifications to the plant glycosylation machinery have been made to render it more human because of the importance of glycosylation for functionality, serum half-life, and the safety profile of the expressed proteins. These modifications include removal of plant-specific β1,2-xylose and core α1,3-fucose, and addition of bisecting N-acetylglucosamine, β1,4-galactoses, and sialic acid residues. Another glycosylation step that is essential for the production of complex human-type glycans is the synthesis of multiantennary structures, which are frequently found on human N-glycans but are not generated by wild-type plants. Here, we report both the magnICON-based transient as well as stable introduction of the α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV isozymes a and b) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) in Nicotiana benthamiana plants. The enzymes were targeted to the Golgi apparatus by fusing their catalytic domains to the plant-specific localization signals of xylosyltransferase and fucosyltransferase. The GnT-IV and -V modifications were tested in the wild-type background, but were also combined with the RNA interference-mediated knockdown of β1,2-xylosyltransferase and α1,3-fucosyltransferase. Results showed that triantennary Gn[GnGn] and [GnGn]Gn N-glycans could be produced according to the expected activities of the respective enzymes. Combination of the two enzymes by crossing stably transformed GnT-IV and GnT-V plants showed that up to 10% tetraantennary [GnGn][GnGn], 25% triantennary, and 35% biantennary N-glycans were synthesized. All transgenic plants were viable and showed no aberrant phenotype under standard growth conditions.
Solid-phase glycan isolation for glycomics analysis.
Yang, Shuang; Zhang, Hui
2012-12-01
Glycosylation is one of the most significant protein PTMs. The biological activities of proteins are dramatically changed by the glycans associated with them. Thus, structural analysis of the glycans of glycoproteins in complex biological or clinical samples is critical in correlation with the functions of glycans with diseases. Profiling of glycans by HPLC-MS is a commonly used technique in analyzing glycan structures and quantifying their relative abundance in different biological systems. Methods relied on MS require isolation of glycans from negligible salts and other contaminant ions since salts and ions may interfere with the glycans, resulting in poor glycan ionization. To accomplish those objectives, glycan isolation and clean-up methods including SPE, liquid-phase extraction, chromatography, and electrophoresis have been developed. Traditionally, glycans are isolated from proteins or peptides using a combination of hydrophobic and hydrophilic columns: proteins and peptides remain on hydrophobic absorbent while glycans, salts, and other hydrophilic reagents are collected as flowthrough. The glycans in the flowthrough are then purified through graphite-activated carbon column by hydrophilic interaction LC. Yet, the drawback in these affinity-based approaches is nonspecific binding. As a result, chemical methods by hydrazide or oxime have been developed for solid-phase isolation of glycans with high specificity and yield. Combined with high-resolution MS, specific glycan isolation techniques provide tremendous potentials as useful tools for glycomics analysis. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Chemoenzymatic assembly of mammalian O-mannose glycans.
Cao, Hongzhi; Meng, Caicai; Sasmal, Aniruddha; Zhang, Yan; Gao, Tian; Liu, Chang-Cheng; Khan, Naazneen; Varki, Ajit; Wang, Fengshan
2018-05-26
O-Mannose glycans account up to 30% of total O-glycans in brain. Previous synthesis and functional studies only focused on the Core M3 O-mannose glycans of α-dystroglycan which are a causative factor for various muscular diseases. In this study, a highly efficient chemoenzymatic strategy was developed that enabled the first collective synthesis of 63 Core M1 and Core M2 O-mannose glycans. This chemoenzymatic strategy features the gram-scale chemical synthesis of 5 judiciously designed core structures, and the diversity-oriented modification of the core structures with 3 enzyme modules to provide 58 complex O-mannose glycans in a linear sequence that does not exceed 4 steps. The binding profiles of synthetic O-mannose glycans with a panel of lectins, antibodies and brain proteins were also explored using the printed O-mannose glycan array. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Automated Glycan Assembly of Complex Oligosaccharides Related to Blood Group Determinants.
Hahm, Heung Sik; Liang, Chien-Fu; Lai, Chian-Hui; Fair, Richard J; Schuhmacher, Frank; Seeberger, Peter H
2016-07-15
Lactotetraosyl (Lc4) and neo-lactotetraosyl (nLc4) are backbones that are common to many glycans. Using automated glycan assembly, these common core structures were constructed and elaborated to access synthetically challenging glycans of biological relevance. The incorporation of α-fucoses is demonstrated for H-type I and II; α(1,3)-galactose epitopes were prepared, and the pentasaccharide HNK-1 required incorporation of a 3-O-sulfate. In addition to preparing the target structures, essential insights were gained regarding the relationships of glycosylating agents and nucleophiles as well as the linker stability.
Ceroni, Alessio; Dell, Anne; Haslam, Stuart M
2007-08-07
Carbohydrates play a critical role in human diseases and their potential utility as biomarkers for pathological conditions is a major driver for characterization of the glycome. However, the additional complexity of glycans compared to proteins and nucleic acids has slowed the advancement of glycomics in comparison to genomics and proteomics. The branched nature of carbohydrates, the great diversity of their constituents and the numerous alternative symbolic notations, make the input and display of glycans not as straightforward as for example the amino-acid sequence of a protein. Every glycoinformatic tool providing a user interface would benefit from a fast, intuitive, appealing mechanism for input and output of glycan structures in a computer readable format. A software tool for building and displaying glycan structures using a chosen symbolic notation is described here. The "GlycanBuilder" uses an automatic rendering algorithm to draw the saccharide symbols and to place them on the drawing board. The information about the symbolic notation is derived from a configurable graphical model as a set of rules governing the aspect and placement of residues and linkages. The algorithm is able to represent a structure using only few traversals of the tree and is inherently fast. The tool uses an XML format for import and export of encoded structures. The rendering algorithm described here is able to produce high-quality representations of glycan structures in a chosen symbolic notation. The automated rendering process enables the "GlycanBuilder" to be used both as a user-independent component for displaying glycans and as an easy-to-use drawing tool. The "GlycanBuilder" can be integrated in web pages as a Java applet for the visual editing of glycans. The same component is available as a web service to render an encoded structure into a graphical format. Finally, the "GlycanBuilder" can be integrated into other applications to create intuitive and appealing user
Directory of Open Access Journals (Sweden)
Dell Anne
2007-08-01
Full Text Available Abstract Background Carbohydrates play a critical role in human diseases and their potential utility as biomarkers for pathological conditions is a major driver for characterization of the glycome. However, the additional complexity of glycans compared to proteins and nucleic acids has slowed the advancement of glycomics in comparison to genomics and proteomics. The branched nature of carbohydrates, the great diversity of their constituents and the numerous alternative symbolic notations, make the input and display of glycans not as straightforward as for example the amino-acid sequence of a protein. Every glycoinformatic tool providing a user interface would benefit from a fast, intuitive, appealing mechanism for input and output of glycan structures in a computer readable format. Results A software tool for building and displaying glycan structures using a chosen symbolic notation is described here. The "GlycanBuilder" uses an automatic rendering algorithm to draw the saccharide symbols and to place them on the drawing board. The information about the symbolic notation is derived from a configurable graphical model as a set of rules governing the aspect and placement of residues and linkages. The algorithm is able to represent a structure using only few traversals of the tree and is inherently fast. The tool uses an XML format for import and export of encoded structures. Conclusion The rendering algorithm described here is able to produce high-quality representations of glycan structures in a chosen symbolic notation. The automated rendering process enables the "GlycanBuilder" to be used both as a user-independent component for displaying glycans and as an easy-to-use drawing tool. The "GlycanBuilder" can be integrated in web pages as a Java applet for the visual editing of glycans. The same component is available as a web service to render an encoded structure into a graphical format. Finally, the "GlycanBuilder" can be integrated into other
Qrator: A web-based curation tool for glycan structures
Eavenson, Matthew; Kochut, Krys J; Miller, John A; Ranzinger, René; Tiemeyer, Michael; Aoki, Kazuhiro; York, William S
2015-01-01
Most currently available glycan structure databases use their own proprietary structure representation schema and contain numerous annotation errors. These cause problems when glycan databases are used for the annotation or mining of data generated in the laboratory. Due to the complexity of glycan structures, curating these databases is often a tedious and labor-intensive process. However, rigorously validating glycan structures can be made easier with a curation workflow that incorporates a structure-matching algorithm that compares candidate glycans to a canonical tree that embodies structural features consistent with established mechanisms for the biosynthesis of a particular class of glycans. To this end, we have implemented Qrator, a web-based application that uses a combination of external literature and database references, user annotations and canonical trees to assist and guide researchers in making informed decisions while curating glycans. Using this application, we have started the curation of large numbers of N-glycans, O-glycans and glycosphingolipids. Our curation workflow allows creating and extending canonical trees for these classes of glycans, which have subsequently been used to improve the curation workflow. PMID:25165068
Directory of Open Access Journals (Sweden)
Erica E Rosenbaum
2014-05-01
Full Text Available As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II, α-mannosidase-IIb (α-Man-IIb, a β-N-acetylglucosaminidase called fused lobes (Fdl, and hexosaminidase 1 (Hexo1. We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights
Rosenbaum, Erica E.; Vasiljevic, Eva; Brehm, Kimberley S.; Colley, Nansi Jo
2014-01-01
As newly synthesized glycoproteins move through the secretory pathway, the asparagine-linked glycan (N-glycan) undergoes extensive modifications involving the sequential removal and addition of sugar residues. These modifications are critical for the proper assembly, quality control and transport of glycoproteins during biosynthesis. The importance of N-glycosylation is illustrated by a growing list of diseases that result from defects in the biosynthesis and processing of N-linked glycans. The major rhodopsin in Drosophila melanogaster photoreceptors, Rh1, is highly unique among glycoproteins, as the N-glycan appears to be completely removed during Rh1 biosynthesis and maturation. However, much of the deglycosylation pathway for Rh1 remains unknown. To elucidate the key steps in Rh1 deglycosylation in vivo, we characterized mutant alleles of four Drosophila glycosyl hydrolases, namely α-mannosidase-II (α-Man-II), α-mannosidase-IIb (α-Man-IIb), a β-N-acetylglucosaminidase called fused lobes (Fdl), and hexosaminidase 1 (Hexo1). We have demonstrated that these four enzymes play essential and unique roles in a highly coordinated pathway for oligosaccharide trimming during Rh1 biosynthesis. Our results reveal that α-Man-II and α-Man-IIb are not isozymes like their mammalian counterparts, but rather function at distinct stages in Rh1 maturation. Also of significance, our results indicate that Hexo1 has a biosynthetic role in N-glycan processing during Rh1 maturation. This is unexpected given that in humans, the hexosaminidases are typically lysosomal enzymes involved in N-glycan catabolism with no known roles in protein biosynthesis. Here, we present a genetic dissection of glycoprotein processing in Drosophila and unveil key steps in N-glycan trimming during Rh1 biosynthesis. Taken together, our results provide fundamental advances towards understanding the complex and highly regulated pathway of N-glycosylation in vivo and reveal novel insights into the
ANALYSIS OF GLYCANS DERIVED FROM GLYCOCONJUGATES BY CAPILLARY ELECTROPHORESIS-MASS SPECTROMETRY
Mechref, Yehia
2012-01-01
The high structural variation of glycan derived from glycoconjugates, which substantially increases with the molecular size of a protein, contributes to the complexity of glycosylation patterns commonly associated with glycoconjugates. In the case of glycoproteins, such variation originates from the multiple glycosylation sites of proteins and the number of glycan structures associated with each site (microheterogeneity). The ability to comprehensively characterize highly complex mixture of glycans has been analytically stimulating and challenging. Although the most powerful mass spectrometric (MS) and tandem MS techniques are capable of providing a wealth of structural information, they are still not able to readily identify isomeric glycan structures without high order tandem MS (MSn). The analysis of isomeric glycan structures has been attained using several separation methods, including high-pH anion exchange chromatography (HPAEC), hydrophilic interaction chromatography (HILIC) and gas chromatography (GC). However, capillary electrophoresis (CE) and microfluidics capillary electrophoresis (MCE) offer high separation efficiency and resolutions, allowing the separation of closely related glycan structures. Therefore, interfacing CE and MCE to MS is a powerful analytical approach, allowing potentially comprehensive and sensitive analysis of complex glycan samples. This review describes and discusses the utility of different CE and MCE approaches in the structural characterization of glycoproteins and the feasibility of interfacing these approaches to mass spectrometry. PMID:22180203
Glycan array data management at Consortium for Functional Glycomics.
Venkataraman, Maha; Sasisekharan, Ram; Raman, Rahul
2015-01-01
Glycomics or the study of structure-function relationships of complex glycans has reshaped post-genomics biology. Glycans mediate fundamental biological functions via their specific interactions with a variety of proteins. Recognizing the importance of glycomics, large-scale research initiatives such as the Consortium for Functional Glycomics (CFG) were established to address these challenges. Over the past decade, the Consortium for Functional Glycomics (CFG) has generated novel reagents and technologies for glycomics analyses, which in turn have led to generation of diverse datasets. These datasets have contributed to understanding glycan diversity and structure-function relationships at molecular (glycan-protein interactions), cellular (gene expression and glycan analysis), and whole organism (mouse phenotyping) levels. Among these analyses and datasets, screening of glycan-protein interactions on glycan array platforms has gained much prominence and has contributed to cross-disciplinary realization of the importance of glycomics in areas such as immunology, infectious diseases, cancer biomarkers, etc. This manuscript outlines methodologies for capturing data from glycan array experiments and online tools to access and visualize glycan array data implemented at the CFG.
Production of Complex Multiantennary N-Glycans in Nicotiana benthamiana Plants1[W][OA
Nagels, Bieke; Van Damme, Els J.M.; Pabst, Martin; Callewaert, Nico; Weterings, Koen
2011-01-01
In recent years, plants have been developed as an alternative expression system to mammalian hosts for the production of therapeutic proteins. Many modifications to the plant glycosylation machinery have been made to render it more human because of the importance of glycosylation for functionality, serum half-life, and the safety profile of the expressed proteins. These modifications include removal of plant-specific β1,2-xylose and core α1,3-fucose, and addition of bisecting N-acetylglucosamine, β1,4-galactoses, and sialic acid residues. Another glycosylation step that is essential for the production of complex human-type glycans is the synthesis of multiantennary structures, which are frequently found on human N-glycans but are not generated by wild-type plants. Here, we report both the magnICON-based transient as well as stable introduction of the α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV isozymes a and b) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) in Nicotiana benthamiana plants. The enzymes were targeted to the Golgi apparatus by fusing their catalytic domains to the plant-specific localization signals of xylosyltransferase and fucosyltransferase. The GnT-IV and -V modifications were tested in the wild-type background, but were also combined with the RNA interference-mediated knockdown of β1,2-xylosyltransferase and α1,3-fucosyltransferase. Results showed that triantennary Gn[GnGn] and [GnGn]Gn N-glycans could be produced according to the expected activities of the respective enzymes. Combination of the two enzymes by crossing stably transformed GnT-IV and GnT-V plants showed that up to 10% tetraantennary [GnGn][GnGn], 25% triantennary, and 35% biantennary N-glycans were synthesized. All transgenic plants were viable and showed no aberrant phenotype under standard growth conditions. PMID:21233332
The hydroxyl-functionalized magnetic particles for purification of glycan-binding proteins.
Sun, Xiuxuan; Yang, Ganglong; Sun, Shisheng; Quan, Rui; Dai, Weiwei; Li, Bin; Chen, Chao; Li, Zheng
2009-12-01
Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.
Directory of Open Access Journals (Sweden)
M Kristen Hall
Full Text Available The intrinsic electrical properties of a neuron depend on expression of voltage gated potassium (Kv channel isoforms, as well as their distribution and density in the plasma membrane. Recently, we showed that N-glycosylation site occupancy of Kv3.1b modulated its placement in the cell body and neurites of a neuronal-derived cell line, B35 neuroblastoma cells. To extrapolate this mechanism to other N-glycosylated Kv channels, we evaluated the impact of N-glycosylation occupancy of Kv3.1a and Kv1.1 channels. Western blots revealed that wild type Kv3.1a and Kv1.1 α-subunits had complex and oligomannose N-glycans, respectively, and that abolishment of the N-glycosylation site(s generated Kv proteins without N-glycans. Total internal reflection fluorescence microscopy images revealed that N-glycans of Kv3.1a contributed to its placement in the cell membrane while N-glycans had no effect on the distribution of Kv1.1. Based on particle analysis of EGFP-Kv proteins in the adhered membrane, glycosylated forms of Kv3.1a, Kv1.1, and Kv3.1b had differences in the number, size or density of Kv protein clusters in the cell membrane of neurites and cell body of B35 cells. Differences were also observed between the unglycosylated forms of the Kv proteins. Cell dissociation assays revealed that cell-cell adhesion was increased by the presence of complex N-glycans of Kv3.1a, like Kv3.1b, whereas cell adhesion was similar in the oligomannose and unglycosylated Kv1.1 subunit containing B35 cells. Our findings provide direct evidence that N-glycans of Kv3.1 splice variants contribute to the placement of these glycoproteins in the plasma membrane of neuronal-derived cells while those of Kv1.1 were absent. Further when the cell membrane distribution of the Kv channel was modified by N-glycans then the cell-cell adhesion properties were altered. Our study demonstrates that N-glycosylation of Kv3.1a, like Kv3.1b, provides a mechanism for the distribution of these
Mathew, Mohit P; Donaldson, Julie G
2018-05-11
Clathrin-independent endocytosis (CIE) is a form of endocytosis that lacks a defined cytoplasmic machinery. Here, we asked whether glycan interactions, acting from the outside, could be a part of that endocytic machinery. We show that the perturbation of global cellular patterns of protein glycosylation by modulation of metabolic flux affects CIE. Interestingly, these changes in glycosylation had cargo-specific effects. For example, in HeLa cells, GlcNAc treatment, which increases glycan branching, increased major histocompatibility complex class I (MHCI) internalization but inhibited CIE of the glycoprotein CD59 molecule (CD59). The effects of knocking down the expression of galectin 3, a carbohydrate-binding protein and an important player in galectin-glycan interactions, were also cargo-specific and stimulated CD59 uptake. By contrast, inhibition of all galectin-glycan interactions by lactose inhibited CIE of both MHCI and CD59. None of these treatments affected clathrin-mediated endocytosis, implying that glycosylation changes specifically affect CIE. We also found that the galectin lattice tailors membrane fluidity and cell spreading. Furthermore, changes in membrane dynamics mediated by the galectin lattice affected macropinocytosis, an altered form of CIE, in HT1080 cells. Our results suggest that glycans play an important and nuanced role in CIE, with each cargo being affected uniquely by alterations in galectin and glycan profiles and their interactions. We conclude that galectin-driven effects exist on a continuum from stimulatory to inhibitory, with distinct CIE cargo proteins having unique response landscapes and with different cell types starting at different positions on these conceptual landscapes.
Glycan arrays and other tools produced by automated glycan assembly
Directory of Open Access Journals (Sweden)
Peter H. Seeberger
2017-01-01
Full Text Available Carbohydrates are the dominant biopolymer on earth and play important roles ranging from building material for plants to function in many biological systems. Glycans remain poorly studied due to a lack of synthetic tools. The goal of my laboratory has been to develop a general method for the automated assembly of glycans. The general protocols we developed resulted in the commercialisation of the Glyconeer 2.1™ synthesizer as well as the building blocks and all reagents. Oligosaccharides as long as 50-mers are now accessible within days. Rapid access to defined oligosaccharides has been the foundation to many applications including synthetic tools such as glycan microarrays, glycan nanoparticles and anti-glycan antibodies. The platform technology is helping to address real-life problems by the creation of new vaccines and diagnostics. After addressing mainly mammalian glycobiology earlier, material science and plant biology are benefitting increasingly from synthetic glycans.
DEFF Research Database (Denmark)
Yin, Bojiao; Gao, Yuan; Chung, Cheng-yu
2015-01-01
Sialic acid, a terminal residue on complex N-glycans, and branching or antennarity can play key roles in both the biological activity and circulatory lifetime of recombinant glycoproteins of therapeutic interest. In order to examine the impact of glycosyltransferase expression on the N-glycosylat......Sialic acid, a terminal residue on complex N-glycans, and branching or antennarity can play key roles in both the biological activity and circulatory lifetime of recombinant glycoproteins of therapeutic interest. In order to examine the impact of glycosyltransferase expression on the N...... increased by 26%. The increase in sialic acid content was further verified by detailed profiling of the N-glycan structures using mass spectra (MS) analysis. In order to enhance antennarity/branching, UDP-N-acetylglucosamine: α-1,3-D-mannoside β1,4-N-acetylglucosaminyltransferase (GnTIV/Mgat4) and UDP...... a mean for enhancing both N-glycan branching complexity and sialylation with opportunities to generate tailored complex N-glycan structures on therapeutic glycoproteins in the future....
Processing of complex N-glycans in IgG Fc-region is affected by core fucosylation
Castilho, Alexandra; Gruber, Clemens; Thader, Andreas; Oostenbrink, Chris; Pechlaner, Maria; Steinkellner, Herta; Altmann, Friedrich
2015-01-01
We investigated N-glycan processing of immunoglobulin G1 using the monoclonal antibody cetuximab (CxMab), which has a glycosite in the Fab domain in addition to the conserved Fc glycosylation, as a reporter. Three GlcNAc (Gn) terminating bi-antennary glycoforms of CxMab differing in core fucosylation (α1,3- and α1,6-linkage) were generated in a plant-based expression platform. These GnGn, GnGnF3, and GnGnF6 CxMab variants were subjected in vivo to further processing toward sialylation and GlcNAc diversification (bisected and branching structures). Mass spectrometry-based glycan analyses revealed efficient processing of Fab glycans toward envisaged structures. By contrast, Fc glycan processing largely depend on the presence of core fucose. A particularly strong support of glycan processing in the presence of plant-specific core α1,3-fucose was observed. Consistently, molecular modeling suggests changes in the interactions of the Fc carbohydrate chain depending on the presence of core fucose, possibly changing the accessibility. Here, we provide data that reveal molecular mechanisms of glycan processing of IgG antibodies, which may have implications for the generation of glycan-engineered therapeutic antibodies with improved efficacies. PMID:26067753
MCAW-DB: A glycan profile database capturing the ambiguity of glycan recognition patterns.
Hosoda, Masae; Takahashi, Yushi; Shiota, Masaaki; Shinmachi, Daisuke; Inomoto, Renji; Higashimoto, Shinichi; Aoki-Kinoshita, Kiyoko F
2018-05-11
Glycan-binding protein (GBP) interaction experiments, such as glycan microarrays, are often used to understand glycan recognition patterns. However, oftentimes the interpretation of glycan array experimental data makes it difficult to identify discrete GBP binding patterns due to their ambiguity. It is known that lectins, for example, are non-specific in their binding affinities; the same lectin can bind to different monosaccharides or even different glycan structures. In bioinformatics, several tools to mine the data generated from these sorts of experiments have been developed. These tools take a library of predefined motifs, which are commonly-found glycan patterns such as sialyl-Lewis X, and attempt to identify the motif(s) that are specific to the GBP being analyzed. In our previous work, as opposed to using predefined motifs, we developed the Multiple Carbohydrate Alignment with Weights (MCAW) tool to visualize the state of the glycans being recognized by the GBP under analysis. We previously reported on the effectiveness of our tool and algorithm by analyzing several glycan array datasets from the Consortium of Functional Glycomics (CFG). In this work, we report on our analysis of 1081 data sets which we collected from the CFG, the results of which we have made publicly and freely available as a database called MCAW-DB. We introduce this database, its usage and describe several analysis results. We show how MCAW-DB can be used to analyze glycan-binding patterns of GBPs amidst their ambiguity. For example, the visualization of glycan-binding patterns in MCAW-DB show how they correlate with the concentrations of the samples used in the array experiments. Using MCAW-DB, the patterns of glycans found to bind to various GBP-glycan binding proteins are visualized, indicating the binding "environment" of the glycans. Thus, the ambiguity of glycan recognition is numerically represented, along with the patterns of monosaccharides surrounding the binding region. The
Harvey, David J; Crispin, Max; Moffatt, Beryl E; Smith, Sylvia L; Sim, Robert B; Rudd, Pauline M; Dwek, Raymond A
2009-11-01
MALDI-TOF mass spectrometry, negative ion nano-electrospray MS/MS and exoglycosidase digestion were used to identify 36 N-linked glycans from 19S IgM heavy chain derived from the nurse shark (Ginglymostoma cirratum). The major glycan was the high-mannose compound, Man(6)GlcNAc(2) accompanied by small amounts of Man(5)GlcNAc(2), Man(7)GlcNAc(2) and Man(8)GlcNAc(2). Bi- and tri-antennary (isomer with a branched 3-antenna) complex-type glycans were also abundant, most contained a bisecting GlcNAc residue (beta1-->4-linked to the central mannose) and with varying numbers of alpha-galactose residues capping the antennae. Small amounts of monosialylated glycans were also found. This appears to be the first comprehensive study of glycosylation in this species of animal. The glycosylation pattern has implications for the mechanism of activation of the complement system by nurse shark IgM.
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Shunji Natsuka
Full Text Available Glycan Atlas is a set of glycan maps over the whole body of an organism. The glycan map that includes data of glycan structure and quantity displays micro-heterogeneity of the glycans in a tissue, an organ, or cells. The two-dimensional glycan mapping is widely used for structure analysis of N-linked oligosaccharides on glycoproteins. In this study we developed a comprehensive method for the mapping of both N- and O-glycans with and without sialic acid. The mapping data of 150 standard pyridylaminated glycans were collected. The empirical additivity rule which was proposed in former reports was able to adapt for this extended glycan map. The adapted rule is that the elution time of pyridylamino glycans on high performance liquid chromatography (HPLC is expected to be the simple sum of the partial elution times assigned to each monosaccharide residue. The comprehensive mapping method developed in this study is a powerful tool for describing the micro-heterogeneity of the glycans. Furthermore, we prepared 42 pyridylamino (PA- glycans from human serum and were able to draw the map of human serum N- and O-glycans as an initial step of Glycan Atlas editing.
Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides.
Pan, Xuefang; De Aragão, Camila De Britto Pará; Velasco-Martin, Juan P; Priestman, David A; Wu, Harry Y; Takahashi, Kohta; Yamaguchi, Kazunori; Sturiale, Luisella; Garozzo, Domenico; Platt, Frances M; Lamarche-Vane, Nathalie; Morales, Carlos R; Miyagi, Taeko; Pshezhetsky, Alexey V
2017-08-01
Gangliosides (sialylated glycolipids) play an essential role in the CNS by regulating recognition and signaling in neurons. Metabolic blocks in processing and catabolism of gangliosides result in the development of severe neurologic disorders, including gangliosidoses manifesting with neurodegeneration and neuroinflammation. We demonstrate that 2 mammalian enzymes, neuraminidases 3 and 4, play important roles in catabolic processing of brain gangliosides by cleaving terminal sialic acid residues in their glycan chains. In neuraminidase 3 and 4 double-knockout mice, G M3 ganglioside is stored in microglia, vascular pericytes, and neurons, causing micro- and astrogliosis, neuroinflammation, accumulation of lipofuscin bodies, and memory loss, whereas their cortical and hippocampal neurons have lower rate of neuritogenesis in vitro Double-knockout mice also have reduced levels of G M1 ganglioside and myelin in neuronal axons. Furthermore, neuraminidase 3 deficiency drastically increased storage of G M2 in the brain tissues of an asymptomatic mouse model of Tay-Sachs disease, a severe human gangliosidosis, indicating that this enzyme is responsible for the metabolic bypass of β-hexosaminidase A deficiency. Together, our results provide the first in vivo evidence that neuraminidases 3 and 4 have important roles in CNS function by catabolizing gangliosides and preventing their storage in lipofuscin bodies.-Pan, X., De Britto Pará De Aragão, C., Velasco-Martin, J. P., Priestman, D. A., Wu, H. Y., Takahashi, K., Yamaguchi, K., Sturiale, L., Garozzo, D., Platt, F. M., Lamarche-Vane, N., Morales, C. R., Miyagi, T., Pshezhetsky, A. V. Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides. © FASEB.
Jo, Sunhwan; Lee, Hui Sun; Skolnick, Jeffrey; Im, Wonpil
2013-01-01
Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures.
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Sunhwan Jo
Full Text Available Understanding glycan structure and dynamics is central to understanding protein-carbohydrate recognition and its role in protein-protein interactions. Given the difficulties in obtaining the glycan's crystal structure in glycoconjugates due to its flexibility and heterogeneity, computational modeling could play an important role in providing glycosylated protein structure models. To address if glycan structures available in the PDB can be used as templates or fragments for glycan modeling, we present a survey of the N-glycan structures of 35 different sequences in the PDB. Our statistical analysis shows that the N-glycan structures found on homologous glycoproteins are significantly conserved compared to the random background, suggesting that N-glycan chains can be confidently modeled with template glycan structures whose parent glycoproteins share sequence similarity. On the other hand, N-glycan structures found on non-homologous glycoproteins do not show significant global structural similarity. Nonetheless, the internal substructures of these N-glycans, particularly, the substructures that are closer to the protein, show significantly similar structures, suggesting that such substructures can be used as fragments in glycan modeling. Increased interactions with protein might be responsible for the restricted conformational space of N-glycan chains. Our results suggest that structure prediction/modeling of N-glycans of glycoconjugates using structure database could be effective and different modeling approaches would be needed depending on the availability of template structures.
Physiological significance of Fuc and Sialic acid containing glycans in the body
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Muhammad Ramzan Manwar Hussain
2016-09-01
Full Text Available Complex biomolecular machinery carrying diverse glycan chains are involved in a wide range of physiological activities including blood group determination, cancer recognition protein stabilization and sperm–egg interaction. Diversity of glycan chains, linked to lipids and proteins is due to isomeric and conformational modifications of various sugar residues, giving rise to unique carbohydrate structures with a wide range of anomeric linkages. This unique and significant structural diversity of naturally occurring oligosaccharide structures make them the best recognition markers for countless physiological activities. This is a challenging task to explore the relationship between biological processes and stereochemical behavior of sugar residues. Current review article is related with the physiological significance of glycans carrying fucose and/or sialic residues in complex biomolecular assemblies. Both the sugar units have a diverse range of anomery and linkages with the penultimate sugars. The existing literature and databases did not contain comprehensive information regarding structure–function relationship of glycans. Therefore, the current study is scheduled to debate on the structure–function relationship of glycans carrying Fuc and sialic acid in their backbone structures.
Glycans in Medicinal Chemistry: An Underexploited Resource.
Fernández-Tejada, Alberto; Cañada, F Javier; Jiménez-Barbero, Jesús
2015-08-01
The biological relevance of glycans as mediators of key physiological processes, including disease-related mechanisms, makes them attractive targets for a wide range of medical applications. Despite their important biological roles, especially as molecular recognition elements, carbohydrates have not been fully exploited as therapeutics mainly due to the scarcity of structure-activity correlations and their non-drug-like properties. A more detailed understanding of the complex carbohydrate structures and their associated functions should contribute to the development of new glycan-based pharmaceuticals. Recent significant progress in oligosaccharide synthesis and chemical glycobiology has renewed the interest of the medicinal chemistry community in carbohydrates. This promises to increase our possibilities to harness them in drug discovery efforts for the development of new and more effective, synthetic glycan-based therapeutics and vaccines. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Yamada, Keita; Hirabayashi, Jun; Kakehi, Kazuaki
2013-03-19
A method is proposed for the analysis of O-glycans as 9-fluorenylmethyl (Fmoc) derivatives. After releasing the O-glycans from the protein backbone in the presence of ammonia-based media, the glycosylamines thus formed are conveniently labeled with Fmoc-Cl and analyzed by HPLC and MALDI-TOF MS after easy purification. Fmoc labeled O-glycans showed 3.5 times higher sensitivities than those labeled with 2-aminobenzoic acid in fluorescent detection. Various types of O-glycans having sialic acids, fucose, and/or sulfate residues were successfully labeled with Fmoc and analyzed by HPLC and MALDI-TOF MS. The method was applied to the comprehensive analysis of O-glycans expressed on MKN45 cells (human gastric adenocarcinoma). In addition, Fmoc-derivatized O-glycans were easily converted to free hemiacetal or glycosylamine-form glycans that are available for fabrication of glycan array and neoglycoproteins. To demonstrate the availability of our methods, we fabricate the glycan array with Fmoc labeled glycans derived from mucin samples and cancer cells. The model studies using the glycan array showed clear interactions between immobilized glycans and some lectins.
Global site-specific analysis of glycoprotein N-glycan processing.
Cao, Liwei; Diedrich, Jolene K; Ma, Yuanhui; Wang, Nianshuang; Pauthner, Matthias; Park, Sung-Kyu Robin; Delahunty, Claire M; McLellan, Jason S; Burton, Dennis R; Yates, John R; Paulson, James C
2018-06-01
N-glycans contribute to the folding, stability and functions of the proteins they decorate. They are produced by transfer of the glycan precursor to the sequon Asn-X-Thr/Ser, followed by enzymatic trimming to a high-mannose-type core and sequential addition of monosaccharides to generate complex-type and hybrid glycans. This process, mediated by the concerted action of multiple enzymes, produces a mixture of related glycoforms at each glycosite, making analysis of glycosylation difficult. To address this analytical challenge, we developed a robust semiquantitative mass spectrometry (MS)-based method that determines the degree of glycan occupancy at each glycosite and the proportion of N-glycans processed from high-mannose type to complex type. It is applicable to virtually any glycoprotein, and a complete analysis can be conducted with 30 μg of protein. Here, we provide a detailed description of the method that includes procedures for (i) proteolytic digestion of glycoprotein(s) with specific and nonspecific proteases; (ii) denaturation of proteases by heating; (iii) sequential treatment of the glycopeptide mixture with two endoglycosidases, Endo H and PNGase F, to create unique mass signatures for the three glycosylation states; (iv) LC-MS/MS analysis; and (v) data analysis for identification and quantitation of peptides for the three glycosylation states. Full coverage of site-specific glycosylation of glycoproteins is achieved, with up to thousands of high-confidence spectra hits for each glycosite. The protocol can be performed by an experienced technician or student/postdoc with basic skills for proteomics experiments and takes ∼7 d to complete.
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M Kristen Hall
Full Text Available Glycoconjugates at the cell surface are crucial for cells to communicate with each other and the extracellular microenvironment. While it is generally accepted that glycans are vectorial biopolymers, their information content is unclear. This report provides evidence that distinct N-glycan structures influence the spatial arrangement of two integral membrane glycoproteins, Kv3.1 and E-cadherin, at the adherent membrane which in turn alter cellular properties. Distinct N-glycan structures were generated by heterologous expression of these glycoproteins in parental and glycosylation mutant Chinese hamster ovary cell lines. Unlike the N-linked glycans, the O-linked glycans of the mutant cell lines are similar to those of the parental cell line. Western and lectin blots of total membranes and GFP immunopurified samples, combined with glycosidase digestion reactions, were employed to verify the glycoproteins had predominantly complex, oligomannose, and bisecting type N-glycans from Pro(-5, Lec1, and Lec10B cell lines, respectively. Based on total internal reflection fluorescence and differential interference contrast microscopy techniques, and cellular assays of live parental and glycosylation mutant CHO cells, we propose that glycoproteins with complex, oligomannose or bisecting type N-glycans relay information for localization of glycoproteins to various regions of the plasma membrane in both a glycan-specific and protein-specific manner, and furthermore cell-cell interactions are required for deciphering much of this information. These distinct spatial arrangements also impact cell adhesion and migration. Our findings provide direct evidence that N-glycan structures of glycoproteins contribute significantly to the information content of cells.
Self-recognition of high-mannose type glycans mediating adhesion of embryonal fibroblasts.
Yoon, Seon-Joo; Utkina, Natalia; Sadilek, Martin; Yagi, Hirokazu; Kato, Koichi; Hakomori, Sen-itiroh
2013-07-01
High-mannose type N-linked glycan with 6 mannosyl residues, termed "M6Gn2", displayed clear binding to the same M6Gn2, conjugated with ceramide mimetic (cer-m) and incorporated in liposome, or coated on polystyrene plates. However, the conjugate of M6Gn2-cer-m did not interact with complex-type N-linked glycan with various structures having multiple GlcNAc termini, conjugated with cer-m. The following observations indicate that hamster embryonic fibroblast NIL-2 K cells display homotypic autoadhesion, mediated through the self-recognition capability of high-mannose type glycans expressed on these cells: (i) NIL-2 K cells display clear binding to lectins capable of binding to high-mannose type glycans (e.g., ConA), but not to other lectins capable of binding to other carbohydrates (e.g. GS-II). (ii) NIL-2 K cells adhere strongly to plates coated with M6Gn2-cer-m, but not to plates coated with complex-type N-linked glycans having multiple GlcNAc termini, conjugated with cer-m; (iii) degree of NIL-2 K cell adhesion to plates coated with M6Gn2-cer-m showed a clear dose-dependence on the amount of M6Gn2-cer-m; and (iv) the degree of NIL-2 K adhesion to plates coated with M6Gn2-cer-m was inhibited in a dose-dependent manner by α1,4-L-mannonolactone, the specific inhibitor in high-mannose type glycans addition. These data indicate that adhesion of NIL-2 K is mediated by self-aggregation of high mannose type glycan. Further studies are to be addressed on auto-adhesion of other types of cells based on self interaction of high mannose type glycans.
Improve accuracy and sensibility in glycan structure prediction by matching glycan isotope abundance
International Nuclear Information System (INIS)
Xu Guang; Liu Xin; Liu Qingyan; Zhou Yanhong; Li Jianjun
2012-01-01
Highlights: ► A glycan isotope pattern recognition strategy for glycomics. ► A new data preprocessing procedure to detect ion peaks in a giving MS spectrum. ► A linear soft margin SVM classification for isotope pattern recognition. - Abstract: Mass Spectrometry (MS) is a powerful technique for the determination of glycan structures and is capable of providing qualitative and quantitative information. Recent development in computational method offers an opportunity to use glycan structure databases and de novo algorithms for extracting valuable information from MS or MS/MS data. However, detecting low-intensity peaks that are buried in noisy data sets is still a challenge and an algorithm for accurate prediction and annotation of glycan structures from MS data is highly desirable. The present study describes a novel algorithm for glycan structure prediction by matching glycan isotope abundance (mGIA), which takes isotope masses, abundances, and spacing into account. We constructed a comprehensive database containing 808 glycan compositions and their corresponding isotope abundance. Unlike most previously reported methods, not only did we take into count the m/z values of the peaks but also their corresponding logarithmic Euclidean distance of the calculated and detected isotope vectors. Evaluation against a linear classifier, obtained by training mGIA algorithm with datasets of three different human tissue samples from Consortium for Functional Glycomics (CFG) in association with Support Vector Machine (SVM), was proposed to improve the accuracy of automatic glycan structure annotation. In addition, an effective data preprocessing procedure, including baseline subtraction, smoothing, peak centroiding and composition matching for extracting correct isotope profiles from MS data was incorporated. The algorithm was validated by analyzing the mouse kidney MS data from CFG, resulting in the identification of 6 more glycan compositions than the previous annotation
CROSSWORK for Glycans: Glycan Identificatin Through Mass Spectrometry and Bioinformatics
DEFF Research Database (Denmark)
Rasmussen, Morten; Thaysen-Andersen, Morten; Højrup, Peter
We have developed "GLYCANthrope " - CROSSWORKS for glycans: a bioinformatics tool, which assists in identifying N-linked glycosylated peptides as well as their glycan moieties from MS2 data of enzymatically digested glycoproteins. The program runs either as a stand-alone application or as a plug...
Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis.
Mun, Ji-Young; Lee, Kyung Jin; Seo, Hoon; Sung, Min-Sun; Cho, Yee Sook; Lee, Seung-Goo; Kwon, Ohsuk; Oh, Doo-Byoung
2013-08-06
Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.
Reductive Alkaline Release of N-Glycans Generates a Variety of Unexpected, Useful Products.
Figl, Rudolf; Altmann, Friedrich
2018-02-01
Release of O-glycans by reductive β-elimination has become routine in many glyco-analytical laboratories and concomitant release of N-glycans has repeatedly been observed. Revisiting this somewhat forgotten mode of N-glycan release revealed that all kinds of N-glycans including oligomannosidic and complex-type N-glycans from plants with 3-linked fucose and from mammals with or without 6-linked fucose and with sialic acid could be recovered. However, the mass spectra of the obtained products revealed very surprising facts. Even after 16 h incubation in 1 M sodium borohydride, a large part of the glycans occurred in reducing form. Moreover, about one third emerged in the form of the stable amino-functionalized 1-amino-1-deoxy-glycitol. When avoiding acidic conditions, considerable amounts of glycosylamine were observed. In addition, a compound with a reduced asparagine and de-N-acetylation products, in particular of sialylated glycans, was seen. The relative yields of the products reducing glycosylamine, reducing N-glycan, 1-amino-1-deoxy-glycitol or glycitol could be controlled by the release conditions, foremost by temperature and borohydride concentration. Thus, chemical release of N-glycans constitutes a cost-saving alternative to enzymatic hydrolysis for the preparation of precursors for the production of reference compounds for various formats of N-glycan analysis. Moreover, it allows to obtain a stable amino-functionalized glycan derivative, which can be employed to construct glycan arrays or affinity matrices. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Song, Xuezheng; Johns, Brian A.; Ju, Hong; Lasanajak, Yi; Zhao, Chunmei; Smith, David F.; Cummings, Richard D.
2013-01-01
Glycans that are fluorescently tagged by reductive amination have been useful for functional glycomic studies. However, the existing tags can introduce unwanted properties to the glycans and complicate structural and functional studies. Here we describe a facile method using N-bromosuccinimide (NBS) to remove the tags and efficiently regenerate free reducing glycans. The regenerated free reducing glycans can be easily analyzed by routine mass spectrometry or re-tagged with different tags for ...
van Diepen, Angela; van der Plas, Arend-Jan; Kozak, Radoslaw P; Royle, Louise; Dunne, David W; Hokke, Cornelis H
2015-06-01
Upon infection with Schistosoma, antibody responses are mounted that are largely directed against glycans. Over the last few years significant progress has been made in characterising the antigenic properties of N-glycans of Schistosoma mansoni. Despite also being abundantly expressed by schistosomes, much less is understood about O-glycans and antibody responses to these have not yet been systematically analysed. Antibody binding to schistosome glycans can be analysed efficiently and quantitatively using glycan microarrays, but O-glycan array construction and exploration is lagging behind because no universal O-glycanase is available, and release of O-glycans has been dependent on chemical methods. Recently, a modified hydrazinolysis method has been developed that allows the release of O-glycans with free reducing termini and limited degradation, and we applied this method to obtain O-glycans from different S. mansoni life stages. Two-dimensional HPLC separation of 2-aminobenzoic acid-labelled O-glycans generated 362 O-glycan-containing fractions that were printed on an epoxide-modified glass slide, thereby generating the first shotgun O-glycan microarray containing naturally occurring schistosome O-glycans. Monoclonal antibodies and mass spectrometry showed that the O-glycan microarray contains well-known antigenic glycan motifs as well as numerous other, potentially novel, antibody targets. Incubations of the microarrays with sera from Schistosoma-infected humans showed substantial antibody responses to O-glycans in addition to those observed to the previously investigated N- and glycosphingolipid glycans. This underlines the importance of the inclusion of these often schistosome-specific O-glycans in glycan antigen studies and indicates that O-glycans contain novel antigenic motifs that have potential for use in diagnostic methods and studies aiming at the discovery of vaccine targets. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights
Disruption of O-GlcNAc cycling in C. elegans perturbs Nucleotide Sugar pools and Complex Glycans
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Salil K Ghosh
2014-11-01
Full Text Available The carbohydrate modification of serine and threonine residues with O-linked beta-N-acetylglucosamine (O-GlcNAc is ubiquitous and governs cellular processes ranging from cell signaling to apoptosis. The O-GlcNAc modification along with other carbohydrate modifications, including N-linked and O-linked glycans, glycolipids, and sugar polymers, all require the use of the nucleotide sugar UDP-GlcNAc, the end product of the hexosamine biosynthetic pathway. In this paper, we describe the biochemical consequences resulting from perturbation of the O-GlcNAc pathway in C. elegans lacking O-GlcNAc transferase and O-GlcNAcase activities. In ogt-1 null animals, steady-state levels of UDP-GlcNAc/UDP-GalNAc and UDP-glucose were substantially elevated. Transcripts of genes encoding for key members in the Hexosamine Biosynthetic Pathway (gfat-2, gna-2, C36A4.4 and trehalose metabolism (tre-1, tre-2, and tps-2 were elevated in ogt-1 null animals. While there is no evidence to suggest changes in the profile of N-linked glycans in the ogt-1 and oga-1 mutants, glycans insensitive to PNGase digestion (including O-linked glycans, glycolipids, and glycopolymers were altered in these strains. Our data supports that changes in O-GlcNAcylation alters nucleotide sugar production, overall glycan composition, and transcription of genes encoding glycan processing enzymes. These data along with our previous findings that disruption in O-GlcNAc cycling alters macronutrient storage underscores the noteworthy influence this posttranslational modification plays in nutrient sensing.
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Feng, Hua-tao [Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 (Singapore); NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, Singapore 117411 (Singapore); Su, Min; Rifai, Farida Nur [Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 (Singapore); Li, Pingjing [NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, Singapore 117411 (Singapore); Li, Sam F.Y., E-mail: chmlifys@nus.edu.sg [Department of Chemistry, National University of Singapore, 3 Science Drive 3, Singapore 117543 (Singapore); NUS Environmental Research Institute, 5A Engineering Drive 1, T-Lab Building, Singapore 117411 (Singapore)
2017-02-08
The deep involvement of glycans or carbohydrate moieties in biological processes makes glycan patterns an important direction for the clinical and medicine researches. A multiplexing CE mapping method for glycan analysis was developed in this study. By applying different CE separation mechanisms, the potential of combined parallel applications of capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) and capillary gel electrophoresis (CGE) for rapid and accurate identification of glycan was investigated. The combination of CZE and MEKC demonstrated enhancing chromatography separation capacity without the compromises of sample pre-treatment and glycan concentration. The separation mechanisms for multiplexing platform were selected based on the orthogonalities of the separation of glycan standards. MEKC method exhibited promising ability for the analysis of small GU value glycans and thus complementing the unavailability of CZE. The method established required only small amount of samples, simple instrument and single fluorescent labelling for sensitive detection. This integrated method can be used to search important glycan patterns appearing in biopharmaceutical products and other glycoproteins with clinical importance. - Highlights: • Cross-validation of analytes in complex samples was done with different CE separation mechanisms. • A simple strategy is used to confirm peak identification and extend capacity of CE separation. • The method uses small amount of sample, simple instrument and single fluorescent labeling. • Selection of mechanisms is based on orthogonalities of GU values of glycan standards. • Micellar electrokinetic chromatography was suitable for analysis of small or highly sialylated glycans.
International Nuclear Information System (INIS)
Feng, Hua-tao; Su, Min; Rifai, Farida Nur; Li, Pingjing; Li, Sam F.Y.
2017-01-01
The deep involvement of glycans or carbohydrate moieties in biological processes makes glycan patterns an important direction for the clinical and medicine researches. A multiplexing CE mapping method for glycan analysis was developed in this study. By applying different CE separation mechanisms, the potential of combined parallel applications of capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC) and capillary gel electrophoresis (CGE) for rapid and accurate identification of glycan was investigated. The combination of CZE and MEKC demonstrated enhancing chromatography separation capacity without the compromises of sample pre-treatment and glycan concentration. The separation mechanisms for multiplexing platform were selected based on the orthogonalities of the separation of glycan standards. MEKC method exhibited promising ability for the analysis of small GU value glycans and thus complementing the unavailability of CZE. The method established required only small amount of samples, simple instrument and single fluorescent labelling for sensitive detection. This integrated method can be used to search important glycan patterns appearing in biopharmaceutical products and other glycoproteins with clinical importance. - Highlights: • Cross-validation of analytes in complex samples was done with different CE separation mechanisms. • A simple strategy is used to confirm peak identification and extend capacity of CE separation. • The method uses small amount of sample, simple instrument and single fluorescent labeling. • Selection of mechanisms is based on orthogonalities of GU values of glycan standards. • Micellar electrokinetic chromatography was suitable for analysis of small or highly sialylated glycans.
Song, Xuezheng; Johns, Brian A; Ju, Hong; Lasanajak, Yi; Zhao, Chunmei; Smith, David F; Cummings, Richard D
2013-11-15
Glycans that are fluorescently tagged by reductive amination have been useful for functional glycomic studies. However, the existing tags can introduce unwanted properties to the glycans and complicate structural and functional studies. Here, we describe a facile method using N-bromosuccinimide (NBS) to remove the tags and efficiently regenerate free reducing glycans. The regenerated free reducing glycans can be easily analyzed by routine mass spectrometry or retagged with different tags for further studies. This new method can be used to efficiently remove a variety of fluorescent tags installed by reductive amination, including 2-aminobenzoic acid and 2-aminopyridine. NBS treatment essentially transforms the commonly used 2-aminobenzoic linkage to a cleavable linkage. It can be used to cleave printed glycans from microarrays and cleave neoglycopeptides containing a 2-aminobenzoic linker.
Effects of lipopolysaccharide on the catabolic activity of macrophages
International Nuclear Information System (INIS)
Cluff, C.; Ziegler, H.K.
1986-01-01
The ability of macrophages to degrade and catabolize antigens is of relevance both as a means to process complex antigens prior to presentation to T cells, as well as a way to down regulate immune responses by destroying the antigenicity of polypeptides. With these considerations, the authors have investigated the regulation of macrophage catabolic activity by lipopolysaccharide (LPS). Catabolic activity was quantitated by following the distribution and molecular form of 125 -I labelled surface components of heat-killed Listeria monocytogenes (HKLM) subsequent to their uptake by macrophages. They have compared the catabolic activity of macrophages from peritoneal exudates of mice injected i.p. with saline or LPS and have found that LPS-elicited macrophages display a greatly enhanced (3 fold) rate of catabolism. This increase in catabolic activity peaks 3 days after LPS injection and steadily declines thereafter, approaching a baseline level after 3 weeks. The enhancement of catabolic activity is under LPS gene control. LPS-elicited macrophages rapidly destroy the antigenicity of bacterial antigens and function poorly as antigen presenting cells in vitro. These results suggest that LPS elicits a macrophage population specialized for antigen degradation functions with negative regulatory effects on the induction of specific immune responses
Structural basis for diverse N-glycan recognition by HIV-1-neutralizing V1-V2-directed antibody PG16
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Pancera, Marie; Shahzad-ul-Hussan, Syed; Doria-Rose, Nicole A.; McLellan, Jason S.; Bailer, Robert T.; Dai, Kaifan; Loesgen, Sandra; Louder, Mark K.; Staupe, Ryan P.; Yang, Yongping; Zhang, Baoshan; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Blinn, Julie; Alam, S. Munir; Haynes, Barton F.; Amin, Mohammed N.; Wang, Lai-Xi; Burton, Dennis R.; Koff, Wayne C.; Nabel, Gary J.; Mascola, John R.; Bewley, Carole A; Kwong, Peter D. [NIH; (Scripps); (Duke); (Maryland-MED); (IAVI)
2013-08-05
HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1–V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1–V2, showing an epitope comprising both high mannose–type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1–glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.
Mucin glycan foraging in the human gut microbiome
Tailford, Louise E.; Crost, Emmanuelle H.; Kavanaugh, Devon; Juge, Nathalie
2015-01-01
The availability of host and dietary carbohydrates in the gastrointestinal (GI) tract plays a key role in shaping the structure-function of the microbiota. In particular, some gut bacteria have the ability to forage on glycans provided by the mucus layer covering the GI tract. The O-glycan structures present in mucin are diverse and complex, consisting predominantly of core 1-4 mucin-type O-glycans containing α- and β- linked N-acetyl-galactosamine, galactose and N-acetyl-glucosamine. These core structures are further elongated and frequently modified by fucose and sialic acid sugar residues via α1,2/3/4 and α2,3/6 linkages, respectively. The ability to metabolize these mucin O-linked oligosaccharides is likely to be a key factor in determining which bacterial species colonize the mucosal surface. Due to their proximity to the immune system, mucin-degrading bacteria are in a prime location to influence the host response. However, despite the growing number of bacterial genome sequences available from mucin degraders, our knowledge on the structural requirements for mucin degradation by gut bacteria remains fragmented. This is largely due to the limited number of functionally characterized enzymes and the lack of studies correlating the specificity of these enzymes with the ability of the strain to degrade and utilize mucin and mucin glycans. This review focuses on recent findings unraveling the molecular strategies used by mucin-degrading bacteria to utilize host glycans, adapt to the mucosal environment, and influence human health. PMID:25852737
Nishikaze, Takashi; Kaneshiro, Kaoru; Kawabata, Shin-ichirou; Tanaka, Koichi
2012-11-06
Negative-ion fragmentation of underivatized N-glycans has been proven to be more informative than positive-ion fragmentation. Fluorescent labeling via reductive amination is often employed for glycan analysis, but little is known about the influence of the labeling group on negative-ion fragmentation. We previously demonstrated that the on-target glycan-labeling method using 3-aminoquinoline/α-cyano-4-hydroxycinnamic acid (3AQ/CHCA) liquid matrix enables highly sensitive, rapid, and quantitative N-glycan profiling analysis. The current study investigates the suitability of 3AQ-labeled N-glycans for structural analysis based on negative-ion collision-induced dissociation (CID) spectra. 3AQ-labeled N-glycans exhibited simple and informative CID spectra similar to those of underivatized N-glycans, with product ions due to cross-ring cleavages of the chitobiose core and ions specific to two antennae (D and E ions). The interpretation of diagnostic fragment ions suggested for underivatized N-glycans could be directly applied to the 3AQ-labeled N-glycans. However, fluorescently labeled N-glycans by conventional reductive amination, such as 2-aminobenzamide (2AB)- and 2-pyrydilamine (2PA)-labeled N-glycans, exhibited complicated CID spectra consisting of numerous signals formed by dehydration and multiple cleavages. The complicated spectra of 2AB- and 2PA-labeled N-glycans was found to be due to their open reducing-terminal N-acetylglucosamine (GlcNAc) ring, rather than structural differences in the labeling group in the N-glycan derivative. Finally, as an example, the on-target 3AQ labeling method followed by negative-ion CID was applied to structurally analyze neutral N-glycans released from human epidermal growth factor receptor type 2 (HER2) protein. The glycan-labeling method using 3AQ-based liquid matrix should facilitate highly sensitive quantitative and qualitative analyses of glycans.
21 CFR 172.898 - Bakers yeast glycan.
2010-04-01
... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Bakers yeast glycan. 172.898 Section 172.898 Food... Multipurpose Additives § 172.898 Bakers yeast glycan. Bakers yeast glycan may be safely used in food in accordance with the following conditions: (a) Bakers yeast glycan is the comminuted, washed, pasteurized, and...
Pochechueva, Tatiana; Chinarev, Alexander; Schoetzau, Andreas; Fedier, André; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola
2016-01-01
Altered levels of naturally occurring anti-glycan antibodies (AGA) circulating in human blood plasma are found in different pathologies including cancer. Here the levels of AGA directed against 22 negatively charged (sialylated and sulfated) glycans were assessed in high-grade serous ovarian cancer (HGSOC, n = 22) patients and benign controls (n = 31) using our previously developed suspension glycan array (SGA). Specifically, the ability of AGA to differentiate between controls and HGSOC, the most common and aggressive type of ovarian cancer with a poor outcome was determined. Results were compared to CA125, the commonly used ovarian cancer biomarker. AGA to seven glycans that significantly (P<0.05) differentiated between HGSOC and control were identified: AGA to top candidates SiaTn and 6-OSulfo-TF (both IgM) differentiated comparably to CA125. The area under the curve (AUC) of a panel of AGA to 5 glycans (SiaTn, 6-OSulfo-TF, 6-OSulfo-LN, SiaLea, and GM2) (0.878) was comparable to CA125 (0.864), but it markedly increased (0.985) when combined with CA125. AGA to SiaTn and 6-OSulfo-TF were also valuable predictors for HGSOC when CA125 values appeared inconclusive, i.e. were below a certain threshold. AGA-glycan binding was in some cases isotype-dependent and sensitive to glycosidic linkage switch (α2-6 vs. α2-3), to sialylation, and to sulfation of the glycans. In conclusion, plasma-derived AGA to sialylated and sulfated glycans including SiaTn and 6-OSulfo-TF detected by SGA present a valuable alternative to CA125 for differentiating controls from HGSOC patients and for predicting the likelihood of HGSOC, and may be potential HGSOC tumor markers.
Pochechueva, Tatiana; Jacob, Francis; Goldstein, Darlene R; Huflejt, Margaret E; Chinarev, Alexander; Caduff, Rosemarie; Fink, Daniel; Hacker, Neville; Bovin, Nicolai V; Heinzelmann-Schwarz, Viola
2011-12-01
Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection. © The Author(s) 2011. This article is published with open access at Springerlink.com
Prien, Justin M; Prater, Bradley D; Qin, Qiang; Cockrill, Steven L
2010-02-15
Fast, sensitive, robust methods for "high-level" glycan screening are necessary during various stages of a biotherapeutic product's lifecycle, including clone selection, process changes, and quality control for lot release testing. Traditional glycan screening involves chromatographic or electrophoretic separation-based methods, and, although reproducible, these methods can be time-consuming. Even ultrahigh-performance chromatographic and microfluidic integrated LC/MS systems, which work on the tens of minute time scale, become lengthy when hundreds of samples are to be analyzed. Comparatively, a direct infusion mass spectrometry (MS)-based glycan screening method acquires data on a millisecond time scale, exhibits exquisite sensitivity and reproducibility, and is amenable to automated peak annotation. In addition, characterization of glycan species via sequential mass spectrometry can be performed simultaneously. Here, we demonstrate a quantitative high-throughput MS-based mapping approach using stable isotope 2-aminobenzoic acid (2-AA) for rapid "high-level" glycan screening.
Comprehensive functional analysis of N-linked glycans on Ebola virus GP1.
Lennemann, Nicholas J; Rhein, Bethany A; Ndungo, Esther; Chandran, Kartik; Qiu, Xiangguo; Maury, Wendy
2014-01-28
Ebola virus (EBOV) entry requires the virion surface-associated glycoprotein (GP) that is composed of a trimer of heterodimers (GP1/GP2). The GP1 subunit contains two heavily glycosylated domains, the glycan cap and the mucin-like domain (MLD). The glycan cap contains only N-linked glycans, whereas the MLD contains both N- and O-linked glycans. Site-directed mutagenesis was performed on EBOV GP1 to systematically disrupt N-linked glycan sites to gain an understanding of their role in GP structure and function. All 15 N-glycosylation sites of EBOV GP1 could be removed without compromising the expression of GP. The loss of these 15 glycosylation sites significantly enhanced pseudovirion transduction in Vero cells, which correlated with an increase in protease sensitivity. Interestingly, exposing the receptor-binding domain (RBD) by removing the glycan shield did not allow interaction with the endosomal receptor, NPC1, indicating that the glycan cap/MLD domains mask RBD residues required for binding. The effects of the loss of GP1 N-linked glycans on Ca(2+)-dependent (C-type) lectin (CLEC)-dependent transduction were complex, and the effect was unique for each of the CLECs tested. Surprisingly, EBOV entry into murine peritoneal macrophages was independent of GP1 N-glycans, suggesting that CLEC-GP1 N-glycan interactions are not required for entry into this important primary cell. Finally, the removal of all GP1 N-glycans outside the MLD enhanced antiserum and antibody sensitivity. In total, our results provide evidence that the conserved N-linked glycans on the EBOV GP1 core protect GP from antibody neutralization despite the negative impact the glycans have on viral entry efficiency. Filovirus outbreaks occur sporadically throughout central Africa, causing high fatality rates among the general public and health care workers. These unpredictable hemorrhagic fever outbreaks are caused by multiple species of Ebola viruses, as well as Marburg virus. While filovirus
Wang, Xiaoning; Deng, Zaian; Huang, Chuncui; Zhu, Tong; Lou, Jiatao; Wang, Lin; Li, Yan
2018-02-10
N-glycan profiling is a powerful approach for analyzing the functional relationship between N-glycosylation and cancer. Current methods rely on either serum or fresh tissue samples; however, N-glycan patterns may differ between serum and tissue, as the proteins of serum originate from a variety of tissues. Furthermore, fresh tissue samples are difficult to ship and store. Here, we used a profiling method based on formalin-fixed paraffin-embedded (FFPE) tissue sections from lung adenocarcinoma patients. We found that our method was highly reproducible. We identified 58 N-glycan compositions from lung adenocarcinoma FFPE samples, 51 of which were further used for MS n -based structure prediction. We show that high mannose type N-glycans are upregulated, while sialylated N-glycans are downregulated in our FFPE lung adenocarcinoma samples, compared to the control samples. Our receiver operating characteristic (ROC) curve analysis shows that high mannose type and sialylated N-glycans are useful discriminators to distinguish between lung adenocarcinoma and control tissue. Together, our results indicate that expression levels of specific N-glycans correlate well with lung adenocarcinoma, and strongly suggest that our FFPE-based method will be useful for N-glycan profiling of cancer tissues. Glycosylation is one of the most important post-translational protein modifications, and is associated with several physiopathological processes, including carcinogenesis. In this study, we tested the feasibility of using formalin-fixed paraffin-embedded (FFPE) tissue sections to identify changes in N-glycan patterns and identified the differentially expressed N-glycans of lung adenocarcinoma. Our study shows that the FFPE-based N-glycan profiling method is useful for clinical diagnosis as well as identification of potential biomarkers, and our data expand current knowledge of differential N-glycan patterns of lung adenocarcinoma. Copyright © 2017 Elsevier B.V. All rights reserved.
Smit, Cornelis H; van Diepen, Angela; Nguyen, D Linh; Wuhrer, Manfred; Hoffmann, Karl F; Deelder, André M; Hokke, Cornelis H
2015-07-01
Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles of glycans and antigenic glycan-motifs during a range of critical stages of the complex schistosome lifecycle. We performed a longitudinal profiling study covering schistosome glycosylation throughout worm- and egg-development using a mass spectrometry-based glycomics approach. Our study revealed that during worm development N-glycans with Galβ1-4(Fucα1-3)GlcNAc (LeX) and core-xylose motifs were rapidly lost after cercariae to schistosomula transformation, whereas GalNAcβ1-4GlcNAc (LDN)-motifs gradually became abundant and predominated in adult worms. LeX-motifs were present on glycolipids up to 2 weeks of schistosomula development, whereas glycolipids with mono- and multifucosylated LDN-motifs remained present up to the adult worm stage. In contrast, expression of complex O-glycans diminished to undetectable levels within days after transformation. During egg development, a rich diversity of N-glycans with fucosylated motifs was expressed, but with α3-core fucose and a high degree of multifucosylated antennae only in mature eggs and miracidia. N-glycan antennae were exclusively LDN-based in miracidia. O-glycans in the mature eggs were also diverse and contained LeX- and multifucosylated LDN, but none of these were associated with miracidia in which we detected only the Galβ1-3(Galβ1-6)GalNAc core glycan. Immature eggs also exhibited short O-glycan core structures only, suggesting that complex fucosylated O-glycans of schistosome eggs are derived primarily from glycoproteins produced by the subshell envelope in the developed egg. Lipid glycans with multifucosylated GlcNAc repeats were present throughout egg development, but with the longer highly fucosylated
Protein-linked glycans in periodontal bacteria: prevalence and role at the immune interface
Settem, Rajendra P.; Honma, Kiyonobu; Stafford, Graham P.; Sharma, Ashu
2013-01-01
Protein modification with complex glycans is increasingly being recognized in many pathogenic and non-pathogenic bacteria, and is now thought to be central to the successful life-style of those species in their respective hosts. This review aims to convey current knowledge on the extent of protein glycosylation in periodontal pathogenic bacteria and its role in the modulation of the host immune responses. The available data show that surface glycans of periodontal bacteria orchestrate dendrit...
Kato, Tatsuya; Kikuta, Kotaro; Kanematsu, Ayumi; Kondo, Sachiko; Yagi, Hirokazu; Kato, Koichi; Park, Enoch Y
2017-09-01
To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae. Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man 3 GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins. Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.
Park, Sang-Jun; Lee, Jumin; Patel, Dhilon S; Ma, Hongjing; Lee, Hui Sun; Jo, Sunhwan; Im, Wonpil
2017-10-01
Glycans play a central role in many essential biological processes. Glycan Reader was originally developed to simplify the reading of Protein Data Bank (PDB) files containing glycans through the automatic detection and annotation of sugars and glycosidic linkages between sugar units and to proteins, all based on atomic coordinates and connectivity information. Carbohydrates can have various chemical modifications at different positions, making their chemical space much diverse. Unfortunately, current PDB files do not provide exact annotations for most carbohydrate derivatives and more than 50% of PDB glycan chains have at least one carbohydrate derivative that could not be correctly recognized by the original Glycan Reader. Glycan Reader has been improved and now identifies most sugar types and chemical modifications (including various glycolipids) in the PDB, and both PDB and PDBx/mmCIF formats are supported. CHARMM-GUI Glycan Reader is updated to generate the simulation system and input of various glycoconjugates with most sugar types and chemical modifications. It also offers a new functionality to edit the glycan structures through addition/deletion/modification of glycosylation types, sugar types, chemical modifications, glycosidic linkages, and anomeric states. The simulation system and input files can be used for CHARMM, NAMD, GROMACS, AMBER, GENESIS, LAMMPS, Desmond, OpenMM, and CHARMM/OpenMM. Glycan Fragment Database in GlycanStructure.Org is also updated to provide an intuitive glycan sequence search tool for complex glycan structures with various chemical modifications in the PDB. http://www.charmm-gui.org/input/glycan and http://www.glycanstructure.org. wonpil@lehigh.edu. Supplementary data are available at Bioinformatics online. © The Author (2017). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com
Kashlan, Ossama B; Kinlough, Carol L; Myerburg, Michael M; Shi, Shujie; Chen, Jingxin; Blobner, Brandon M; Buck, Teresa M; Brodsky, Jeffrey L; Hughey, Rebecca P; Kleyman, Thomas R
2018-03-01
Epithelial Na + channel (ENaC) subunits undergo N-linked glycosylation in the endoplasmic reticulum where they assemble into an αβγ complex. Six, 13, and 5 consensus sites (Asn-X-Ser/Thr) for N-glycosylation reside in the extracellular domains of the mouse α-, β-, and γ-subunits, respectively. Because the importance of ENaC N-linked glycans has not been fully addressed, we examined the effect of preventing N-glycosylation of specific subunits on channel function, expression, maturation, and folding. Heterologous expression in Xenopus oocytes or Fischer rat thyroid cells with αβγ-ENaC lacking N-linked glycans on a single subunit reduced ENaC activity as well as the inhibitory response to extracellular Na + . The lack of N-linked glycans on the β-subunit also precluded channel activation by trypsin. However, channel activation by shear stress was N-linked glycan independent, regardless of which subunit was modified. We also discovered that the lack of N-linked glycans on any one subunit reduced the total and surface levels of cognate subunits. The lack of N-linked glycans on the β-subunit had the largest effect on total levels, with the lack of N-linked glycans on the γ- and α-subunits having intermediate and modest effects, respectively. Finally, channels with wild-type β-subunits were more sensitive to limited trypsin proteolysis than channels lacking N-linked glycans on the β-subunit. Our results indicate that N-linked glycans on each subunit are required for proper folding, maturation, surface expression, and function of the channel.
Structural Analysis of N- and O-glycans Using ZIC-HILIC/Dialysis Coupled to NMR Detection
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Qu, Yi; Feng, Ju; Deng, Shuang; Cao, Li; Zhang, Qibin; Zhao, Rui; Zhang, Zhaorui; Jiang, Yuxuan; Zink, Erika M.; Baker, Scott E.; Lipton, Mary S.; Pasa-Tolic, Ljiljana; Hu, Jian Z.; Wu, Si
2014-11-19
Protein glycosylation, an important and complex post-translational modification (PTM), is involved in various biological processes including the receptor-ligand and cell-cell interaction, and plays a crucial role in many biological functions. However, little is known about the glycan structures of important biological complex samples, and the conventional glycan enrichment strategy (i.e., size-exclusion column [SEC] separation,) prior to nuclear magnetic resonance (NMR) detection is time-consuming and tedious. In this study, we employed SEC, Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), and ZIC-HILIC coupled with dialysis strategies to enrich the glycopeptides from the pronase E digests of RNase B, followed by NMR analysis of the glycoconjugate. Our results suggest that the ZIC-HILIC enrichment coupled with dialysis is the most efficient, which was thus applied to the analysis of biological complex sample, the pronase E digest of the secreted proteins from the fungi Aspergillus niger. The NMR spectra revealed that the secreted proteins from A. niger contain both N-linked glycans with a high-mannose core and O-linked glycans bearing mannose and glucose with 1->3 and 1->6 linkages. In all, our study provides compelling evidence that ZIC-HILIC separation coupled to dialysis is superior to the commonly used SEC separation to prepare glycopeptides for the downstream NMR analysis, which could greatly facilitate the future NMR-based glycoproteomics research.
Notable Aspects of Glycan-Protein Interactions
Directory of Open Access Journals (Sweden)
Miriam Cohen
2015-09-01
Full Text Available This mini review highlights several interesting aspects of glycan-mediated interactions that are common between cells, bacteria, and viruses. Glycans are ubiquitously found on all living cells, and in the extracellular milieu of multicellular organisms. They are known to mediate initial binding and recognition events of both immune cells and pathogens with their target cells or tissues. The host target tissues are hidden under a layer of secreted glycosylated decoy targets. In addition, pathogens can utilize and display host glycans to prevent identification as foreign by the host’s immune system (molecular mimicry. Both the host and pathogens continually evolve. The host evolves to prevent infection and the pathogens evolve to evade host defenses. Many pathogens express both glycan-binding proteins and glycosidases. Interestingly, these proteins are often located at the tip of elongated protrusions in bacteria, or in the leading edge of the cell. Glycan-protein interactions have low affinity and, as a result, multivalent interactions are often required to achieve biologically relevant binding. These enable dynamic forms of adhesion mechanisms, reviewed here, and include rolling (cells, stick and roll (bacteria or surfacing (viruses.
Akune, Yukie; Lin, Chi-Hung; Abrahams, Jodie L; Zhang, Jingyu; Packer, Nicolle H; Aoki-Kinoshita, Kiyoko F; Campbell, Matthew P
2016-08-05
Glycan structures attached to proteins are comprised of diverse monosaccharide sequences and linkages that are produced from precursor nucleotide-sugars by a series of glycosyltransferases. Databases of these structures are an essential resource for the interpretation of analytical data and the development of bioinformatics tools. However, with no template to predict what structures are possible the human glycan structure databases are incomplete and rely heavily on the curation of published, experimentally determined, glycan structure data. In this work, a library of 45 human glycosyltransferases was used to generate a theoretical database of N-glycan structures comprised of 15 or less monosaccharide residues. Enzyme specificities were sourced from major online databases including Kyoto Encyclopedia of Genes and Genomes (KEGG) Glycan, Consortium for Functional Glycomics (CFG), Carbohydrate-Active enZymes (CAZy), GlycoGene DataBase (GGDB) and BRENDA. Based on the known activities, more than 1.1 million theoretical structures and 4.7 million synthetic reactions were generated and stored in our database called UniCorn. Furthermore, we analyzed the differences between the predicted glycan structures in UniCorn and those contained in UniCarbKB (www.unicarbkb.org), a database which stores experimentally described glycan structures reported in the literature, and demonstrate that UniCorn can be used to aid in the assignment of ambiguous structures whilst also serving as a discovery database. Copyright © 2016 Elsevier Ltd. All rights reserved.
Directory of Open Access Journals (Sweden)
Y Lei
Full Text Available DENV envelope glycoprotein (E is responsible for interacting with host cell receptors and is the main target for the development of a dengue vaccine based on an induction of neutralizing antibodies. It is well known that DENV E glycoprotein has two potential N-linked glycosylation sites at Asn67 and Asn153. The N-glycans of E glycoprotein have been shown to influence the proper folding of the protein, its cellular localization, its interactions with receptors and its immunogenicity. However, the precise structures of the N-glycans that are attached to E glycoprotein remain elusive, although the crystal structure of DENV E has been determined. This study characterized the structures of envelope protein N-linked glycans on mature DENV-2 particles derived from insect cells via an integrated method that used both lectin microarray and MALDI-TOF-MS. By combining these methods, a high heterogeneity of DENV N-glycans was found. Five types of N-glycan were identified on DENV-2, including mannose, GalNAc, GlcNAc, fucose and sialic acid; high mannose-type N-linked oligosaccharides and the galactosylation of N-glycans were the major structures that were found. Furthermore, a complex between a glycan on DENV and the carbohydrate recognition domain (CRD of DC-SIGN was mimicked with computational docking experiments. For the first time, this study provides a comprehensive understanding of the N-linked glycan profile of whole DENV-2 particles derived from insect cells.
A spin column-free approach to sodium hydroxide-based glycan permethylation.
Hu, Yueming; Borges, Chad R
2017-07-24
Glycan permethylation was introduced as a tool to facilitate the study of glycans in 1903. Since that time, permethylation procedures have been continually modified to improve permethylation efficiency and qualitative applicability. Typically, however, either laborious preparation steps or cumbersome and uneconomical spin columns have been needed to obtain decent permethylation yields on small glycan samples. Here we describe a spin column-free (SCF) glycan permethylation procedure that is applicable to both O- and N-linked glycans and can be employed upstream to intact glycan analysis by MALDI-MS, ESI-MS, or glycan linkage analysis by GC-MS. The SCF procedure involves neutralization of NaOH beads by acidified phosphate buffer, which eliminates the risk of glycan oxidative degradation and avoids the use of spin columns. Optimization of the new permethylation procedure provided high permethylation efficiency for both hexose (>98%) and HexNAc (>99%) residues-yields which were comparable to (or better than) those of some widely-used spin column-based procedures. A light vs. heavy labelling approach was employed to compare intact glycan yields from a popular spin-column based approach to the SCF approach. Recovery of intact N-glycans was significantly better with the SCF procedure (p 0.75; p < 0.01). In summary, the SCF permethylation procedure expedites and economizes both intact glycan analysis and linkage analysis of glycans from whole biospecimens.
A spin column-free approach to sodium hydroxide-based glycan permethylation†
Hu, Yueming; Borges, Chad R.
2018-01-01
Glycan permethylation was introduced as a tool to facilitate the study of glycans in 1903. Since that time, permethylation procedures have been continually modified to improve permethylation efficiency and qualitative applicability. Typically, however, either laborious preparation steps or cumbersome and uneconomical spin columns have been needed to obtain decent permethylation yields on small glycan samples. Here we describe a spin column-free (SCF) glycan permethylation procedure that is applicable to both O- and N-linked glycans and can be employed upstream to intact glycan analysis by MALDI-MS, ESI-MS, or glycan linkage analysis by GC-MS. The SCF procedure involves neutralization of NaOH beads by acidified phosphate buffer, which eliminates the risk of glycan oxidative degradation and avoids the use of spin columns. Optimization of the new permethylation procedure provided high permethylation efficiency for both hexose (>98%) and HexNAc (>99%) residues—yields which were comparable to (or better than) those of some widely-used spin column-based procedures. A light vs. heavy labelling approach was employed to compare intact glycan yields from a popular spin-column based approach to the SCF approach. Recovery of intact N-glycans was significantly better with the SCF procedure (p 0.75; p < 0.01). In summary, the SCF permethylation procedure expedites and economizes both intact glycan analysis and linkage analysis of glycans from whole biospecimens. PMID:28635997
Glycotherapy: new advances inspire a reemergence of glycans in medicine.
Hudak, Jason E; Bertozzi, Carolyn R
2014-01-16
The beginning of the 20(th) century marked the dawn of modern medicine with glycan-based therapies at the forefront. However, glycans quickly became overshadowed as DNA- and protein-focused treatments became readily accessible. The recent development of new tools and techniques to study and produce structurally defined carbohydrates has spurred renewed interest in the therapeutic applications of glycans. This review focuses on advances within the past decade that are bringing glycan-based treatments back to the forefront of medicine and the technologies that are driving these efforts. These include the use of glycans themselves as therapeutic molecules as well as engineering protein and cell surface glycans to suit clinical applications. Glycan therapeutics offer a rich and promising frontier for developments in the academic, biopharmaceutical, and medical fields. Copyright © 2014 Elsevier Ltd. All rights reserved.
A Capping Step During Automated Glycan Assembly Enables Access to Complex Glycans in High Yield.
Yu, Yang; Kononov, Andrew; Delbianco, Martina; Seeberger, Peter H
2018-04-20
The products of multi-step automated solid phase syntheses are purified after release from the resin. Capping of unreacted nucleophiles is commonplace in automated oligonucleotide synthesis to minimize accumulation of deletion sequences. To date, capping was not used routinely during automated glycan assembly (AGA) since previous capping protocols suffered from long reaction times and conditions incompatible with some protective groups. Here, a method using methanesulfonic acid and acetic anhydride for the fast and quantitative capping of hydroxyl groups that failed to be glycosylated is reported. Commonly used protective groups in AGA are stable under these capping conditions. The introduction of a capping step into the coupling cycle drastically improved overall yields by decreasing side-products and simplifying purification, while reducing building block consumption. To illustrate the method, the biologically important tetrasaccharide Lc4, as well as a 50-mer polymannoside were prepared. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Structural Feature Ions for Distinguishing N- and O-Linked Glycan Isomers by LC-ESI-IT MS/MS
Everest-Dass, Arun V.; Abrahams, Jodie L.; Kolarich, Daniel; Packer, Nicolle H.; Campbell, Matthew P.
2013-06-01
Glycomics is the comprehensive study of glycan expression in an organism, cell, or tissue that relies on effective analytical technologies to understand glycan structure-function relationships. Owing to the macro- and micro-heterogeneity of oligosaccharides, detailed structure characterization has required an orthogonal approach, such as a combination of specific exoglycosidase digestions, LC-MS/MS, and the development of bioinformatic resources to comprehensively profile a complex biological sample. Liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) has emerged as a key tool in the structural analysis of oligosaccharides because of its high sensitivity, resolution, and robustness. Here, we present a strategy that uses LC-ESI-MS/MS to characterize over 200 N- and O-glycans from human saliva glycoproteins, complemented by sequential exoglycosidase treatment, to further verify the annotated glycan structures. Fragment-specific substructure diagnostic ions were collated from an extensive screen of the literature available on the detailed structural characterization of oligosaccharides and, together with other specific glycan structure feature ions derived from cross-ring and glycosidic-linkage fragmentation, were used to characterize the glycans and differentiate isomers. The availability of such annotated mass spectrometric fragmentation spectral libraries of glycan structures, together with such substructure diagnostic ions, will be key inputs for the future development of the automated elucidation of oligosaccharide structures from MS/MS data.
Harvey, David J.; Sobott, Frank; Crispin, Max; Wrobel, Antoni; Bonomelli, Camille; Vasiljevic, Snezana; Scanlan, Christopher N.; Scarff, Charlotte A.; Thalassinos, Konstantinos; Scrivens, James H.
2011-03-01
The analysis of glycosylation from native biological sources is often frustrated by the low abundances of available material. Here, ion mobility combined with electrospray ionization mass spectrometry have been used to extract the spectra of N-glycans released with PNGase F from a serial titration of recombinantly expressed envelope glycoprotein, gp120, from the human immunodeficiency virus (HIV). Analysis was also performed on gp120 expressed in the α-mannosidase inhibitor, and in a matched mammalian cell line deficient in GlcNAc transferase I. Without ion mobility separation, ESI spectra frequently contained no observable ions from the glycans whereas ions from other compounds such as detergents and residual buffer salts were abundant. After ion mobility separation on a Waters T-wave ion mobility mass spectrometer, the N-glycans fell into a unique region of the ion mobility/ m/z plot allowing their profiles to be extracted with good signal:noise ratios. This method allowed N-glycan profiles to be extracted from crude incubation mixtures with no clean-up even in the presence of surfactants such as NP40. Furthermore, this technique allowed clear profiles to be obtained from sub-microgram amounts of glycoprotein. Glycan profiles were similar to those generated by MALDI-TOF MS although they were more susceptible to double charging and fragmentation. Structural analysis could be accomplished by MS/MS experiments in either positive or negative ion mode but negative ion mode gave the most informative spectra and provided a reliable approach to the analysis of glycans from small amounts of glycoprotein.
Inhibition of AMPK catabolic action by GSK3
Suzuki, Tsukasa; Bridges, Dave; Nakada, Daisuke; Skiniotis, Georgios; Morrison, Sean J.; Lin, Jiandie; Saltiel, Alan R.; Inoki, Ken
2013-01-01
SUMMARY AMP-activated protein kinase (AMPK) regulates cellular energy homeostasis by inhibiting anabolic and activating catabolic processes. While AMPK activation has been extensively studied, mechanisms that inhibit AMPK remain elusive. Here we report that glycogen synthase kinase 3 (GSK3) inhibits AMPK function. GSK3 forms a stable complex with AMPK through interactions with the AMPK β regulatory subunit and phosphorylates the AMPK α catalytic subunit. This phosphorylation enhances the accessibility of the activation loop of the α subunit to phosphatases, thereby inhibiting AMPK kinase activity. Surprisingly, PI3K-Akt signaling, which is a major anabolic signaling and normally inhibits GSK3 activity, promotes GSK3 phosphorylation and inhibition of AMPK, thus revealing how AMPK senses anabolic environments in addition to cellular energy levels. Consistently, disrupting GSK3 function within the AMPK complex sustains higher AMPK activity and cellular catabolic processes even under anabolic conditions, indicating that GSK3 acts as a critical sensor for anabolic signaling to regulate AMPK. PMID:23623684
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Shan Li
Full Text Available BACKGROUND AND OBJECTIVE: Due to recurrence and metastasis, the mortality of Hepatocellular carcinoma (HCC is high. It is well known that the epithelial mesenchymal transition (EMT and glycan of cell surface glycoproteins play pivotal roles in tumor metastasis. The goal of this study was to identify HCC metastasis related differential glycan pattern and their enzymatic basis using a HGF induced EMT model. METHODOLOGY: HGF was used to induce HCC EMT model. Lectin microarray was used to detect the expression of cell surface glycan and the difference was validated by lectin blot and fluorescence cell lectin-immunochemistry. The mRNA expression levels of glycotransferases were determined by qRT-PCR. RESULTS: After HGF treatment, the Huh7 cell lost epithelial characteristics and obtained mesenchymal markers. These changes demonstrated that HGF could induce a typical cell model of EMT. Lectin microarray analysis identified a decreased affinity in seven lectins ACL, BPL, JAC, MPL, PHA-E, SNA, and SBA to the glycan of cell surface glycoproteins. This implied that glycan containing T/Tn-antigen, NA2 and bisecting GlcNAc, Siaα2-6Gal/GalNAc, terminal α or βGalNAc structures were reduced. The binding ability of thirteen lectins, AAL, LCA, LTL, ConA, NML, NPL, DBA, HAL, PTL II, WFL, ECL, GSL II and PHA-L to glycan were elevated, and a definite indication that glycan containing terminal αFuc and ± Sia-Le, core fucose, α-man, gal-β(α GalNAc, β1,6 GlcNAc branching and tetraantennary complex oligosaccharides structures were increased. These results were further validated by lectin blot and fluorescence cell lectin-immunochemistry. Furthermore, the mRNA expression level of Mgat3 decreased while that of Mgat5, FucT8 and β3GalT5 increased. Therefore, cell surface glycan alterations in the EMT process may coincide with the expression of glycosyltransferase. CONCLUSIONS: The findings of this study systematically clarify the alterations of cell surface
N-glycan sialylation in a silkworm-baculovirus expression system.
Suganuma, Masatoshi; Nomura, Tsuyoshi; Higa, Yukiko; Kataoka, Yukiko; Funaguma, Shunsuke; Okazaki, Hironobu; Suzuki, Takeo; Fujiyama, Kazuhito; Sezutsu, Hideki; Tatematsu, Ken-Ichiro; Tamura, Toshiki
2018-02-09
A silkworm-baculovirus system is particularly effective for producing recombinant proteins, including glycoproteins. However, N-glycan structures in silkworm differ from those in mammals. Glycoproteins in silkworm are secreted as pauci-mannose type N-glycans without sialic acid or galactose residues. Sialic acid on N-glycans plays important roles in protein functions. Therefore, we developed pathways for galactosylation and sialylation in silkworm. Sialylated N-glycans on proteins were successfully produced in silkworm by co-expressing galactosyltransferase and sialyltransferase and providing an external supply of a sialylation-related substrate. α2,3/α2,6 Sialylation to N-glycans was controlled by changing the type of sialyltransferase expressed in silkworm. Furthermore, the co-expression of N-acetylglucosaminyltransferase II facilitated the formation of additional di-sialylated N-glycan structures. Our results provide new information on the control of N-glycosylation in silkworm. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
Imaging specific cellular glycan structures using glycosyltransferases via click chemistry.
Wu, Zhengliang L; Person, Anthony D; Anderson, Matthew; Burroughs, Barbara; Tatge, Timothy; Khatri, Kshitij; Zou, Yonglong; Wang, Lianchun; Geders, Todd; Zaia, Joseph; Sackstein, Robert
2018-02-01
Heparan sulfate (HS) is a polysaccharide fundamentally important for biologically activities. T/Tn antigens are universal carbohydrate cancer markers. Here, we report the specific imaging of these carbohydrates using a mesenchymal stem cell line and human umbilical vein endothelial cells (HUVEC). The staining specificities were demonstrated by comparing imaging of different glycans and validated by either removal of target glycans, which results in loss of signal, or installation of target glycans, which results in gain of signal. As controls, representative key glycans including O-GlcNAc, lactosaminyl glycans and hyaluronan were also imaged. HS staining revealed novel architectural features of the extracellular matrix (ECM) of HUVEC cells. Results from T/Tn antigen staining suggest that O-GalNAcylation is a rate-limiting step for O-glycan synthesis. Overall, these highly specific approaches for HS and T/Tn antigen imaging should greatly facilitate the detection and functional characterization of these biologically important glycans. © The Author(s) 2017. Published by Oxford University Press.
Glycan-deficient PrP stimulates VEGFR2 signaling via glycosaminoglycan.
Gao, Zhenxing; Zhang, Huixia; Hu, Fei; Yang, Liheng; Yang, Xiaowen; Zhu, Ying; Sy, Man-Sun; Li, Chaoyang
2016-06-01
Whether the two N-linked glycans are important in prion, PrP, biology is unresolved. In Chinese hamster ovary (CHO) cells, the two glycans are clearly not important in the cell surface expression of transfected human PrP. Compared to fully-glycosylated PrP, glycan-deficient PrP preferentially partitions to lipid raft. In CHO cells glycan-deficient PrP also interacts with glycosaminoglycan (GAG) and vascular endothelial growth factor receptor 2 (VEGFR2), resulting in VEGFR2 activation and enhanced Akt phosphorylation. Accordingly, CHO cells expressing glycan-deficient PrP lacking the GAG binding motif or cells treated with heparinase to remove GAG show diminished Akt signaling. Being in lipid raft is critical, chimeric glycan-deficient PrP with CD4 transmembrane and cytoplasmic domains is absent in lipid raft and does not activate Akt signaling. CHO cells bearing glycan-deficient PrP also exhibit enhanced cellular adhesion and migration. Based on these findings, we propose a model in which glycan-deficient PrP, GAG, and VEGFR2 interact, activating VEGFR2 and resulting in changes in cellular behavior. Copyright © 2016 Elsevier Inc. All rights reserved.
Yuan, Jiangbei; Wang, Chengjian; Sun, Yujiao; Huang, Linjuan; Wang, Zhongfu
2014-10-01
A novel strategy is proposed, using cost-saving chemical reactions to generate intact free reducing N-glycans and their fluorescent derivatives from glycoproteins for subsequent analysis. N-Glycans without core α-1,3-linked fucose are released in reducing form by selective hydrolysis of the N-type carbohydrate-peptide bond of glycoproteins under a set of optimized mild alkaline conditions and are comparable to those released by commonly used peptide-N-glycosidase (PNGase) F in terms of yield without any detectable side reaction (peeling or deacetylation). The obtained reducing glycans can be routinely derivatized with 2-aminobenzoic acid (2-AA), 1-phenyl-3-methyl-5-pyrazolone (PMP), and potentially some other fluorescent reagents for comprehensive analysis. Alternatively, the core α-1,3-fucosylated N-glycans are released in mild alkaline medium and derivatized with PMP in situ, and their yields are comparable to those obtained using commonly used PNGase A without conspicuous peeling reaction or any detectable deacetylation. Using this new technique, the N-glycans of a series of purified glycoproteins and complex biological samples were successfully released and analyzed by electrospray ionization mass spectrometry (ESI-MS) and tandem mass spectrometry (MS/MS), demonstrating its general applicability to glycomic studies. Copyright © 2014 Elsevier Inc. All rights reserved.
Hehemann, Jan-Hendrik; Kelly, Amelia G; Pudlo, Nicholas A; Martens, Eric C; Boraston, Alisdair B
2012-11-27
Humans host an intestinal population of microbes--collectively referred to as the gut microbiome--which encode the carbohydrate active enzymes, or CAZymes, that are absent from the human genome. These CAZymes help to extract energy from recalcitrant polysaccharides. The question then arises as to if and how the microbiome adapts to new carbohydrate sources when modern humans change eating habits. Recent metagenome analysis of microbiomes from healthy American, Japanese, and Spanish populations identified putative CAZymes obtained by horizontal gene transfer from marine bacteria, which suggested that human gut bacteria evolved to degrade algal carbohydrates-for example, consumed in form of sushi. We approached this hypothesis by studying such a polysaccharide utilization locus (PUL) obtained by horizontal gene transfer by the gut bacterium Bacteroides plebeius. Transcriptomic and growth experiments revealed that the PUL responds to the polysaccharide porphyran from red algae, enabling growth on this carbohydrate but not related substrates like agarose and carrageenan. The X-ray crystallographic and biochemical analysis of two proteins encoded by this PUL, BACPLE_01689 and BACPLE_01693, showed that they are β-porphyranases belonging to glycoside hydrolase families 16 and 86, respectively. The product complex of the GH86 at 1.3 Å resolution highlights the molecular details of porphyran hydrolysis by this new porphyranase. Combined, these data establish experimental support for the argument that CAZymes and associated genes obtained from extrinsic microbes add new catabolic functions to the human gut microbiome.
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Ewa M. Kratz
2015-01-01
Full Text Available Carbohydrates are known to mediate some events involved in successful fertilization. Although some studies on the glycosylation of seminal plasma proteins are available, the total glycan profile was rarely analyzed as a feature influencing fertilization potential. In this work we aimed to compare some glycosylation traits in seminal plasma glycoproteins of fertile and infertile men. The following findings emerge from our studies: (1 in human seminal plasma the presence and alterations of O-linked glycans were observed; (2 the expression of SNA-reactive sialic acid significantly differs between asthenozoospermia and both normozoospermic (fertile and infertile groups; (3 the expression of PHA-L-reactive highly branched N-glycans was significantly lower in oligozoospermic patients than in both normozoospermic groups. Indication of the appropriate lectins that would enable the possibly precise determination of the glycan profile seems to be a good supplement to mass spectrum analysis. Extension of the lectin panel is useful for the further research.
Huang, Yining; Orlando, Ron
2017-12-01
The biologic activity of IgG molecules is modulated by its crystallizable fragment N-glycosylation, and thus, the analysis of IgG glycosylation is critical. A standard approach to analyze glycosylation of IgGs involves the release of the N-glycans by the enzyme peptide N-glycosidase F, which cleaves the linkage between the asparagine residue and innermost N-acetylglucosamine (GlcNAc) of all N-glycans except those containing a 3-linked fucose attached to the reducing terminal GlcNAc residue. The importance of obtaining complete glycan release for accurate quantitation led us to investigate the kinetics of this de-glycosylation reaction for IgG glycopeptides and to determine the effect of glycan structure and amino acid sequence on the rate of glycan release from glycopeptides of IgGs. This study revealed that the slight differences in amino acid sequences did not lead to a statistically different deglycosylation rate. However, statistically significant differences in the deglycosylation rate constants were observed between glycopeptides differing only in glycan structure ( i.e. , nonfucosylated, fucosylated, bisecting-GlcNAc, sialylated, etc .). For example, a single sialic acid residue was found to decrease the rate by a factor of 3. Similar reductions in rate were associated with the presence of a bisecting-GlcNAc. We predict the differences in release kinetics can lead to significant quantitative variations of the glycosylation study of IgGs.
Link-Lenczowski, Paweł; Bubka, Monika; Balog, Crina I A; Koeleman, Carolien A M; Butters, Terry D; Wuhrer, Manfred; Lityńska, Anna
2018-04-01
N-acetylglucosaminyltransferase III (GnT-III) is known to catalyze N-glycan "bisection" and thereby modulate the formation of highly branched complex structures within the Golgi apparatus. While active, it inhibits the action of other GlcNAc transferases such as GnT-IV and GnT-V. Moreover, GnT-III is considered as an inhibitor of the metastatic potential of cancer cells both in vitro and in vivo. However, the effects of GnT-III may be more diverse and depend on the cellular context. We describe the detailed glycomic analysis of the effect of GnT-III overexpression in WM266-4-GnT-III metastatic melanoma cells. We used MALDI-TOF and ESI-ion-trap-MS/MS together with HILIC-HPLC of 2-AA labeled N-glycans to study the N-glycome of membrane-attached and secreted proteins. We found that the overexpression of GnT-III in melanoma leads to the modification of a broad range of N-glycan types by the introduction of the "bisecting" GlcNAc residue with highly branched complex structures among them. The presence of these unusual complex N-glycans resulted in stronger interactions of cellular glycoproteins with the PHA-L. Based on the data presented here we conclude that elevated activity of GnT-III in cancer cells does not necessarily lead to a total abrogation of the formation of highly branched glycans. In addition, the modification of pre-existing N-glycans by the introduction of "bisecting" GlcNAc can modulate their capacity to interact with carbohydrate-binding proteins such as plant lectins. Our results suggest further studies on the biological function of "bisected" oligosaccharides in cancer cell biology and their interactions with carbohydrate-binding proteins.
Genetically engineered tissue to screen for glycan function in tissue formation
DEFF Research Database (Denmark)
M., Adamopoulou; E.M., Pallesen; A., Levann
2017-01-01
engineered GlycoSkin tissue models can be used to study biological interactions involving glycan structure on lipids, or glycosaminoglycans. This engineering approach will allow us to investigate the functions of glycans in homeostasis and elucidate the role of glycans in normal epithelial formation....... We use genetic engineering with CRISPR/Cas9 combined with 3D organotypic skin models to examine how distinct glycans influence epithelial formation. We have performed knockout and knockin of more than 100 select genes in the genome of human immortalized human keratinocytes, enabling a systematic...... analysis of the impact of specific glycans in the formation and transformation of the human skin. The genetic engineered human skin models (GlycoSkin) was designed with and without all major biosynthetic pathways in mammalian glycan biosynthesis, including GalNAc-O-glycans, O-fucosylation, O...
Patel, Kashyap R; Roberts, Jacob T; Subedi, Ganesh P; Barb, Adam W
2018-03-09
CD16a/Fc γ receptor IIIa is the most abundant antibody Fc receptor expressed on human natural killer (NK) cells and activates a protective cytotoxic response following engagement with antibody clustered on the surface of a pathogen or diseased tissue. Therapeutic monoclonal antibodies (mAbs) with greater Fc-mediated affinity for CD16a show superior therapeutic outcome; however, one significant factor that promotes antibody-CD16a interactions, the asparagine-linked carbohydrates ( N -glycans), remains undefined. Here, we purified CD16a from the primary NK cells of three donors and identified a large proportion of hybrid (22%) and oligomannose N -glycans (23%). These proportions indicated restricted N -glycan processing and were unlike those of the recombinant CD16a forms, which have predominantly complex-type N -glycans (82%). Tethering recombinant CD16a to the membrane by including the transmembrane and intracellular domains and via coexpression with the Fc ϵ receptor γ-chain in HEK293F cells was expected to produce N -glycoforms similar to NK cell-derived CD16a but yielded N -glycoforms different from NK cell-derived CD16a and recombinant soluble CD16a. Of note, these differences in CD16a N -glycan composition affected antibody binding: CD16a with oligomannose N -glycans bound IgG1 Fc with 12-fold greater affinity than did CD16a having primarily complex-type and highly branched N -glycans. The changes in binding activity mirrored changes in NMR spectra of the two CD16a glycoforms, indicating that CD16a glycan composition also affects the glycoprotein's structure. These results indicated that CD16a from primary human NK cells is compositionally, and likely also functionally, distinct from commonly used recombinant forms. Furthermore, our study provides critical evidence that cell lineage determines CD16a N -glycan composition and antibody-binding affinity. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.
Bahar, M; de Majnik, J; Wexler, M; Fry, J; Poole, P S; Murphy, P J
1998-11-01
Rhizopines are nodule-specific compounds that confer an intraspecies competitive nodulation advantage to strains that can catabolize them. The rhizopine (3-O-methyl-scyllo-inosamine, 3-O-MSI) catabolic moc gene cluster mocCABRDE(F) in Rhizobium leguminosarum bv. viciae strain 1a is located on the Sym plasmid. MocCABR are homologous to the mocCABR gene products from Sinorhizobium meliloti. MocD and MocE contain motifs corresponding to a TOL-like oxygenase and a [2Fe-2S] Rieske-like ferredoxin, respectively. The mocF gene encodes a ferredoxin reductase that would complete the oxygenase system, but is not essential for rhizopine catabolism. We propose a rhizopine catabolic model whereby MocB transports rhizopine into the cell and MocDE and MocF (or a similar protein elsewhere in the genome), under the regulation of MocR, act in concert to form a ferredoxin oxygenase system that demethylates 3-O-MSI to form scyllo-inosamine (SI). MocA, an NAD(H)-dependent dehydrogenase, and MocC continue the catabolic process. Compounds formed then enter the inositol catabolic pathway.
Glycans: bioactive signals decoded by lectins.
Gabius, Hans-Joachim
2008-12-01
The glycan part of cellular glycoconjugates affords a versatile means to build biochemical signals. These oligosaccharides have an exceptional talent in this respect. They surpass any other class of biomolecule in coding capacity within an oligomer (code word). Four structural factors account for this property: the potential for variability of linkage points, anomeric position and ring size as well as the aptitude for branching (first and second dimensions of the sugar code). Specific intermolecular recognition is favoured by abundant potential for hydrogen/co-ordination bonds and for C-H/pi-interactions. Fittingly, an array of protein folds has developed in evolution with the ability to select certain glycans from the natural diversity. The thermodynamics of this reaction profits from the occurrence of these ligands in only a few energetically favoured conformers, comparing favourably with highly flexible peptides (third dimension of the sugar code). Sequence, shape and local aspects of glycan presentation (e.g. multivalency) are key factors to regulate the avidity of lectin binding. At the level of cells, distinct glycan determinants, a result of enzymatic synthesis and dynamic remodelling, are being defined as biomarkers. Their presence gains a functional perspective by co-regulation of the cognate lectin as effector, for example in growth regulation. The way to tie sugar signal and lectin together is illustrated herein for two tumour model systems. In this sense, orchestration of glycan and lectin expression is an efficient means, with far-reaching relevance, to exploit the coding potential of oligosaccharides physiologically and medically.
Dong, Xue; Zhou, Shiyue; Mechref, Yehia
2016-06-01
Oligosaccharides in milk not only provide nutrition to the infants but also have significant immune biofunctions such as inhibition of pathogen binding to the host cell. The main component in milk oligosaccharides is free oligosaccharides. Since the proteins in milk are highly glycosylated, N-glycans in milk also play an import role. In this study, we investigated the permethylated free oligosaccharides and N-glycans extracted from bovine, goat, and human milks using LC-MS/MS. Quantitation profiles of free oligosaccharides and N-glycans were reported. The number of free oligosaccharides observed in bovine, goat, and human milk samples (without isomeric consideration) were 11, 8, and 11, respectively. Human milk had more complex free oligosaccharides structures than the other two milk samples. Totally 58, 21, and 43 N-glycan structures (without isomeric consideration) were associated with whey proteins extracted from bovine, goat, and human milk samples, respectively. Bovine milk free oligosaccharides and N-glycans from whey proteins were highly sialylated and to a lesser extend fucosylated. Goat and human milk free oligosaccharides and N-glycans from whey proteins were both highly fucosylated. Also, the isomeric glycans in milk samples were determined by porous graphitic carbon LC at elevated temperatures. For example, separation of human milk free oligosaccharide Gal-GlcNAc-(Fuc)-Gal-Glc and Gal-GlcNAc-Gal-Glc-Fuc isomers was achieved using porous graphitic carbon column. Permethylation of the glycan structures facilitated the interpretation of MS/MS. For example, internal cleavage and glycosidic bond cleavage are readily distinguished in the tandem mass spectra of permethylated glycans. This feature resulted in the identification of several isomers. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Ashwood, Christopher; Lin, Chi-Hung; Thaysen-Andersen, Morten; Packer, Nicolle H.
2018-03-01
Profiling cellular protein glycosylation is challenging due to the presence of highly similar glycan structures that play diverse roles in cellular physiology. As the anomericity and the exact linkage type of a single glycosidic bond can influence glycan function, there is a demand for improved and automated methods to confirm detailed structural features and to discriminate between structurally similar isomers, overcoming a significant bottleneck in the analysis of data generated by glycomics experiments. We used porous graphitized carbon-LC-ESI-MS/MS to separate and detect released N- and O-glycan isomers from mammalian model glycoproteins using negative mode resonance activation CID-MS/MS. By interrogating similar fragment spectra from closely related glycan isomers that differ only in arm position and sialyl linkage, product fragment ions for discrimination between these features were discovered. Using the Skyline software, at least two diagnostic fragment ions of high specificity were validated for automated discrimination of sialylation and arm position in N-glycan structures, and sialylation in O-glycan structures, complementing existing structural diagnostic ions. These diagnostic ions were shown to be useful for isomer discrimination using both linear and 3D ion trap mass spectrometers when analyzing complex glycan mixtures from cell lysates. Skyline was found to serve as a useful tool for automated assessment of glycan isomer discrimination. This platform-independent workflow can potentially be extended to automate the characterization and quantitation of other challenging glycan isomers. [Figure not available: see fulltext.
Shimomura, Yoshiharu; Murakami, Taro; Nakai, Naoya; Nagasaki, Masaru; Harris, Robert A
2004-06-01
Branched-chain amino acids (BCAAs) are essential amino acids that can be oxidized in skeletal muscle. It is known that BCAA oxidation is promoted by exercise. The mechanism responsible for this phenomenon is attributed to activation of the branched-chain alpha-keto acid dehydrogenase (BCKDH) complex, which catalyzes the second-step reaction of the BCAA catabolic pathway and is the rate-limiting enzyme in the pathway. This enzyme complex is regulated by a phosphorylation-dephosphorylation cycle. The BCKDH kinase is responsible for inactivation of the complex by phosphorylation, and the activity of the kinase is inversely correlated with the activity state of the BCKDH complex, which suggests that the kinase is the primary regulator of the complex. We found recently that administration of ligands for peroxisome proliferator-activated receptor-alpha (PPARalpha) in rats caused activation of the hepatic BCKDH complex in association with a decrease in the kinase activity, which suggests that promotion of fatty acid oxidation upregulates the BCAA catabolism. Long-chain fatty acids are ligands for PPARalpha, and the fatty acid oxidation is promoted by several physiological conditions including exercise. These findings suggest that fatty acids may be one of the regulators of BCAA catabolism and that the BCAA requirement is increased by exercise. Furthermore, BCAA supplementation before and after exercise has beneficial effects for decreasing exercise-induced muscle damage and promoting muscle-protein synthesis; this suggests the possibility that BCAAs are a useful supplement in relation to exercise and sports.
Expression of LacdiNAc Groups on N-Glycans among Human Tumors Is Complex
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Kiyoko Hirano
2014-01-01
Full Text Available Aberrant glycosylation of proteins and lipids is one of the characteristic features of malignantly transformed cells. The GalNAcβ1 → 4GlcNAc (LacdiNAc or LDN group at the nonreducing termini of both N- and O-glycans is not generally found in mammalian cells. We previously showed that the expression level of the LacdiNAc group in N-glycans decreases dramatically during the progression of human breast cancer. In contrast, the enhanced expression of the LacdiNAc group has been shown to be associated with the progression of human prostate, ovarian, and pancreatic cancers. Therefore, the expression of the disaccharide group appears to be dependent on types of tumors. The mechanism of formation of the LacdiNAc group in human tumors and cancer cells has been studied, and two β4-N-acetylgalacto-saminyltransferases (β4GalNAcTs, β4GalNAcT3 and β4GalNAcT4, have been shown to be involved in the biosynthesis of this disaccharide group in a tissue-dependent manner. Transfection of the β4GalNAcT3 gene brought about significant changes in the malignant phenotypes of human neuroblastoma, indicating that this disaccharide group is important for suppressing the tumor growth.
DEFF Research Database (Denmark)
Rasmussen, Morten
, and observed that the glyco-peptides were identified correctly in 11/11 cases and the glycan moieties were annotated in 11/11 cases. Finally, glycan structures were proposed in 10/11 cases, all of which were in agreement with previously reported structures. As a stand-alone program, GLYCANthrope is a useful...
Protein-linked glycans in periodontal bacteria: prevalence and role at the immune interface.
Settem, Rajendra P; Honma, Kiyonobu; Stafford, Graham P; Sharma, Ashu
2013-10-17
Protein modification with complex glycans is increasingly being recognized in many pathogenic and non-pathogenic bacteria, and is now thought to be central to the successful life-style of those species in their respective hosts. This review aims to convey current knowledge on the extent of protein glycosylation in periodontal pathogenic bacteria and its role in the modulation of the host immune responses. The available data show that surface glycans of periodontal bacteria orchestrate dendritic cell cytokine responses to drive T cell immunity in ways that facilitate bacterial persistence in the host and induce periodontal inflammation. In addition, surface glycans may help certain periodontal bacteria protect against serum complement attack or help them escape immune detection through glycomimicry. In this review we will focus mainly on the generalized surface-layer protein glycosylation system of the periodontal pathogen Tannerella forsythia in shaping innate and adaptive host immunity in the context of periodontal disease. In addition, we will also review the current state of knowledge of surface protein glycosylation and its potential for immune modulation in other periodontal pathogens.
Neutral glycans from sandfish skin can reduce friction of polymers
Vihar, Boštjan; Hanisch, Franz Georg; Baumgartner, Werner
2016-01-01
The lizard Scincus scincus, also known as sandfish, can move through aeolian desert sand in a swimming-like manner. A prerequisite for this ability is a special integument, i.e. scales with a very low friction for sand and a high abrasion resistance. Glycans in the scales are causally related to the low friction. Here, we analysed the glycans and found that neutral glycans with five to nine mannose residues are important. If these glycans were covalently bound to acrylic polymers like poly(methyl methacrylate) or acrylic car coatings at a density of approximately one molecule per 4 nm², friction for and adhesion of sand particles could be reduced to levels close to those observed with sandfish scales. This was also found true, if the glycans were isolated from sources other than sandfish scales like plants such as almonds or mistletoe. We speculate that these neutral glycans act as low density spacers separating sand particles from the dense scales thereby reducing van der Waals forces. PMID:27030038
Bayón, Carlos; He, Ning; Deir-Kaspar, Mario; Blasco, Pilar; André, Sabine; Gabius, Hans-Joachim; Rumbero, Ángel; Jiménez-Barbero, Jesús; Fessner, Wolf-Dieter; Hernáiz, María J
2017-01-31
The sequence of a glycan and its topology of presentation team up to determine the specificity and selectivity of recognition by saccharide receptors (lectins). Structure-activity analysis would be furthered if the glycan part of a glycocluster could be efficiently elaborated in situ while keeping all other parameters constant. By using a bacterial α2,6-sialyltransferase and a small library of bi- to tetravalent glycoclusters, we illustrate the complete conversion of scaffold-presented lactoside units into two different sialylated ligands based on N-acetyl/glycolyl-neuraminic acid incorporation. We assess the ensuing effect on their bioactivity for a plant toxin, and present an analysis of the noncovalent substrate binding contacts that the added sialic acid moiety makes to the lectin. Enzymatic diversification of a scaffold-presented glycan can thus be brought to completion in situ, offering a versatile perspective for rational glycocluster engineering. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Glycobiology Modifications in Intratumoral Hypoxia: The Breathless Side of Glycans Interaction
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Antônio F. Silva-filho
2017-04-01
Full Text Available Post-translational and co-translational enzymatic addition of glycans (glycosylation to proteins, lipids, and other carbohydrates, is a powerful regulator of the molecular machinery involved in cell cycle, adhesion, invasion, and signal transduction, and is usually seen in both in vivo and in vitro cancer models. Glycosyltransferases can alter the glycosylation pattern of normal cells, subsequently leading to the establishment and progression of several diseases, including cancer. Furthermore, a growing amount of research has shown that different oxygen tensions, mainly hypoxia, leads to a markedly altered glycosylation, resulting in altered glycan-receptor interactions. Alteration of intracellular glucose metabolism, from aerobic cellular respiration to anaerobic glycolysis, inhibition of integrin 3α1β translocation to the plasma membrane, decreased 1,2-fucosylation of cell-surface glycans, and galectin overexpression are some consequences of the hypoxic tumor microenvironment. Additionally, increased expression of gangliosides carrying N-glycolyl sialic acid can also be significantly affected by hypoxia. For all these reasons, it is possible to realize that hypoxia strongly alters glycobiologic events within tumors, leading to changes in their behavior. This review aims to analyze the complexity and importance of glycoconjugates and their molecular interaction network in the hypoxic context of many solid tumors.
Wu, Yike; Sha, Qiuyue; Du, Juan; Wang, Chang; Zhang, Liang; Liu, Bi-Feng; Lin, Yawei; Liu, Xin
2018-02-02
Robust, efficient identification and accurate quantification of N-glycans are of great significance in N-glycomics analysis. Here, a simple and rapid derivatization method, based on the combination of microwave-assisted deglycosylation and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) labeling, was developed for the analysis of N-glycan by high performance liquid chromatography with fluorescence detection (HPLC-FLD). After optimizing various parameters affecting deglycosylation and derivatization by RNase B, the time for N-glycan labeling was shortened to 50 min with ∼10-fold enhancement in detection sensitivity comparing to conventional 2-aminobenzoic acid (2-AA) labeling method. Additionally, the method showed good linearity (correlation coefficients > 0.991) and reproducibility (RSD < 8.7%). These advantages of the proposed method were further validated by the analysis of complex samples, including fetuin and human serum. Investigation of serum N-glycome for preliminary diagnosis of human lung cancer was conducted, where significant changes of several N-glycans corresponding to core-fucosylated, mono- and disialylated glycans have been evidenced by a series of statistical analysis. Copyright © 2018 Elsevier B.V. All rights reserved.
Amino acid catabolism by Lactobacillus helveticus in cheese
DEFF Research Database (Denmark)
Kananen, Soila Kaarina
Amino acid catabolism is the final step in the conversion of caseins to flavour compounds and a part of a complex combination of biochemical pathways in cheese flavour formation. Lactobacillus helveticus is a thermophilic lactic acid bacterium that is used in cheese manufacture as a primary starter...... culture or as an adjunct culture. It has shown high proteolytic activities in conversion of caseins to peptides and further to amino acids and flavour compounds. Better understanding of the enzyme activity properties and the influence of different properties on final cheese flavour is favourable...... for developing new cheese products with enhanced flavour. The aim of this Ph.D. study was to investigate the importance of strain variation of Lb. helveticus in relation flavour formation in cheese related to amino acid catabolism. Aspects of using Lb. helveticus as starter as well as adjunct culture in cheese...
Human Milk Contains Novel Glycans That Are Potential Decoy Receptors for Neonatal Rotaviruses*
Yu, Ying; Lasanajak, Yi; Song, Xuezheng; Hu, Liya; Ramani, Sasirekha; Mickum, Megan L.; Ashline, David J.; Prasad, B. V. Venkataram; Estes, Mary K.; Reinhold, Vernon N.; Cummings, Richard D.; Smith, David F.
2014-01-01
Human milk contains a rich set of soluble, reducing glycans whose functions and bioactivities are not well understood. Because human milk glycans (HMGs) have been implicated as receptors for various pathogens, we explored the functional glycome of human milk using shotgun glycomics. The free glycans from pooled milk samples of donors with mixed Lewis and Secretor phenotypes were labeled with a fluorescent tag and separated via multidimensional HPLC to generate a tagged glycan library containing 247 HMG targets that were printed to generate the HMG shotgun glycan microarray (SGM). To investigate the potential role of HMGs as decoy receptors for rotavirus (RV), a leading cause of severe gastroenteritis in children, we interrogated the HMG SGM with recombinant forms of VP8* domains of the RV outer capsid spike protein VP4 from human neonatal strains N155(G10P[11]) and RV3(G3P[6]) and a bovine strain, B223(G10P[11]). Glycans that were bound by RV attachment proteins were selected for detailed structural analyses using metadata-assisted glycan sequencing, which compiles data on each glycan based on its binding by antibodies and lectins before and after exo- and endo-glycosidase digestion of the SGM, coupled with independent MSn analyses. These complementary structural approaches resulted in the identification of 32 glycans based on RV VP8* binding, many of which are novel HMGs, whose detailed structural assignments by MSn are described in a companion report. Although sialic acid has been thought to be important as a surface receptor for RVs, our studies indicated that sialic acid is not required for binding of glycans to individual VP8* domains. Remarkably, each VP8* recognized specific glycan determinants within a unique subset of related glycan structures where specificity differences arise from subtle differences in glycan structures. PMID:25048705
Improving N-Glycan Coverage using HPLC-MS with Electrospray Ionization at Subambient Pressure
Energy Technology Data Exchange (ETDEWEB)
Marginean, Ioan; Kronewitter, Scott R.; Moore, Ronald J.; Slysz, Gordon W.; Monroe, Matthew E.; Anderson, Gordon A.; Tang, Keqi; Smith, Richard D.
2012-10-01
Human serum glycan profiling with mass spectrometry (MS) has been employed to study several disease conditions and is demonstrating promise for e.g. clinical biomarker discovery. However, the poor glycan ionization efficiency and the large dynamic range of glycan concentrations in human sera hinder comprehensive profiling. In particular, large glycans are problematic because they are present at low concentrations and prone to fragmentation. Here we show that the sub-ambient pressure ionization with nanoelectrospray (SPIN)-MS can expand the serum glycome profile when compared with the conventional atmospheric pressure electrospray ionization (ESI)-MS with a heated capillary inlet. Notably, the ions generated by the SPIN interface were observed at higher charge states for 50% of the annotated glycans. Out of a total of 130 detected glycans, 34 were only detected with the SPIN-MS, resulting in improved coverage of glycan families as well as of glycans with larger numbers of labile monosaccharides.
Wang, Chengjian; Lu, Yu; Han, Jianli; Jin, Wanjun; Li, Lingmei; Zhang, Ying; Song, Xuezheng; Huang, Linjuan; Wang, Zhongfu
2018-05-24
Most glycoproteins and biological protein samples undergo both O- and N-glycosylation, making characterization of their structures very complicated and time-consuming. Nevertheless, to fully understand the biological functions of glycosylation, both the glycosylation forms need to be analyzed. Herein we report a versatile, convenient one-pot method in which O- and N-glycans are simultaneously released from glycoproteins and chromogenically labeled in situ and thus available for further characterization. In this procedure, glycoproteins are incubated with 1-phenyl-3-methyl-5-pyrazolone (PMP) in aqueous ammonium hydroxide, making O-glycans released from protein backbones by β-elimination and N-glycans liberated by alkaline hydrolysis. The released glycans are promptly derivatized with PMP in situ by Knoevenagel condensation and Michael addition, with peeling degradation almost completely prevented. The recovered mixture of O- and N-glycans as bis-PMP derivatives features strong ultraviolet (UV) absorbing ability and hydrophobicity, allowing for high-resolution chromatographic separation and high-sensitivity spectrometric detection. Using this technique, O- and N-glycans were simultaneously prepared from some model glycoproteins and complex biological samples, without significant peeling, desialylation, deacetylation, desulfation or other side-reactions, and then comprehensively analyzed by online HILIC-UV-ESI-MS/MS and RP-HPLC-UV-ESI-MS/MS, with which some novel O- and N-glycan structures were first found. This method provides a simple, versatile strategy for high-throughput glycomics analysis.
Glycan gimmickry by parasitic helminths: a strategy for modulating the host immune response?
van Die, Irma; Cummings, Richard D
2010-01-01
Parasitic helminths (worms) co-evolved with vertebrate immune systems to enable long-term survival of worms in infected hosts. Among their survival strategies, worms use their glycans within glycoproteins and glycolipids, which are abundant on helminth surfaces and in their excretory/ secretory products, to regulate and suppress host immune responses. Many helminths express unusual and antigenic (nonhost-like) glycans, including those containing polyfucose, tyvelose, terminal GalNAc, phosphorylcholine, methyl groups, and sugars in unusual linkages. In addition, some glycan antigens are expressed that share structural features with those in their intermediate and vertebrate hosts (host-like glycans), including Le(X) (Galbeta1-4[Fucalpha1-3]GlcNAc-), LDNF (GalNAcbeta1-4[Fucalpha1-3]GlcNAc-), LDN (GalNAcbeta1-4GlcNAc-), and Tn (GalNAcalpha1-O-Thr/Ser) antigens. The expression of host-like glycan determinants is remarkable and suggests that helminths may gain advantages by synthesizing such glycans. The expression of host-like glycans by parasites previously led to the concept of "molecular mimicry," in which molecules are either derived from the pathogen or acquired from the host to evade recognition by the host immune system. However, recent discoveries into the potential of host glycan-binding proteins (GBPs), such as C-type lectin receptors and galectins, to functionally interact with various host-like helminth glycans provide new insights. Host GBPs through their interactions with worm-derived glycans participate in shaping innate and adaptive immune responses upon infection. We thus propose an alternative concept termed "glycan gimmickry," which is defined as an active strategy of parasites to use their glycans to target GBPs within the host to promote their survival.
Directory of Open Access Journals (Sweden)
Sujata Halder
Full Text Available The recognition of sialic acids by two strains of minute virus of mice (MVM, MVMp (prototype and MVMi (immunosuppressive, is an essential requirement for successful infection. To understand the potential for recognition of different modifications of sialic acid by MVM, three types of capsids, virus-like particles, wild type empty (no DNA capsids, and DNA packaged virions, were screened on a sialylated glycan microarray (SGM. Both viruses demonstrated a preference for binding to 9-O-methylated sialic acid derivatives, while MVMp showed additional binding to 9-O-acetylated and 9-O-lactoylated sialic acid derivatives, indicating recognition differences. The glycans recognized contained a type-2 Galβ1-4GlcNAc motif (Neu5Acα2-3Galβ1-4GlcNAc or 3'SIA-LN and were biantennary complex-type N-glycans with the exception of one. To correlate the recognition of the 3'SIA-LN glycan motif as well as the biantennary structures to their natural expression in cell lines permissive for MVMp, MVMi, or both strains, the N- and O-glycans, and polar glycolipids present in three cell lines used for in vitro studies, A9 fibroblasts, EL4 T lymphocytes, and the SV40 transformed NB324K cells, were analyzed by MALDI-TOF/TOF mass spectrometry. The cells showed an abundance of the sialylated glycan motifs recognized by the viruses in the SGM and previous glycan microarrays supporting their role in cellular recognition by MVM. Significantly, the NB324K showed fucosylation at the non-reducing end of their biantennary glycans, suggesting that recognition of these cells is possibly mediated by the Lewis X motif as in 3'SIA-Le(X identified in a previous glycan microarray screen.
Jiao, Jing; Yang, Lijun; Zhang, Ying; Lu, Haojie
2015-08-21
The analysis of glycan is important for understanding cell biology and disease processes because the glycans play a key role in many important biological behaviors, such as cell division, cellular localization, tumor immunology and inflammation. Nevertheless, it is still hard work to analyze glycans by MALDI-MS, which generally stems from the inherent low abundance and the low ionization efficiency of glycans. Moreover, the difficulty in generating informative fragmentations further hinders glycans structure characterization. In this work, hydrazinonicotinic acid (HYNIC) was used as a novel derivatized reagent for improved and selective detection of glycans. Through tagging the reducing terminus of glycans with the diazanyl group of HYNIC, significant enhancement of the ionization efficiency of glycans was achieved. After derivatization, the signal to noise ratio (S/N) of the maltoheptaose was improved by more than one order of magnitude in positive mode. HYNIC derivatization also allowed the sensitive detection of sialylated glycan in negative mode, with a 15 fold enhancement of S/N. Interestingly, it is noteworthy that the HYNIC reagent not only effectively labeled the reducing end of glycans in the presence of tryptic peptides, but also suppressed the ionization of peptides, enabling the direct detection of glycans from glycoprotein without separation. Therefore, analysis of glycans became easier due to the omission of a pre-separation step. Importantly, by using different acid reagents as the catalyst, derivatized product signals corresponding to [M + Na](+) or [M + H](+) were obtained respectively, which yield complementary fragmentation patterns for the structure elucidation of glycans. Finally, more than 40 N-glycans were successfully detected in 10 μL human serum using this method.
Determination of site-specific glycan heterogeneity on glycoproteins
DEFF Research Database (Denmark)
Kolarich, Daniel; Jensen, Pia Hønnerup; Altmann, Friedrich
2012-01-01
and the determination of site-specific glycan heterogeneity. The described workflow takes approximately 3-5 d, including sample preparation and data analysis. The data obtained from analyzing released glycans of rHuEPO and IgG, described in the second protocol of this series (10.1038/nprot.2012.063), provide...
Infection's Sweet Tooth: How Glycans Mediate Infection and Disease Susceptibility.
Taylor, Steven L; McGuckin, Michael A; Wesselingh, Steve; Rogers, Geraint B
2018-02-01
Glycans form a highly variable constituent of our mucosal surfaces and profoundly affect our susceptibility to infection and disease. The diversity and importance of these surface glycans can be seen in individuals who lack a functional copy of the fucosyltransferase gene, FUT2. Representing around one-fifth of the population, these individuals have an altered susceptibility to many bacterial and viral infections and diseases. The mediation of host-pathogen interactions by mucosal glycans, such as those added by FUT2, is poorly understood. We highlight, with specific examples, important mechanisms by which host glycans influence infection dynamics, including by: acting as pathogen receptors (or receptor-decoys), promoting microbial stability, altering the physical characteristics of mucus, and acting as immunological markers. We argue that the effect glycans have on infection dynamics has profound implications for many aspects of healthcare and policy, including clinical management, outbreak control, and vaccination policy. Copyright © 2017 Elsevier Ltd. All rights reserved.
MALDI Mass Spectrometry Imaging of N-Linked Glycans in Cancer Tissues.
Drake, R R; Powers, T W; Jones, E E; Bruner, E; Mehta, A S; Angel, P M
2017-01-01
Glycosylated proteins account for a majority of the posttranslation modifications of cell surface, secreted, and circulating proteins. Within the tumor microenvironment, the presence of immune cells, extracellular matrix proteins, cell surface receptors, and interactions between stroma and tumor cells are all processes mediated by glycan binding and recognition reactions. Changes in glycosylation during tumorigenesis are well documented to occur and affect all of these associated adhesion and regulatory functions. A MALDI imaging mass spectrometry (MALDI-IMS) workflow for profiling N-linked glycan distributions in fresh/frozen tissues and formalin-fixed paraffin-embedded tissues has recently been developed. The key to the approach is the application of a molecular coating of peptide-N-glycosidase to tissues, an enzyme that cleaves asparagine-linked glycans from their protein carrier. The released N-linked glycans can then be analyzed by MALDI-IMS directly on tissue. Generally 40 or more individual glycan structures are routinely detected, and when combined with histopathology localizations, tumor-specific glycans are readily grouped relative to nontumor regions and other structural features. This technique is a recent development and new approach in glycobiology and mass spectrometry imaging research methodology; thus, potential uses such as tumor-specific glycan biomarker panels and other applications are discussed. © 2017 Elsevier Inc. All rights reserved.
Glycan-mediated modification of the immune response
DEFF Research Database (Denmark)
Madsen, Caroline B; Pedersen, Anders E; Wandall, Hans H
2013-01-01
Aberrantly glycosylated tumor antigens represent promising targets for the development of anti-cancer vaccines, yet how glycans influence immune responses is poorly understood. Recent studies have demonstrated that GalNAc-glycosylation enhances antigen uptake by dendritic cells as well as CD4(+) T......-cell and humoral responses, but prevents CD8(+) T-cell activation. Here, we briefly discuss the relevance of glycans as candidate targets for anti-cancer vaccines....
Clark, Emily L; Emmadi, Madhu; Krupp, Katharine L; Podilapu, Ananda R; Helble, Jennifer D; Kulkarni, Suvarn S; Dube, Danielle H
2016-12-16
Bacterial glycans contain rare, exclusively bacterial monosaccharides that are frequently linked to pathogenesis and essentially absent from human cells. Therefore, bacterial glycans are intriguing molecular targets. However, systematic discovery of bacterial glycoproteins is hampered by the presence of rare deoxy amino sugars, which are refractory to traditional glycan-binding reagents. Thus, the development of chemical tools that label bacterial glycans is a crucial step toward discovering and targeting these biomolecules. Here, we explore the extent to which metabolic glycan labeling facilitates the studying and targeting of glycoproteins in a range of pathogenic and symbiotic bacterial strains. We began with an azide-containing analog of the naturally abundant monosaccharide N-acetylglucosamine and discovered that it is not broadly incorporated into bacterial glycans, thus revealing a need for additional azidosugar substrates to broaden the utility of metabolic glycan labeling in bacteria. Therefore, we designed and synthesized analogs of the rare deoxy amino d-sugars N-acetylfucosamine, bacillosamine, and 2,4-diacetamido-2,4,6-trideoxygalactose and established that these analogs are differentially incorporated into glycan-containing structures in a range of pathogenic and symbiotic bacterial species. Further application of these analogs will refine our knowledge of the glycan repertoire in diverse bacteria and may find utility in treating a variety of infectious diseases with selectivity.
High-throughput profiling of anti-glycan humoral responses to SIV vaccination and challenge.
Directory of Open Access Journals (Sweden)
Christopher T Campbell
Full Text Available Recent progress toward an HIV vaccine highlights both the potential of vaccines to end the AIDS pandemic and the need to boost efficacy by incorporating additional vaccine strategies. Although many aspects of the immune response can contribute to vaccine efficacy, the key factors have not been defined fully yet. A particular area that may yield new insights is anti-glycan immune responses, such as those against the glycan shield that HIV uses to evade the immune system. In this study, we used glycan microarray technology to evaluate anti-glycan antibody responses induced by SIV vaccination and infection in a non-human primate model of HIV infection. This comprehensive profiling of circulating anti-glycan antibodies found changes in anti-glycan antibody levels after both vaccination with the Ad5hr-SIV vaccine and SIV infection. Notably, SIV infection produced generalized declines in anti-glycan IgM antibodies in a number of animals. Additionally, some infected animals generated antibodies to the Tn antigen, which is a cryptic tumor-associated antigen exposed by premature termination of O-linked glycans; however, the Ad5hr-SIV vaccine did not induce anti-Tn IgG antibodies. Overall, this study demonstrates the potential contributions that glycan microarrays can make for HIV vaccine development.
Directory of Open Access Journals (Sweden)
Anna-Janina Behrens
2016-03-01
Full Text Available The HIV-1 envelope glycoprotein trimer is covered by an array of N-linked glycans that shield it from immune surveillance. The high density of glycans on the trimer surface imposes steric constraints limiting the actions of glycan-processing enzymes, so that multiple under-processed structures remain on specific areas. These oligomannose glycans are recognized by broadly neutralizing antibodies (bNAbs that are not thwarted by the glycan shield but, paradoxically, target it. Our site-specific glycosylation analysis of a soluble, recombinant trimer (BG505 SOSIP.664 maps the extremes of simplicity and diversity of glycan processing at individual sites and reveals a mosaic of dense clusters of oligomannose glycans on the outer domain. Although individual sites usually minimally affect the global integrity of the glycan shield, we identify examples of how deleting some glycans can subtly influence neutralization by bNAbs that bind at distant sites. The network of bNAb-targeted glycans should be preserved on vaccine antigens.
Increased levels of anti-glycan antibodies in patients with cystic fibrosis
Directory of Open Access Journals (Sweden)
Hirche TO
2011-09-01
Full Text Available Abstract Background The prevalence of Crohn's disease (CD is increased in patients with cystic fibrosis (CF. Anti-Saccharomyces cerevisiae antibodies (ASCA have been suggested as a screening tool to detect CD in CF. Recently, several new anti-glycan antibodies have been reported in CD. Materials and methods The sera of 119 CF patients of various age groups were prospectively screened for ASCA type IgG (gASCA, anti-laminaribioside carbohydrate IgG antibodies (ALCA, anti-chitobioside carbohydrate IgA antibodies (ACCA, and anti-mannobioside carbohydrate IgG antibodies (AMCA. The frequency of these anti-glycan antibodies was then compared in patients with CD, ulcerative colitis, rheumatoid arthritis and healthy volunteers. Results A significant number of CF patients were positive for gASCA (51.3% [41.6-60.6] and up to three other anti-glycan antibodies concurrently. Serum levels of anti-glycan antibodies in CF and CD were not related to parameters of inflammation. Despite the well-documented difference in clinical course between male and female CF patients no gender difference of anti-glycan antibodies was found. In contrast, there was a significant positive correlation between anti-glycan markers and age in CF patients. Conclusions Our findings demonstrate for the first time the increased frequency of a panel of anti-glycan antibodies in CF and provide a link between the presence of these serological biomarkers and patient's age. Anti-glycan antibody profiling may therefore become a valuable tool in the care of patients with CF.
Improved sample preparation for CE-LIF analysis of plant N-glycans.
Nagels, Bieke; Santens, Francis; Weterings, Koen; Van Damme, Els J M; Callewaert, Nico
2011-12-01
In view of glycomics studies in plants, it is important to have sensitive tools that allow one to analyze and characterize the N-glycans present on plant proteins in different species. Earlier methods combined plant-based sample preparations with CE-LIF N-glycan analysis but suffered from background contaminations, often resulting in non-reproducible results. This publication describes a reproducible and sensitive protocol for the preparation and analysis of plant N-glycans, based on a combination of the 'in-gel release method' and N-glycan analysis on a multicapillary DNA sequencer. Our protocol makes it possible to analyze plant N-glycans starting from low amounts of plant material with highly reproducible results. The developed protocol was validated for different plant species and plant cells. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Directory of Open Access Journals (Sweden)
Matthias T Ehebauer
2015-02-01
Full Text Available Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these--the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α-β unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications.
Higel, Fabian; Seidl, Andreas; Demelbauer, Uwe; Sörgel, Fritz; Frieß, Wolfgang
2014-01-01
N-glycosylation is a complex post-translational modification with potential effects on the efficacy and safety of therapeutic proteins and known influence on the effector function of biopharmaceutical monoclonal antibodies (mAbs). Comprehensive characterization of N-glycosylation is therefore important in biopharmaceutical development. In early development, e.g. during pool or clone selection, however, only minute protein amounts of multiple samples are available for analytics. High sensitivity and high throughput methods are thus needed. An approach based on 96-well plate sample preparation and nanoLC-MS of 2- anthranilic acid or 2-aminobenzoic acid (AA) labeled N-glycans for the characterization of biopharmaceuticals in early development is reported here. With this approach, 192 samples can be processed simultaneously from complex matrices (e.g., cell culture supernatant) to purified 2-AA glycans, which are then analyzed by reversed phase nanoLC-MS. Attomolar sensitivity has been achieved by use of nanoelectrospray ionization, resulting in detailed glycan maps of mAbs and fusion proteins that are exemplarily shown in this work. Reproducibility, robustness and linearity of the approach are demonstrated, making use in a routine manner during pool or clone selection possible. Other potential fields of application, such as glycan biomarker discovery from serum samples, are also presented.
Hilliard, Mark; Alley, William R; McManus, Ciara A; Yu, Ying Qing; Hallinan, Sinead; Gebler, John; Rudd, Pauline M
Glycosylation is an important attribute of biopharmaceutical products to monitor from development through production. However, glycosylation analysis has traditionally been a time-consuming process with long sample preparation protocols and manual interpretation of the data. To address the challenges associated with glycan analysis, we developed a streamlined analytical solution that covers the entire process from sample preparation to data analysis. In this communication, we describe the complete analytical solution that begins with a simplified and fast N-linked glycan sample preparation protocol that can be completed in less than 1 hr. The sample preparation includes labelling with RapiFluor-MS tag to improve both fluorescence (FLR) and mass spectral (MS) sensitivities. Following HILIC-UPLC/FLR/MS analyses, the data are processed and a library search based on glucose units has been included to expedite the task of structural assignment. We then applied this total analytical solution to characterize the glycosylation of the NIST Reference Material mAb 8761. For this glycoprotein, we confidently identified 35 N-linked glycans and all three major classes, high mannose, complex, and hybrid, were present. The majority of the glycans were neutral and fucosylated; glycans featuring N-glycolylneuraminic acid and those with two galactoses connected via an α1,3-linkage were also identified.
Stable Isotope Quantitative N-Glycan Analysis by Liquid Separation Techniques and Mass Spectrometry.
Mittermayr, Stefan; Albrecht, Simone; Váradi, Csaba; Millán-Martín, Silvia; Bones, Jonathan
2017-01-01
Liquid phase separation analysis and subsequent quantitation remains a challenging task for protein-derived oligosaccharides due to their inherent structural complexity and diversity. Incomplete resolution or co-detection of multiple glycan species complicates peak area-based quantitation and associated statistical analysis when optical detection methods are used. The approach outlined herein describes the utilization of stable isotope variants of commonly used fluorescent tags that allow for mass-based glycan identification and relative quantitation following separation by liquid chromatography (LC) or capillary electrophoresis (CE). Comparability assessment of glycoprotein-derived oligosaccharides is performed by derivatization with commercially available isotope variants of 2-aminobenzoic acid or aniline and analysis by LC- and CE-mass spectrometry. Quantitative information is attained from the extracted ion chromatogram/electropherogram ratios generated from the light and heavy isotope clusters.
Metabolic control analysis of xylose catabolism in Aspergillus
Prathumpai, W.; Gabelgaard, J.B.; Wanchanthuek, P.; Vondervoort, van de P.J.I.; Groot, de M.J.L.; McIntyre, M.; Nielsen, J.
2003-01-01
A kinetic model for xylose catabolism in Aspergillus is proposed. From a thermodynamic analysis it was found that the intermediate xylitol will accumulate during xylose catabolism. Use of the kinetic model allowed metabolic control analysis (MCA) of the xylose catabolic pathway to be carried out,
Jo, Sunhwan; Song, Kevin C.; Desaire, Heather; MacKerell, Alexander D.; Im, Wonpil
2011-01-01
Understanding how glycosylation affects protein structure, dynamics, and function is an emerging and challenging problem in biology. As a first step toward glycan modeling in the context of structural glycobiology, we have developed Glycan Reader and integrated it into the CHARMM-GUI, http://www.charmm-gui.org/input/glycan. Glycan Reader greatly simplifies the reading of PDB structure files containing glycans through (i) detection of carbohydrate molecules, (ii) automatic annotation of carbohydrates based on their three-dimensional structures, (iii) recognition of glycosidic linkages between carbohydrates as well as N-/O-glycosidic linkages to proteins, and (iv) generation of inputs for the biomolecular simulation program CHARMM with the proper glycosidic linkage setup. In addition, Glycan Reader is linked to other functional modules in CHARMM-GUI, allowing users to easily generate carbohydrate or glycoprotein molecular simulation systems in solution or membrane environments and visualize the electrostatic potential on glycoprotein surfaces. These tools are useful for studying the impact of glycosylation on protein structure and dynamics. PMID:21815173
Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags.
Kang, Ji-Yeon; Kwon, Ohsuk; Gil, Jin Young; Oh, Doo-Byoung
2016-06-01
Mannose-6-phosphate (M-6-P) glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino)-6-aminoacridine [AA-Ac]) were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps). The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in "Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans" (Kang et al., 2016 [1]).
Glycan involvement in the adhesion of Pseudomonas aeruginosa to tears.
Kautto, Liisa; Nguyen-Khuong, Terry; Everest-Dass, Arun; Leong, Andrea; Zhao, Zhenjun; Willcox, Mark D P; Packer, Nicolle H; Peterson, Robyn
2016-04-01
The human eye is constantly bathed by tears, which protect the ocular surface via a variety of mechanisms. The O-linked glycans of tear mucins have long been considered to play a role in binding to pathogens and facilitating their removal in the tear flow. Other conjugated glycans in tears could similarly contribute to pathogen binding and removal but have received less attention. In the work presented here we assessed the contribution of glycan moieties, in particular the protein attached N-glycans, presented by the broad complement of tear proteins to the adhesion of the opportunistic pathogen Pseudomonas aeruginosa, a leading cause of microbial keratitis and ulceration of the cornea. Our adhesion assay involved immobilising the macromolecular components of tears into the wells of a polyvinyl difluoride (PVDF) microtitre filter plate and probing the binding of fluorescently labelled bacteria. Three P. aeruginosa strains were studied: a cytotoxic strain (6206) and an invasive strain (6294) from eye infections, and an invasive strain (320) from a urinary tract infection (UTI). The ocular isolates adhered two to three times more to human tears than to human saliva or porcine gastric mucin, suggesting ocular niche-specific adaptation. Support for the role of the N-glycans carried by human tear proteins in the binding and removal of P. aeruginosa from the eye was shown by: 1) pre-incubation of the bacteria with free component sugars, galactose, mannose, fucose and sialyl lactose (or combination thereof) inhibiting adhesion of all the P. aeruginosa strains to the immobilised tear proteins, with the greatest inhibition of binding of the ocular cytotoxic 6206 and least for the invasive 6294 strain; 2) pre-incubation of the bacteria with N-glycans released from the commercially available human milk lactoferrin, an abundant protein that carries N-linked glycans in tears, inhibiting the adhesion to tears of the ocular bacteria by up to 70%, which was significantly more
A synthetic glycan microarray enables epitope mapping of plant cell wall glycan-directed antibodies
DEFF Research Database (Denmark)
Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah
2017-01-01
In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories world-wide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types......, and developmental stages. Despite their importance and broad use, the precise binding epitope for only a few of these antibodies has been determined. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies....... Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall...
Glycan cross-feeding activities between bifidobacteria under in vitro conditions
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Francesca eTurroni
2015-09-01
Full Text Available Bifidobacteria colonize the gut of various mammals, including humans, where they may metabolize complex, diet- and host-derived carbohydrates. The glycan-associated metabolic features encoded by bifidobacteria are believed to be strongly influenced by cross-feeding activities due to the co-existence of strains with different glycan-degrading properties. In this study, we observed an enhanced growth yield of Bifidobacterium bifidum PRL2010 when co-cultivated with Bifidobacterium breve 12L, Bifidobacterium adolescentis 22L or Bifidobacterium thermophilum JCM1207. This enhanced growth phenomenon was confirmed by whole genome transcriptome analyses, which revealed co-cultivation-associated transcriptional induction of PRL2010 genes involved in carbohydrate metabolism, such as those encoding for carbohydrate transporters and associated energy production, and genes reuired for translation, ribosomal structure and biogenesis, thus supporting the idea that co-cultivation of certain bifidobacterial strains with B. bifidum PRL2010 causes enhanced metabolic activity, and consequently increased lactate and/or acetate production. Overall, these data suggest that PRL2010 cells benefit from the presence of other bifidobacterial strains.
Terkelsen, Thilde; Haakensen, Vilde D; Saldova, Radka; Gromov, Pavel; Hansen, Merete Kjaer; Stöckmann, Henning; Lingjaerde, Ole Christian; Børresen-Dale, Anne-Lise; Papaleo, Elena; Helland, Åslaug; Rudd, Pauline M; Gromova, Irina
2018-04-26
Particular N-glycan structures are known to be associated with breast malignancies by coordinating various regulatory events within the tumor and corresponding microenvironment, thus implying that N-glycan patterns may be used for cancer stratification and as predictive or prognostic biomarkers. However, the association between N-glycans secreted by breast tumor and corresponding clinical relevance remain to be elucidated. We profiled N-glycans by HILIC UPLC across a discovery dataset composed of tumor interstitial fluids (TIF, n=85), paired normal interstitial fluids (NIF, n=54) and serum samples (n=28) followed by independent evaluation, with the ultimate goal of identifying tumor-related N-glycan patterns in blood of breast cancer patients. The segregation of N-linked oligosaccharides revealed 33 compositions, which exhibited differential abundances between TIF and NIF. TIFs were depleted of bisecting N-glycans, which are known to play essential roles in tumor suppression. An increased level of simple high mannose N-glycans in TIF strongly correlated with the presence of tumor infiltrating lymphocytes within tumor. At the same time, a low level of highly complex N-glycans in TIF inversely correlated with the presence of infiltrating lymphocytes within tumor. Survival analysis showed that patients exhibiting increased TIF abundance of GP24 had better outcomes, whereas low levels of GP10, GP23, GP38, and coreF were associated with poor prognosis. Levels of GP1, GP8, GP9, GP14, GP23, GP28, GP37, GP38, and coreF were significantly correlated between TIF and paired serum samples. Cross-validation analysis using an independent serum dataset supported the observed correlation between TIF and serum, for five out of nine N-glycan groups: GP8, GP9, GP14, GP23, and coreF. Collectively, our results imply that profiling of N-glycans from proximal breast tumor fluids is a promising strategy for determining tumor-derived glyco-signature(s) in the blood. N-glycans structures
Dual modifications strategy to quantify neutral and sialylated N-glycans simultaneously by MALDI-MS.
Zhou, Hui; Warren, Peter G; Froehlich, John W; Lee, Richard S
2014-07-01
Differences in ionization efficiency among neutral and sialylated glycans prevent direct quantitative comparison by their respective mass spectrometric signals. To overcome this challenge, we developed an integrated chemical strategy, Dual Reactions for Analytical Glycomics (DRAG), to quantitatively compare neutral and sialylated glycans simultaneously by MALDI-MS. Initially, two glycan samples to be compared undergo reductive amination with 2-aminobenzoic acid and 2-(13)[C6]-aminobenzoic acid, respectively. The different isotope-incorporated glycans are then combined and subjected to the methylamidation of the sialic acid residues in one mixture, homogenizing the ionization responses for all neutral and sialylated glycans. By this approach, the expression change of relevant glycans between two samples is proportional to the ratios of doublet signals with a static 6 Da mass difference in MALDI-MS and the change in relative abundance of any glycan within samples can also be determined. The strategy was chemically validated using well-characterized N-glycans from bovine fetuin and IgG from human serum. By comparing the N-glycomes from a first morning (AM) versus an afternoon (PM) urine sample obtained from a single donor, we further demonstrated the ability of DRAG strategy to measure subtle quantitative differences in numerous urinary N-glycans.
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Xiang, Yong; Karaveg, Khanita; Moremen, Kelley W.
2016-11-17
Asn-linked glycosylation of newly synthesized polypeptides occurs in the endoplasmic reticulum of eukaryotic cells. Glycan structures are trimmed and remodeled as they transit the secretory pathway, and processing intermediates play various roles as ligands for folding chaperones and signals for quality control and intracellular transport. Key steps for the generation of these trimmed intermediates are catalyzed by glycoside hydrolase family 47 (GH47) α-mannosidases that selectively cleave α1,2-linked mannose residues. Despite the sequence and structural similarities among the GH47 enzymes, the molecular basis for residue-specific cleavage remains obscure. The present studies reveal enzyme–substrate complex structures for two related GH47 α-mannosidases and provide insights into how these enzymes recognize the same substrates differently and catalyze the complementary glycan trimming reactions necessary for glycan maturation.
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Pratick Khara
2014-01-01
Full Text Available Sphingobium sp. PNB, like other sphingomonads, has multiple ring-hydroxylating oxygenase (RHO genes. Three different fosmid clones have been sequenced to identify the putative genes responsible for the degradation of various aromatics in this bacterial strain. Comparison of the map of the catabolic genes with that of different sphingomonads revealed a similar arrangement of gene clusters that harbors seven sets of RHO terminal components and a sole set of electron transport (ET proteins. The presence of distinctly conserved amino acid residues in ferredoxin and in silico molecular docking analyses of ferredoxin with the well characterized terminal oxygenase components indicated the structural uniqueness of the ET component in sphingomonads. The predicted substrate specificities, derived from the phylogenetic relationship of each of the RHOs, were examined based on transformation of putative substrates and their structural homologs by the recombinant strains expressing each of the oxygenases and the sole set of available ET proteins. The RHO AhdA1bA2b was functionally characterized for the first time and was found to be capable of transforming ethylbenzene, propylbenzene, cumene, p-cymene and biphenyl, in addition to a number of polycyclic aromatic hydrocarbons. Overexpression of aromatic catabolic genes in strain PNB, revealed by real-time PCR analyses, is a way forward to understand the complex regulation of degradative genes in sphingomonads.
Morrison, Kelsey A.; Clowers, Brian H.
2017-06-01
The alternative dissociation pathways initiated by ultraviolet photodissociation (UVPD) compared with collision-induced dissociation (CID) may provide useful diagnostic fragments for biomolecule identification, including glycans. However, underivatized glycans do not commonly demonstrate strong UV absorbance, resulting in low fragmentation yields for UVPD spectra. In contrast to UVPD experiments that leverage covalent modification of glycans, we detail the capacity of metal adduction to yield comparatively rich UVPD fragmentation patterns and enhance separation factors for an isomeric glycan set in a drift tube ion mobility system. Ion mobility and UVPD-MS spectra for two N-acetyl glycan isomers were examined, each adducted with sodium or cobalt cations, with the latter providing fragment yield gains of an order of magnitude versus sodium adducts. Furthermore, our glycan analysis incorporated front-end ion mobility separation such that the structural glycan isomers could still be identified even as a mixture and not simply composite spectra of isomeric standards. Cobalt adduction proved influential in the glycan separation by yielding an isomer resolution of 0.78 when analyzed simultaneously versus no discernable separation obtained with the sodium adducts. It is the combined enhancement of both isomeric drift time separation and isomer distinction with improved UVPD fragment ion yields that further bolster multivalent metal adduction for advancing glycan IM-MS experiments. [Figure not available: see fulltext.
Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans.
Kang, Ji-Yeon; Kwon, Ohsuk; Gil, Jin Young; Oh, Doo-Byoung
2016-05-15
Mannose-6-phosphate (M-6-P) glycan analysis is important for quality control of therapeutic enzymes for lysosomal storage diseases. Here, we found that the analysis of glycans containing two M-6-Ps was highly affected by the hydrophilicity of the elution solvent used in high-performance liquid chromatography (HPLC). In addition, the performances of three fluorescent tags--2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), and 3-(acetyl-amino)-6-aminoacridine (AA-Ac)--were compared with each other for M-6-P glycan analysis using HPLC and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The best performance for analyzing M-6-P glycans was shown by 2-AA labeling in both analyses. Copyright © 2016 Elsevier Inc. All rights reserved.
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Akihiko Kameyama
Full Text Available Glycan analysis may result in exploitation of glycan biomarkers and evaluation of heterogeneity of glycosylation of biopharmaceuticals. For N-linked glycan analysis, we investigated alkaline hydrolysis of the asparagine glycosyl carboxamide of glycoproteins as a deglycosylation reaction. By adding hydroxylamine into alkaline de-N-glycosylation, we suppressed the degradation of released glycans and obtained a mixture of oximes, free glycans, and glycosylamines. The reaction was completed within 1 h, and the mixture containing oximes was easily tagged with 2-aminobenzamide by reductive amination. Here, we demonstrated N-linked glycan analysis using this method for a monoclonal antibody, and examined whether this method could liberate glycans without degradation from apo-transferrin containing NeuAc and NeuGc and horseradish peroxidase containing Fuc α1-3 GlcNAc at the reducing end. Furthermore, we compared glycan recoveries between conventional enzymatic glycan release and this method. Increasing the reaction temperature and reaction duration led to degradation, whereas decreasing these parameters resulted in lower release. Considering this balance, we proposed to carry out the reaction at 80°C for 1 h for asialo glycoproteins from mammals and at 50°C for 1 h for sialoglycoproteins.
Anumula, Kalyan Rao
2012-08-31
Typical clinical grade human IgG (intravenous immunoglobulin, IVIG), used for carbohydrate analysis, is derived from thousands of healthy donors. Quantitative high-resolution glycan profiles of IgG and its Fc-Fab fragments are presented here. Glycan profiles were established following digestions with Fc specific endoglycosidase S and generic PNGase F under denaturing and non-denaturing (native) conditions. The native PNGase F glycan profile of IgG was similar (but not identical) to that of Endo S. Endo S profiles did not contain the glycans with bisecting GlcNAc. PNGase F glycan profiles were the same for Fc fragments that were isolated from pepsin and Ide S protease digests. Both isolated Fab fragments and the previously deglycosylated IVIG (native conditions) yielded the same glycan profile. Glycan profiles were established using high resolution HPLC with 2-aminobenzoic acid (2AA) labeling. An accurate determination of sialylation levels can be made by this method. Carbohydrate content in Fc and Fab was determined using an internal standard and corrected for both protein and glycan recoveries. Fab portion contained about 14% of the total carbohydrate which translates to 2.3 sugar chains per mol in IVIG where 2 chains are located in the CH2 domain of the Fc. Fc glycans consisted of neutral (N) 84.5%; mono-sialylated (S1) 15% and di-sialylated (S2) 0.5%. In contrast, Fab contained N, 21%; S1, 43% and S2, 36%. The distribution of bisecting N-acetylglucosamine and fucose was found to be very different in various glycans (N, S1 and S2) found in Fab and Fc. Total IgG glycan profile (Fab plus Fc) contained N, 78.5%; S1, 17% and S2, 4.5%. Percent distribution of glycans G0, G1 and G2 (with 0, 1 and 2 two galactoses) was 26, 49 and 25 respectively within the 78% of the neutral glycans. Glycan profiles were nearly the same for various clinical grade IVIG preparations from various manufacturers. A fast HPLC profiling method was developed for the separation and quantitation
Hexose rearrangements upon fragmentation of N-glycopeptides and reductively aminated N-glycans.
Wuhrer, Manfred; Koeleman, Carolien A M; Deelder, André M
2009-06-01
Tandem mass spectrometry of glycans and glycoconjugates in protonated form is known to result in rearrangement reactions leading to internal residue loss. Here we studied the occurrence of hexose rearrangements in tandem mass spectrometry of N-glycopeptides and reductively aminated N-glycans by MALDI-TOF/TOF-MS/MS and ESI-ion trap-MS/MS. Fragmentation of proton adducts of oligomannosidic N-glycans of ribonuclease B that were labeled with 2-aminobenzamide and 2-aminobenzoic acid resulted in transfer of one to five hexose residues to the fluorescently tagged innermost N-acetylglucosamine. Glycopeptides from various biological sources with oligomannosidic glycans were likewise shown to undergo hexose rearrangement reactions, resulting in chitobiose cleavage products that have acquired one or two hexose moieties. Tryptic immunoglobulin G Fc-glycopeptides with biantennary N-glycans likewise showed hexose rearrangements resulting in hexose transfer to the peptide moiety retaining the innermost N-acetylglucosamine. Thus, as a general phenomenon, tandem mass spectrometry of reductively aminated glycans as well as glycopeptides may result in hexose rearrangements. This characteristic of glycopeptide MS/MS has to be considered when developing tools for de novo glycopeptide structural analysis.
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Yunus E. Tuncil
2017-10-01
Full Text Available When presented with nutrient mixtures, several human gut Bacteroides species exhibit hierarchical utilization of glycans through a phenomenon that resembles catabolite repression. However, it is unclear how closely these observed physiological changes, often measured by altered transcription of glycan utilization genes, mirror actual glycan depletion. To understand the glycan prioritization strategies of two closely related human gut symbionts, Bacteroides ovatus and Bacteroides thetaiotaomicron, we performed a series of time course assays in which both species were individually grown in a medium with six different glycans that both species can degrade. Disappearance of the substrates and transcription of the corresponding polysaccharide utilization loci (PULs were measured. Each species utilized some glycans before others, but with different priorities per species, providing insight into species-specific hierarchical preferences. In general, the presence of highly prioritized glycans repressed transcription of genes involved in utilizing lower-priority nutrients. However, transcriptional sensitivity to some glycans varied relative to the residual concentration in the medium, with some PULs that target high-priority substrates remaining highly expressed even after their target glycan had been mostly depleted. Coculturing of these organisms in the same mixture showed that the hierarchical orders generally remained the same, promoting stable coexistence. Polymer length was found to be a contributing factor for glycan utilization, thereby affecting its place in the hierarchy. Our findings not only elucidate how B. ovatus and B. thetaiotaomicron strategically access glycans to maintain coexistence but also support the prioritization of carbohydrate utilization based on carbohydrate structure, advancing our understanding of the relationships between diet and the gut microbiome.
Glycosylation with ribitol-phosphate in mammals: New insights into the O-mannosyl glycan.
Manya, Hiroshi; Endo, Tamao
2017-10-01
O-mannosyl glycans have been found in a limited number of glycoproteins of the brain, nerves, and skeletal muscles, particularly in α-dystroglycan (α-DG). Defects in O-mannosyl glycan on α-DG are the primary cause of a group of congenital muscular dystrophies, which are collectively termed α-dystroglycanopathy. Recent studies have revealed various O-mannosyl glycan structures, which can be classified as core M1, core M2, and core M3 glycans. Although many dystroglycanopathy genes are involved in core M3 processing, the structure and biosynthesis of core M3 glycan remains only partially understood. This review presents recent findings about the structure, biosynthesis, and pathology of O-mannosyl glycans. Recent studies have revealed that the entire structure of core M3 glycan, including ribitol-5-phosphate, is a novel structure in mammals; its unique biosynthetic pathway has been elucidated by the identification of new causative genes for α-dystroglycanopathies and their functions. O-mannosyl glycan has a novel, unique structure that is important for the maintenance of brain and muscle functions. These findings have opened up a new field in glycoscience. These studies will further contribute to the understanding of the pathomechanism of α-dystroglycanopathy and the development of glycotherapeutics. This article is part of a Special Issue entitled Neuro-glycoscience, edited by Kenji Kadomatsu and Hiroshi Kitagawa. Copyright © 2017 Elsevier B.V. All rights reserved.
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Ropartz, David; Giuliani, Alexandre; Fanuel, Mathieu; Hervé, Cécile; Czjzek, Mirjam; Rogniaux, Hélène
2016-01-01
The activation of ions by extreme-energy photons (XUV) produced by a synchrotron radiation beamline is a powerful method for characterizing complex glycans using tandem mass spectrometry (MS). As previously described, this activation method leads to rich fragmentation spectra with many structurally valuable cross-ring cleavages while maintaining labile modifications on the glycan structures. However, until now, the tandem MS event was too long to be compatible with liquid chromatography elution times. In this work, the duty cycle of the activation and detection of fragments was shortened, and the background signal on the spectra was drastically reduced. Both improvements allowed, for the first time, the successful coupling of a UHPLC system to XUV-activated tandem MS. The approach was used to characterize a complex mixture of oligo-porphyrans, which are a class of highly sulfated oligosaccharides, in a fully automated way. Due to an enhanced dynamic range and an increased sensitivity, some hypothetical structures of low abundance have been unequivocally confirmed in this study and others have been revised. Some previously undescribed species of oligo-porphyrans that exhibit lateral branching have been fully resolved. This work contributes to the scarce knowledge of the structure of porphyrans in red algae and pushes the current capacities of XUV-activation tandem MS by demonstrating the possibility of a direct coupling with UHPLC. This study will considerably broaden the applicability and practicality of this method in many fields of analytical biology. - Highlights: • For the first time, XUV photon activation tandem MS was coupled to UHPLC. • The approach was used to characterize a complex mixture of biomolecules. • The MSMS duty cycle was compatible with elution times of UHPLC without compromised. • Minor species were characterized with an enhanced sensitivity and dynamic range. • These results broaden the application of the technique in many field of
Energy Technology Data Exchange (ETDEWEB)
Ropartz, David, E-mail: David.Ropartz@nantes.inra.fr [INRA, UR1268 Biopolymers Interactions Assemblies F-44316 Nantes (France); Giuliani, Alexandre [Synchrotron SOLEIL, L' Orme des Merisiers, F-91190 Gif-sur-Yvette (France); UAR 1008 CEPIA, INRA, F-44316 Nantes (France); Fanuel, Mathieu [INRA, UR1268 Biopolymers Interactions Assemblies F-44316 Nantes (France); Hervé, Cécile; Czjzek, Mirjam [Sorbonne Universités, Université Pierre et Marie Curie, Paris VI, CNRS, Integrative Biology of Marine Models, UMR 8227, Station Biologique, Place George Teissier, F29688 Roscoff Cedex (France); Rogniaux, Hélène [INRA, UR1268 Biopolymers Interactions Assemblies F-44316 Nantes (France)
2016-08-24
The activation of ions by extreme-energy photons (XUV) produced by a synchrotron radiation beamline is a powerful method for characterizing complex glycans using tandem mass spectrometry (MS). As previously described, this activation method leads to rich fragmentation spectra with many structurally valuable cross-ring cleavages while maintaining labile modifications on the glycan structures. However, until now, the tandem MS event was too long to be compatible with liquid chromatography elution times. In this work, the duty cycle of the activation and detection of fragments was shortened, and the background signal on the spectra was drastically reduced. Both improvements allowed, for the first time, the successful coupling of a UHPLC system to XUV-activated tandem MS. The approach was used to characterize a complex mixture of oligo-porphyrans, which are a class of highly sulfated oligosaccharides, in a fully automated way. Due to an enhanced dynamic range and an increased sensitivity, some hypothetical structures of low abundance have been unequivocally confirmed in this study and others have been revised. Some previously undescribed species of oligo-porphyrans that exhibit lateral branching have been fully resolved. This work contributes to the scarce knowledge of the structure of porphyrans in red algae and pushes the current capacities of XUV-activation tandem MS by demonstrating the possibility of a direct coupling with UHPLC. This study will considerably broaden the applicability and practicality of this method in many fields of analytical biology. - Highlights: • For the first time, XUV photon activation tandem MS was coupled to UHPLC. • The approach was used to characterize a complex mixture of biomolecules. • The MSMS duty cycle was compatible with elution times of UHPLC without compromised. • Minor species were characterized with an enhanced sensitivity and dynamic range. • These results broaden the application of the technique in many field of
Rup, Bonita; Alon, Sari; Amit-Cohen, Bat-Chen; Brill Almon, Einat; Chertkoff, Raul; Rudd, Pauline M.
2017-01-01
Plants are a promising alternative for the production of biotherapeutics. Manufacturing in-planta adds plant specific glycans. To understand immunogenic potential of these glycans, we developed a validated method to detect plant specific glycan antibodies in human serum. Using this assay, low prevalence of pre-existing anti-plant glycan antibodies was found in healthy humans (13.5%) and in glucocerebrosidase-deficient Gaucher disease (GD) patients (5%). A low incidence (9% in naïve patient and none in treatment experienced patients) of induced anti-plant glycan antibodies was observed in GD patients after up to 30 months replacement therapy treatment with taliglucerase alfa, a version of human glucocerebrosidase produced in plant cells. Detailed evaluation of clinical safety and efficacy endpoints indicated that anti-plant glycan antibodies did not affect the safety or efficacy of taliglucerase alfa in patients. This study shows the benefit of using large scale human trials to evaluate the immunogenicity risk of plant derived glycans, and indicates no apparent risk related to anti-plant glycan antibodies. PMID:29088235
A nonself sugar mimic of the HIV glycan shield shows enhanced antigenicity
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Doores, Katie J.; Fulton, Zara; Hong, Vu; Patel, Mitul K.; Scanlan, Christopher N.; Wormald, Mark R.; Finn, M.G.; Burton, Dennis R.; Wilson, Ian A.; Davis, Benjamin G. (Scripps); (Oxford)
2011-08-24
Antibody 2G12 uniquely neutralizes a broad range of HIV-1 isolates by binding the high-mannose glycans on the HIV-1 surface glycoprotein, gp120. Antigens that resemble these natural epitopes of 2G12 would be highly desirable components for an HIV-1 vaccine. However, host-produced (self)-carbohydrate motifs have been unsuccessful so far at eliciting 2G12-like antibodies that cross-react with gp120. Based on the surprising observation that 2G12 binds nonproteinaceous monosaccharide D-fructose with higher affinity than D-mannose, we show here that a designed set of nonself, synthetic monosaccharides are potent antigens. When introduced to the terminus of the D1 arm of protein glycans recognized by 2G12, their antigenicity is significantly enhanced. Logical variation of these unnatural sugars pinpointed key modifications, and the molecular basis of this increased antigenicity was elucidated using high-resolution crystallographic analyses. Virus-like particle protein conjugates containing such nonself glycans are bound more tightly by 2G12. As immunogens they elicit higher titers of antibodies than those immunogenic conjugates containing the self D1 glycan motif. These antibodies generated from nonself immunogens also cross-react with this self motif, which is found in the glycan shield, when it is presented in a range of different conjugates and glycans. However, these antibodies did not bind this glycan motif when present on gp120.
Glycan characterization of biopharmaceuticals: Updates and perspectives
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Planinc, Ana [Analytical Platform of the Faculty of Pharmacy and Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Universite Libre de Bruxelles (ULB), Brussels (Belgium); Bones, Jonathan [Characterisation and Comparability Laboratory, NIBRT – The National Institute for Bioprocessing Research and Training, Foster Avenue, Mount Merrion, Blackrock, Co. Dublin (Ireland); Dejaegher, Bieke [Laboratory of Instrumental Analysis and Bioelectrochemistry, Faculty of Pharmacy, Université Libre de Bruxelles (ULB), Boulevard du Triomphe, B-1050 Brussels (Belgium); Department of Analytical Chemistry and Pharmaceutical Technology (FABI), Center for Pharmaceutical Research (CePhaR), Faculty of Medicines and Pharmacy, Vrije Universiteit Brussel - VUB, Laarbeeklaan 103, B-1090 Brussels (Belgium); Van Antwerpen, Pierre [Analytical Platform of the Faculty of Pharmacy and Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Universite Libre de Bruxelles (ULB), Brussels (Belgium); Delporte, Cédric, E-mail: cedric.delporte@ulb.ac.be [Analytical Platform of the Faculty of Pharmacy and Laboratory of Pharmaceutical Chemistry, Faculty of Pharmacy, Universite Libre de Bruxelles (ULB), Brussels (Belgium)
2016-05-19
Therapeutic proteins are rapidly becoming the most promising class of pharmaceuticals on the market due to their successful treatment of a vast array of serious diseases, such as cancers and immune disorders. Therapeutic proteins are produced using recombinant DNA technology. More than 60% of therapeutic proteins are posttranslationally modified following biosynthesis by the addition of N- or O-linked glycans. Glycosylation is the most common posttranslational modifications of proteins. However, it is also the most demanding and complex posttranslational modification from the analytical point of view. Moreover, research has shown that glycosylation significantly impacts stability, half-life, mechanism of action and safety of a therapeutic protein. Considering the exponential growth of biotherapeutics, this present review of the literature (2009–2015) focuses on the characterization of protein glycosylation, which has witnessed an improvement in methodology. Furthermore, it discusses current issues in the fields of production and characterization of therapeutic proteins. This review also highlights the problem of non-standard requirements for the approval of biosimilars with regard to their glycosylation and discusses recent developments and perspectives for improved glycan characterization. - Highlights: • Biopharmaceuticals have emerged as the new class of blockbuster drugs in the pharmaceutical industry. • More than 60% of the approved biopharmaceuticals are glycosylated. • Glycosylation has an effect on the efficacy and the safety of therapeutic glycoproteins. • N-glycosylation characterization of therapeutic glycoproteins is a regulatory requirement. • Biosimilar releases are increasing and demonstration of comparability poses challenges for N-glycosylation characterization.
Glycan characterization of biopharmaceuticals: Updates and perspectives
International Nuclear Information System (INIS)
Planinc, Ana; Bones, Jonathan; Dejaegher, Bieke; Van Antwerpen, Pierre; Delporte, Cédric
2016-01-01
Therapeutic proteins are rapidly becoming the most promising class of pharmaceuticals on the market due to their successful treatment of a vast array of serious diseases, such as cancers and immune disorders. Therapeutic proteins are produced using recombinant DNA technology. More than 60% of therapeutic proteins are posttranslationally modified following biosynthesis by the addition of N- or O-linked glycans. Glycosylation is the most common posttranslational modifications of proteins. However, it is also the most demanding and complex posttranslational modification from the analytical point of view. Moreover, research has shown that glycosylation significantly impacts stability, half-life, mechanism of action and safety of a therapeutic protein. Considering the exponential growth of biotherapeutics, this present review of the literature (2009–2015) focuses on the characterization of protein glycosylation, which has witnessed an improvement in methodology. Furthermore, it discusses current issues in the fields of production and characterization of therapeutic proteins. This review also highlights the problem of non-standard requirements for the approval of biosimilars with regard to their glycosylation and discusses recent developments and perspectives for improved glycan characterization. - Highlights: • Biopharmaceuticals have emerged as the new class of blockbuster drugs in the pharmaceutical industry. • More than 60% of the approved biopharmaceuticals are glycosylated. • Glycosylation has an effect on the efficacy and the safety of therapeutic glycoproteins. • N-glycosylation characterization of therapeutic glycoproteins is a regulatory requirement. • Biosimilar releases are increasing and demonstration of comparability poses challenges for N-glycosylation characterization.
Steentoft, Catharina; Migliorini, Denis; King, Tiffany R; Mandel, Ulla; June, Carl H; Posey, Avery D
2018-01-23
Cancer immunotherapy is rapidly advancing in the treatment of a variety of hematopoietic cancers, including pediatric acute lymphoblastic leukemia and diffuse large B cell lymphoma, with chimeric antigen receptor (CAR)-T cells. CARs are genetically encoded artificial T cell receptors that combine the antigen specificity of an antibody with the machinery of T cell activation. However, implementation of CAR technology in the treatment of solid tumors has been progressing much slower. Solid tumors are characterized by a number of challenges that need to be overcome, including cellular heterogeneity, immunosuppressive tumor microenvironment (TME), and, in particular, few known cancer-specific targets. Post-translational modifications that differentially occur in malignant cells generate valid cell surface, cancer-specific targets for CAR-T cells. We previously demonstrated that CAR-T cells targeting an aberrant O-glycosylation of MUC1, a common cancer marker associated with changes in cell adhesion, tumor growth, and poor prognosis, could control malignant growth in mouse models. Here, we discuss the field of glycan-directed CAR-T cells and review the different classes of antibodies specific for glycan-targeting, including the generation of high affinity O-glycopeptide antibodies. Finally, we discuss historic and recently investigated glycan targets for CAR-T cells and provide our perspective on how targeting the tumor glycoproteome and/or glycome will improve CAR-T immunotherapy. © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Hoogenboom, Jorin; Berghuis, Nathalja; Cramer, Dario; Geurts, Rene; Zuilhof, Han; Wennekes, Tom
2016-10-10
Carbohydrates, also called glycans, play a crucial but not fully understood role in plant health and development. The non-template driven formation of glycans makes it impossible to image them in vivo with genetically encoded fluorescent tags and related molecular biology approaches. A solution to this problem is the use of tailor-made glycan analogs that are metabolically incorporated by the plant into its glycans. These metabolically incorporated probes can be visualized, but techniques documented so far use toxic copper-catalyzed labeling. To further expand our knowledge of plant glycobiology by direct imaging of its glycans via this method, there is need for novel click-compatible glycan analogs for plants that can be bioorthogonally labelled via copper-free techniques. Arabidopsis seedlings were incubated with azido-containing monosaccharide analogs of N-acetylglucosamine, N-acetylgalactosamine, L-fucose, and L-arabinofuranose. These azido-monosaccharides were metabolically incorporated in plant cell wall glycans of Arabidopsis seedlings. Control experiments indicated active metabolic incorporation of the azido-monosaccharide analogs into glycans rather than through non-specific absorption of the glycan analogs onto the plant cell wall. Successful copper-free labeling reactions were performed, namely an inverse-electron demand Diels-Alder cycloaddition reaction using an incorporated N-acetylglucosamine analog, and a strain-promoted azide-alkyne click reaction. All evaluated azido-monosaccharide analogs were observed to be non-toxic at the used concentrations under normal growth conditions. Our results for the metabolic incorporation and fluorescent labeling of these azido-monosaccharide analogs expand the possibilities for studying plant glycans by direct imaging. Overall we successfully evaluated five azido-monosaccharide analogs for their ability to be metabolically incorporated in Arabidopsis roots and their imaging after fluorescent labeling. This expands
Kronewitter, Scott R; An, Hyun Joo; de Leoz, Maria Lorna; Lebrilla, Carlito B; Miyamoto, Suzanne; Leiserowitz, Gary S
2009-06-01
Annotation of the human serum N-linked glycome is a formidable challenge but is necessary for disease marker discovery. A new theoretical glycan library was constructed and proposed to provide all possible glycan compositions in serum. It was developed based on established glycobiology and retrosynthetic state-transition networks. We find that at least 331 compositions are possible in the serum N-linked glycome. By pairing the theoretical glycan mass library with a high mass accuracy and high-resolution MS, human serum glycans were effectively profiled. Correct isotopic envelope deconvolution to monoisotopic masses and the high mass accuracy instruments drastically reduced the amount of false composition assignments. The high throughput capacity enabled by this library permitted the rapid glycan profiling of large control populations. With the use of the library, a human serum glycan mass profile was developed from 46 healthy individuals. This paper presents a theoretical N-linked glycan mass library that was used for accurate high-throughput human serum glycan profiling. Rapid methods for evaluating a patient's glycome are instrumental for studying glycan-based markers.
Subramanian, Sabarinath Peruvemba; Babu, Ponnusamy; Palakodeti, Dasaradhi; Subramanian, Ramaswamy
2018-05-04
Cell surface-associated glycans mediate many cellular processes, including adhesion, migration, signaling, and extracellular matrix organization. The galactosylation of core fucose (GalFuc epitope) in paucimannose and complex-type N -glycans is characteristic of protostome organisms, including flatworms (planarians). Although uninvestigated, the structures of these glycans may play a role in planarian regeneration. Whole-organism MALDI-MS analysis of N -linked oligosaccharides from the planarian Schmidtea mediterranea revealed the presence of multiple isomeric high-mannose and paucimannose structures with unusual mono-, di-, and polygalactosylated ( n = 3-5) core fucose structures; the latter structures have not been reported in other systems. Di- and trigalactosylated core fucoses were the most dominant glycomers. N -Glycans showed extensive, yet selective, methylation patterns, ranging from non-methylated to polymethylated glycoforms. Although the majority of glycoforms were polymethylated, a small fraction also consisted of non-methylated glycans. Remarkably, monogalactosylated core fucose remained unmethylated, whereas its polygalactosylated forms were methylated, indicating structurally selective methylation. Using database searches, we identified two potential homologs of the Galβ1-4Fuc-synthesizing enzyme from nematodes (GALT-1) that were expressed in the prepharyngeal, pharyngeal, and mesenchymal regions in S. mediterranea. The presence of two GALT-1 homologs suggests different requirements for mono- and polygalactosylation of core fucose for the formation of multiple isomers. Furthermore, we observed variations in core fucose glycosylation patterns in different planarian strains, suggesting evolutionary adaptation in fucose glycosylation. The various core chitobiose modifications and methylations create >60 different glycoforms in S. mediterranea. These results contribute greatly to our understanding of N -glycan biosynthesis and suggest the presence of a
Metabolic signature of sun exposed skin suggests catabolic pathway overweighs anabolic pathway.
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Manpreet Randhawa
Full Text Available Skin chronically exposed to sun results in phenotypic changes referred as photoaging. This aspect of aging has been studied extensively through genomic and proteomic tools. Metabolites, the end product are generated as a result of biochemical reactions are often studied as a culmination of complex interplay of gene and protein expression. In this study, we focused exclusively on the metabolome to study effects from sun-exposed and sun-protected skin sites from 25 human subjects. We generated a highly accurate metabolomic signature for the skin that is exposed to sun. Biochemical pathway analysis from this data set showed that sun-exposed skin resides under high oxidative stress and the chains of reactions to produce these metabolites are inclined toward catabolism rather than anabolism. These catabolic activities persuade the skin cells to generate metabolites through the salvage pathway instead of de novo synthesis pathways. Metabolomic profile suggests catabolic pathways and reactive oxygen species operate in a feed forward fashion to alter the biology of sun exposed skin.
DEFF Research Database (Denmark)
Madsen, Caroline B; Lavrsen, Kirstine; Steentoft, Catharina
2013-01-01
are recognized as cancer associated truncated glycans, and are expressed in many adenocarcinomas, e.g. breast- and pancreatic cancer cells. To investigate the role of the cancer associated glycan truncations in immune-mediated killing we created glyco-engineered breast- and pancreatic cancer cells expressing...... only the shortest possible mucin-like glycans (Tn and STn). Glyco-engineering was performed by zinc finger nuclease (ZFN) knockout (KO) of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast...
Quantification of the Impact of the HIV-1-Glycan Shield on Antibody Elicitation
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Tongqing Zhou
2017-04-01
Full Text Available While the HIV-1-glycan shield is known to shelter Env from the humoral immune response, its quantitative impact on antibody elicitation has been unclear. Here, we use targeted deglycosylation to measure the impact of the glycan shield on elicitation of antibodies against the CD4 supersite. We engineered diverse Env trimers with select glycans removed proximal to the CD4 supersite, characterized their structures and glycosylation, and immunized guinea pigs and rhesus macaques. Immunizations yielded little neutralization against wild-type viruses but potent CD4-supersite neutralization (titers 1: >1,000,000 against four-glycan-deleted autologous viruses with over 90% breadth against four-glycan-deleted heterologous strains exhibiting tier 2 neutralization character. To a first approximation, the immunogenicity of the glycan-shielded protein surface was negligible, with Env-elicited neutralization (ID50 proportional to the exponential of the protein-surface area accessible to antibody. Based on these high titers and exponential relationship, we propose site-selective deglycosylated trimers as priming immunogens to increase the frequency of site-targeting antibodies.
Quantification of the Impact of the HIV-1-Glycan Shield on Antibody Elicitation
Energy Technology Data Exchange (ETDEWEB)
Zhou, Tongqing; Doria-Rose, Nicole A.; Cheng, Cheng; Stewart-Jones, Guillaume B. E.; Chuang, Gwo-Yu; Chambers, Michael; Druz, Aliaksandr; Geng, Hui; McKee, Krisha; Kwon, Young Do; O’Dell, Sijy; Sastry, Mallika; Schmidt, Stephen D.; Xu, Kai; Chen, Lei; Chen, Rita E.; Louder, Mark K.; Pancera, Marie; Wanninger, Timothy G.; Zhang, Baoshan; Zheng, Anqi; Farney, S. Katie; Foulds, Kathryn E.; Georgiev, Ivelin S.; Joyce, M. Gordon; Lemmin, Thomas; Narpala, Sandeep; Rawi, Reda; Soto, Cinque; Todd, John-Paul; Shen, Chen-Hsiang; Tsybovsky, Yaroslav; Yang, Yongping; Zhao, Peng; Haynes, Barton F.; Stamatatos, Leonidas; Tiemeyer, Michael; Wells, Lance; Scorpio, Diana G.; Shapiro, Lawrence; McDermott, Adrian B.; Mascola, John R.; Kwong, Peter D.
2017-04-01
While the HIV-1-glycan shield is known to shelter Env from the humoral immune response, its quantitative impact on antibody elicitation has been unclear. Here, we use targeted deglycosylation to measure the impact of the glycan shield on elicitation of antibodies against the CD4 supersite. We engineered diverse Env trimers with select glycans removed proximal to the CD4 supersite, characterized their structures and glycosylation, and immunized guinea pigs and rhesus macaques. Immunizations yielded little neutralization against wild-type viruses but potent CD4-supersite neutralization (titers 1: >1,000,000 against four-glycan-deleted autologous viruses with over 90% breadth against four-glycan-deleted heterologous strains exhibiting tier 2 neutralization character). To a first approximation, the immunogenicity of the glycan-shielded protein surface was negligible, with Env-elicited neutralization (ID50) proportional to the exponential of the protein-surface area accessible to antibody. Based on these high titers and exponential relationship, we propose site-selective deglycosylated trimers as priming immunogens to increase the frequency of site-targeting antibodies.
Mutations in HNF1A Result in Marked Alterations of Plasma Glycan Profile
Thanabalasingham, Gaya; Huffman, Jennifer E.; Kattla, Jayesh J.; Novokmet, Mislav; Rudan, Igor; Gloyn, Anna L.; Hayward, Caroline; Adamczyk, Barbara; Reynolds, Rebecca M.; Muzinic, Ana; Hassanali, Neelam; Pucic, Maja; Bennett, Amanda J.; Essafi, Abdelkader; Polasek, Ozren; Mughal, Saima A.; Redzic, Irma; Primorac, Dragan; Zgaga, Lina; Kolcic, Ivana; Hansen, Torben; Gasperikova, Daniela; Tjora, Erling; Strachan, Mark W.J.; Nielsen, Trine; Stanik, Juraj; Klimes, Iwar; Pedersen, Oluf B.; Njølstad, Pål R.; Wild, Sarah H.; Gyllensten, Ulf; Gornik, Olga; Wilson, James F.; Hastie, Nicholas D.; Campbell, Harry; McCarthy, Mark I.; Rudd, Pauline M.; Owen, Katharine R.; Lauc, Gordan; Wright, Alan F.
2013-01-01
A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167), and nondiabetic controls (n = 98). The DG9-glycan index was markedly lower in HNF1A-MODY than in controls or other diabetes subtypes, offered good discrimination between HNF1A-MODY and both type 1 and type 2 diabetes (C statistic ≥0.90), and enabled us to detect three previously undetected HNF1A mutations in patients with diabetes. In conclusion, glycan profiles are altered substantially in HNF1A-MODY, and the DG9-glycan index has potential clinical value as a diagnostic biomarker of HNF1A dysfunction. PMID:23274891
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Reeja Maria Cherian
2015-08-01
Full Text Available Sialylated glycans serve as key elements of receptors for many viruses, bacteria, and bacterial toxins. The microbial recognition and their binding specificity can be affected by the linkage of the terminal sugar residue, types of underlying sugar chains, and the nature of the entire glycoconjugate. Owing to the pathobiological significance of sialylated glycans, we have engineered Chinese hamster ovary (CHO cells to secrete mucin-type immunoglobulin-fused proteins carrying terminal α2,3- or α2,6-linked sialic acid on defined O-glycan core saccharide chains. Besides stably expressing P-selectin glycoprotein ligand-1/mouse immunoglobulin G2b cDNA (PSGL-1/mIgG2b, CHO cells were stably transfected with plasmids encoding glycosyltransferases to synthesize core 2 (GCNT1, core 3 (B3GNT6, core 4 (GCNT1 and B3GNT6, or extended core 1 (B3GNT3 chains with or without the type 1 chain-encoding enzyme B3GALT5 and ST6GAL1. Western blot and liquid chromatography-mass spectrometry analysis confirmed the presence of core 1, 2, 3, 4, and extended core 1 chains carrying either type 1 (Galb3GlcNAc or type 2 (Galb4GlcNAc outer chains with or without α2,6-linked sialic acids. This panel of recombinant mucins carrying a repertoire of sialylated O-glycans will be important tools in studies aiming at determining the fine O-glycan binding specificity of sialic acid-specific microbial adhesins and mammalian lectins.
Directory of Open Access Journals (Sweden)
Xue Sun
2017-04-01
Full Text Available The glycosylation of proteins is responsible for their structural and functional roles in many cellular activities. This work describes a strategy that combines an efficient release, labeling and liquid chromatography-mass spectral analysis with the use of a comprehensive database to analyze N-glycans. The analytical method described relies on a recently commercialized kit in which quick deglycosylation is followed by rapid labeling and cleanup of labeled glycans. This greatly improves the separation, mass spectrometry (MS analysis and fluorescence detection of N-glycans. A hypothetical database, constructed using GlycResoft, provides all compositional possibilities of N-glycans based on the common sugar residues found in N-glycans. In the initial version this database contains >8,700 N-glycans, and is compatible with MS instrument software and expandable. N-glycans from four different well-studied glycoproteins were analyzed by this strategy. The results provided much more accurate and comprehensive data than had been previously reported. This strategy was then used to analyze the N-glycans present on the membrane glycoproteins of gastric carcinoma cells with different degrees of differentiation. Accurate and comprehensive N-glycan data from those cells was obtained efficiently and their differences compared corresponding to their differentiation states. Thus, the novel strategy developed greatly improves accuracy, efficiency and comprehensiveness of N-glycan analysis.
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Fleur S. van de Bovenkamp
2018-04-01
Full Text Available Immunoglobulin G (IgG can contain N-linked glycans in the variable domains, the so-called Fab glycans, in addition to the Fc glycans in the CH2 domains. These Fab glycans are acquired following introduction of N-glycosylation sites during somatic hypermutation and contribute to antibody diversification. We investigated whether Fab glycans may—in addition to affecting antigen binding—contribute to antibody stability. By analyzing thermal unfolding profiles of antibodies with or without Fab glycans, we demonstrate that introduction of Fab glycans can improve antibody stability. Strikingly, removal of Fab glycans naturally acquired during antigen-specific immune responses can deteriorate antibody stability, suggesting in vivo selection of stable, glycosylated antibodies. Collectively, our data show that variable domain N-linked glycans acquired during somatic hypermutation can contribute to IgG antibody stability. These findings indicate that introducing Fab glycans may represent a mechanism to improve therapeutic/diagnostic antibody stability.
The Impact of O-Glycan Chemistry on the Stability of Intrinsically Disordered Proteins
Energy Technology Data Exchange (ETDEWEB)
Beckham, Gregg T [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Prates, Erica T [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Crowley, Michael F [National Renewable Energy Laboratory (NREL), Golden, CO (United States); Guan, Xiaoyang [University of Colorado; Li, Yaohao [University of Colorado; Wang, Xinfeng [University of Colorado; Chaffey, Patrick K. [University of Colorado; Skaf, Munir S. [University of Campinas; Tan, Zhongping [University of Colorado
2018-03-02
Protein glycosylation is a diverse post-translational modification that serves myriad biological functions. O-linked glycans in particular vary widely in extent and chemistry in eukaryotes, with secreted proteins from fungi and yeast commonly exhibiting O-mannosylation in intrinsically disordered regions of proteins, likely for proteolysis protection, among other functions. However, it is not well understood why mannose is often the preferred glycan, and more generally, if the neighboring protein sequence and glycan have coevolved to protect against proteolysis in glycosylated intrinsically disordered proteins (IDPs). Here, we synthesized variants of a model IDP, specifically a natively O-mannosylated linker from a fungal enzyme, with a-O-linked mannose, glucose, and galactose moieties, along with a non-glycosylated linker. Upon exposure to thermolysin, O-mannosylation, by far, provides the highest extent of proteolysis protection. To explain this observation, extensive molecular dynamics simulations were conducted, revealing that the axial configuration of the C2-hydroxyl group (2-OH) of a-mannose adjacent to the glycan-peptide bond strongly influences the conformational features of the linker. Specifically, a-mannose restricts the torsions of the IDP main chain more than other glycans whose equatorial 2-OH groups exhibit interactions that favor perpendicular glycan-protein backbone orientation. We suggest that IDP stiffening due to O-mannosylation impairs protease action, with contributions from protein-glycan interactions, protein flexibility, and protein stability. Our results further imply that resistance to proteolysis is an important driving force for evolutionary selection of a-mannose in eukaryotic IDPs, and more broadly, that glycan motifs for proteolysis protection likely coevolve with the protein sequence to which they attach.
Data for analysis of mannose-6-phosphate glycans labeled with fluorescent tags
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Ji-Yeon Kang
2016-06-01
Full Text Available Mannose-6-phosphate (M-6-P glycan plays an important role in lysosomal targeting of most therapeutic enzymes for treatment of lysosomal storage diseases. This article provides data for the analysis of M-6-P glycans by high-performance liquid chromatography (HPLC and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF mass spectrometry. The identities of M-6-P glycan peaks in HPLC profile were confirmed by measuring the masses of the collected peak eluates. The performances of three fluorescent tags (2-aminobenzoic acid [2-AA], 2-aminobenzamide [2-AB], and 3-(acetyl-amino-6-aminoacridine [AA-Ac] were compared focusing on the analysis of bi-phosphorylated glycan (containing two M-6-Ps. The bi-phosphorylated glycan analysis is highly affected by the attached fluorescent tag and the hydrophilicity of elution solvent used in HPLC. The data in this article is associated with the research article published in “Comparison of fluorescent tags for analysis of mannose-6-phosphate glycans” (Kang et al., 2016 [1].
Borges, Chad R; Rehder, Douglas S
2016-09-15
Disagreement exists regarding the O-glycan structure attached to human vitamin D binding protein (DBP). Previously reported evidence indicated that the O-glycan of the Gc1S allele product is the linear core 1 NeuNAc-Gal-GalNAc-Thr trisaccharide. Here, glycan structural evidence is provided from glycan linkage analysis and over 30 serial glycosidase-digestion experiments which were followed by analysis of the intact protein by electrospray ionization mass spectrometry (ESI-MS). Results demonstrate that the O-glycan from the Gc1F protein is the same linear trisaccharide found on the Gc1S protein and that the hexose residue is galactose. In addition, the putative anti-cancer derivative of DBP known as Gc Protein-derived Macrophage Activating Factor (GcMAF, which is formed by the combined action of β-galactosidase and neuraminidase upon DBP) was analyzed intact by ESI-MS, revealing that the activating E. coli β-galactosidase cleaves nothing from the protein-leaving the glycan structure of active GcMAF as a Gal-GalNAc-Thr disaccharide, regardless of the order in which β-galactosidase and neuraminidase are applied. Moreover, glycosidase digestion results show that α-N-Acetylgalactosamindase (nagalase) lacks endoglycosidic function and only cleaves the DBP O-glycan once it has been trimmed down to a GalNAc-Thr monosaccharide-precluding the possibility of this enzyme removing the O-glycan trisaccharide from cancer-patient DBP in vivo. Copyright © 2016 Elsevier Inc. All rights reserved.
Energy Technology Data Exchange (ETDEWEB)
Zhang, Ying [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032 (China); Peng, Ye; Bin, Zhichao [Department of Chemistry, Fudan University, Shanghai 200032 (China); Wang, Huijie [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Lu, Haojie, E-mail: luhaojie@fudan.edu.cn [Shanghai Cancer Center and Institutes of Biomedical Sciences, Fudan University, Shanghai 200032 (China); Department of Chemistry, Fudan University, Shanghai 200032 (China); Key Laboratory of Glycoconjugates Research Ministry of Public Health, Fudan University, Shanghai 200032 (China)
2016-08-31
N-linked protein glycosylation is involved in regulation of a wide variety of cellular processes and associated with numerous diseases. Highly specific identification of N-glycome remains a challenge while its biological significance is acknowledged. The relatively low abundance of glycan in complex biological mixtures, lack of basic sites for protonation, and suppression by other highly abundant proteins/peptides lead to the particularly poor detection sensitivity of N-glycans in the MS analysis. Therefore, the highly specific purification procedure becomes a crucial step prior to MS analysis of the N-glycome. Herein, a novel N-glycans enrichment approach based on phosphate derivatization combined with Ti{sup 4+}-SPE (solid phase extraction) was developed. Briefly, in this strategy, N-glycans were chemically labeled with a phospho-group at their reducing ends, such that the Ti{sup 4+}-SPE microspheres were able to capture the phospho-containing glycans. The enrichment method was developed and optimized using model oligosaccharides (maltoheptaose DP7 and sialylated glycan A1) and also glycans from a standard glycoprotein (asialofetuin, ASF). This method experimentally showed high derivatization efficiency (almost 100%), excellent selectivity (analyzing DP7 in the digests of bovine serum albumin at a mass ratio of 1:100), high enriching recovery (90%), good reproducibility (CV<15%) as well as high sensitivity (LOD at fmol level). At last, the proposed method was successfully applied in the profiling of N-glycome in human serum, in which a total of 31 N-glycan masses were identified. - Graphical abstract: A selective enrichment method for the N-glycome is reported. N-glycans were chemically labeled with a phosphate derivatization reagent (AMS), then the phospho-containing glycans were enriched using Ti{sup 4+}-microspheres. - Highlights: • A highly specific N-glycans purification method based on phosphate derivatization combined with Ti{sup 4+}-SPE was developed
Lin, Chi-Hung; Krisp, Christoph; Packer, Nicolle H; Molloy, Mark P
2018-02-10
Glycoproteomics investigates glycan moieties in a site specific manner to reveal the functional roles of protein glycosylation. Identification of glycopeptides from data-dependent acquisition (DDA) relies on high quality MS/MS spectra of glycopeptide precursors and often requires manual validation to ensure confident assignments. In this study, we investigated pseudo-MRM (MRM-HR) and data-independent acquisition (DIA) as alternative acquisition strategies for glycopeptide analysis. These approaches allow data acquisition over the full MS/MS scan range allowing data re-analysis post-acquisition, without data re-acquisition. The advantage of MRM-HR over DDA for N-glycopeptide detection was demonstrated from targeted analysis of bovine fetuin where all three N-glycosylation sites were detected, which was not the case with DDA. To overcome the duty cycle limitation of MRM-HR acquisition needed for analysis of complex samples such as plasma we trialed DIA. This allowed development of a targeted DIA method to identify N-glycopeptides without pre-defined knowledge of the glycan composition, thus providing the potential to identify N-glycopeptides with unexpected structures. This workflow was demonstrated by detection of 59 N-glycosylation sites from 41 glycoproteins from a HILIC enriched human plasma tryptic digest. 21 glycoforms of IgG1 glycopeptides were identified including two truncated structures that are rarely reported. We developed a data-independent mass spectrometry workflow to identify specific glycopeptides from complex biological mixtures. The novelty is that this approach does not require glycan composition to be pre-defined, thereby allowing glycopeptides carrying unexpected glycans to be identified. This is demonstrated through the analysis of immunoglobulins in human plasma where we detected two IgG1 glycoforms that are rarely observed. Copyright © 2017 Elsevier B.V. All rights reserved.
Mutations in HNF1A result in marked alterations of plasma glycan profile
DEFF Research Database (Denmark)
Thanabalasingham, Gaya; Huffman, Jennifer E; Kattla, Jayesh J
2013-01-01
A recent genome-wide association study identified hepatocyte nuclear factor 1-α (HNF1A) as a key regulator of fucosylation. We hypothesized that loss-of-function HNF1A mutations causal for maturity-onset diabetes of the young (MODY) would display altered fucosylation of N-linked glycans on plasma...... proteins and that glycan biomarkers could improve the efficiency of a diagnosis of HNF1A-MODY. In a pilot comparison of 33 subjects with HNF1A-MODY and 41 subjects with type 2 diabetes, 15 of 29 glycan measurements differed between the two groups. The DG9-glycan index, which is the ratio of fucosylated...... to nonfucosylated triantennary glycans, provided optimum discrimination in the pilot study and was examined further among additional subjects with HNF1A-MODY (n = 188), glucokinase (GCK)-MODY (n = 118), hepatocyte nuclear factor 4-α (HNF4A)-MODY (n = 40), type 1 diabetes (n = 98), type 2 diabetes (n = 167...
Jeong, Yeong Ran; Kim, Sun Young; Park, Young Sam; Lee, Gyun Min
2018-03-21
N-glycans of therapeutic glycoproteins are critical quality attributes that should be monitored throughout all stages of biopharmaceutical development. To reduce both the time for sample preparation and the variations in analytical results, we have developed an N-glycan analysis method that includes improved 2-aminobenzoic acid (2-AA) labeling to easily remove deglycosylated proteins. Using this analytical method, 15 major 2-AA-labeled N-glycans of Enbrel ® were separated into single peaks in hydrophilic interaction chromatography mode and therefore could be quantitated. 2-AA-labeled N-glycans were also highly compatible with in-line quadrupole time-of-flight mass spectrometry (MS) for structural identification. The structures of 15 major and 18 minor N-glycans were identified from their mass values determined by quadrupole time-of-flight MS. Furthermore, the structures of 14 major N-glycans were confirmed by interpreting the MS/MS data of each N-glycan. This analytical method was also successfully applied to neutral N-glycans of Humira ® and highly sialylated N-glycans of NESP ® . Furthermore, the analysis data of Enbrel ® that were accumulated for 2.5 years demonstrated the high-level consistency of this analytical method. Taken together, the results show that a wide repertoire of N-glycans of therapeutic glycoproteins can be analyzed with high efficiency and consistency using the improved 2-AA labeling-based N-glycan analysis method. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Levy-Ontman, Oshrat; Arad, Shoshana (Malis); Harvey, David J.; Parsons, Thomas B.; Fairbanks, Antony; Tekoah, Yoram
2011-01-01
We report here the structural determination of the N-linked glycans in the 66-kDa glycoprotein, part of the unique sulfated complex cell wall polysaccharide of the red microalga Porphyridium sp. Structures were elucidated by a combination of normal phase/reverse phase HPLC, positive ion MALDI-TOF MS, negative ion electrospray ionization, and MS/MS. The sugar moieties of the glycoprotein consisted of at least four fractions of N-linked glycans, each composed of the same four monosaccharides, GlcNAc, Man, 6-O-MeMan, and Xyl, with compositions Man8–9Xyl1–2Me3GlcNAc2. The present study is the first report of N-glycans with the terminal Xyl attached to the 6-mannose branch of the 6-antenna and to the 3-oxygen of the penultimate (core) GlcNAc. Another novel finding was that all four glycans contain three O-methylmannose residues in positions that have never been reported before. Although it is known that some lower organisms are able to methylate terminal monosaccharides in glycans, the present study on Porphyridium sp. is the first describing an organism that is able to methylate non-terminal mannose residues. This study will thus contribute to understanding of N-glycosylation in algae and might shed light on the evolutionary development from prokaryotes to multicellular organisms. It also may contribute to our understanding of the red algae polysaccharide formation. The additional importance of this research lies in its potential for biotechnological applications, especially in evaluating the use of microalgae as cell factories for the production of therapeutic proteins. PMID:21515680
Toonstra, Christian; Wu, Lisa; Li, Chao; Wang, Denong; Wang, Lai-Xi
2018-05-22
High-mannose-type N-glycans are an important component of neutralizing epitopes on HIV-1 envelope glycoprotein gp120. They also serve as signals for protein folding, trafficking, and degradation in protein quality control. A number of lectins and antibodies recognize high-mannose-type N-glycans, and glycan array technology has provided an avenue to probe these oligomannose-specific proteins. We describe in this paper a top-down chemoenzymatic approach to synthesize a library of high-mannose N-glycans and related neoglycoproteins for glycan microarray analysis. The method involves the sequential enzymatic trimming of two readily available natural N-glycans, the Man 9 GlcNAc 2 Asn prepared from soybean flour and the sialoglycopeptide (SGP) isolated from chicken egg yolks, coupled with chromatographic separation to obtain a collection of a full range of natural high-mannose N-glycans. The Asn-linked N-glycans were conjugated to bovine serum albumin (BSA) to provide neoglycoproteins containing the oligomannose moieties. The glycoepitopes displayed were characterized using an array of glycan-binding proteins, including the broadly virus-neutralizing agents, glycan-specific antibody 2G12, Galanthus nivalis lectin (GNA), and Narcissus pseudonarcissus lectin (NPA).
Glycan Markers as Potential Immunological Targets in Circulating Tumor Cells.
Wang, Denong; Wu, Lisa; Liu, Xiaohe
2017-01-01
We present here an experimental approach for exploring a new class of tumor biomarkers that are overexpressed by circulating tumor cells (CTCs) and are likely targetable in immunotherapy against tumor metastasis. Using carbohydrate microarrays, anti-tumor monoclonal antibodies (mAbs) were scanned against a large panel of carbohydrate antigens to identify potential tumor glycan markers. Subsequently, flow cytometry and fiber-optic array scanning technology (FAST) were applied to determine whether the identified targets are tumor-specific cell-surface markers and are, therefore, likely suitable for targeted immunotherapy. Finally, the tumor glycan-specific antibodies identified were validated using cancer patients' blood samples for their performance in CTC-detection and immunotyping analysis. In this article, identifying breast CTC-specific glycan markers and targeting mAbs serve as examples to illustrate this tumor biomarker discovery strategy.
Glycan bioengineering in immunogen design for tumor T antigen immunotargeting
DEFF Research Database (Denmark)
Sendra, Victor G; Zlocowski, Natacha; Ditamo, Yanina
2009-01-01
MM2 energy function showed that pentalysine (Lys5) linker and benzyl (Bzl) residue enhance TFD rigidity of the glycosidic bond. Antibodies raised against BzlalphaTFD-Lys5 immunogen recognize tumor T antigen. Competitive assays confirm that TFD-related structures are the main glycan epitope...... to the bioengineered glycoconjugate inhibited CT26 tumor cell proliferation and reduced tumor growth in an in vivo mouse model. These results show that TFD bioengineering is a useful immunogenic strategy with potential application in cancer therapy. The same approach can be extended to other glycan immunogens......Bioengineering of Galbeta3GalNAcalpha, known as Thomsen-Friedenreich disaccharide (TFD), is studied to promote glycan immunogenicity and immunotargeting to tumor T antigen (Galbeta3GalNAcalpha-O-Ser/Thr). Theoretical studies on disaccharide conformations by energy minimization of structures using...
Glutamine alimentation in catabolic state.
Boelens, P G; Nijveldt, R J; Houdijk, A P; Meijer, S; van Leeuwen, P A
2001-09-01
Glutamine should be reclassified as a conditionally essential amino acid in the catabolic state because the body's glutamine expenditures exceed synthesis and low glutamine levels in plasma are associated with poor clinical outcome. After severe stress, several amino acids are mobilized from muscle tissue to supply energy and substrate to the host. Glutamine is one of the most important amino acids that provide this function. Glutamine acts as the preferred respiratory fuel for lymphocytes, hepatocytes and intestinal mucosal cells and is metabolized in the gut to citrulline, ammonium and other amino acids. Low concentrations of glutamine in plasma reflect reduced stores in muscle and this reduced availability of glutamine in the catabolic state seems to correlate with increased morbidity and mortality. Adding glutamine to the nutrition of clinical patients, enterally or parenterally, may reduce morbidity. Several excellent clinical trials have been performed to prove efficacy and feasibility of the use of glutamine supplementation in parenteral and enteral nutrition. The increased intake of glutamine has resulted in lower septic morbidity in certain critically ill patient populations. This review will focus on the efficacy and the importance of glutamine supplementation in diverse catabolic states.
Isomer Information from Ion Mobility Separation of High-Mannose Glycan Fragments.
Harvey, David J; Seabright, Gemma E; Vasiljevic, Snezana; Crispin, Max; Struwe, Weston B
2018-05-01
Extracted arrival time distributions of negative ion CID-derived fragments produced prior to traveling-wave ion mobility separation were evaluated for their ability to provide structural information on N-linked glycans. Fragmentation of high-mannose glycans released from several glycoproteins, including those from viral sources, provided over 50 fragments, many of which gave unique collisional cross-sections and provided additional information used to assign structural isomers. For example, cross-ring fragments arising from cleavage of the reducing terminal GlcNAc residue on Man 8 GlcNAc 2 isomers have unique collision cross-sections enabling isomers to be differentiated in mixtures. Specific fragment collision cross-sections enabled identification of glycans, the antennae of which terminated in the antigenic α-galactose residue, and ions defining the composition of the 6-antenna of several of the glycans were also found to have different cross-sections from isomeric ions produced in the same spectra. Potential mechanisms for the formation of the various ions are discussed and the estimated collisional cross-sections are tabulated. Graphical Abstract ᅟ.
Body Weight Independently Affects Articular Cartilage Catabolism
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W. Matt Denning, Jason G. Winward, Michael Becker Pardo, J. Ty Hopkins, Matthew K. Seeley
2015-06-01
Full Text Available Although obesity is associated with osteoarthritis, it is unclear whether body weight (BW independently affects articular cartilage catabolism (i.e., independent from physiological factors that also accompany obesity. The primary purpose of this study was to evaluate the independent effect of BW on articular cartilage catabolism associated with walking. A secondary purpose was to determine how decreased BW influenced cardiovascular response due to walking. Twelve able-bodied subjects walked for 30 minutes on a lower-body positive pressure treadmill during three sessions: control (unadjusted BW, +40%BW, and -40%BW. Serum cartilage oligomeric matrix protein (COMP was measured immediately before (baseline and after, and 15 and 30 minutes after the walk. Heart rate (HR and rate of perceived exertion (RPE were measured every three minutes during the walk. Relative to baseline, average serum COMP concentration was 13% and 5% greater immediately after and 15 minutes after the walk. Immediately after the walk, serum COMP concentration was 14% greater for the +40%BW session than for the -40%BW session. HR and RPE were greater for the +40%BW session than for the other two sessions, but did not differ between the control and -40%BW sessions. BW independently influences acute articular cartilage catabolism and cardiovascular response due to walking: as BW increases, so does acute articular cartilage catabolism and cardiovascular response. These results indicate that lower-body positive pressure walking may benefit certain individuals by reducing acute articular cartilage catabolism, due to walking, while maintaining cardiovascular response.
Nakano, Miyako; Saldanha, Rohit; Göbel, Anja; Kavallaris, Maria; Packer, Nicolle H
2011-11-01
Resistance to tubulin-binding agents used in cancer is often multifactorial and can include changes in drug accumulation and modified expression of tubulin isotypes. Glycans on cell membrane proteins play important roles in many cellular processes such as recognition and apoptosis, and this study investigated whether changes to the glycan structures on cell membrane proteins occur when cells become resistant to drugs. Specifically, we investigated the alteration of glycan structures on the cell membrane proteins of human T-cell acute lymphoblastic leukemia (CEM) cells that were selected for resistance to desoxyepothilone B (CEM/dEpoB). The glycan profile of the cell membrane glycoproteins was obtained by sequential release of N- and O-glycans from cell membrane fraction dotted onto polyvinylidene difluoride membrane with PNGase F and β-elimination respectively. The released glycan alditols were analyzed by liquid chromatography (graphitized carbon)-electrospray ionization tandem MS. The major N-glycan on CEM cell was the core fucosylated α2-6 monosialo-biantennary structure. Resistant CEM/dEpoB cells had a significant decrease of α2-6 linked sialic acid on N-glycans. The lower α2-6 sialylation was caused by a decrease in activity of β-galactoside α2-6 sialyltransferase (ST6Gal), and decreased expression of the mRNA. It is clear that the membrane glycosylation of leukemia cells changes during acquired resistance to dEpoB drugs and that this change occurs globally on all cell membrane glycoproteins. This is the first identification of a specific glycan modification on the surface of drug resistant cells and the mechanism of this downstream effect on microtubule targeting drugs may offer a route to new interventions to overcome drug resistance.
DEFF Research Database (Denmark)
Scott, Nichollas E; Nothaft, Harald; Edwards, Alistair V G
2012-01-01
. Interrogation of these data allowed the identification of a phosphoethanolamine (pEtN)-modified variant of the N-glycan that was attached to multiple proteins. The pEtN moiety was attached to the terminal GalNAc of the canonical N-glycan. Deletion of the pEtN transferase eptC removed all evidence of the p......, yet above background levels of pEtN-glycan were also observed in E. coli not expressing eptC, suggesting that endogenous E. coli pEtN transferases can mediate the addition of pEtN to N-glycans. The addition of pEtN must be considered in the context of glycoengineering and may alter C. jejuni glycan...
Organic Electrochemical Transistors for the Detection of Cell Surface Glycans.
Chen, Lizhen; Fu, Ying; Wang, Naixiang; Yang, Anneng; Li, Yuanzhe; Wu, Jie; Ju, Huangxian; Yan, Feng
2018-05-23
Cell surface glycans play critical roles in diverse biological processes, such as cell-cell communication, immunity, infection, development, and differentiation. Their expressions are closely related to cancer growth and metastasis. This work demonstrates an organic electrochemical transistor (OECT)-based biosensor for the detection of glycan expression on living cancer cells. Herein, mannose on human breast cancer cells (MCF-7) as the target glycan model, poly dimethyl diallyl ammonium chloride-multiwall carbon nanotubes (PDDA-MWCNTs) as the loading interface, concanavalin A (Con A) with active mannose binding sites, aptamer and horseradish peroxidase co-immobilized gold nanoparticles (HRP-aptamer-Au NPs) as specific nanoprobes are used to fabricate the OECT biosensor. In this strategy, PDDA-MWCNT interfaces can enhance the loading of Con A, and the target cells can be captured through Con A via active mannose binding sites. Thus, the expression of cell surface can be reflected by the amount of cells captured on the gate. Specific nanoprobes are introduced to the captured cells to produce an OECT signal because of the reduction of hydrogen peroxide catalyzed by HRP conjugated on Au nanoparticles, while the aptamer on nanoprobes can selectively recognize the MCF-7 cells. It is reasonable that more target cells are captured on the gate electrode, more HRP-nanoprobes are loaded thus a larger signal response. The device shows an obvious response to MCF-7 cells down to 10 cells/μL and can be used to selectively monitor the change of mannose expression on cell surfaces upon a treatment with the N-glycan inhibitor. The OECT-based biosensor is promising for the analysis of glycan expressions on the surfaces of different types of cells.
Microarray Glycan Profiling Reveals Algal Fucoidan Epitopes in Diverse Marine Metazoans
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Armando A. Salmeán
2017-09-01
Full Text Available Despite the biological importance and pharmacological potential of glycans from marine organisms, there are many unanswered questions regarding their distribution, function, and evolution. Here we describe microarray-based glycan profiling of a diverse selection of marine animals using antibodies raised against fucoidan isolated from a brown alga. We demonstrate the presence of two fucoidan epitopes in six animals belonging to three phyla including Porifera, Molusca, and Chordata. We studied the spatial distribution of these epitopes in Cliona celata (“boring sponge” and identified their restricted localization on the surface of internal chambers. Our results show the potential of high-throughput screening and probes commonly used in plant and algal cell wall biology to study the diversity and distribution of glycan structures in metazoans.
Shared strategies for β-lactam catabolism in the soil microbiome
DEFF Research Database (Denmark)
Crofts, Terence S.; Wang, Bin; Spivak, Aaron
2018-01-01
The soil microbiome can produce, resist, or degrade antibiotics and even catabolize them. While resistance genes are widely distributed in the soil, there is a dearth of knowledge concerning antibiotic catabolism. Here we describe a pathway for penicillin catabolism in four isolates. Genomic......, respectively. Elucidation of additional pathways may allow bioremediation of antibiotic-contaminated soils and discovery of antibiotic-remodeling enzymes with industrial utility....
Shah, Bhavana; Jiang, Xinzhao Grace; Chen, Louise; Zhang, Zhongqi
2014-06-01
Protein N-Glycan analysis is traditionally performed by high pH anion exchange chromatography (HPAEC), reversed phase liquid chromatography (RPLC), or hydrophilic interaction liquid chromatography (HILIC) on fluorescence-labeled glycans enzymatically released from the glycoprotein. These methods require time-consuming sample preparations and do not provide site-specific glycosylation information. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) peptide mapping is frequently used for protein structural characterization and, as a bonus, can potentially provide glycan profile on each individual glycosylation site. In this work, a recently developed glycopeptide fragmentation model was used for automated identification, based on their MS/MS, of N-glycopeptides from proteolytic digestion of monoclonal antibodies (mAbs). Experimental conditions were optimized to achieve accurate profiling of glycoforms. Glycan profiles obtained from LC-MS/MS peptide mapping were compared with those obtained from HPAEC, RPLC, and HILIC analyses of released glycans for several mAb molecules. Accuracy, reproducibility, and linearity of the LC-MS/MS peptide mapping method for glycan profiling were evaluated. The LC-MS/MS peptide mapping method with fully automated data analysis requires less sample preparation, provides site-specific information, and may serve as an alternative method for routine profiling of N-glycans on immunoglobulins as well as other glycoproteins with simple N-glycans.
Pentose phosphates in nucleoside interconversion and catabolism.
Tozzi, Maria G; Camici, Marcella; Mascia, Laura; Sgarrella, Francesco; Ipata, Piero L
2006-03-01
Ribose phosphates are either synthesized through the oxidative branch of the pentose phosphate pathway, or are supplied by nucleoside phosphorylases. The two main pentose phosphates, ribose-5-phosphate and ribose-1-phosphate, are readily interconverted by the action of phosphopentomutase. Ribose-5-phosphate is the direct precursor of 5-phosphoribosyl-1-pyrophosphate, for both de novo and 'salvage' synthesis of nucleotides. Phosphorolysis of deoxyribonucleosides is the main source of deoxyribose phosphates, which are interconvertible, through the action of phosphopentomutase. The pentose moiety of all nucleosides can serve as a carbon and energy source. During the past decade, extensive advances have been made in elucidating the pathways by which the pentose phosphates, arising from nucleoside phosphorolysis, are either recycled, without opening of their furanosidic ring, or catabolized as a carbon and energy source. We review herein the experimental knowledge on the molecular mechanisms by which (a) ribose-1-phosphate, produced by purine nucleoside phosphorylase acting catabolically, is either anabolized for pyrimidine salvage and 5-fluorouracil activation, with uridine phosphorylase acting anabolically, or recycled for nucleoside and base interconversion; (b) the nucleosides can be regarded, both in bacteria and in eukaryotic cells, as carriers of sugars, that are made available though the action of nucleoside phosphorylases. In bacteria, catabolism of nucleosides, when suitable carbon and energy sources are not available, is accomplished by a battery of nucleoside transporters and of inducible catabolic enzymes for purine and pyrimidine nucleosides and for pentose phosphates. In eukaryotic cells, the modulation of pentose phosphate production by nucleoside catabolism seems to be affected by developmental and physiological factors on enzyme levels.
Amino Acid Catabolism in Multiple Sclerosis Affects Immune Homeostasis.
Negrotto, Laura; Correale, Jorge
2017-03-01
Amino acid catabolism has been implicated in immunoregulatory mechanisms present in several diseases, including autoimmune disorders. Our aims were to assess expression and activity of enzymes involved in Trp and Arg catabolism, as well as to investigate amino acid catabolism effects on the immune system of multiple sclerosis (MS) patients. To this end, 40 MS patients, 30 healthy control subjects, and 30 patients with other inflammatory neurological diseases were studied. Expression and activity of enzymes involved in Trp and Arg catabolism (IDO1, IDO2, Trp 2,3-dioxygenase [TDO], arginase [ARG] 1, ARG2, inducible NO synthetase) were evaluated in PBMCs. Expression of general control nonrepressed 2 serine/threonine kinase and mammalian target of rapamycin (both molecules involved in sensing amino acid levels) was assessed in response to different stimuli modulating amino acid catabolism, as were cytokine secretion levels and regulatory T cell numbers. The results demonstrate that expression and activity of IDO1 and ARG1 were significantly reduced in MS patients compared with healthy control subjects and other inflammatory neurological diseases. PBMCs from MS patients stimulated with a TLR-9 agonist showed reduced expression of general control nonrepressed 2 serine/threonine kinase and increased expression of mammalian target of rapamycin, suggesting reduced amino acid catabolism in MS patients. Functionally, this reduction resulted in a decrease in regulatory T cells, with an increase in myelin basic protein-specific T cell proliferation and secretion of proinflammatory cytokines. In contrast, induction of IDO1 using CTLA-4 or a TLR-3 ligand dampened proinflammatory responses. Overall, these results highlight the importance of amino acid catabolism in the modulation of the immunological responses in MS patients. Molecules involved in these pathways warrant further exploration as potential new therapeutic targets in MS. Copyright © 2017 by The American Association of
Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides
DEFF Research Database (Denmark)
Luis, Ana S.; Briggs, Jonathon; Zhang, Xiaoyang
2018-01-01
The major nutrients available to human colonic Bacteroides species are glycans, exemplified by pectins, a network of covalently linked plant cell wall polysaccharides containing galacturonic acid (GalA). Metabolism of complex carbohydrates by the Bacteroides genus is orchestrated by polysaccharid...... PULs ensuring a continuous supply of inducing molecules throughout growth. The contribution of Bacteroides spp. to metabolism of the pectic network is illustrated by cross-feeding between organisms....
Wang, Chengjian; Zhang, Ping; Jin, Wanjun; Li, Lingmei; Qiang, Shan; Zhang, Ying; Huang, Linjuan; Wang, Zhongfu
2017-01-06
O-glycomic comparison between perch and salmon eggs by ESI-MS, MS/MS and online RP-HPLC-UV-ESI-MS/MS, demonstrating its excellent applicability to various complex biological samples. O-Linked glycoproteins, generated via a widely existing glycosylation modification process on serine (Ser) or threonine (Thr) residues of nascent proteins, play essential roles in a series of biological processes. As a type of informational molecule, the O-glycans of these glycoproteins participate directly in these biological mechanisms. Thus, the characteristic differences or changes of O-glycans in expression level usually relate to pathologies of many diseases and represent an important opportunity to uncover the functional mechanisms of various glycoprotein O-glycans. The novel strategy introduced here provides a simple and versatile analytical method for the precise quantitation of glycoprotein O-glycans by mass spectrometry, enabling rapid evaluation of the differences or changes of O-glycans in expression level. It is attractive for the field of quantitative/comparative O-glycomics, which has great significance for exploring the complex structure-function relationship of O-glycans, as well as for the search of O-glycan biomarkers of some major diseases and O-glycan related targets of some drugs. Copyright © 2016 Elsevier B.V. All rights reserved.
Newburg, D S
2009-04-01
This review discusses the role of human milk glycans in protecting infants, but the conclusion that the human milk glycans constitute an innate immune system whereby the mother protects her offspring may have general applicability in all mammals, including species of commercial importance. Infants that are not breastfed have a greater incidence of severe diarrhea and respiratory diseases than those who are breastfed. In the past, this had been attributed primarily to human milk secretory antibodies. However, the oligosaccharides are major components of human milk, and milk is also rich in other glycans, including glycoproteins, mucins, glycosaminoglycans, and glycolipids. These milk glycans, especially the oligosaccharides, are composed of thousands of components. The milk factor that promotes gut colonization by Bifidobacterium bifidum was found to be a glycan, and such prebiotic characteristics may contribute to protection against infectious agents. However, the ability of human milk glycans to protect the neonate seems primarily to be due to their inhibition of pathogen binding to their host cell target ligands. Many such examples include specific fucosylated oligosaccharides and glycans that inhibit specific pathogens. Most human milk oligosaccharides are fucosylated, and their production depends on fucosyltransferase enzymes; mutations in these fucosyltransferase genes are common and underlie the various Lewis blood types in humans. Variable expression of specific fucosylated oligosaccharides in milk, also a function of these genes (and maternal Lewis blood type), is significantly associated with the risk of infectious disease in breastfed infants. Human milk also contains major quantities and large numbers of sialylated oligosaccharides, many of which are also present in bovine colostrum. These could similarly inhibit several common viral pathogens. Moreover, human milk oligosaccharides strongly attenuate inflammatory processes in the intestinal mucosa. These
Enzymes for N-Glycan Branching and Their Genetic and Nongenetic Regulation in Cancer
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Yasuhiko Kizuka
2016-04-01
Full Text Available N-glycan, a fundamental and versatile protein modification in mammals, plays critical roles in various physiological and pathological events including cancer progression. The formation of N-glycan branches catalyzed by specific N-acetylglucosaminyltransferases [GnT-III, GnT-IVs, GnT-V, GnT-IX (Vb] and a fucosyltransferase, Fut8, provides functionally diverse N-glycosylated proteins. Aberrations of these branches are often found in cancer cells and are profoundly involved in cancer growth, invasion and metastasis. In this review, we focus on the GlcNAc and fucose branches of N-glycans and describe how their expression is dysregulated in cancer by genetic and nongenetic mechanisms including epigenetics and nucleotide sugar metabolisms. We also survey the roles that these N-glycans play in cancer progression and therapeutics. Finally, we discuss possible applications of our knowledge on basic glycobiology to the development of medicine and biomarkers for cancer therapy.
A novel core 1 O-linked glycan-specific binding lectin from the fruiting body of Hericium erinaceus.
Kim, Seonghun
2018-02-01
Mucin-type O-glycans are involved in biological functions on the cell surface as well as the glycoproteins and can also be used as specific carbohydrate biomarkers of many diseases. In this study, I purified a novel core 1 O-linked glycan specific lectin, Hericium erinaceus lecin (HeL), from the fruiting body of the mushroom Hericium erinaceus, which is known as the natural source for a sialic acid-binding lectin. Upon optimization of the purification conditions, a sequence of ion exchange, affinity, ion exchange, and size-exclusion chromatography resulted in the highest yield and best quality of lectin without protease activity. The resulting purified HeL is an apparent hexameric protein with a subunit molecular weight of 15kDa, and a pI of 4.3. In hemagglutination inhibition assay, the purified lectin was only inhibited by glycoproteins containing mucin-type O-glycans and reacted weakly with Galβ(1,3)GalNAc. Glycan array analyses showed that HeL specifically interacts with core 1 O-linked glycans as well as extended O-glycan structures containing sialylation or fucosylation. The glycan binding specificity of HeL is comparable to that of peanut agglutinin for detection of a broader range of extended core 1 O-glycan structures. Taken together, these results provide an efficient and optimized procedure for the purification of HeL from the fruiting body of the mushroom Hericium erinaceus. Moreover, HeL represents a powerful tool for analyzing core 1 and extended core 1 O- glycan structures in diagnosis assays. Copyright © 2017 Elsevier B.V. All rights reserved.
Biochemistry of Catabolic Reductive Dehalogenation.
Fincker, Maeva; Spormann, Alfred M
2017-06-20
A wide range of phylogenetically diverse microorganisms couple the reductive dehalogenation of organohalides to energy conservation. Key enzymes of such anaerobic catabolic pathways are corrinoid and Fe-S cluster-containing, membrane-associated reductive dehalogenases. These enzymes catalyze the reductive elimination of a halide and constitute the terminal reductases of a short electron transfer chain. Enzymatic and physiological studies revealed the existence of quinone-dependent and quinone-independent reductive dehalogenases that are distinguishable at the amino acid sequence level, implying different modes of energy conservation in the respective microorganisms. In this review, we summarize current knowledge about catabolic reductive dehalogenases and the electron transfer chain they are part of. We review reaction mechanisms and the role of the corrinoid and Fe-S cluster cofactors and discuss physiological implications.
Anaerobic catabolism of aromatic compounds: a genetic and genomic view.
Carmona, Manuel; Zamarro, María Teresa; Blázquez, Blas; Durante-Rodríguez, Gonzalo; Juárez, Javier F; Valderrama, J Andrés; Barragán, María J L; García, José Luis; Díaz, Eduardo
2009-03-01
Aromatic compounds belong to one of the most widely distributed classes of organic compounds in nature, and a significant number of xenobiotics belong to this family of compounds. Since many habitats containing large amounts of aromatic compounds are often anoxic, the anaerobic catabolism of aromatic compounds by microorganisms becomes crucial in biogeochemical cycles and in the sustainable development of the biosphere. The mineralization of aromatic compounds by facultative or obligate anaerobic bacteria can be coupled to anaerobic respiration with a variety of electron acceptors as well as to fermentation and anoxygenic photosynthesis. Since the redox potential of the electron-accepting system dictates the degradative strategy, there is wide biochemical diversity among anaerobic aromatic degraders. However, the genetic determinants of all these processes and the mechanisms involved in their regulation are much less studied. This review focuses on the recent findings that standard molecular biology approaches together with new high-throughput technologies (e.g., genome sequencing, transcriptomics, proteomics, and metagenomics) have provided regarding the genetics, regulation, ecophysiology, and evolution of anaerobic aromatic degradation pathways. These studies revealed that the anaerobic catabolism of aromatic compounds is more diverse and widespread than previously thought, and the complex metabolic and stress programs associated with the use of aromatic compounds under anaerobic conditions are starting to be unraveled. Anaerobic biotransformation processes based on unprecedented enzymes and pathways with novel metabolic capabilities, as well as the design of novel regulatory circuits and catabolic networks of great biotechnological potential in synthetic biology, are now feasible to approach.
DEFF Research Database (Denmark)
Thygesen, Mikkel Boas; Sørensen, Kasper Kildegaard; Cló, Emiliano
2009-01-01
Chemoselective oxime coupling was used for facile conjugation of unprotected, reducing glycans and glycopeptide aldehydes with core-shell gold nanoparticles carrying reactive aminooxy groups on the organic shell.......Chemoselective oxime coupling was used for facile conjugation of unprotected, reducing glycans and glycopeptide aldehydes with core-shell gold nanoparticles carrying reactive aminooxy groups on the organic shell....
Lopes, Ana M; Breiman, Adrien; Lora, Mónica; Le Moullac-Vaidye, Béatrice; Galanina, Oxana; Nyström, Kristina; Marchandeau, Stephane; Le Gall-Reculé, Ghislaine; Strive, Tanja; Neimanis, Aleksija; Bovin, Nicolai V; Ruvoën-Clouet, Nathalie; Esteves, Pedro J; Abrantes, Joana; Le Pendu, Jacques
2017-11-29
The rabbit hemorrhagic disease virus (RHDV) and the European brown hare syndrome virus (EBHSV) are two lagoviruses from the family Caliciviridae that cause fatal diseases in two leporid genera, Oryctolagus and Lepus , respectively. In the last few years, several examples of host jumps of lagoviruses among leporids were recorded. In addition, a new pathogenic genotype of RHDV emerged and many non-pathogenic strains of lagoviruses have been described. The molecular mechanisms behind host shifts and the emergence of virulence are unknown. Since RHDV uses glycans of the histo-blood group antigen type as attachment factors to initiate infection, we studied if glycan specificities of the new pathogenic RHDV genotype, non-pathogenic lagoviruses and EBHSV potentially play a role in determining host range and virulence of lagoviruses. We observed binding to A, B or H antigens of the histo-blood group family for all strains known to primarily infect European rabbits ( Oryctolagus cuniculus ), that have recently been classified as GI strains. Yet, we could not explain the emergence of virulence since similar glycan specificities were found between several pathogenic and non-pathogenic strains. By contrast, EBHSV, recently classified as GII.1, bound to terminal β-linked N-acetylglucosamine residues of O-glycans. Expression of these attachment factors in the upper respiratory and digestive tracts in three lagomorph species ( Oryctolagus cuniculus, Lepus europaeus and Sylvilagus floridanus ) showed species-specific patterns regarding the susceptibility to infection by these viruses, indicating that species-specific glycan expression is likely a major contributor to lagoviruses host specificity and range. IMPORTANCE Lagoviruses constitute a genus of the Caliciviridae family, comprising highly pathogenic viruses, RHDV and EBHSV, which infect rabbits and hares, respectively. Recently, non-pathogenic strains were discovered and new pathogenic strains have emerged. In addition, host
The Atg1-Tor pathway regulates yolk catabolism in Drosophila embryos.
Kuhn, Hallie; Sopko, Richelle; Coughlin, Margaret; Perrimon, Norbert; Mitchison, Tim
2015-11-15
Yolk provides an important source of nutrients during the early development of oviparous organisms. It is composed mainly of vitellogenin proteins packed into membrane-bound compartments called yolk platelets. Catabolism of yolk is initiated by acidification of the yolk platelet, leading to the activation of Cathepsin-like proteinases, but it is unknown how this process is triggered. Yolk catabolism initiates at cellularization in Drosophila melanogaster embryos. Using maternal shRNA technology we found that yolk catabolism depends on the Tor pathway and on the autophagy-initiating kinase Atg1. Whereas Atg1 was required for a burst of spatially regulated autophagy during late cellularization, autophagy was not required for initiating yolk catabolism. We propose that the conserved Tor metabolic sensing pathway regulates yolk catabolism, similar to Tor-dependent metabolic regulation on the lysosome. © 2015. Published by The Company of Biologists Ltd.
Re-Factoring Glycolytic Genes for Targeted Engineering of Catabolism in Gram-Negative Bacteria
DEFF Research Database (Denmark)
Sánchez-Pascuala, Alberto; Nikel, Pablo I.; de Lorenzo, Víctor
2018-01-01
the potential applications of such a portable tool for targeted pathway engineering, in the present protocol we describe how the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals......The Embden-Meyerhof-Parnas (EMP) pathway is widely accepted to be the biochemical standard of glucose catabolism. The well-characterized glycolytic route of Escherichia coli, based on the EMP catabolism, is an example of an intricate pathway in terms of genomic organization of the genes involved...... and patterns of gene expression and regulation. This intrinsic genetic and metabolic complexity renders it difficult to engineer glycolytic activities and transfer them onto other microbial cell factories, thus limiting the biotechnological potential of bacterial hosts that lack the route. Taking into account...
The Role of Conserved N-Linked Glycans on Ebola Virus Glycoprotein 2.
Lennemann, Nicholas J; Walkner, Madeline; Berkebile, Abigail R; Patel, Neil; Maury, Wendy
2015-10-01
N-linked glycosylation is a common posttranslational modification found on viral glycoproteins (GPs) and involved in promoting expression, cellular attachment, protection from proteases, and antibody evasion. The GP subunit GP2 of filoviruses contains 2 completely conserved N-linked glycosylation sites (NGSs) at N563 and N618, suggesting that they have been maintained through selective pressures. We assessed mutants lacking these glycans for expression and function to understand the role of these sites during Ebola virus entry. Elimination of either GP2 glycan individually had a modest effect on GP expression and no impact on antibody neutralization of vesicular stomatitis virus pseudotyped with Ebola virus GP. However, loss of the N563 glycan enhanced entry by 2-fold and eliminated GP detection by a well-characterized monoclonal antibody KZ52. Loss of both sites dramatically decreased GP expression and abolished entry. Surprisingly, a GP that retained a single NGS at N563, eliminating the remaining 16 NGSs from GP1 and GP2, had detectable expression, a modest increase in entry, and pronounced sensitivity to antibody neutralization. Our findings support the importance of the GP2 glycans in GP expression/structure, transduction efficiency, and antibody neutralization, particularly when N-linked glycans are also removed from GP1. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Regulation of cytokine receptors by Golgi N-glycan processing and endocytosis.
Partridge, Emily A; Le Roy, Christine; Di Guglielmo, Gianni M; Pawling, Judy; Cheung, Pam; Granovsky, Maria; Nabi, Ivan R; Wrana, Jeffrey L; Dennis, James W
2004-10-01
The Golgi enzyme beta1,6 N-acetylglucosaminyltransferase V (Mgat5) is up-regulated in carcinomas and promotes the substitution of N-glycan with poly N-acetyllactosamine, the preferred ligand for galectin-3 (Gal-3). Here, we report that expression of Mgat5 sensitized mouse cells to multiple cytokines. Gal-3 cross-linked Mgat5-modified N-glycans on epidermal growth factor and transforming growth factor-beta receptors at the cell surface and delayed their removal by constitutive endocytosis. Mgat5 expression in mammary carcinoma was rate limiting for cytokine signaling and consequently for epithelial-mesenchymal transition, cell motility, and tumor metastasis. Mgat5 also promoted cytokine-mediated leukocyte signaling, phagocytosis, and extravasation in vivo. Thus, conditional regulation of N-glycan processing drives synchronous modification of cytokine receptors, which balances their surface retention against loss via endocytosis.
Energy Technology Data Exchange (ETDEWEB)
Koropatkin, Nicole M.; Martens, Eric C.; Gordon, Jeffrey I.; Smith, Thomas J. (WU); (Danforth)
2009-01-12
The human gut microbiota performs functions that are not encoded in our Homo sapiens genome, including the processing of otherwise undigestible dietary polysaccharides. Defining the structures of proteins involved in the import and degradation of specific glycans by saccharolytic bacteria complements genomic analysis of the nutrient-processing capabilities of gut communities. Here, we describe the atomic structure of one such protein, SusD, required for starch binding and utilization by Bacteroides thetaiotaomicron, a prominent adaptive forager of glycans in the distal human gut microbiota. The binding pocket of this unique {alpha}-helical protein contains an arc of aromatic residues that complements the natural helical structure of starch and imposes this conformation on bound maltoheptaose. Furthermore, SusD binds cyclic oligosaccharides with higher affinity than linear forms. The structures of several SusD/oligosaccharide complexes reveal an inherent ligand recognition plasticity dominated by the three-dimensional conformation of the oligosaccharides rather than specific interactions with the composite sugars.
Diversity in RotavirusâHost Glycan Interactions: A âSweetâ SpectrumSummary
Directory of Open Access Journals (Sweden)
Sasirekha Ramani
2016-05-01
Full Text Available Interaction with cellular glycans is a critical initial step in the pathogenesis of many infectious agents. Technological advances in glycobiology have expanded the repertoire of studies delineating host glycanâpathogen interactions. For rotavirus, the VP8* domain of the outer capsid spike protein VP4 is known to interact with cellular glycans. Sialic acid was considered the key cellular attachment factor for rotaviruses for decades. Although this is true for many rotavirus strains causing infections in animals, glycan array screens show that many human rotavirus strains bind nonsialylated glycoconjugates, called histo-blood group antigens, in a strain-specific manner. The expression of histo-blood group antigens is determined genetically and is regulated developmentally. Variations in glycan binding between different rotavirus strains are biologically relevant and provide new insights into multiple aspects of virus pathogenesis such as interspecies transmission, host range restriction, and tissue tropism. The genetics of glycan expression may affect susceptibility to different rotavirus strains and vaccine viruses, and impact the efficacy of rotavirus vaccination in different populations. A multidisciplinary approach to understanding rotavirusâhost glycan interactions provides molecular insights into the interaction between microbial pathogens and glycans, and opens up new avenues to translate findings from the bench to the human population. Keywords: Rotavirus, VP8*, Glycans, Sia, Histo-Blood Group Antigens
Functional network of glycan-related molecules: Glyco-Net in Glycoconjugate Data Bank
Directory of Open Access Journals (Sweden)
Miura Nobuaki
2010-06-01
Full Text Available Abstract Background Glycans are involved in a wide range of biological process, and they play an essential role in functions such as cell differentiation, cell adhesion, pathogen-host recognition, toxin-receptor interactions, signal transduction, cancer metastasis, and immune responses. Elucidating pathways related to post-translational modifications (PTMs such as glycosylation are of growing importance in post-genome science and technology. Graphical networks describing the relationships among glycan-related molecules, including genes, proteins, lipids and various biological events are considered extremely valuable and convenient tools for the systematic investigation of PTMs. However, there is no database which dynamically draws functional networks related to glycans. Description We have created a database called Glyco-Net http://www.glycoconjugate.jp/functions/, with many binary relationships among glycan-related molecules. Using search results, we can dynamically draw figures of the functional relationships among these components with nodes and arrows. A certain molecule or event corresponds to a node in the network figures, and the relationship between the molecule and the event are indicated by arrows. Since all components are treated equally, an arrow is also a node. Conclusions In this paper, we describe our new database, Glyco-Net, which is the first database to dynamically show networks of the functional profiles of glycan related molecules. The graphical networks will assist in the understanding of the role of the PTMs. In addition, since various kinds of bio-objects such as genes, proteins, and inhibitors are equally treated in Glyco-Net, we can obtain a large amount of information on the PTMs.
Poiroux, Guillaume; Barre, Annick; van Damme, Els J. M.; Benoist, Hervé; Rougé, Pierre
2017-01-01
Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors. PMID:28598369
Zhou, Shiyue; Tello, Nadia; Harvey, Alex; Boyes, Barry; Orlando, Ron; Mechref, Yehia
2016-06-01
Glycans have numerous functions in various biological processes and participate in the progress of diseases. Reliable quantitative glycomic profiling techniques could contribute to the understanding of the biological functions of glycans, and lead to the discovery of potential glycan biomarkers for diseases. Although LC-MS is a powerful analytical tool for quantitative glycomics, the variation of ionization efficiency and MS intensity bias are influencing quantitation reliability. Internal standards can be utilized for glycomic quantitation by MS-based methods to reduce variability. In this study, we used stable isotope labeled IgG2b monoclonal antibody, iGlycoMab, as an internal standard to reduce potential for errors and to reduce variabililty due to sample digestion, derivatization, and fluctuation of nanoESI efficiency in the LC-MS analysis of permethylated N-glycans released from model glycoproteins, human blood serum, and breast cancer cell line. We observed an unanticipated degradation of isotope labeled glycans, tracked a source of such degradation, and optimized a sample preparation protocol to minimize degradation of the internal standard glycans. All results indicated the effectiveness of using iGlycoMab to minimize errors originating from sample handling and instruments. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Walker, S Hunter; Taylor, Amber D; Muddiman, David C
2013-06-30
Traditionally, free oligosaccharide internal standards are used to account for variability in glycan relative quantification experiments by mass spectrometry. However, a more suitable internal standard would be a glycoprotein, which could also control for enzymatic cleavage efficiency, allowing for more accurate quantitative experiments. Hydrophobic, hydrazide N-linked glycan reagents (both native and stable-isotope labeled) are used to derivatize and differentially label N-linked glycan samples for relative quantification, and the samples are analyzed by a reversed-phase liquid chromatography chip system coupled online to a Q-Exactive mass spectrometer. The inclusion of two internal standards, maltoheptaose (previously used) and horseradish peroxidase (HRP) (novel), is studied to demonstrate the effectiveness of using a glycoprotein as an internal standard in glycan relative quantification experiments. HRP is a glycoprotein containing a xylosylated N-linked glycan, which is unique from mammalian N-linked glycans. Thus, the internal standard xylosylated glycan could be detected without interference to the sample. Additionally, it was shown that differences in cleavage efficiency can be detected by monitoring the HRP glycan. In a sample where cleavage efficiency variation is minimal, the HRP glycan performs as well as maltoheptaose. Because the HRP glycan performs as well as maltoheptaose but is also capable of correcting and accounting for cleavage variability, it is a more versatile internal standard and will be used in all subsequent biological studies. Because of the possible lot-to-lot variation of an enzyme, differences in biological matrix, and variable enzyme activity over time, it is a necessity to account for glycan cleavage variability in glycan relative quantification experiments. Copyright © 2013 John Wiley & Sons, Ltd.
Microarray glycan profiling reveals algal fucoidan epitopes in diverse marine metazoans
DEFF Research Database (Denmark)
Asunción Salmeán, Armando; Hervé, Cécile; Jørgensen, Bodil
2017-01-01
Despite the biological importance and pharmacological potential of glycans from marine organisms, there are many unanswered questions regarding their distribution, function, and evolution. Here we describe microarray-based glycan profiling of a diverse selection of marine animals using antibodies...... raised against fucoidan isolated from a brown alga. We demonstrate the presence of two fucoidan epitopes in six animals belonging to three phyla including Porifera, Molusca, and Chordata. We studied the spatial distribution of these epitopes in Cliona celata ("boring sponge") and identified...
Walker, S. Hunter; Carlisle, Brandon C.; Muddiman, David C.
2013-01-01
Due to the hydrophilic nature of glycans, reverse phase chromatography has not been widely used as a glycomic separation technique coupled to mass spectrometry. Other approaches such as hydrophilic interaction chromatography and porous graphitized carbon chromatography are often employed, though these strategies frequently suffer from decreased chromatographic resolution, long equilibration times, indefinite retention, and column bleed. Herein, it is shown that through an efficient hydrazone formation derivatization of N-linked glycans (∼4 hr of additional sample preparation time which is carried out in parallel), numerous experimental and practical advantages are gained when analyzing the glycans by online reverse phase chromatography. These benefits include an increased number of glycans detected, increased peak capacity of the separation, and the ability to analyze glycans on the identical liquid chromatography-mass spectrometry platform commonly used for proteomic analyses. The data presented show that separation of derivatized N-linked glycans by reverse phase chromatography significantly out-performs traditional separation of native or derivatized glycans by hydrophilic interaction chromatography. Furthermore, the movement to a more ubiquitous separation technique will afford numerous research groups the opportunity to analyze both proteomic and glycomic samples on the same platform with minimal time and physical change between experiments, increasing the efficiency of ‘multi-omic’ biological approaches. PMID:22954204
Energy Technology Data Exchange (ETDEWEB)
Posen, Paulette [School of Environmental Sciences, University of East Anglia, Earlham Road, Norwich NR4 7TJ (United Kingdom)]. E-mail: p.posen@uea.ac.uk; Lovett, Andrew [School of Environmental Sciences, University of East Anglia, Earlham Road, Norwich NR4 7TJ (United Kingdom); Hiscock, Kevin [School of Environmental Sciences, University of East Anglia, Earlham Road, Norwich NR4 7TJ (United Kingdom); Evers, Sarah [Environment Agency, Olton Court, 10 Warwick Road, Olton, Solihull, B92 7HX (United Kingdom); Ward, Rob [Environment Agency, Olton Court, 10 Warwick Road, Olton, Solihull, B92 7HX (United Kingdom); Reid, Brian [School of Environmental Sciences, University of East Anglia, Earlham Road, Norwich NR4 7TJ (United Kingdom)
2006-08-31
The catabolic activity of incumbent microorganisms in soil samples of eleven dissimilar soil series was investigated, with respect to the herbicide isoproturon. Soils were collected from a 30 x 37 km area of river catchment to the north-west of London, England. Catabolic activity in each soil type during a 500 h assay was determined by {sup 14}C-radiorespirometry. Results showed four soils that exhibited high levels of catabolic activity (33-44% mineralisation) while the remaining seven soils showed lower levels of catabolic activity (12-16% mineralisation). There was evidence to suggest that soils exhibiting high catabolic activity had low (< 22%) clay content and tended towards lower organic carbon content (< 2.7%), but that these higher levels of catabolic activity were also related to pre-exposure to isoproturon. The {sup 14}C-radiorespirometric results were used to produce a GIS layer representing levels of catabolic activity for the dissimilar soils across the study area. This layer was combined with other GIS layers relating to pesticide attenuation, including soil organic carbon content, depth to groundwater and hydrogeology, to produce a map showing risk of groundwater contamination by isoproturon. The output from this approach was compared with output from an attenuation-only approach and differences appraised. Inclusion of the catabolism layer resulted in a lowering of risk in the model in 15% of the study area. Although there appears to be limited benefit in including pesticide catabolic activity in this regional-scale groundwater risk model, this type of addition could be useful in a site-specific risk assessment.
International Nuclear Information System (INIS)
Posen, Paulette; Lovett, Andrew; Hiscock, Kevin; Evers, Sarah; Ward, Rob; Reid, Brian
2006-01-01
The catabolic activity of incumbent microorganisms in soil samples of eleven dissimilar soil series was investigated, with respect to the herbicide isoproturon. Soils were collected from a 30 x 37 km area of river catchment to the north-west of London, England. Catabolic activity in each soil type during a 500 h assay was determined by 14 C-radiorespirometry. Results showed four soils that exhibited high levels of catabolic activity (33-44% mineralisation) while the remaining seven soils showed lower levels of catabolic activity (12-16% mineralisation). There was evidence to suggest that soils exhibiting high catabolic activity had low ( 14 C-radiorespirometric results were used to produce a GIS layer representing levels of catabolic activity for the dissimilar soils across the study area. This layer was combined with other GIS layers relating to pesticide attenuation, including soil organic carbon content, depth to groundwater and hydrogeology, to produce a map showing risk of groundwater contamination by isoproturon. The output from this approach was compared with output from an attenuation-only approach and differences appraised. Inclusion of the catabolism layer resulted in a lowering of risk in the model in 15% of the study area. Although there appears to be limited benefit in including pesticide catabolic activity in this regional-scale groundwater risk model, this type of addition could be useful in a site-specific risk assessment
Major O-glycans from the nest of Vespula germanica contain phospho-ethanolamine.
Maes, Emmanuel; Garénaux, Estelle; Strecker, Gérard; Leroy, Yves; Wieruszeski, Jean-Michel; Brassart, Colette; Guérardel, Yann
2005-08-15
We describe here the structural deciphering of four wasp O-glycans. Following purification of a mixture of glycoproteins from nests of the common wasp Vespula germanica L. (Hymenoptera), their substituting O-glycans were liberated by reducing beta-elimination and characterised using a combination of high resolution NMR and mass spectrometry analyses. Besides ubiquitously found in the insect cells GalNAc-ol and Gal(beta1-3)GalNAc-ol compounds, two novel O-glycans carrying a 2-aminoethyl phosphate group were described for the first time here. We suggest that they present the following structures: Etn-P-(O-->6)-GalNAc-ol and Etn-P-(O-->6)-[Gal(beta1-3)]GalNAc-ol. In conjunction with previous studies, these results suggest that a 2-aminoethyl phosphate group may act as an alternative to sialic acid for conferring charges to glycoproteins.
LENUS (Irish Health Repository)
Coss, K P
2012-02-01
N-glycan processing and assembly defects have been demonstrated in untreated and partially treated patients with Classical Galactosaemia. These defects may contribute to the ongoing pathophysiology of this disease. The aim of this study was to develop an informative method of studying differential galactose tolerance levels and diet control in individuals with Galactosaemia, compared to the standard biochemical markers. Ten Galactosaemia adults with normal intellectual outcomes were analyzed in the study. Five subjects followed galactose liberalization, increments of 300 mg to 4000 mg\\/day over 16 weeks, and were compared to five adult Galactosaemia controls on a galactose restricted diet. All study subjects underwent clinical and biochemical monitoring of red blood cell galactose-1-phosphate (RBC Gal-1-P) and urinary galactitol levels. Serum N-glycans were isolated and analyzed by normal phase high-performance liquid chromatography (NP-HPLC) with galactosylation of IgG used as a specific biomarker of galactose tolerance. IgG N-glycan profiles showed consistent individual alterations in response to diet liberalization. The individual profiles were improved for all, but one study subject, at a galactose intake of 1000 mg\\/day, with decreases in agalactosylated (G0) and increases in digalactosylated (G2) N-glycans. We conclude that IgG N-glycan profiling is an improved method of monitoring variable galactosylation and determining individual galactose tolerance in Galactosaemia compared to the standard methods.
Zhang, Peilan; Li, Kunhua; Yang, Guang; Xia, Changqing; Polston, Jane E; Li, Gengnan; Li, Shiwu; Lin, Zhao; Yang, Li-Jun; Bruner, Steven D; Ding, Yousong
2017-08-22
Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan-GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Coprinus comatus Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcβ1-4(Fucα1-3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αβα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel β-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment.
Association of Anti-glycan Antibodies and Inflammatory Bowel Disease Course.
Paul, S; Boschetti, G; Rinaudo-Gaujous, M; Moreau, A; Del Tedesco, E; Bonneau, J; Presles, E; Mounsef, F; Clavel, L; Genin, C; Flourié, B; Phelip, J-M; Nancey, S; Roblin, X
2015-06-01
The usefulness of anti-glycan antibodies alone or combined with anti-Saccharomyces cerevisiae [ASCA] or perinuclear antineutrophil cytoplasmic [pANCA] antibodies for diagnosis of inflammatory bowel disease [IBD], differentiation between Crohn's disease [CD] and ulcerative colitis [UC], disease stratification including IBD phenotype, and also for determination of the course of the disease, remain unclear. A large panel of serological anti-glycan carbohydrate antibodies, including anti-mannobioside IgG antibodies [AMCA], anti-chitobioside IgA [ACCA], anti-laminaribioside IgG antibodies [ALCA], anti-laminarin [anti-L] and anti-chitine [anti-C] were measured in the serum from a cohort of 195 patients with IBD] [107 CD and 88 UC]. The respective accuracy of isolated or combined markers for diagnosis, disease differentiation, stratification disease phenotype, and severity of the disease course, defined by a wide panel of criteria obtained from the past medical history, was assessed. The positivity of at least one anti-glycan antibody was detected in a significant higher proportion of CD and UC compared with healthy controls [p ACCA [> 51U/ml] and anti-laminarin [> 31U/ml] were significantly linked with a higher association with steroid dependency (odds ratio [OR] =2.0 [1.0-4.0], p = 0.03 and OR = 2.4 [1.1-5.2], p = 0.02, respectively]. We further defined the respective performance of anti-glycan antibodies to discriminate between patients with severe or not severe CD and UC course and determined the associated optimal cut-off values: severe CD course was significantly more likely in case of AMCA > 77U/ml [OR = 4.3; p = 0.002], ASCA > 63U/ml [OR = 3.5; p ACCA > 50U/ml [OR = 2.8; p 52U/ml [OR = 3.4; p = 0.04] and ACCA > 25U/ml [OR = 3.0; p < 0.04]. Anti-glycan antibodies are valuable serological markers, especially AMCA antibodies that may help clinicians to promptly classify patients into high risk for severe disease. Copyright © 2015 European Crohn’s and Colitis
Cell surface N-glycans influence the level of functional E-cadherin at the cell–cell border
Directory of Open Access Journals (Sweden)
M. Kristen Hall
2014-01-01
Full Text Available E-cadherin is crucial for adhesion of cells to each other and thereby development and maintenance of tissue. While it is has been established that N-glycans inside the cell impact the level of E-cadherin at the cell surface of epithelial-derived cells, it is unclear whether N-glycans outside the cell control the clustering of E-cadherin at the cell–cell border. Here, we demonstrate reduction of N-glycans at the cell surface weakened the recruitment and retention of E-cadherin at the cell–cell border, and consequently reduced the strength of cell–cell interactions. We conclude that N-glycans at the cell surface are tightly linked to the placement of E-cadherin at the cell–cell border and thereby control E-cadherin mediated cell–cell adhesion.
Xu, Minjun; Kitaura, Yasuyuki; Ishikawa, Takuya; Kadota, Yoshihiro; Terai, Chihaya; Shindo, Daichi; Morioka, Takashi; Ota, Miki; Morishita, Yukako; Ishihara, Kengo; Shimomura, Yoshiharu
2017-01-01
It is known that the catabolism of branched-chain amino acids (BCAAs) in skeletal muscle is suppressed under normal and sedentary conditions but is promoted by exercise. BCAA catabolism in muscle tissues is regulated by the branched-chain α-keto acid (BCKA) dehydrogenase complex, which is inactivated by phosphorylation by BCKA dehydrogenase kinase (BDK). In the present study, we used muscle-specific BDK deficient mice (BDK-mKO mice) to examine the effect of uncontrolled BCAA catabolism on endurance exercise performance and skeletal muscle energy metabolism. Untrained control and BDK-mKO mice showed the same performance; however, the endurance performance enhanced by 2 weeks of running training was somewhat, but significantly less in BDK-mKO mice than in control mice. Skeletal muscle of BDK-mKO mice had low levels of glycogen. Metabolome analysis showed that BCAA catabolism was greatly enhanced in the muscle of BDK-mKO mice and produced branched-chain acyl-carnitine, which induced perturbation of energy metabolism in the muscle. These results suggest that the tight regulation of BCAA catabolism in muscles is important for homeostasis of muscle energy metabolism and, at least in part, for adaptation to exercise training.
Directory of Open Access Journals (Sweden)
Minjun Xu
Full Text Available It is known that the catabolism of branched-chain amino acids (BCAAs in skeletal muscle is suppressed under normal and sedentary conditions but is promoted by exercise. BCAA catabolism in muscle tissues is regulated by the branched-chain α-keto acid (BCKA dehydrogenase complex, which is inactivated by phosphorylation by BCKA dehydrogenase kinase (BDK. In the present study, we used muscle-specific BDK deficient mice (BDK-mKO mice to examine the effect of uncontrolled BCAA catabolism on endurance exercise performance and skeletal muscle energy metabolism. Untrained control and BDK-mKO mice showed the same performance; however, the endurance performance enhanced by 2 weeks of running training was somewhat, but significantly less in BDK-mKO mice than in control mice. Skeletal muscle of BDK-mKO mice had low levels of glycogen. Metabolome analysis showed that BCAA catabolism was greatly enhanced in the muscle of BDK-mKO mice and produced branched-chain acyl-carnitine, which induced perturbation of energy metabolism in the muscle. These results suggest that the tight regulation of BCAA catabolism in muscles is important for homeostasis of muscle energy metabolism and, at least in part, for adaptation to exercise training.
Quantitative characterization of glycan-receptor binding of H9N2 influenza A virus hemagglutinin.
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Karunya Srinivasan
Full Text Available Avian influenza subtypes such as H5, H7 and H9 are yet to adapt to the human host so as to establish airborne transmission between humans. However, lab-generated reassorted viruses possessing hemagglutinin (HA and neuraminidase (NA genes from an avian H9 isolate and other genes from a human-adapted (H3 or H1 subtype acquired two amino acid changes in HA and a single amino acid change in NA that confer respiratory droplet transmission in ferrets. We previously demonstrated for human-adapted H1, H2 and H3 subtypes that quantitative binding affinity of their HA to α2→6 sialylated glycan receptors correlates with respiratory droplet transmissibility of the virus in ferrets. Such a relationship remains to be established for H9 HA. In this study, we performed a quantitative biochemical characterization of glycan receptor binding properties of wild-type and mutant forms of representative H9 HAs that were previously used in context of reassorted viruses in ferret transmission studies. We demonstrate here that distinct molecular interactions in the glycan receptor-binding site of different H9 HAs affect the glycan-binding specificity and affinity. Further we show that α2→6 glycan receptor-binding affinity of a mutant H9 HA carrying Thr-189→Ala amino acid change correlates with the respiratory droplet transmission in ferrets conferred by this change. Our findings contribute to a framework for monitoring the evolution of H9 HA by understanding effects of molecular changes in HA on glycan receptor-binding properties.
B S, Gnanesh Kumar; Surolia, Avadhesha
2017-05-01
Carboxypeptidase Y from Saccharomyces cerivisiae was characterized for its site specific N-glycosylation through mass spectrometry. The N-glycopeptides were derived using non specific proteases and are analysed directly on liquid chromatography coupled to ion trap mass spectrometer in tandem mode. The evaluation of glycan fragment ions and the Y 1 ions (peptide+HexNAc) +n revealed the glycan sequence and the corresponding site of attachment. We observed the microheterogeneity in N-glycans such as Man 11-15 GlcNAc 2 at Asn 13 , Man 8-12 GlcNAc 2 at Asn 87 , Man 9-14 GlcNAc 2 at Asn 168 and phosphorylated Man 12-17 GlcNAc 2 as well as Man 11-16 GlcNAc 2 at Asn 368 . The presence of N-glycans with Man <18 GlcNAc 2 indicated that in vacuoles the steady release of mannose/phospho mannose residues from glycans occurs initially at Asn 13 or Asn 168 followed by at Asn 368 . However, glycans at Asn 87 which comprises Man 8-12 residues as reported earlier remain intact suggesting its inaccessibility for a similar processing. This in turn indicates the interaction of the glycan at Asn 87 with the polypeptide chain implicating it in the folding of the protein. Copyright © 2017 Elsevier B.V. All rights reserved.
Immunosuppressive Tryptophan Catabolism and Gut Mucosal Dysfunction Following Early HIV Infection
Jenabian, Mohammad-Ali; El-Far, Mohamed; Vyboh, Kishanda; Kema, Ido; Costiniuk, Cecilia T.; Thomas, Rejean; Baril, Jean-Guy; LeBlanc, Roger; Kanagaratham, Cynthia; Radzioch, Danuta; Allam, Ossama; Ahmad, Ali; Lebouche, Bertrand; Tremblay, Cecile; Ancuta, Petronela; Routy, Jean-Pierre
2015-01-01
Background. Tryptophan (Trp) catabolism into kynurenine (Kyn) contributes to immune dysfunction in chronic human immunodeficiency virus (HIV) infection. To better define the relationship between Trp catabolism, inflammation, gut mucosal dysfunction, and the role of early antiretroviral therapy
C-terminus glycans with critical functional role in the maturation of secretory glycoproteins.
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Daniela Cioaca
Full Text Available The N-glycans of membrane glycoproteins are mainly exposed to the extracellular space. Human tyrosinase is a transmembrane glycoprotein with six or seven bulky N-glycans exposed towards the lumen of subcellular organelles. The central active site region of human tyrosinase is modeled here within less than 2.5 Å accuracy starting from Streptomyces castaneoglobisporus tyrosinase. The model accounts for the last five C-terminus glycosylation sites of which four are occupied and indicates that these cluster in two pairs--one in close vicinity to the active site and the other on the opposite side. We have analyzed and compared the roles of all tyrosinase N-glycans during tyrosinase processing with a special focus on the proximal to the active site N-glycans, s6:N337 and s7:N371, versus s3:N161 and s4:N230 which decorate the opposite side of the domain. To this end, we have constructed mutants of human tyrosinase in which its seven N-glycosylation sites were deleted. Ablation of the s6:N337 and s7:N371 sites arrests the post-translational productive folding process resulting in terminally misfolded mutants subjected to degradation through the mannosidase driven ERAD pathway. In contrast, single mutants of the other five N-glycans located either opposite to the active site or into the N-terminus Cys1 extension of tyrosinase are temperature-sensitive mutants and recover enzymatic activity at the permissive temperature of 31°C. Sites s3 and s4 display selective calreticulin binding properties. The C-terminus sites s7 and s6 are critical for the endoplasmic reticulum retention and intracellular disposal. Results herein suggest that individual N-glycan location is critical for the stability, regional folding control and secretion of human tyrosinase and explains some tyrosinase gene missense mutations associated with oculocutaneous albinism type I.
Gnanou, Yves
2016-10-20
Provided herein are methods for synthesizing cyclic carbonates, glycocarbonates, and polyglycocarbonates by reacting polyol glycans with carbon dioxide. Synthesis can include selective polycondensation of polyol glycan hydroxyl moieties.
Gnanou, Yves; Pati, Debasis; Feng, Xiaoshuang; Hadjichristidis, Nikolaos
2016-01-01
Provided herein are methods for synthesizing cyclic carbonates, glycocarbonates, and polyglycocarbonates by reacting polyol glycans with carbon dioxide. Synthesis can include selective polycondensation of polyol glycan hydroxyl moieties.
Sładek, Małgorzata; Wasilewska, Agata; Swiat, Agnieszka; Cmiel, Adam
2014-01-01
Antibodies reacting with various microbial epitopes have been described in inflammatory bowel disease (IBD) and are associated with a specific diagnosis and clinical presentation. To evaluate the profile of new anti-glycan antibodies, their potential association with disease phenotype and diagnostic accuracy in paediatric Crohn's disease (CD). Blood samples from 134 paediatric IBD patients (109 CD, 25 ulcerative colitis (UC)) and 67 controls were blindly analysed for anti-Saccharomyces cerevisiae (ASCA), anti-chitobioside carbohydrate (ACCA), anti-laminaribioside carbohydrate (ALCA), and anti-mannobioside carbohydrate (AMCA) antibodies using commercially available assays. The serological response to glycans was correlated with clinical disease characteristics. At least one of the tested anti-glycan antibodies was present in 75% of CD patients. Despite the high frequency of reactivity to glycan epitopes, a limited overlap of serological markers was observed. In total, 49% of ASCA-negative patients presented with one of the following: ACCA, ALCA, or AMCA. The occurrence of one antibody from the anti-glycan panel was independently associated with complicated disease phenotype and ileocolonic disease location. A higher level of immune response as assessed by the quartile sum scores for ACCA, ALCA, and AMCA was linked with older age at diagnosis (10-17 years) and ileocolonic disease location. The ASCA had the greatest accuracy for diagnosis and differentiation of CD. Qualitative and quantitative serologicalal response to glycan epitopes was associated with distinct clinical presentation in paediatric CD patients. This raises the possibility for the use of these markers to differentiate subgroups of CD patients with more sever clinical presentation. The ASCA was the most accurate serological marker for CD; however, testing for the new anti-glycan antibodies may constitute an adjunctive tool in a specific group of patients to aid in the differentiation of CD with absent
Directory of Open Access Journals (Sweden)
Caroline B Madsen
Full Text Available Membrane bound mucins are up-regulated and aberrantly glycosylated during malignant transformation in many cancer cells. This results in a negatively charged glycoprotein coat which may protect cancer cells from immune surveillance. However, only limited data have so far demonstrated the critical steps in glycan elongation that make aberrantly glycosylated mucins affect the interaction between cancer cells and cytotoxic effector cells of the immune system. Tn (GalNAc-Ser/Thr, STn (NeuAcα2-6GalNAc-Ser/Thr, T (Galβ1-3GalNAc-Ser/Thr, and ST (NeuAcα2-6Galβ1-3GalNAc-Ser/Thr antigens are recognized as cancer associated truncated glycans, and are expressed in many adenocarcinomas, e.g. breast- and pancreatic cancer cells. To investigate the role of the cancer associated glycan truncations in immune-mediated killing we created glyco-engineered breast- and pancreatic cancer cells expressing only the shortest possible mucin-like glycans (Tn and STn. Glyco-engineering was performed by zinc finger nuclease (ZFN knockout (KO of the Core 1 enzyme chaperone COSMC, thereby preventing glycan elongation beyond the initial GalNAc residue in O-linked glycans. We find that COSMC KO in the breast and pancreatic cancer cell lines T47D and Capan-1 increases sensitivity to both NK cell mediated antibody-dependent cellular-cytotoxicity (ADCC and cytotoxic T lymphocyte (CTL-mediated killing. In addition, we investigated the association between total cell surface expression of MUC1/MUC16 and NK or CTL mediated killing, and observed an inverse correlation between MUC16/MUC1 expression and the sensitivity to ADCC and CTL-mediated killing. Together, these data suggest that up-regulation of membrane bound mucins protects cells from immune mediated killing, and that particular glycosylation steps, as demonstrated for glycan elongation beyond Tn and STn, can be important for fine tuning of the immune escape mechanisms in cancer cells.
Variable carbon catabolism among Salmonella enterica serovar Typhi isolates.
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Lay Ching Chai
Full Text Available BACKGROUND: Salmonella enterica serovar Typhi (S. Typhi is strictly a human intracellular pathogen. It causes acute systemic (typhoid fever and chronic infections that result in long-term asymptomatic human carriage. S. Typhi displays diverse disease manifestations in human infection and exhibits high clonality. The principal factors underlying the unique lifestyle of S. Typhi in its human host during acute and chronic infections remain largely unknown and are therefore the main objective of this study. METHODOLOGY/PRINCIPAL FINDINGS: To obtain insight into the intracellular lifestyle of S. Typhi, a high-throughput phenotypic microarray was employed to characterise the catabolic capacity of 190 carbon sources in S. Typhi strains. The success of this study lies in the carefully selected library of S. Typhi strains, including strains from two geographically distinct areas of typhoid endemicity, an asymptomatic human carrier, clinical stools and blood samples and sewage-contaminated rivers. An extremely low carbon catabolic capacity (27% of 190 carbon substrates was observed among the strains. The carbon catabolic profiles appeared to suggest that S. Typhi strains survived well on carbon subtrates that are found abundantly in the human body but not in others. The strains could not utilise plant-associated carbon substrates. In addition, α-glycerolphosphate, glycerol, L-serine, pyruvate and lactate served as better carbon sources to monosaccharides in the S. Typhi strains tested. CONCLUSION: The carbon catabolic profiles suggest that S. Typhi could survive and persist well in the nutrient depleted metabolic niches in the human host but not in the environment outside of the host. These findings serve as caveats for future studies to understand how carbon catabolism relates to the pathogenesis and transmission of this pathogen.
Miura, Yuri; Endo, Tamao
2016-08-01
Since glycosylation depends on glycosyltransferases, glycosidases, and sugar nucleotide donors, it is susceptible to the changes associated with physiological and pathological conditions. Therefore, alterations in glycan structures may be good targets and biomarkers for monitoring health conditions. Since human aging and longevity are affected by genetic and environmental factors such as diseases, lifestyle, and social factors, a scale that reflects various environmental factors is required in the study of human aging and longevity. We herein focus on glycosylation changes elucidated by glycomic and glycoproteomic studies on aging, longevity, and age-related diseases including cognitive impairment, diabetes mellitus, and frailty. We also consider the potential of glycan structures as biomarkers and/or targets for monitoring physiological and pathophysiological changes. Glycan structures are altered in age-related diseases. These glycans and glycoproteins may be involved in the pathophysiology of these diseases and, thus, be useful diagnostic markers. Age-dependent changes in N-glycans have been reported previously in cohort studies, and characteristic N-glycans in extreme longevity have been proposed. These findings may lead to a deeper understanding of the mechanisms underlying aging as well as the factors influencing longevity. Alterations in glycosylation may be good targets and biomarkers for monitoring health conditions, and be applicable to studies on age-related diseases and healthy aging. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. Copyright © 2016 Elsevier B.V. All rights reserved.
Optimization of the Small Glycan Presentation for Binding a Tumor-Associated Antibody
DEFF Research Database (Denmark)
Kveton, Filip; Blšáková, Anna; Hushegyi, Andras
2017-01-01
on the immobilization of the Tn antigen on a mixed self-assembled monolayer (SAM) (2D biosensor) and the third one utilizing a layer of a human serum albumin (HSA) for the immobilization of a glycan forming a 3D interface. Results showed that the 3D interface with the immobilized Tn antigen is the most effective...... bioreceptive surface for binding its analyte. The 3D impedimetric glycan biosensor exhibited a limit of detection of 1.4 aM, a wide linear range (6 orders of magnitude), and high assay reproducibility with an average relative standard deviation of 4%. The buildup of an interface was optimized using various...... techniques with the visualization of the glycans on the biosensor surface by atomic force microscopy. The study showed that the 3D biosensor is not only the most sensitive compared to other two biosensor platforms but that the Tn antigen on the 3D biosensor surface is more accessible for antibody binding...
Chocholova, Erika; Bertok, Tomas; Jane, Eduard; Lorencova, Lenka; Holazova, Alena; Belicka, Ludmila; Belicky, Stefan; Mislovicova, Danica; Vikartovska, Alica; Imrich, Richard; Kasak, Peter; Tkac, Jan
2018-06-01
In this study, one hundred serum samples from healthy people and patients with rheumatoid arthritis (RA) were analyzed. Standard immunoassays for detection of 10 different RA markers and analysis of glycan markers on antibodies in 10 different assay formats with several lectins were applied for each serum sample. A dataset containing 2000 data points was data mined using artificial neural networks (ANN). We identified key RA markers, which can discriminate between healthy people and seropositive RA patients (serum containing autoantibodies) with accuracy of 83.3%. Combination of RA markers with glycan analysis provided much better discrimination accuracy of 92.5%. Immunoassays completely failed to identify seronegative RA patients (serum not containing autoantibodies), while glycan analysis correctly identified 43.8% of these patients. Further, we revealed other critical parameters for successful glycan analysis such as type of a sample, format of analysis and orientation of captured antibodies for glycan analysis. Copyright © 2018 Elsevier B.V. All rights reserved.
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Jae-Han Kim
Full Text Available Bifidobacterium longum subsp. infantis is a common member of the intestinal microbiota in breast-fed infants and capable of metabolizing human milk oligosaccharides (HMO. To investigate the bacterial response to different prebiotics, we analyzed both cell wall associated and whole cell proteins in B. infantis. Proteins were identified by LC-MS/MS followed by comparative proteomics to deduce the protein localization within the cell. Enzymes involved in the metabolism of lactose, glucose, galactooligosaccharides, fructooligosaccharides and HMO were constitutively expressed exhibiting less than two-fold change regardless of the sugar used. In contrast, enzymes in N-Acetylglucosamine and sucrose catabolism were induced by HMO and fructans, respectively. Galactose-metabolizing enzymes phosphoglucomutase, UDP-glucose 4-epimerase and UTP glucose-1-P uridylytransferase were expressed constitutively, while galactokinase and galactose-1-phosphate uridylyltransferase, increased their expression three fold when HMO and lactose were used as substrates for cell growth. Cell wall-associated proteomics also revealed ATP-dependent sugar transport systems associated with consumption of different prebiotics. In addition, the expression of 16 glycosyl hydrolases revealed the complete metabolic route for each substrate. Mucin, which possesses O-glycans that are structurally similar to HMO did not induced the expression of transport proteins, hydrolysis or sugar metabolic pathway indicating B. infantis do not utilize these glycoconjugates.
Xu, Hui; Chakrabarty, Yindrila; Philmus, Benjamin; Mehta, Angad P.; Bhandari, Dhananjay; Hohmann, Hans-Peter; Begley, Tadhg P.
2016-01-01
Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its catabolic pathway, despite intriguing hints in a few distinct organisms, has never been established. This article describes the isolation of a Microbacterium maritypicum riboflavin catabolic strain, and the cloning of the riboflavin catabolic genes. RcaA, RcaB, RcaD, and RcaE were overexpressed and biochemically characterized as riboflavin kinase, riboflavin reductase, ribokinase, and riboflavin hydrolase, respectively. Based on these activities, a pathway for riboflavin catabolism is proposed. PMID:27590337
Li, Xiuru; Martin, Sharon J H; Chinoy, Zoeisha S; Liu, Lin; Rittgers, Brandon; Dluhy, Richard A; Boons, Geert-Jan
2016-01-01
A glyco-array platform has been developed, in which glycans are attached to plasmonic nanoparticles through strain-promoted azide-alkyne cycloaddition. Glycan-protein binding events can then be detected in a label-free manner employing surface-enhanced Raman spectroscopy (SERS). As proof of concept,
The identification and characterization of novel N-glycan-based biomarkers in gastric cancer.
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Long Liu
Full Text Available To identify and validate N-glycan biomarkers in gastric cancer (GC and to elucidate their underlying molecular mechanism of action.In total, 347 individuals, including patients with GC (gastric cancer or atrophic gastritis and healthy controls, were randomly divided into a training group (n=287 and a retrospective validation group (n=60. Serum N-glycan profiling was achieved with DNA sequencer-assisted/fluorophore-assisted carbohydrate electrophoresis (DSA-FACE. Two diagnostic models were constructed based on the N-glycan profiles using logistic stepwise regression. The diagnostic performance of each model was assessed in retrospective, prospective (n=60, and follow-up (n=40 cohorts. Lectin blotting was performed to determine total core-fucosylation, and the expression of genes involved in core-fucosylation in GC was analyzed by reverse transcriptase-polymerase chain reaction.We identified at least 9 N-glycan structures (peaks and the levels of core fucose residues and fucosyltransferase were significantly decreased in GC. Two diagnostic models, designated GCglycoA and GCglycoB, were constructed to differentiate GC from control and atrophic gastritis. The areas under the receiver operating characteristic (ROC curves (AUC for both GCglycoA and GCglycoB were higher than those for CEA, CA19-9, CA125 and CA72-4. Compared with CEA, CA19-9, CA125 and CA72-4, the sensitivity of GCglycoA increased 29.66%, 37.28%, 56.78% and 61.86%, respectively, and the accuracy increased 10.62%, 16.82%, 25.67% and 28.76%, respectively. For GCglycoB, the sensitivity increased 27.97%, 35.59%, 55.09% and 60.17% and the accuracy increased 21.26%, 24.64%, 31.40% and 34.30% compared with CEA, CA19-9, CA125 and CA72-4, respectively. After curative surgery, the core fucosylated peak (peak 3 and the total core fucosylated N-glycans (sumfuc were reversed.The results indicated that the diagnostic models based on N-glycan markers are valuable and noninvasive alternatives for
Xu, Hui; Chakrabarty, Yindrila; Philmus, Benjamin; Mehta, Angad P; Bhandari, Dhananjay; Hohmann, Hans-Peter; Begley, Tadhg P
2016-11-04
Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its catabolic pathway, despite intriguing hints in a few distinct organisms, has never been established. This article describes the isolation of a Microbacterium maritypicum riboflavin catabolic strain, and the cloning of the riboflavin catabolic genes. RcaA, RcaB, RcaD, and RcaE were overexpressed and biochemically characterized as riboflavin kinase, riboflavin reductase, ribokinase, and riboflavin hydrolase, respectively. Based on these activities, a pathway for riboflavin catabolism is proposed. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Pochechueva, Tatiana; Alam, Shahidul; Schötzau, Andreas; Chinarev, Alexander; Bovin, Nicolai V; Hacker, Neville F; Jacob, Francis; Heinzelmann-Schwarz, Viola
2017-02-10
Glycosphingolipids are important compounds of the plasma membrane of mammalian cells and a number of them have been associated with malignant transformation and progression, reinforcing tumour aggressiveness and metastasis. Here we investigated the levels of naturally occurring anti-glycan antibodies to Globo H in blood plasma obtained from high-grade serous ovarian cancer patients (SOC) and women without gynaecological malignancies (control) using suspension glycan array technology employing chemically synthesized glycans as antibody targets. We found that anti-human Globo H IgG antibodies were able to significantly discriminate SOC from controls (P anti-Globo H antibodies highly correlated (r = 0.992). The incubation of plasma-derived anti-glycan antibodies with chemically synthesized (presented on fluorescence microspheres) and native Globo H (expressed on Globo H-positive cell lines) revealed strong reactivity of naturally occurring human anti-Globo H antibodies towards its antigen expressed on ovarian cancer cells. Our data demonstrate that human plasma-derived antibodies to Globo H as well as the presence of the antigen might be considered as therapeutic option in ovarian cancer.
Antitumor Active Protein-containing Glycans from the Body of Ganoderma tsugae
Institute of Scientific and Technical Information of China (English)
LIU Ying; LI Yue-fei; ZHENG Ke-yan; FEI Xiao-fang
2012-01-01
To explore the effects of traditional herbal medicine Ganoderma tsugae(G.tsugae) on immunomodulatory and antitumor activities,the crude polysaccharides ofG.tsugae were purified by filtration,diethylaminoethyl(DEAE)sepharose-fast flow chromatography and sephadex G-100 size-exclusion chromatography.Two main fractions,protein-containing glycans CSSLP-I and CSSLP-2,were obtained via the gradient elution.The protein content,molecular weight,and monosaccharide composition of the two fractions were analyzed.Furthermore,the influence of the protein-containing glycans from G.tsugae on the activation of human acute monocytic leukemia cell line(THP-1 ) and their antitumor activities to the human hepatocellular liver carcinoma cell(HepG-2) in vitro were evaluated.The results indicate that CSSLP-I and CSSLP-2 could increase the pinocytic activity of THP-1 cells and induce THP-1 cells to produce the cytokines of TNFa and IL-2,significantly.CSSLP-1 and CSSLP-2 also played an inhibiting effect on the cancer cell(NepG-2).Moreover,the anti-proliferation activity of CSSLP-1 and CSSLP-2 increased with the participation of TNFa and 1L-2 or other antitumor factors induced from THP-1 cclls by G.tsugae protein-containing glycan fractions.
Transient glyco-engineering to produce recombinant IgA1 with defined N- and O-glycans in plants
Directory of Open Access Journals (Sweden)
Martina eDicker
2016-01-01
Full Text Available The production of therapeutic antibodies to combat pathogens and treat diseases such as cancer is of great interest for the biotechnology industry. The recent development of plant-based expression systems has demonstrated that plants are well-suited for the production of recombinant monoclonal antibodies with defined glycosylation. Compared to immunoglobulin G (IgG, less effort has been undertaken to express immunoglobulin A (IgA, which is the most prevalent antibody class at mucosal sites and a promising candidate for novel recombinant biopharmaceuticals with enhanced anti-tumour activity. Here, we transiently expressed recombinant human IgA1 against the VP8* rotavirus antigen in glyco-engineered deltaXT/FT Nicotiana benthamiana plants. Mass spectrometric analysis of IgA1 glycopeptides revealed the presence of complex biantennary N-glycans with terminal N-acetylglucosamine present on the N-glycosylation site of the CH2 domain in the IgA1 alpha chain. Analysis of the peptide carrying nine potential O-glycosylation sites in the IgA1 alpha chain hinge region showed the presence of plant-specific modifications including hydroxyproline formation and the attachment of pentoses. By co-expression of enzymes required for initiation and elongation of human O-glycosylation it was possible to generate disialylated mucin-type core 1 O-glycans on plant-produced IgA1. Our data demonstrate that deltaXT/FT Nicotiana benthamiana plants can be engineered towards the production of recombinant IgA1 with defined human-type N- and O-linked glycans.
Kurogochi, Masaki; Mori, Masako; Osumi, Kenji; Tojino, Mami; Sugawara, Shu-Ichi; Takashima, Shou; Hirose, Yuriko; Tsukimura, Wataru; Mizuno, Mamoru; Amano, Junko; Matsuda, Akio; Tomita, Masahiro; Takayanagi, Atsushi; Shoda, Shin-Ichiro; Shirai, Takashi
2015-01-01
Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain), and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC). To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases), one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2), high-mannose type (Man4-9GlcNAc2), and complex type (Man3GlcNAc3-4) N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL), the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1) were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q), and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2) was performed using SKBR-3 and BT-474 as target cells, and
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Masaki Kurogochi
Full Text Available Many therapeutic antibodies have been developed, and IgG antibodies have been extensively generated in various cell expression systems. IgG antibodies contain N-glycans at the constant region of the heavy chain (Fc domain, and their N-glycosylation patterns differ during various processes or among cell expression systems. The Fc N-glycan can modulate the effector functions of IgG antibodies, such as antibody-dependent cellular cytotoxicity (ADCC and complement dependent cytotoxicity (CDC. To control Fc N-glycans, we performed a rearrangement of Fc N-glycans from a heterogeneous N-glycosylation pattern to homogeneous N-glycans using chemoenzymatic approaches with two types of endo-β-N-acetyl glucosaminidases (ENG'ases, one that works as a hydrolase to cleave all heterogeneous N-glycans, another that is used as a glycosynthase to generate homogeneous N-glycans. As starting materials, we used an anti-Her2 antibody produced in transgenic silkworm cocoon, which consists of non-fucosylated pauci-mannose type (Man2-3GlcNAc2, high-mannose type (Man4-9GlcNAc2, and complex type (Man3GlcNAc3-4 N-glycans. As a result of the cleavage of several ENG'ases (endoS, endoM, endoD, endoH, and endoLL, the heterogeneous glycans on antibodies were fully transformed into homogeneous-GlcNAc by a combination of endoS, endoD, and endoLL. Next, the desired N-glycans (M3; Man3GlcNAc1, G0; GlcNAc2Man3GlcNAc1, G2; Gal2GlcNAc2Man3GlcNAc1, A2; NeuAc2Gal2GlcNAc2Man3GlcNAc1 were transferred from the corresponding oxazolines to the GlcNAc residue on the intact anti-Her2 antibody with an ENG'ase mutant (endoS-D233Q, and the glycoengineered anti-Her2 antibody was obtained. The binding assay of anti-Her2 antibody with homogenous N-glycans with FcγRIIIa-V158 showed that the glycoform influenced the affinity for FcγRIIIa-V158. In addition, the ADCC assay for the glycoengineered anti-Her2 antibody (mAb-M3, mAb-G0, mAb-G2, and mAb-A2 was performed using SKBR-3 and BT-474 as target
Aich, Udayanath; Liu, Aston; Lakbub, Jude; Mozdzanowski, Jacek; Byrne, Michael; Shah, Nilesh; Galosy, Sybille; Patel, Pramthesh; Bam, Narendra
2016-03-01
Consistent glycosylation in therapeutic monoclonal antibodies is a major concern in the biopharmaceutical industry as it impacts the drug's safety and efficacy and manufacturing processes. Large numbers of samples are created for the analysis of glycans during various stages of recombinant proteins drug development. Profiling and quantifying protein N-glycosylation is important but extremely challenging due to its microheterogeneity and more importantly the limitations of existing time-consuming sample preparation methods. Thus, a quantitative method with fast sample preparation is crucial for understanding, controlling, and modifying the glycoform variance in therapeutic monoclonal antibody development. Presented here is a rapid and highly quantitative method for the analysis of N-glycans from monoclonal antibodies. The method comprises a simple and fast solution-based sample preparation method that uses nontoxic reducing reagents for direct labeling of N-glycans. The complete work flow for the preparation of fluorescently labeled N-glycans takes a total of 3 h with less than 30 min needed for the release of N-glycans from monoclonal antibody samples. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.
Role of siglecs and related glycan-binding proteins in immune responses and immunoregulation.
Bochner, Bruce S; Zimmermann, Nives
2015-03-01
Virtually all cells and extracellular material are heavily decorated by various glycans, yet our understanding of the structure and function of these moieties lags behind the understanding of nucleic acids, lipids, and proteins. Recent years have seen a tremendous acceleration of knowledge in the field of glycobiology, revealing many intricacies and functional contributions that were previously poorly appreciated or even unrecognized. This review highlights several topics relevant to glycoimmunology in which mammalian and pathogen-derived glycans displayed on glycoproteins and other scaffolds are recognized by specific glycan-binding proteins (GBPs), leading to a variety of proinflammatory and anti-inflammatory cellular responses. The focus for this review is mainly on 2 families of GBPs, sialic acid-binding immunoglobulin-like lectins (siglecs) and selectins, that are involved in multiple steps of the immune response, including distinguishing pathogens from self, cell trafficking to sites of inflammation, fine-tuning of immune responses leading to activation or tolerance, and regulation of cell survival. Importantly for the clinician, accelerated rates of discovery in the field of glycoimmunology are being translated into innovative medical approaches that harness the interaction of glycans and GBPs to the benefit of the host and might soon lead to novel diagnostics and therapeutics. Copyright © 2014 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.
Upton, Rosie; Bell, Leonard; Guy, Colin; Caldwell, Paul; Estdale, Sian; Barran, Perdita E; Firth, David
2016-10-18
In the development of therapeutic antibodies and biosimilars, an appropriate biopharmaceutical CMC control strategy that connects critical quality attributes with mechanism of action should enable product assessment at an early stage of development in order to mitigate risk. Here we demonstrate a new analytical workflow using trastuzumab which comprises "middle-up" analysis using a combination of IdeS and the endoglycosidases EndoS and EndoS2 to comprehensively map the glycan content. Enzymatic cleavage between the two N-acetyl glucosamine residues of the chitobiose core of N-glycans significantly simplifies the oligosaccharide component enabling facile distinction of GlcNAc from GlcNAc with core fucose. This approach facilitates quantitative determination of total Fc-glycan core-afucosylation, which was in turn correlated with receptor binding affinity by surface plasmon resonance and in vitro ADCC potency with a cell based bioassay. The strategy also quantifies Fc-glycan occupancy and the relative contribution from high mannose glycans.
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Yusuke Takeuchi
Full Text Available The AMPA-type glutamate receptor (AMPAR, which is a tetrameric complex composed of four subunits (GluA1-4 with several combinations, mediates the majority of rapid excitatory synaptic transmissions in the nervous system. Cell surface expression levels of AMPAR modulate synaptic plasticity, which is considered one of the molecular bases for learning and memory formation. To date, a unique trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc, human natural killer-1 (HNK-1 carbohydrate, was found expressed specifically on N-linked glycans of GluA2 and regulated the cell surface expression of AMPAR and the spine maturation process. However, evidence that the HNK-1 epitope on N-glycans of GluA2 directly affects these phenomena is lacking. Moreover, it is thought that other N-glycans on GluA2 also have potential roles in the regulation of AMPAR functions. In the present study, using a series of mutants lacking potential N-glycosylation sites (N256, N370, N406, and N413 within GluA2, we demonstrated that the mutant lacking the N-glycan at N370 strongly suppressed the intracellular trafficking of GluA2 from the endoplasmic reticulum (ER in HEK293 cells. Cell surface expression of GluA1, which is a major subunit of AMPAR in neurons, was also suppressed by co-expression of the GluA2 N370S mutant. The N370S mutant and wild-type GluA2 were co-immunoprecipitated with GluA1, suggesting that N370S was properly associated with GluA1. Moreover, we found that N413 was the main potential site of the HNK-1 epitope that promoted the interaction of GluA2 with N-cadherin, resulting in enhanced cell surface expression of GluA2. The HNK-1 epitope on N-glycan at the N413 of GluA2 was also involved in the cell surface expression of GluA1. Thus, our data suggested that site-specific N-glycans on GluA2 regulate the intracellular trafficking and cell surface expression of AMPAR.
The effects of acetaldehyde and acrolein on muscle catabolism in C2 myotubes.
Rom, Oren; Kaisari, Sharon; Aizenbud, Dror; Reznick, Abraham Z
2013-12-01
The toxic aldehydes acetaldehyde and acrolein were previously suggested to damage skeletal muscle. Several conditions in which exposure to acetaldehyde and acrolein is increased were associated with muscle wasting and dysfunction. These include alcoholic myopathy, renal failure, oxidative stress, and inflammation. A main exogenous source of both acetaldehyde and acrolein is cigarette smoking, which was previously associated with increased muscle catabolism. Recently, we have shown that exposure of skeletal myotubes to cigarette smoke stimulated muscle catabolism via increased oxidative stress, activation of p38 MAPK, and upregulation of muscle-specific E3 ubiquitin ligases. In this study, we aimed to investigate the effects of acetaldehyde and acrolein on catabolism of skeletal muscle. Skeletal myotubes differentiated from the C2 myoblast cell line were exposed to acetaldehyde or acrolein and their effects on signaling pathways related to muscle catabolism were studied. Exposure of myotubes to acetaldehyde did not promote muscle catabolism. However, exposure to acrolein caused increased generation of free radicals, activation of p38 MAPK, upregulation of the muscle-specific E3 ligases atrogin-1 and MuRF1, degradation of myosin heavy chain, and atrophy of myotubes. Inhibition of p38 MAPK by SB203580 abolished acrolein-induced muscle catabolism. Our findings demonstrate that acrolein but not acetaldehyde activates a signaling cascade resulting in muscle catabolism in skeletal myotubes. Although within the limitations of an in vitro study, these findings indicate that acrolein may promote muscle wasting in conditions of increased exposure to this aldehyde. Copyright © 2013 Elsevier Inc. All rights reserved.
International Nuclear Information System (INIS)
Reid, Brian J.; Papanikolaou, Niki D.; Wilcox, Ronah K.
2005-01-01
The catabolic activity with respect to the systemic herbicide isoproturon was determined in soil samples by 14 C-radiorespirometry. The first experiment assessed levels of intrinsic catabolic activity in soil samples that represented three dissimilar soil series under arable cultivation. Results showed average extents of isoproturon mineralisation (after 240 h assay time) in the three soil series to be low. A second experiment assessed the impact of addition of isoproturon (0.05 μg kg -1 ) into these soils on the levels of catabolic activity following 28 days of incubation. Increased catabolic activity was observed in all three soils. A third experiment assessed levels of intrinsic catabolic activity in soil samples representing a single soil series managed under either conventional agricultural practice (including the use of isoproturon) or organic farming practice (with no use of isoproturon). Results showed higher (and more consistent) levels of isoproturon mineralisation in the soil samples collected from conventional land use. The final experiment assessed the impact of isoproturon addition on the levels of inducible catabolic activity in these soils. The results showed no significant difference in the case of the conventional farm soil samples while the induction of catabolic activity in the organic farm soil samples was significant. - Dissimilar levels of isoproturon catabolic activity in dissimilar soils and soils under dissimilar land use influence inferred risk
Energy Technology Data Exchange (ETDEWEB)
Reid, Brian J. [School of Environmental Sciences, University of East Anglia, Norwich NR4 7TJ (United Kingdom)]. E-mail: b.reid@uea.ac.uk; Papanikolaou, Niki D. [School of Environmental Sciences, University of East Anglia, Norwich NR4 7TJ (United Kingdom); Wilcox, Ronah K. [School of Environmental Sciences, University of East Anglia, Norwich NR4 7TJ (United Kingdom)
2005-02-01
The catabolic activity with respect to the systemic herbicide isoproturon was determined in soil samples by {sup 14}C-radiorespirometry. The first experiment assessed levels of intrinsic catabolic activity in soil samples that represented three dissimilar soil series under arable cultivation. Results showed average extents of isoproturon mineralisation (after 240 h assay time) in the three soil series to be low. A second experiment assessed the impact of addition of isoproturon (0.05 {mu}g kg{sup -1}) into these soils on the levels of catabolic activity following 28 days of incubation. Increased catabolic activity was observed in all three soils. A third experiment assessed levels of intrinsic catabolic activity in soil samples representing a single soil series managed under either conventional agricultural practice (including the use of isoproturon) or organic farming practice (with no use of isoproturon). Results showed higher (and more consistent) levels of isoproturon mineralisation in the soil samples collected from conventional land use. The final experiment assessed the impact of isoproturon addition on the levels of inducible catabolic activity in these soils. The results showed no significant difference in the case of the conventional farm soil samples while the induction of catabolic activity in the organic farm soil samples was significant. - Dissimilar levels of isoproturon catabolic activity in dissimilar soils and soils under dissimilar land use influence inferred risk.
Metabolic control analysis of Aspergillus niger L-arabinose catabolism
DEFF Research Database (Denmark)
de Groot, M.J.L.; Prathumpai, Wai; Visser, J.
2005-01-01
A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography...... aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes...
Galactose-extended glycans of antibodies produced by transgenic plants
Bakker, H.; Bardor, M.; Molthoff, J.W.; Gomord, V.; Elbers, I.; Stevens, L.H.; Jordi, W.; Lommen, A.; Faye, L.; Lerouge, P.; Bosch, D.
2001-01-01
Plant-specific N-glycosylation can represent an important limitation for the use of recombinant glycoproteins of mammalian origin produced by transgenic plants. Comparison of plant and mammalian N-glycan biosynthesis indicates that β1,4-galactosyltransferase is the most important enzyme that is
Bhat, Ganapati; Hothpet, Vishwanath-Reddy; Lin, Ming-Fong; Cheng, Pi-Wan
2017-11-01
There is a pressing need for biomarkers that can distinguish indolent from aggressive prostate cancer to prevent over-treatment of patients with indolent tumor. Golgi targeting of glycosyltransferases was characterized by confocal microscopy after knockdown of GM130, giantin, or both. N-glycans on a trans-Golgi enzyme β4galactosyltransferase-1 isolated by immunoprecipitation from androgen-sensitive and independent prostate cancer cells were determined by matrix-assisted laser desorption-time of flight-mass spectrometry. In situ proximity ligation assay was employed to determine co-localization of (a) α-mannosidase IA, an enzyme required for processing Man 8 GlcNAc 2 down to Man 5 GlcNAc 2 to enable synthesis of complex-type N-glycans, with giantin, GM130, and GRASP65, and (b) trans-Golgi glycosyltransferases with high mannose N-glycans terminated with α3-mannose. Defective giantin in androgen-independent prostate cancer cells results in a shift of Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65. Consequently, trans-Golgi enzymes and cell surface glycoproteins acquire high mannose N-glycans, which are absent in cells with functional giantin. In situ proximity ligation assays of co-localization of α-mannosidase IA with GM130 and GRASP65, and trans-Golgi glycosyltransferases with high mannose N-glycans are negative in androgen-sensitive LNCaP C-33 cells but positive in androgen-independent LNCaP C-81 and DU145 cells, and LNCaP C-33 cells devoid of giantin. In situ proximity ligation assays of Golgi localization of α-mannosidase IA at giantin versus GM130-GRASP65 site, and absence or presence of N-glycans terminated with α3-mannose on trans-Golgi glycosyltransferases may be useful for distinguishing indolent from aggressive prostate cancer cells. Copyright © 2017 Elsevier B.V. All rights reserved.
Chai, Wengang; Zhang, Yibing; Mauri, Laura; Ciampa, Maria G.; Mulloy, Barbara; Sonnino, Sandro; Feizi, Ten
2018-05-01
Gangliosides, as plasma membrane-associated sialylated glycolipids, are antigenic structures and they serve as ligands for adhesion proteins of pathogens, for toxins of bacteria, and for endogenous proteins of the host. The detectability by carbohydrate-binding proteins of glycan antigens and ligands on glycolipids can be influenced by the differing lipid moieties. To investigate glycan sequences of gangliosides as recognition structures, we have underway a program of work to develop a "gangliome" microarray consisting of isolated natural gangliosides and neoglycolipids (NGLs) derived from glycans released from them, and each linked to the same lipid molecule for arraying and comparative microarray binding analyses. Here, in the first phase of our studies, we describe a strategy for high-sensitivity assignment of the tetrasaccharide backbones and application to identification of eight of monosialylated glycans released from bovine brain gangliosides. This approach is based on negative-ion electrospray mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) of the desialylated glycans. Using this strategy, we have the data on backbone regions of four minor components among the monosialo-ganglioside-derived glycans; these are of the ganglio-, lacto-, and neolacto-series.
Zhang, Peilan; Yang, Guang; Xia, Changqing; Polston, Jane E.; Li, Gengnan; Li, Shiwu; Lin, Zhao; Yang, Li-jun; Bruner, Steven D.
2017-01-01
Glycans possess significant chemical diversity; glycan binding proteins (GBPs) recognize specific glycans to translate their structures to functions in various physiological and pathological processes. Therefore, the discovery and characterization of novel GBPs and characterization of glycan–GBP interactions are significant to provide potential targets for therapeutic intervention of many diseases. Here, we report the biochemical, functional, and structural characterization of a 130-amino-acid protein, Y3, from the mushroom Coprinus comatus. Biochemical studies of recombinant Y3 from a yeast expression system demonstrated the protein is a unique GBP. Additionally, we show that Y3 exhibits selective and potent cytotoxicity toward human T-cell leukemia Jurkat cells compared with a panel of cancer cell lines via inducing caspase-dependent apoptosis. Screening of a glycan array demonstrated GalNAcβ1–4(Fucα1–3)GlcNAc (LDNF) as a specific Y3-binding ligand. To provide a structural basis for function, the crystal structure was solved to a resolution of 1.2 Å, revealing a single-domain αβα-sandwich motif. Two monomers were dimerized to form a large 10-stranded, antiparallel β-sheet flanked by α-helices on each side, representing a unique oligomerization mode among GBPs. A large glycan binding pocket extends into the dimeric interface, and docking of LDNF identified key residues for glycan interactions. Disruption of residues predicted to be involved in LDNF/Y3 interactions resulted in the significant loss of binding to Jurkat T-cells and severely impaired their cytotoxicity. Collectively, these results demonstrate Y3 to be a GBP with selective cytotoxicity toward human T-cell leukemia cells and indicate its potential use in cancer diagnosis and treatment. PMID:28784797
Extrinsic functions of lectin domains in O-N-acetylgalactosamine glycan biosynthesis
DEFF Research Database (Denmark)
Lorenz, Virginia; Ditamo, Yanina; Cejas, Romina B
2016-01-01
during O-GalNAc glycan biosynthesis. The presence of lectin domain T3lec or T4lec during ppGalNAc-T2 and ppGalNAc-T3 catalytic reaction had a clear inhibitory effect on GalNAc-T activity. Interaction of T3lec or T4lec with ppGalNAc-T2 catalytic domain was not mediated by carbohydrate. T3lec, but not T2......Glycan biosynthesis occurs mainly in Golgi. Molecular organization and functional regulation of this process are not well understood. We evaluated the extrinsic effect of lectin domains (β-trefoil fold) of polypeptide GalNAc-transferases (ppGalNAc-Ts) on catalytic activity of glycosyltransferases...
Choi, Ji Yu; Park, Matthew; Cho, Hyeoncheol; Kim, Mi-Hee; Kang, Kyungtae; Choi, Insung S
2017-12-20
Glycans are intimately involved in several facets of neuronal development and neuropathology. However, the metabolic labeling of surface glycans in primary neurons is a difficult task because of the neurotoxicity of unnatural monosaccharides that are used as a metabolic precursor, hindering the progress of metabolic engineering in neuron-related fields. Therefore, in this paper, we report a neurosupportive, neuron-astrocyte coculture system that neutralizes the neurotoxic effects of unnatural monosaccharides, allowing for the long-term observation and characterization of glycans in primary neurons in vitro. Polysialic acids in neurons are selectively imaged, via the metabolic labeling of sialoglycans with peracetylated N-azidoacetyl-d-mannosamine (Ac 4 ManNAz), for up to 21 DIV. Two-color labeling shows that neuronal activities, such as neurite outgrowth and recycling of membrane components, are highly dynamic and change over time during development. In addition, the insertion sites of membrane components are suggested to not be random, but be predominantly localized in developing neurites. This work provides a new research platform and also suggests advanced 3D systems for metabolic-labeling studies of glycans in primary neurons.
Reid, Brian J; Papanikolaou, Niki D; Wilcox, Ronah K
2005-02-01
The catabolic activity with respect to the systemic herbicide isoproturon was determined in soil samples by (14)C-radiorespirometry. The first experiment assessed levels of intrinsic catabolic activity in soil samples that represented three dissimilar soil series under arable cultivation. Results showed average extents of isoproturon mineralisation (after 240 h assay time) in the three soil series to be low. A second experiment assessed the impact of addition of isoproturon (0.05 microg kg(-1)) into these soils on the levels of catabolic activity following 28 days of incubation. Increased catabolic activity was observed in all three soils. A third experiment assessed levels of intrinsic catabolic activity in soil samples representing a single soil series managed under either conventional agricultural practice (including the use of isoproturon) or organic farming practice (with no use of isoproturon). Results showed higher (and more consistent) levels of isoproturon mineralisation in the soil samples collected from conventional land use. The final experiment assessed the impact of isoproturon addition on the levels of inducible catabolic activity in these soils. The results showed no significant difference in the case of the conventional farm soil samples while the induction of catabolic activity in the organic farm soil samples was significant.
DEFF Research Database (Denmark)
Brown, Margaret E.; Mukhopadhyay, Aindrila; Keasling, Jay D.
2016-01-01
conversion with a key reaction performed by the acetone carboxylase complex (ACX). We engineered the heterologous expression of the ACX complex from Xanthobacter autotrophicus PY2 to match the naturally occurring subunit stoichiometry and purified the recombinant complex from E. coli for biochemical analysis....... Incorporating this ACX complex and enzymes from diverse organisms, we introduced an isopropanol degradation pathway in E. coli, optimized induction conditions, and decoupled enzyme expression to probe pathway bottlenecks. Our engineered E. coli consumed 65% of isopropanol compared to no-cell controls......We report an engineered strain of Escherichia coli that catabolizes the carbonaceous component of the extremely toxic chemical warfare agent sarin. Enzymatic decomposition of sarin generates isopropanol waste that, with this engineered strain, is then transformed into acetyl-CoA by enzymatic...
Xu, Hui; Chakrabarty, Yindrila; Philmus, Benjamin; Mehta, Angad P.; Bhandari, Dhananjay; Hohmann, Hans-Peter; Begley, Tadhg P.
2016-01-01
Riboflavin is a common cofactor, and its biosynthetic pathway is well characterized. However, its catabolic pathway, despite intriguing hints in a few distinct organisms, has never been established. This article describes the isolation of a Microbacterium maritypicum riboflavin catabolic strain, and the cloning of the riboflavin catabolic genes. RcaA, RcaB, RcaD, and RcaE were overexpressed and biochemically characterized as riboflavin kinase, riboflavin reductase, ribokinase, and riboflavin ...
van Die, I.; Gomord, V.; Kooyman, F. N.; van den Berg, T. K.; Cummings, R. D.; Vervelde, L.
1999-01-01
Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex-type N-glycans from Arabidopsis thaliana, which contain
Catabolic Processes in Cardiosurgical Patients
Directory of Open Access Journals (Sweden)
V. V. Lomivorotov
2007-01-01
Full Text Available Objective: to evaluate catabolic and anabolic processes in cardiosurgical patients during heart operations under extracorporeal circulation.Subjects and methods. Seventy-one patients with coronary heart disease (CHD and acquired cardiac defects (ACD, who had been operated on under extracorporeal circulation, were examined. The plasma levels of cortisol, adrenaline, insulin, growth hormone, and albumin were measured. For determination of daily nitrogen excretion, blood and diurnal urine were sampled at the following stages: 1 before surgery; 2 postoperative (PO day 1; 3 PO day 3; 4 PO day 7; 5 PO day 14; 6 PO day 21.Results. The preoperative daily nitrogen excretion in CHD patients was 10.4±1.0 g/day. By PO day 3, there was a significant increase in nitrogen excretion by 66%, up to 17.3±1.6 g/day (p<0.01. In ACD patients, the baseline daily urinary nitrogen excretion was 11.9±1.7 g/day. By PO day 3, there was a 1.4-fold increase in this index — up to 16.3±2.0 g/day. Daily nitrogen excretion significantly increased up to 17.1±1.2 g/day by the end of the first PO week (p<0.05, by exceeding the baseline values by 44%. Nitrogen excretion peaked by the end of PO days 14 (17.2±1.6 g/day (p<0.05. By hospital discharge, nitrogen excretion was 23% greater than its baseline preoperative level (p>0.05. In cardiosurgical patients, an increase in daily nitrogen excretion occurred with the elevated concentrations of the stress hormones cortisol and adrenaline.Conclusion. The magnitude of catabolic reactions after cardiosurgical interventions depends on the type of cardiac disease. In patients with CHD, the maximum catabolic reactions were recorded on PO day 3 whereas in those with ACD, they continued within three weeks postoperatively.
Evidence for an imidazoline by-product from glycans using tandem mass spectrometry.
Duff, Robert J; Smith, Elaine; Li, Wenzhou; Fodor, Szilan
2017-06-09
Herein is reported the separation and identification of a previously unknown imidazoline by-product originating from the fluorescent labeling procedure when applied to enzymatically released N-linked glycans of a human IgG1. The imidazoline by-product was generated via the reductive amination procedure with either sodium cyanoborohydride or 2-picoline borane. Using ultra performance liquid chromatography (UPLC) in conjunction with hydrophilic interaction-based chromatography (HILIC), the 2-aminobenzoic acid (2-AA)-labeled glycans were well-resolved from imidazoline by-products to facilitate direct identification utilizing electrospray ionization mass spectrometry (ESI-MS) with fragmentation. It was found that this minor species (∼2%) was 18.0105u less than the neighboring peak GlcNAc 2 Man 3 GlcNAc 2 Fuc peak, abbreviated as A2G0F at 1582.5899u. While this mass loss corresponds to the mass of a water molecule, the molecular location of loss of water was not straightforward in consideration of the biantennary A2G0F structure. Model studies were carried out using A2G0F standard and N-acetyllactosamine to identify the impurity as an imidazoline ring structure located at the reducing end of the glycan as confirmed by high resolution mass fragment ions. Imidazoline content decreased when the reductant concentration was increased. To conclude, evidence for the imidazoline structure was accomplished through high resolution, high accuracy mass spectrometry (HRAM), and experiments showing chemical susceptibility and isotopically labeled tracers. This study is the first to identify these minor species which likely impact all N-acetylglucosamine-type N-linked glycans from biologics. Copyright © 2017 Elsevier B.V. All rights reserved.
International Nuclear Information System (INIS)
Leger, D.; Tordera, V.; Spik, G.; Dorland, L.; Haverkamp, J.; Vliegenthart, J.F.G.
1978-01-01
The glycopeptide fraction of rabbit serotransferrin (STF) has been investigated applying an original method for the determination of glycan primary structure combining monosaccharide determination, permethylation and 360 MHz 1 H NMR. It is concluded that the highly purified rabbit transferrin contains only 1 glycan chain/molecule. A heterogeneity of the glycan moiety in the sialic acid residues was observed on isolation by paper electrophoresis of a disialylglycopeptide G-1 and a monosialylglycopeptide 2. The primary structure of glycopeptide G-1 deduced on the basis of the data of carbohydrate composition, permethylation analysis and 360 MHz 1 H NMR spectroscopy is identical to the primary structure of human serotransferrin glycan and the glycopeptide G-2 was shown by 1 H NMR spectroscopy, to be a mixture of two isomeric monosialylglycopeptides. (Auth.)
Introduction of tri-antennary N-glycans in Arabidopsis thaliana plants.
Nagels, Bieke; Van Damme, Els J M; Callewaert, Nico; Weterings, Koen
2012-04-01
Because the pathway for protein synthesis is largely conserved between plants and animals, plants provide an attractive platform for the cost effective and flexible production of biopharmaceuticals. However, there are some differences in glycosylation between plants and humans that need to be considered before plants can be used as an efficient expression platform. In the presented research the human genes encoding α1,3-mannosyl-β1,4-N-acetylglucosaminyltransferase (GnT-IV) and α1,6-mannosyl-β1,6-N-acetylglucosaminyltransferase (GnT-V) were introduced in the fast cycling model plant Arabidopsis thaliana to synthesize tri-antennary N-glycans. The GnT-IV and -V enzymes were targeted to the Golgi apparatus with plant-specific localization signals. The experiments were performed both in a wild type background, as well as in plants lacking β1,2-xylosyltransferase (XylT) and α1,3-fucosyltransferase (FucT) activity. Glycan analysis of endogenous proteins in the transgenic lines using CE-LIF showed that tri-antennary N-glycans could be produced in the XylT/FucT deficient line, while these structures were not found in the wild type background. Since β-N-acetylhexosaminidases, that remove terminal GlcNAcs, are active in A. thaliana plants, the specificity of these enzymes for different GlcNAc linkages was tested. The results showed that there is no pronounced preference of the A. thaliana hexosaminidases for human-type GlcNAc-linkages. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.
Maslen, Sarah; Sadowski, Pawel; Adam, Alex; Lilley, Kathryn; Stephens, Elaine
2006-12-15
The detailed characterization of protein N-glycosylation is very demanding given the many different glycoforms and structural isomers that can exist on glycoproteins. Here we report a fast and sensitive method for the extensive structure elucidation of reducing-end labeled N-glycan mixtures using a combination of capillary normal-phase HPLC coupled off-line to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and TOF/TOF-MS/MS. Using this method, isobaric N-glycans released from honey bee phospholipase A2 and Arabidopsis thaliana glycoproteins were separated by normal-phase chromatography and subsequently identified by key fragment ions in the MALDI-TOF/TOF tandem mass spectra. In addition, linkage and branching information were provided by abundant cross-ring and "elimination" fragment ions in the MALDI-CID spectra that gave extensive structural information. Furthermore, the fragmentation characteristics of N-glycans reductively aminated with 2-aminobenzoic acid and 2-aminobenzamide were compared. The identification of N-glycans containing 3-linked core fucose was facilitated by distinctive ions present only in the MALDI-CID spectra of 2-aminobenzoic acid-labeled oligosaccharides. To our knowledge, this is the first MS/MS-based technique that allows confident identification of N-glycans containing 3-linked core fucose, which is a major allergenic determinant on insect and plant glycoproteins.
Contribution of N-linked glycans on HSV-2 gB to cell–cell fusion and viral entry
Energy Technology Data Exchange (ETDEWEB)
Luo, Sukun [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Hu, Kai [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); He, Siyi; Wang, Ping; Zhang, Mudan; Huang, Xin [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Du, Tao [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Zheng, Chunfu [Soochow University, Institutes of Biology and Medical Sciences, Suzhou 215123 (China); Liu, Yalan [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Hu, Qinxue, E-mail: qhu@wh.iov.cn [State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071 (China); Institute for Infection and Immunity, St George' s University of London, London SW17 0RE (United Kingdom)
2015-09-15
HSV-2 is the major cause of genital herpes and its infection increases the risk of HIV-1 acquisition and transmission. HSV-2 glycoprotein B together with glycoproteins D, H and L are indispensable for viral entry, of which gB, as a class III fusogen, plays an essential role. HSV-2 gB has seven potential N-linked glycosylation (N-CHO) sites, but their significance has yet to be determined. For the first time, we systematically analyzed the contributions of N-linked glycans on gB to cell–cell fusion and viral entry. Our results demonstrated that, of the seven potential N-CHO sites on gB, mutation at N390, N483 or N668 decreased cell–cell fusion and viral entry, while mutation at N133 mainly affected protein expression and the production of infectious virus particles by blocking the transport of gB from the endoplasmic reticulum to Golgi. Our findings highlight the significance of N-linked glycans on HSV-2 gB expression and function. - Highlights: • N-linked glycan at N133 is important for gB intracellular trafficking and maturation. • N-linked glycans at N390, N483 and N668 on gB are necessary for optimal cell–cell fusion. • N-linked glycans at N390, N483 and N668 on gB are necessary for optimal viral entry.
Contribution of N-linked glycans on HSV-2 gB to cell–cell fusion and viral entry
International Nuclear Information System (INIS)
Luo, Sukun; Hu, Kai; He, Siyi; Wang, Ping; Zhang, Mudan; Huang, Xin; Du, Tao; Zheng, Chunfu; Liu, Yalan; Hu, Qinxue
2015-01-01
HSV-2 is the major cause of genital herpes and its infection increases the risk of HIV-1 acquisition and transmission. HSV-2 glycoprotein B together with glycoproteins D, H and L are indispensable for viral entry, of which gB, as a class III fusogen, plays an essential role. HSV-2 gB has seven potential N-linked glycosylation (N-CHO) sites, but their significance has yet to be determined. For the first time, we systematically analyzed the contributions of N-linked glycans on gB to cell–cell fusion and viral entry. Our results demonstrated that, of the seven potential N-CHO sites on gB, mutation at N390, N483 or N668 decreased cell–cell fusion and viral entry, while mutation at N133 mainly affected protein expression and the production of infectious virus particles by blocking the transport of gB from the endoplasmic reticulum to Golgi. Our findings highlight the significance of N-linked glycans on HSV-2 gB expression and function. - Highlights: • N-linked glycan at N133 is important for gB intracellular trafficking and maturation. • N-linked glycans at N390, N483 and N668 on gB are necessary for optimal cell–cell fusion. • N-linked glycans at N390, N483 and N668 on gB are necessary for optimal viral entry
The anti-catabolic role of bovine lactoferricin in cartilage.
Ahmadinia, Kasra; Yan, Dongyao; Ellman, Michael; Im, Hee-Jeong
2013-10-01
Bovine lactoferricin (LfcinB) is a multifunctional peptide derived from bovine lactoferrin that demonstrates antibacterial, antifungal, antiviral, antitumor, and immunomodulatory activities. Recently, studies have focused on the anti-catabolic and anti-inflammatory potential of LfcinB. LfcinB is able to modulate the effects cytokines such as IL-1 and fibroblast growth factor 2 as well as promote specific cartilage anabolic factors. These properties are particularly important in maintaining cartilage homeostasis and preventing a catabolic state, which leads to clinical pathology. This review focuses on the recent literature elucidating the role of LfcinB in preventing cartilage degradation.
Sjöwall, C; Zapf, J; von Löhneysen, S; Magorivska, I; Biermann, M; Janko, C; Winkler, S; Bilyy, R; Schett, G; Herrmann, M; Muñoz, L E
2015-05-01
In addition to the redundancy of the receptors for the Fc portion of immunoglobulins, glycans result in potential ligands for a plethora of lectin receptors found in immune effector cells. Here we analysed the exposure of glycans containing fucosyl residues and the fucosylated tri-mannose N-type core by complexed native IgG in longitudinal serum samples of well-characterized patients with systemic lupus erythematosus. Consecutive serum samples of a cohort of 15 patients with systemic lupus erythematosus during periods of increased disease activity and remission were analysed. All patients fulfilled the 1982 American College of Rheumatology classification criteria. Sera of 15 sex- and age-matched normal healthy blood donors served as controls. The levels and type of glycosylation of complexed random IgG was measured with lectin enzyme-immunosorbent assays. After specifically gathering IgG complexes from sera, biotinylated lectins Aleuria aurantia lectin and Lens culinaris agglutinin were employed to detect IgG-associated fucosyl residues and the fucosylated tri-mannose N-glycan core, respectively. In sandwich-ELISAs, IgG-associated IgM, IgA, C1q, C3c and C-reactive protein (CRP) were detected as candidates for IgG immune complex constituents. We studied associations of the glycan of complexed IgG and disease activity according to the physician's global assessment of disease activity and the systemic lupus erythematosus disease activity index 2000 documented at the moment of blood taking. Our results showed significantly higher levels of Aleuria aurantia lectin and Lens culinaris agglutinin binding sites exposed on IgG complexes of patients with systemic lupus erythematosus than on those of normal healthy blood donors. Disease activity in systemic lupus erythematosus correlated with higher exposure of Aleuria aurantia lectin-reactive fucosyl residues by immobilized IgG complexes. Top levels of Aleuria aurantia lectin-reactivity were found in samples taken during the
Viral hemagglutinin-esterases: Mediators of dynamic virion-glycan interactions
Langereis, M.A.|info:eu-repo/dai/nl/304823597
2011-01-01
The sialic acids (Sias), a diverse family of 9-carbon sugars, are among the most important molecules of life. Commonly occurring as terminal residues of glycans on proteins and lipids, they are key elements of glycotopes of cellular lectins and there is accumulating evidence for them to act as
Park, Youmie; Zhang, Zhenqing; Laremore, Tatiana N; Li, Boyangzi; Sim, Joon-Soo; Im, A-Rang; Ahn, Mi Young; Kim, Yeong Shik; Linhardt, Robert J
2008-12-01
Acharan sulfate content from African giant snail (Achatina fulica) was compared in eggs and snails of different ages. Acharan sulfate was not found in egg. Acharan sulfate disaccharide -->4)-alpha-D-GlcNpAc (1-->4)-alpha-L-IdoAp2S(1-->, analyzed by SAX (strong-anion exchange)-HPLC was observed soon after hatching and increases as the snails grow. Monosaccharide compositional analysis showed that mole % of glucosamine, a major monosaccharide of acharan sulfate, increased with age while mole % of galactose decreased with age. These results suggest that galactans represent a major energy source during development, while acharan sulfate appearing immediately after hatching, is essential for the snail growth. The structures of neutral N-glycans released from eggs by peptide N-glycosidase F (PNGase F), were next elucidated using ESI-MS/MS, MALDI-MS/MS, enzyme digestion, and monosaccharide composition analysis. Three types of neutral N-glycan structures were observed, truncated (Hex(2-4)-HexNAc(2)), high mannose (Hex(5-9)-HexNAc(2)), and complex (Hex(3)-HexNAc(2-10)) types. None showed core fucosylation.
Giannone, Chiara; Fagioli, Claudio; Valetti, Caterina; Sitia, Roberto; Anelli, Tiziana
2017-02-03
The polymeric structure of secretory IgM allows efficient antigen binding and complement fixation. The available structural models place the N-glycans bound to asparagines 402 and 563 of Ig-μ chains within a densely packed core of native IgM. These glycans are found in the high mannose state also in secreted IgM, suggesting that polymerization hinders them to Golgi processing enzymes. Their absence alters polymerization. Here we investigate their role following the fate of aggregation-prone mutant μ chains lacking the Cμ1 domain (μ∆). Our data reveal that μ∆ lacking 563 glycans (μ∆5) form larger intracellular aggregates than μ∆ and are not secreted. Like μ∆, they sequester ERGIC-53, a lectin previously shown to promote polymerization. In contrast, μ∆ lacking 402 glycans (μ∆4) remain detergent soluble and accumulate in the ER, as does a double mutant devoid of both (μ∆4-5). These results suggest that the two C-terminal Ig-μ glycans shape the polymerization-dependent aggregation by engaging lectins and acting as spacers in the alignment of individual IgM subunits in native polymers.
Poly (ADP-ribose) catabolism in mammalian cells exposed to DNA-damaging agents
International Nuclear Information System (INIS)
Alvarez-Gonzalez, R.; Althaus, F.R.
1989-01-01
DNA damage inflicted by the alkylating agens N-methyl-N-nitro-N-nitrosoquanidine, or by UV stimulated the catabolism of protein-bound poly (ADP-ribose) in the chromatin of cultured hepatocytes. The stimulation was highest at the largest doses of DNA-damaging treatment. As a consequence, the half-life of ADP-ribosyl polymers may drop to less than 41 s. This rapid turnover contrasts with the slow catabolism of a constitutive fraction of polymers exhibiting a half-life of 7.7 h. These data suggest that post-incisional stimulation of poly (ADP-ribose) biosynthesis in DNA-excision repair is coupled with an adaptation of poly (ADP-ribose) catabolism in mammalian cells. (Author). 37 refs.; 3 figs
Yan, Xin; Gu, Tao; Yi, Zhongquan; Huang, Junwei; Liu, Xiaowei; Zhang, Ji; Xu, Xihui; Xin, Zhihong; Hong, Qing; He, Jian; Spain, Jim C; Li, Shunpeng; Jiang, Jiandong
2016-12-01
The worldwide use of the phenylurea herbicide, isoproturon (IPU), has resulted in considerable concern about its environmental fate. Although many microbial metabolites of IPU are known and IPU-mineralizing bacteria have been isolated, the molecular mechanism of IPU catabolism has not been elucidated yet. In this study, complete genes that encode the conserved IPU catabolic pathway were revealed, based on comparative analysis of the genomes of three IPU-mineralizing sphingomonads and subsequent experimental validation. The complete genes included a novel hydrolase gene ddhA, which is responsible for the cleavage of the urea side chain of the IPU demethylated products; a distinct aniline dioxygenase gene cluster adoQTA1A2BR, which has a broad substrate range; and an inducible catechol meta-cleavage pathway gene cluster adoXEGKLIJC. Furthermore, the initial mono-N-demethylation genes pdmAB were further confirmed to be involved in the successive N-demethylation of the IPU mono-N-demethylated product. These IPU-catabolic genes were organized into four transcription units and distributed on three plasmids. They were flanked by multiple mobile genetic elements and highly conserved among IPU-mineralizing sphingomonads. The elucidation of the molecular mechanism of IPU catabolism will enhance our understanding of the microbial mineralization of IPU and provide insights into the evolutionary scenario of the conserved IPU-catabolic pathway. © 2016 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.
Energy Technology Data Exchange (ETDEWEB)
Leger, D; Tordera, V; Spik, G [Lille-1 Univ., 59 - Villeneuve-d' Ascq (France); Dorland, L; Haverkamp, J; Vliegenthart, J F.G. [Rijksuniversiteit Utrecht (Netherlands)
1978-09-15
The glycopeptide fraction of rabbit serotransferrin (STF) has been investigated applying an original method for the determination of glycan primary structure combining monosaccharide determination, permethylation and 360 MHz /sup 1/H NMR. It is concluded that the highly purified rabbit transferrin contains only 1 glycan chain/molecule. A heterogeneity of the glycan moiety in the sialic acid residues was observed on isolation by paper electrophoresis of a disialylglycopeptide G-1 and a monosialylglycopeptide 2. The primary structure of glycopeptide G-1 deduced on the basis of the data of carbohydrate composition, permethylation analysis and 360 MHz /sup 1/H NMR spectroscopy is identical to the primary structure of human serotransferrin glycan and the glycopeptide G-2 was shown by /sup 1/H NMR spectroscopy, to be a mixture of two isomeric monosialylglycopeptides.
Tang, Yang; Wei, Juan; Costello, Catherine E.; Lin, Cheng
2018-04-01
The occurrence of numerous structural isomers in glycans from biological sources presents a severe challenge for structural glycomics. The subtle differences among isomeric structures demand analytical methods that can provide structural details while working efficiently with on-line glycan separation methods. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool for mixture analysis, the commonly utilized collision-induced dissociation (CID) method often does not generate a sufficient number of fragments at the MS2 level for comprehensive structural characterization. Here, we studied the electronic excitation dissociation (EED) behaviors of metal-adducted, permethylated glycans, and identified key spectral features that could facilitate both topology and linkage determinations. We developed an EED-based, nanoscale, reversed phase (RP)LC-MS/MS platform, and demonstrated its ability to achieve complete structural elucidation of up to five structural isomers in a single LC-MS/MS analysis. [Figure not available: see fulltext.
Status Report on the High-Throughput Characterization of Complex Intact O-Glycopeptide Mixtures
Pap, Adam; Klement, Eva; Hunyadi-Gulyas, Eva; Darula, Zsuzsanna; Medzihradszky, Katalin F.
2018-05-01
A very complex mixture of intact, human N- and O-glycopeptides, enriched from the tryptic digest of urinary proteins of three healthy donors using a two-step lectin affinity enrichment, was analyzed by LC-MS/MS, leading to approximately 45,000 glycopeptide EThcD spectra. Two search engines, Byonic and Protein Prospector, were used for the interpretation of the data, and N- and O-linked glycopeptides were assigned from separate searches. The identification rate was very low in all searches, even when results were combined. Thus, we investigated the reasons why was it so, to help to improve the identification success rate. Focusing on O-linked glycopeptides, we noticed that in EThcD, larger glycan oxonium ions better survive the activation than those in HCD. These fragments, combined with reducing terminal Y ions, provide important information about the glycan(s) present, so we investigated whether filtering the peaklists for glycan oxonium ions indicating the presence of a tetra- or hexasaccharide structure would help to reveal all molecules containing such glycans. Our study showed that intact glycans frequently do not survive even mild supplemental activation, meaning one cannot rely on these oxonium ions exclusively. We found that ETD efficiency is still a limiting factor, and for highly glycosylated peptides, the only information revealed in EThcD was related to the glycan structures. The limited overlap of results delivered by the two search engines draws attention to the fact that automated data interpretation of O-linked glycopeptides is not even close to being solved. [Figure not available: see fulltext.
Detection and Isolation of Novel Rhizopine-Catabolizing Bacteria from the Environment
Gardener, Brian B. McSpadden; de Bruijn, Frans J.
1998-01-01
Microbial rhizopine-catabolizing (Moc) activity was detected in serial dilutions of soil and rhizosphere washes. The activity observed generally ranged between 106 and 107 catabolic units per g, and the numbers of nonspecific culture-forming units were found to be approximately 10 times higher. A diverse set of 37 isolates was obtained by enrichment on scyllo-inosamine-containing media. However, none of the bacteria that were isolated were found to contain DNA sequences homologous to the know...
Directory of Open Access Journals (Sweden)
Anne-Sophie eLeprince
2015-01-01
Full Text Available Plant adaptation to abiotic stresses such as drought and salinity involves complex regulatory processes. Deciphering the signalling components that are involved in stress signal transduction and cellular responses is of importance to understand how plants cope with salt stress. Accumulation of osmolytes such as proline is considered to participate in the osmotic adjustment of plant cells to salinity. Proline accumulation results from a tight regulation between its biosynthesis and catabolism. Lipid signal components such as phospholipases C and D have previously been shown to be involved in the regulation of proline metabolism in Arabidopsis thaliana. In this study, we demonstrate that proline metabolism is also regulated by class-III Phosphatidylinositol 3-kinase (PI3K, VPS34, which catalyses the formation of phosphatidylinositol 3-phosphate (PI3P from phosphatidylinositol. Using pharmacological and biochemical approaches, we show that the PI3K inhibitor, LY294002, affects PI3P levels in vivo and that it triggers a decrease in proline accumulation in response to salt treatment of A. thaliana seedlings. The lower proline accumulation is correlated with a lower transcript level of Pyrroline-5-carboxylate synthetase 1 biosynthetic enzyme and higher transcript and protein levels of Proline dehydrogenase 1 (ProDH1, a key-enzyme in proline catabolism. We also found that the ProDH1 expression is induced in a pi3k-hemizygous mutant, further demonstrating that PI3K is involved in the regulation of proline catabolism through transcriptional regulation of ProDH1. A broader metabolomic analysis indicates that LY294002 also reduced other metabolites, such as hydrophobic and aromatic amino acids and sugars like raffinose.
A multi-method approach toward de novo glycan characterization: a Man-5 case study.
Prien, Justin M; Prater, Bradley D; Cockrill, Steven L
2010-05-01
Regulatory agencies' expectations for biotherapeutic approval are becoming more stringent with regard to product characterization, where minor species as low as 0.1% of a given profile are typically identified. The mission of this manuscript is to demonstrate a multi-method approach toward de novo glycan characterization and quantitation, including minor species at or approaching the 0.1% benchmark. Recently, unexpected isomers of the Man(5)GlcNAc(2) (M(5)) were reported (Prien JM, Ashline DJ, Lapadula AJ, Zhang H, Reinhold VN. 2009. The high mannose glycans from bovine ribonuclease B isomer characterization by ion trap mass spectrometry (MS). J Am Soc Mass Spectrom. 20:539-556). In the current study, quantitative analysis of these isomers found in commercial M(5) standard demonstrated that they are in low abundance (2-aminobenzoic acid to detect and chromatographically resolve multiple M(5) isomers in bovine ribonuclease B. With this multi-method approach, we have the capabilities to comprehensively characterize a biotherapeutic's glycan array in a de novo manner, including structural isomers at >/=0.1% of the total chromatographic peak area.
Liu, Yang; Xu, Shenyuan; Woodruff, Andrew L; Xia, Ming; Tan, Ming; Kennedy, Michael A; Jiang, Xi
2017-11-01
Recognition of specific cell surface glycans, mediated by the VP8* domain of the spike protein VP4, is the essential first step in rotavirus (RV) infection. Due to lack of direct structural information of virus-ligand interactions, the molecular basis of ligand-controlled host ranges of the major human RVs (P[8] and P[4]) in P[II] genogroup remains unknown. Here, through characterization of a minor P[II] RV (P[19]) that can infect both animals (pigs) and humans, we made an important advance to fill this knowledge gap by solving the crystal structures of the P[19] VP8* in complex with its ligands. Our data showed that P[19] RVs use a novel binding site that differs from the known ones of other genotypes/genogroups. This binding site is capable of interacting with two types of glycans, the mucin core and type 1 histo-blood group antigens (HBGAs) with a common GlcNAc as the central binding saccharide. The binding site is apparently shared by other P[II] RVs and possibly two genotypes (P[10] and P[12]) in P[I] as shown by their highly conserved GlcNAc-interacting residues. These data provide strong evidence of evolutionary connections among these human and animal RVs, pointing to a common ancestor in P[I] with a possible animal host origin. While the binding properties to GlcNAc-containing saccharides are maintained, changes in binding to additional residues, such as those in the polymorphic type 1 HBGAs may occur in the course of RV evolution, explaining the complex P[II] genogroup that mainly causes diseases in humans but also in some animals.
International Nuclear Information System (INIS)
Oliveira, Joao Ezequiel de
2007-01-01
A novel, fast and practical two-step purification strategy, consisting of a classical ion exchange and a reversed-phase high performance liquid chromatography (RP-HPLC), for rapidly obtaining CHO-derived hTSH, was set up providing r-hTSH with 70% yield and > 99% purity. A consistent increase of ∼ 60% in the secretion yields of r-hTSH-IPEN was observed by changing cell culture CO 2 conditions from 5% CO 2 to air environment (0.03% CO 2 ). The overall quality of the products obtained under both conditions was evaluated for what concerns N-glycan structure, charge isomers and biological activity in comparison with a well known recombinant biopharmaceutical (Thyrogen R ) and with a pituitary reference preparation (p-hTSH) from National Hormone and Pituitary Program (NIDDK, USA). The N-glycans identified in the recombinant preparations were of the complex type, presenting bi-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing ∼ 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more bi-antennary structures obtained in the absence of CO 2 and 7-9% more tri-antennary structures in its presence. In the case of p-hTSH, complex, high-mannose and hybrid N-glycan structures were identified, most of them containing sialic acid and/or sulphate terminal residues. The two most abundant structures were shown to contain one or two sulphate residues, one of which unexpectedly bound to galactose. The sialic acid-galactose linkage was also determined, having found that 68 3 ± 10% was in the α 2,6 and 32 ± 10% in the α2,3 conformation. No remarkable difference in charge isomers was observed between the three recombinant preparations, the isoelectric focusing profiles showing six distinct bands in the 5.39 - 7.35 pl range. A considerably different
Determinants of glycan receptor specificity of H2N2 influenza A virus hemagglutinin.
Viswanathan, Karthik; Koh, Xiaoying; Chandrasekaran, Aarthi; Pappas, Claudia; Raman, Rahul; Srinivasan, Aravind; Shriver, Zachary; Tumpey, Terrence M; Sasisekharan, Ram
2010-10-29
The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA). The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004) that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant) comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58) HA.
Determinants of glycan receptor specificity of H2N2 influenza A virus hemagglutinin.
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Karthik Viswanathan
Full Text Available The H2N2 subtype of influenza A virus was responsible for the Asian pandemic of 1957-58. However, unlike other subtypes that have caused pandemics such as H1N1 and H3N2, which continue to circulate among humans, H2N2 stopped circulating in the human population in 1968. Strains of H2 subtype still continue to circulate in birds and occasionally pigs and could be reintroduced into the human population through antigenic drift or shift. Such an event is a potential global health concern because of the waning population immunity to H2 hemagglutinin (HA. The first step in such a cross-species transmission and human adaptation of influenza A virus is the ability for its surface glycoprotein HA to bind to glycan receptors expressed in the human upper respiratory epithelia. Recent structural and biochemical studies have focused on understanding the glycan receptor binding specificity of the 1957-58 pandemic H2N2 HA. However, there has been considerable HA sequence divergence in the recent avian-adapted H2 strains from the pandemic H2N2 strain. Using a combination of structural modeling, quantitative glycan binding and human respiratory tissue binding methods, we systematically identify mutations in the HA from a recent avian-adapted H2N2 strain (A/Chicken/PA/2004 that make its quantitative glycan receptor binding affinity (defined using an apparent binding constant comparable to that of a prototypic pandemic H2N2 (A/Albany/6/58 HA.
A metabolic pathway for catabolizing levulinic acid in bacteria
International Nuclear Information System (INIS)
Rand, Jacqueline M.; Pisithkul, Tippapha; Clark, Ryan L.; Thiede, Joshua M.; Mehrer, Christopher R.
2017-01-01
Microorganisms can catabolize a wide range of organic compounds and therefore have the potential to perform many industrially relevant bioconversions. One barrier to realizing the potential of biorefining strategies lies in our incomplete knowledge of metabolic pathways, including those that can be used to assimilate naturally abundant or easily generated feedstocks. For instance, levulinic acid (LA) is a carbon source that is readily obtainable as a dehydration product of lignocellulosic biomass and can serve as the sole carbon source for some bacteria. Yet, the genetics and structure of LA catabolism have remained unknown. Here, we report the identification and characterization of a seven-gene operon that enables LA catabolism in Pseudomonas putida KT2440. When the pathway was reconstituted with purified proteins, we observed the formation of four acyl-CoA intermediates, including a unique 4-phosphovaleryl-CoA and the previously observed 3-hydroxyvaleryl-CoA product. Using adaptive evolution, we obtained a mutant of Escherichia coli LS5218 with functional deletions of fadE and atoC that was capable of robust growth on LA when it expressed the five enzymes from the P. putida operon. Here, this discovery will enable more efficient use of biomass hydrolysates and metabolic engineering to develop bioconversions using LA as a feedstock.
Perturbation of polyamine catabolism affects grape ripening of Vitis vinifera cv. Trincadeira.
Agudelo-Romero, Patricia; Ali, Kashif; Choi, Young H; Sousa, Lisete; Verpoorte, Rob; Tiburcio, Antonio F; Fortes, Ana M
2014-01-01
Grapes are economically the most important fruit worldwide. However, the complexity of biological events that lead to ripening of nonclimacteric fruits is not fully understood, particularly the role of polyamines' catabolism. The transcriptional and metabolic profilings complemented with biochemical data were studied during ripening of Trincadeira grapes submitted to guazatine treatment, a potent inhibitor of polyamine oxidase activity. The mRNA expression profiles of one time point (EL 38) corresponding to harvest stage was compared between mock and guazatine treatments using Affymetrix GrapeGen(®) genome array. A total of 2113 probesets (1880 unigenes) were differentially expressed between these samples. Quantitative RT-PCR validated microarrays results being carried out for EL 35 (véraison berries), EL 36 (ripe berries) and EL 38 (harvest stage berries). Metabolic profiling using HPLC and (1)H NMR spectroscopy showed increase of putrescine, proline, threonine and 1-O-ethyl-β-glucoside in guazatine treated samples. Genes involved in amino acid, carbohydrate and water transport were down-regulated in guazatine treated samples suggesting that the strong dehydrated phenotype obtained in guazatine treated samples may be due to impaired transport mechanisms. Genes involved in terpenes' metabolism were differentially expressed between guazatine and mock treated samples. Altogether, results support an important role of polyamine catabolism in grape ripening namely in cell expansion and aroma development. Copyright © 2013 Elsevier Masson SAS. All rights reserved.
DEFF Research Database (Denmark)
Seppälä, Ulla; Selby, David; Monsalve, Rafael
2009-01-01
of the study was to characterize the glycosylation patterns in Ves v 2 isoallergens and to assess their immunological properties regarding antibody binding and T cell activation. The glycosylation sites and the carbohydrate structures were verified by use of tandem mass spectrometry (MS/MS). The immunological....... Non-glycosylated rVes v 2, however, induced T cell and cytokine responses comparable to glycosylated nVes v 2. The present study shows that N-glycan structures are needed for the antibody recognition but not for the T cell reactivity of Ves v 2 in vitro. The occurrences of carbohydrate......-specific antibodies against nVes v 2, however, suggest that non-mammalian glycan structures as in nVes v 2 may provide a link between T cells and other effector cells in allergic responses....
Directory of Open Access Journals (Sweden)
Ema T Crooks
2015-05-01
Full Text Available Eliciting broad tier 2 neutralizing antibodies (nAbs is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs expressing trimers (trimer VLP sera and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera. All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs. Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype rendered 50% or 16.7% (n = 18 of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative "glycan fence" that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.
An adenovirus vector incorporating carbohydrate binding domains utilizes glycans for gene transfer.
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Julius W Kim
Full Text Available Vectors based on human adenovirus serotype 5 (HAdV-5 continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting.As a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4. This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells.These data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.
Powers, Thomas W; Neely, Benjamin A; Shao, Yuan; Tang, Huiyuan; Troyer, Dean A; Mehta, Anand S; Haab, Brian B; Drake, Richard R
2014-01-01
A recently developed matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) method to spatially profile the location and distribution of multiple N-linked glycan species in frozen tissues has been extended and improved for the direct analysis of glycans in clinically derived formalin-fixed paraffin-embedded (FFPE) tissues. Formalin-fixed tissues from normal mouse kidney, human pancreatic and prostate cancers, and a human hepatocellular carcinoma tissue microarray were processed by antigen retrieval followed by on-tissue digestion with peptide N-glycosidase F. The released N-glycans were detected by MALDI-IMS analysis, and the structural composition of a subset of glycans could be verified directly by on-tissue collision-induced fragmentation. Other structural assignments were confirmed by off-tissue permethylation analysis combined with multiple database comparisons. Imaging of mouse kidney tissue sections demonstrates specific tissue distributions of major cellular N-linked glycoforms in the cortex and medulla. Differential tissue distribution of N-linked glycoforms was also observed in the other tissue types. The efficacy of using MALDI-IMS glycan profiling to distinguish tumor from non-tumor tissues in a tumor microarray format is also demonstrated. This MALDI-IMS workflow has the potential to be applied to any FFPE tissue block or tissue microarray to enable higher throughput analysis of the global changes in N-glycosylation associated with cancers.
Kiyoshi, Masato; Caaveiro, Jose M M; Tada, Minoru; Tamura, Hiroko; Tanaka, Toru; Terao, Yosuke; Morante, Koldo; Harazono, Akira; Hashii, Noritaka; Shibata, Hiroko; Kuroda, Daisuke; Nagatoishi, Satoru; Oe, Seigo; Ide, Teruhiko; Tsumoto, Kouhei; Ishii-Watabe, Akiko
2018-03-02
The N-glycan moiety of IgG-Fc has a significant impact on multifaceted properties of antibodies such as in their effector function, structure, and stability. Numerous studies have been devoted to understanding its biological effect since the exact composition of the Fc N-glycan modulates the magnitude of effector functions such as the antibody-dependent cell mediated cytotoxicity (ADCC), and the complement-dependent cytotoxicity (CDC). To date, systematic analyses of the properties and influence of glycan variants have been of great interest. Understanding the principles on how N-glycosylation modulates those properties is important for the molecular design, manufacturing, process optimization, and quality control of therapeutic antibodies. In this study, we have separated a model therapeutic antibody into three fractions according to the composition of the N-glycan by using a novel FcγRIIIa chromatography column. Notably, Fc galactosylation was a major factor influencing the affinity of IgG-Fc to the FcγRIIIa immobilized on the column. Each antibody fraction was employed for structural, biological, and physicochemical analysis, illustrating the mechanism by which galactose modulates the affinity to FcγRIIIa. In addition, we discuss the benefits of the FcγRIIIa chromatography column to assess the heterogeneity of the N-glycan.
Directory of Open Access Journals (Sweden)
Florian Rieder
Full Text Available INTRODUCTION: Anti-glycan antibodies are a promising tool for differential diagnosis and disease stratification of patients with Crohn's disease (CD. We longitudinally assessed level and status changes of anti-glycan antibodies over time in individual CD patients as well as determinants of this phenomenon. METHODS: 859 serum samples derived from a cohort of 253 inflammatory bowel disease (IBD patients (207 CD, 46 ulcerative colitis (UC were tested for the presence of anti-laminarin (Anti-L, anti-chitin (Anti-C, anti-chitobioside (ACCA, anti-laminaribioside (ALCA, anti-mannobioside (AMCA and anti-Saccharomyces cerevisiae (gASCA antibodies by ELISA. All patients had at least two and up to eleven serum samples taken during the disease course. RESULTS: Median follow-up time for CD was 17.4 months (Interquartile range (IQR 8.0, 31.6 months and for UC 10.9 months (IQR 4.9, 21.0 months. In a subgroup of CD subjects marked changes in the overall immune response (quartile sum score and levels of individual markers were observed over time. The marker status (positive versus negative remained widely stable. Neither clinical phenotype nor NOD2 genotype was associated with the observed fluctuations. In a longitudinal analysis neither changes in disease activity nor CD behavior led to alterations in the levels of the glycan markers. The ability of the panel to discriminate CD from UC or its association with CD phenotypes remained stable during follow-up. In the serum of UC patients neither significant level nor status changes were observed. CONCLUSIONS: While the levels of anti-glycan antibodies fluctuate in a subgroup of CD patients the antibody status is widely stable over time.
Mern, Demissew S.; Beierfuß, Anja; Fontana, Johann; Thomé, Claudius; Hegewald, Aldemar A.
2014-01-01
Degenerative disc disease (DDD) of the cervical spine is common after middle age and can cause loss of disc height with painful nerve impingement, bone and joint inflammation. Despite the clinical importance of these problems, in current publications the pathology of cervical disc degeneration has been studied merely from a morphologic view point using magnetic resonance imaging (MRI), without addressing the issue of biological treatment approaches. So far a wide range of endogenously expressed bioactive factors in degenerative cervical disc cells has not yet been investigated, despite its importance for gene therapeutic approaches. Although degenerative lumbar disc cells have been targeted by different biological treatment approaches, the quantities of disc cells and the concentrations of gene therapeutic factors used in animal models differ extremely. These indicate lack of experimentally acquired data regarding disc cell proliferation and levels of target proteins. Therefore, we analysed proliferation and endogenous expression levels of anabolic, catabolic, ant-catabolic, inflammatory cytokines and matrix proteins of degenerative cervical disc cells in three-dimensional cultures. Preoperative MRI grading of cervical discs was used, then grade III and IV nucleus pulposus (NP) tissues were isolated from 15 patients, operated due to cervical disc herniation. NP cells were cultured for four weeks with low-glucose in collagen I scaffold. Their proliferation rates were analysed using 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide. Their protein expression levels of 28 therapeutic targets were analysed using enzyme-linked immunosorbent assay. During progressive grades of degeneration NP cell proliferation rates were similar. Significantly decreased aggrecan and collagen II expressions (P<0.0001) were accompanied by accumulations of selective catabolic and inflammatory cytokines (disintegrin and metalloproteinase with thrombospondin motifs 4 and 5, matrix
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Edyta Kopera
2017-04-01
Full Text Available Hemagglutinin glycoprotein (HA is a principle influenza vaccine antigen. Recombinant HA-based vaccines become a potential alternative for traditional approach. Complexity and variation of HA N-glycosylation are considered as the important factors for the vaccine design. The number and location of glycan moieties in the HA molecule are also crucial. Therefore, we decided to study the effect of N-glycosylation pattern on the H5 antigen structure and its ability to induce immunological response. We also decided to change neither the number nor the position of the HA glycosylation sites but only the glycan length. Two variants of the H5 antigen with high mannose glycosylation (H5hm and with low-mannose glycosylation (H5Man5 were prepared utilizing different Pichia strains. Our structural studies demonstrated that only the highly glycosylated H5 antigen formed high molecular weight oligomers similar to viral particles. Further, the H5hm was much more immunogenic for mice than H5Man5. In summary, our results suggest that high mannose glycosylation of vaccine antigen is superior to the low glycosylation pattern. Our findings have strong implications for the recombinant HA-based influenza vaccine design.
Re-Factoring Glycolytic Genes for Targeted Engineering of Catabolism in Gram-Negative Bacteria.
Sánchez-Pascuala, Alberto; Nikel, Pablo I; de Lorenzo, Víctor
2018-01-01
The Embden-Meyerhof-Parnas (EMP) pathway is widely accepted to be the biochemical standard of glucose catabolism. The well-characterized glycolytic route of Escherichia coli, based on the EMP catabolism, is an example of an intricate pathway in terms of genomic organization of the genes involved and patterns of gene expression and regulation. This intrinsic genetic and metabolic complexity renders it difficult to engineer glycolytic activities and transfer them onto other microbial cell factories, thus limiting the biotechnological potential of bacterial hosts that lack the route. Taking into account the potential applications of such a portable tool for targeted pathway engineering, in the present protocol we describe how the genes encoding all the enzymes of the linear EMP route have been individually recruited from the genome of E. coli K-12, edited in silico to remove their endogenous regulatory signals, and synthesized de novo following a standard (i.e., GlucoBrick) that facilitates their grouping in the form of functional modules that can be combined at the user's will. This novel genetic tool allows for the à la carte implementation or boosting of EMP pathway activities into different Gram-negative bacteria. The potential of the GlucoBrick platform is further illustrated by engineering novel glycolytic activities in the most representative members of the Pseudomonas genus (Pseudomonas putida and Pseudomonas aeruginosa).
Lactoferricin mediates anabolic and anti-catabolic effects in the intervertebral disc.
Kim, Jae-Sung; Ellman, Michael B; An, Howard S; Yan, Dongyao; van Wijnen, Andre J; Murphy, Gillian; Hoskin, David W; Im, Hee-Jeong
2012-04-01
Lactoferricin (LfcinB) antagonizes biological effects mediated by angiogenic and catabolic growth factors, in addition to pro-inflammatory cytokines and chemokines in human endothelial cells and tumor cells. However, the effect of LfcinB on intervertebral disc (IVD) cell metabolism has not yet been investigated. Using bovine nucleus pulposus (NP) cells, we analyzed the effect of LfcinB on proteoglycan (PG) accumulation, PG synthesis, and anabolic gene expression. We assessed expression of genes for matrix-degrading enzymes such as matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family), as well as their endogenous inhibitors, tissue inhibitor of metalloproteases (TIMPs). In order to understand the specific molecular mechanisms by which LfcinB exerts its biological effects, we investigated intracellular signaling pathways in NP cells. LfcinB increased PG accumulation mainly via PG synthesis in a dose-dependent manner. Simultaneously, LfcinB dose-dependently downregulated catabolic enzymes. LfcinB's anti-catabolic effects were further demonstrated by a dose-dependent increase in multiple TIMP family members. Our results demonstrate that ERK and/or p38 mitogen-activated protein kinase pathways are the key signaling cascades that exert the biological effects of LfcinB in NP cells, regulating transcription of aggrecan, SOX-9, TIMP-1, TIMP-2, TIMP-3, and iNOS. Our results suggest that LfcinB has anabolic and potent anti-catabolic biological effects on bovine IVD cells that may have considerable promise in the treatment of disc degeneration in the future. Copyright © 2011 Wiley Periodicals, Inc.
Microbial catabolic activities are naturally selected by metabolic energy harvest rate.
González-Cabaleiro, Rebeca; Ofiţeru, Irina D; Lema, Juan M; Rodríguez, Jorge
2015-12-01
The fundamental trade-off between yield and rate of energy harvest per unit of substrate has been largely discussed as a main characteristic for microbial established cooperation or competition. In this study, this point is addressed by developing a generalized model that simulates competition between existing and not experimentally reported microbial catabolic activities defined only based on well-known biochemical pathways. No specific microbial physiological adaptations are considered, growth yield is calculated coupled to catabolism energetics and a common maximum biomass-specific catabolism rate (expressed as electron transfer rate) is assumed for all microbial groups. Under this approach, successful microbial metabolisms are predicted in line with experimental observations under the hypothesis of maximum energy harvest rate. Two microbial ecosystems, typically found in wastewater treatment plants, are simulated, namely: (i) the anaerobic fermentation of glucose and (ii) the oxidation and reduction of nitrogen under aerobic autotrophic (nitrification) and anoxic heterotrophic and autotrophic (denitrification) conditions. The experimentally observed cross feeding in glucose fermentation, through multiple intermediate fermentation pathways, towards ultimately methane and carbon dioxide is predicted. Analogously, two-stage nitrification (by ammonium and nitrite oxidizers) is predicted as prevailing over nitrification in one stage. Conversely, denitrification is predicted in one stage (by denitrifiers) as well as anammox (anaerobic ammonium oxidation). The model results suggest that these observations are a direct consequence of the different energy yields per electron transferred at the different steps of the pathways. Overall, our results theoretically support the hypothesis that successful microbial catabolic activities are selected by an overall maximum energy harvest rate.
DEFF Research Database (Denmark)
Ley, Daniel; Pereira, Sara; Pedersen, Lasse Ebdrup
2017-01-01
CHO cells primarily utilize amino acids for three processes: biomass synthesis, recombinant protein production and catabolism. In this work, we disrupted 9 amino acid catabolic genes participating in 7 dierent catabolic pathways, to increase synthesis of biomass and recombinant protein, while red...... reducing production of growth-inhibiting metabolic by-products from amino acid catabolism....
Clofibric acid stimulates branched-chain amino acid catabolism by three mechanisms.
Kobayashi, Rumi; Murakami, Taro; Obayashi, Mariko; Nakai, Naoya; Jaskiewicz, Jerzy; Fujiwara, Yoko; Shimomura, Yoshiharu; Harris, Robert A
2002-11-15
Clofibrate promotes catabolism of branched-chain amino acids by increasing the activity of the branched-chain alpha-keto acid dehydrogenase [BCKDH] complex. Depending upon the sex of the rats, nutritional state, and tissue being studied, clofibrate can affect BCKDH complex activity by three different mechanisms. First, by directly inhibiting BCKDH kinase activity, clofibrate can increase the proportion of the BCKDH complex in the active, dephosphorylated state. This occurs in situations in which the BCKDH complex is largely inactive due to phosphorylation, e.g., in the skeletal muscle of chow-fed rats or in the liver of female rats late in the light cycle. Second, by increasing the levels at which the enzyme components of the BCKDH complex are expressed, clofibrate can increase the total enzymatic activity of the BCKDH complex. This is readily demonstrated in livers of rats fed a low-protein diet, a nutritional condition that induces a decrease in the level of expression of the BCKDH complex. Third, by decreasing the amount of BCKDH kinase expressed and therefore its activity, clofibrate induces an increase in the percentage of the BCKDH complex in the active, dephosphorylated state. This occurs in the livers of rats fed a low-protein diet, a nutritional condition that causes inactivation of the BCKDH complex due to upregulation of the amount of BCKDH kinase. WY-14,643, which, like clofibric acid, is a ligand for the peroxisome-proliferator-activated receptor alpha [PPARalpha], does not directly inhibit BCKDH kinase but produces the same long-term effects as clofibrate on expression of the BCKDH complex and its kinase. Thus, clofibrate is unique in its capacity to stimulate BCAA oxidation through inhibition of BCKDH kinase activity, whereas PPARalpha activators in general promote BCAA oxidation by increasing expression of components of the BCKDH complex and decreasing expression of the BCKDH kinase.
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Linling He
2017-08-01
Full Text Available Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development.
He, Linling; Lin, Xiaohe; de Val, Natalia; Saye-Francisco, Karen L; Mann, Colin J; Augst, Ryan; Morris, Charles D; Azadnia, Parisa; Zhou, Bin; Sok, Devin; Ozorowski, Gabriel; Ward, Andrew B; Burton, Dennis R; Zhu, Jiang
2017-01-01
Germline precursors and intermediates of broadly neutralizing antibodies (bNAbs) are essential to the understanding of humoral response to HIV-1 infection and B-cell lineage vaccine design. Using a native-like gp140 trimer probe, we examined antibody libraries constructed from donor-17, the source of glycan-dependent PGT121-class bNAbs recognizing the N332 supersite on the HIV-1 envelope glycoprotein. To facilitate this analysis, a digital panning method was devised that combines biopanning of phage-displayed antibody libraries, 900 bp long-read next-generation sequencing, and heavy/light (H/L)-paired antibodyomics. In addition to single-chain variable fragments resembling the wild-type bNAbs, digital panning identified variants of PGT124 (a member of the PGT121 class) with a unique insertion in the heavy chain complementarity-determining region 1, as well as intermediates of PGT124 exhibiting notable affinity for the native-like trimer and broad HIV-1 neutralization. In a competition assay, these bNAb intermediates could effectively compete with mouse sera induced by a scaffolded BG505 gp140.681 trimer for the N332 supersite. Our study thus reveals previously unrecognized lineage complexity of the PGT121-class bNAbs and provides an array of library-derived bNAb intermediates for evaluation of immunogens containing the N332 supersite. Digital panning may prove to be a valuable tool in future studies of bNAb diversity and lineage development.
Generation of a Mutant Mucor hiemalis Endoglycosidase That Acts on Core-fucosylated N-Glycans.
Katoh, Toshihiko; Katayama, Takane; Tomabechi, Yusuke; Nishikawa, Yoshihide; Kumada, Jyunichi; Matsuzaki, Yuji; Yamamoto, Kenji
2016-10-28
Endo-β-N-acetylglucosaminidase M (Endo-M), an endoglycosidase from the fungus Mucor hiemalis, is a useful tool for chemoenzymatic synthesis of glycoconjugates, including glycoprotein-based therapeutics having a precisely defined glycoform, by virtue of its transglycosylation activity. Although Endo-M has been known to act on various N-glycans, it does not act on core-fucosylated N-glycans, which exist widely in mammalian glycoproteins, thus limiting its application. Therefore, we performed site-directed mutagenesis on Endo-M to isolate mutant enzymes that are able to act on mammalian-type core-α1,6-fucosylated glycans. Among the Endo-M mutant enzymes generated, those in which the tryptophan at position 251 was substituted with alanine or asparagine showed altered substrate specificities. Such mutant enzymes exhibited increased hydrolysis of a synthetic α1,6-fucosylated trimannosyl core structure, whereas their activity on the afucosylated form decreased. In addition, among the Trp-251 mutants, the W251N mutant was most efficient in hydrolyzing the core-fucosylated substrate. W251N mutants could act on the immunoglobulin G-derived core-fucosylated glycopeptides and human lactoferrin glycoproteins. This mutant was also capable of transferring the sialyl glycan from an activated substrate intermediate (sialyl glyco-oxazoline) onto an α1,6-fucosyl-N-acetylglucosaminyl biotin. Furthermore, the W251N mutant gained a glycosynthase-like activity when a N175Q substitution was introduced and it caused accumulation of the transglycosylation products. These findings not only give insights into the substrate recognition mechanism of glycoside hydrolase family 85 enzymes but also widen their scope of application in preparing homogeneous glycoforms of core-fucosylated glycoproteins for the production of potent glycoprotein-based therapeutics. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
Gutternigg, Martin; Bürgmayr, Sabine; Pöltl, Gerald; Rudolf, Judith; Staudacher, Erika
2007-11-01
The N-glycosylation potentials of Limax maximus, Cepaea hortensis, Planorbarius corneus, Arianta arbustorum and Achatina fulica were analysed by investigation of the N-glycan structures of the skin and viscera glycoproteins by a combination of HPLC and mass-spectrometry methods. It is one of the first steps to enlarge the knowledge on the glycosylation abilities of gastropods, which may help to establish new cell culture systems, to uncover new means for pest control for some species, and to identify carbohydrate-epitopes which may be relevant for immune response. All snails analysed contained mainly oligomannosidic and small paucimannosidic structures, often terminated with 3-O-methylated mannoses. The truncated structures carried modifications by beta1-2-linked xylose to the beta-mannose residue, and/or an alpha-fucosylation, mainly alpha1,6-linked to the innermost N-acetylglucosaminyl residue of the core. Many of these structures were missing the terminal N-acetylglucosamine, which has been shown to be a prerequisite for processing to complex N-glycans in the Golgi. In some species (Planorbarius corneus and Achatina fulica) traces of large structures, terminated by 3-O-methylated galactoses and carrying xylose and/or fucose residues, were also detected. In Planorbarius viscera low amounts of terminal alpha1-2-fucosylation were determined. Combining these results, gastropods seem to be capable to produce all kinds of structures ranging from those typical in mammals through to structures similar to those found in plants, insects or nematodes. The detailed knowledge of this very complex glycosylation system of the gastropods will be a valuable tool to understand the principle rules of glycosylation in all organisms.
Bassoy, Esen Yonca; Kasahara, Atsuko; Chiusolo, Valentina; Jacquemin, Guillaume; Boydell, Emma; Zamorano, Sebastian; Riccadonna, Cristina; Pellegatta, Serena; Hulo, Nicolas; Dutoit, Valérie; Derouazi, Madiha; Dietrich, Pierre Yves; Walker, Paul R; Martinvalet, Denis
2017-06-01
Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma. © 2017 The Authors.
Cerliani, Juan P; Stowell, Sean R; Mascanfroni, Iván D; Arthur, Connie M; Cummings, Richard D; Rabinovich, Gabriel A
2011-02-01
Effective immunity relies on the recognition of pathogens and tumors by innate immune cells through diverse pattern recognition receptors (PRRs) that lead to initiation of signaling processes and secretion of pro- and anti-inflammatory cytokines. Galectins, a family of endogenous lectins widely expressed in infected and neoplastic tissues have emerged as part of the portfolio of soluble mediators and pattern recognition receptors responsible for eliciting and controlling innate immunity. These highly conserved glycan-binding proteins can control immune cell processes through binding to specific glycan structures on pathogens and tumors or by acting intracellularly via modulation of selective signaling pathways. Recent findings demonstrate that various galectin family members influence the fate and physiology of different innate immune cells including polymorphonuclear neutrophils, mast cells, macrophages, and dendritic cells. Moreover, several pathogens may actually utilize galectins as a mechanism of host invasion. In this review, we aim to highlight and integrate recent discoveries that have led to our current understanding of the role of galectins in host-pathogen interactions and innate immunity. Challenges for the future will embrace the rational manipulation of galectin-glycan interactions to instruct and shape innate immunity during microbial infections, inflammation, and cancer.
Energy Technology Data Exchange (ETDEWEB)
Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.; Mkhize, Nonhlanhla N.; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D.; Labuschagne, Phillip; Louder, Mark K.; Bailer, Robert T.; Karim, Salim S. Abdool; Mascola, John R.; Williamson, Carolyn; Moore, Penny L.; Kwong, Peter D.; Morris, Lynn (NHLS-South Africa); (NIH); (Witwatersrand); (KwaZulu-Natal)
2016-08-31
All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage.
Taxon- and Site-Specific Melatonin Catabolism
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Rüdiger Hardeland
2017-11-01
Full Text Available Melatonin is catabolized both enzymatically and nonenzymatically. Nonenzymatic processes mediated by free radicals, singlet oxygen, other reactive intermediates such as HOCl and peroxynitrite, or pseudoenzymatic mechanisms are not species- or tissue-specific, but vary considerably in their extent. Higher rates of nonenzymatic melatonin metabolism can be expected upon UV exposure, e.g., in plants and in the human skin. Additionally, melatonin is more strongly nonenzymatically degraded at sites of inflammation. Typical products are several hydroxylated derivatives of melatonin and N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK. Most of these products are also formed by enzymatic catalysis. Considerable taxon- and site-specific differences are observed in the main enzymatic routes of catabolism. Formation of 6-hydroxymelatonin by cytochrome P450 subforms are prevailing in vertebrates, predominantly in the liver, but also in the brain. In pineal gland and non-mammalian retina, deacetylation to 5-methoxytryptamine (5-MT plays a certain role. This pathway is quantitatively prevalent in dinoflagellates, in which 5-MT induces cyst formation and is further converted to 5-methoxyindole-3-acetic acid, an end product released to the water. In plants, the major route is catalyzed by melatonin 2-hydroxylase, whose product is tautomerized to 3-acetamidoethyl-3-hydroxy-5-methoxyindolin-2-one (AMIO, which exceeds the levels of melatonin. Formation and properties of various secondary products are discussed.
A role for TNFα in intervertebral disc degeneration: A non-recoverable catabolic shift
International Nuclear Information System (INIS)
Purmessur, D.; Walter, B.A.; Roughley, P.J.; Laudier, D.M.; Hecht, A.C.; Iatridis, James
2013-01-01
Highlights: ► TNFα induced catabolic changes similar to human intervertebral disc degeneration. ► The metabolic shift induced by TNFα was sustained following removal. ► TNFα induced changes suggestive of cell senescence without affecting cell viability. ► Interventions are required to stimulate anabolism and increase cell proliferation. -- Abstract: This study examines the effect of TNFα on whole bovine intervertebral discs in organ culture and its association with changes characteristic of intervertebral disc degeneration (IDD) in order to inform future treatments to mitigate the chronic inflammatory state commonly found with painful IDD. Pro-inflammatory cytokines such as TNFα contribute to disc pathology and are implicated in the catabolic phenotype associated with painful IDD. Whole bovine discs were cultured to examine cellular (anabolic/catabolic gene expression, cell viability and senescence using β-galactosidase) and structural (histology and aggrecan degradation) changes in response to TNFα treatment. Control or TNFα cultures were assessed at 7 and 21 days; the 21 day group also included a recovery group with 7 days TNFα followed by 14 days in basal media. TNFα induced catabolic and anti-anabolic shifts in the nucleus pulposus (NP) and annulus fibrosus (AF) at 7 days and this persisted until 21 days however cell viability was not affected. Data indicates that TNFα increased aggrecan degradation products and suggests increased β-galactosidase staining at 21 days without any recovery. TNFα treatment of whole bovine discs for 7 days induced changes similar to the degeneration processes that occur in human IDD: aggrecan degradation, increased catabolism, pro-inflammatory cytokines and nerve growth factor expression. TNFα significantly reduced anabolism in cultured IVDs and a possible mechanism may be associated with cell senescence. Results therefore suggest that successful treatments must promote anabolism and cell proliferation in
Xue, Peipei; Zeng, Fanfan; Duan, Qiuhong; Xiao, Juanjuan; Liu, Lin; Yuan, Ping; Fan, Linni; Sun, Huimin; Malyarenko, Olesya S; Lu, Hui; Xiu, Ruijuan; Liu, Shaoqing; Shao, Chen; Zhang, Jianmin; Yan, Wei; Wang, Zhe; Zheng, Jianyong; Zhu, Feng
2017-06-01
Branched-chain amino acids catabolism plays an important role in human cancers. Colorectal cancer is the third most commonly diagnosed cancer in males and the second in females, and the new global incidence is over 1.2 million cases. The branched-chain α-keto acid dehydrogenase kinase (BCKDK) is a rate-limiting enzyme in branched-chain amino acids catabolism, which plays an important role in many serious human diseases. Here we investigated that abnormal branched-chain amino acids catabolism in colorectal cancer is a result of the disease process, with no role in disease initiation; BCKDK is widely expressed in colorectal cancer patients, and those patients that express higher levels of BCKDK have shorter survival times than those with lower levels; BCKDK promotes cell transformation or colorectal cancer ex vivo or in vivo. Mechanistically, BCKDK promotes colorectal cancer by enhancing the MAPK signaling pathway through direct MEK phosphorylation, rather than by branched-chain amino acids catabolism. And the process above could be inhibited by a BCKDK inhibitor, phenyl butyrate. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.
Directory of Open Access Journals (Sweden)
Peipei Xue
2017-06-01
Full Text Available Branched-chain amino acids catabolism plays an important role in human cancers. Colorectal cancer is the third most commonly diagnosed cancer in males and the second in females, and the new global incidence is over 1.2 million cases. The branched-chain α-keto acid dehydrogenase kinase (BCKDK is a rate-limiting enzyme in branched-chain amino acids catabolism, which plays an important role in many serious human diseases. Here we investigated that abnormal branched-chain amino acids catabolism in colorectal cancer is a result of the disease process, with no role in disease initiation; BCKDK is widely expressed in colorectal cancer patients, and those patients that express higher levels of BCKDK have shorter survival times than those with lower levels; BCKDK promotes cell transformation or colorectal cancer ex vivo or in vivo. Mechanistically, BCKDK promotes colorectal cancer by enhancing the MAPK signaling pathway through direct MEK phosphorylation, rather than by branched-chain amino acids catabolism. And the process above could be inhibited by a BCKDK inhibitor, phenyl butyrate.
Directory of Open Access Journals (Sweden)
C Sangavai
2017-12-01
Full Text Available Model-driven systems engineering has been more fascinating process for the microbial production of biofuel and bio-refineries in chemical and pharmaceutical industries. Genome-scale modeling and simulations have been guided for metabolic engineering of Clostridium species for the production of organic solvents and organic acids. Among them, Clostridium sticklandii is one of the potential organisms to be exploited as a microbial cell factory for biofuel production. It is a hyper-ammonia producing bacterium and is able to catabolize amino acids as important carbon and energy sources via Stickland reactions and the development of the specific pathways. Current genomic and metabolic aspects of this bacterium are comprehensively reviewed herein, which provided information for learning about protein catabolism-directed biofuel production. It has a metabolic potential to drive energy and direct solventogenesis as well as acidogenesis from protein catabolism. It produces by-products such as ethanol, acetate, n-butanol, n-butyrate and hydrogen from amino acid catabolism. Model-driven systems engineering of this organism would improve the performance of the industrial sectors and enhance the industrial economy by using protein-based waste in environment-friendly ways. Keywords: Biofuel, Amino acid catabolism, Genome-scale model, Metabolic engineering, Systems biology, ABE fermentation, Clostridium sticklandii
Metabolic control analysis of Aspergillus niger L-arabinose catabolism
Groot, de M.J.L.; Prathumpai, W.; Visser, J.; Ruijter, G.J.G.
2005-01-01
A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their
Sakuraba, Yasuhito; Schelbert, Silvia; Park, So-Yon; Han, Su-Hyun; Lee, Byoung-Doo; Andrès, Céline Besagni; Kessler, Felix; Hörtensteiner, Stefan; Paek, Nam-Chon
2012-01-01
During leaf senescence, plants degrade chlorophyll to colorless linear tetrapyrroles that are stored in the vacuole of senescing cells. The early steps of chlorophyll breakdown occur in plastids. To date, five chlorophyll catabolic enzymes (CCEs), NONYELLOW COLORING1 (NYC1), NYC1-LIKE, pheophytinase, pheophorbide a oxygenase (PAO), and red chlorophyll catabolite reductase, have been identified; these enzymes catalyze the stepwise degradation of chlorophyll to a fluorescent intermediate, pFCC, which is then exported from the plastid. In addition, STAY-GREEN (SGR), Mendel’s green cotyledon gene encoding a chloroplast protein, is required for the initiation of chlorophyll breakdown in plastids. Senescence-induced SGR binds to light-harvesting complex II (LHCII), but its exact role remains elusive. Here, we show that all five CCEs also specifically interact with LHCII. In addition, SGR and CCEs interact directly or indirectly with each other at LHCII, and SGR is essential for recruiting CCEs in senescing chloroplasts. PAO, which had been attributed to the inner envelope, is found to localize in the thylakoid membrane. These data indicate a predominant role for the SGR-CCE-LHCII protein interaction in the breakdown of LHCII-located chlorophyll, likely to allow metabolic channeling of phototoxic chlorophyll breakdown intermediates upstream of nontoxic pFCC. PMID:22366162
International Nuclear Information System (INIS)
Milcic-Terzic, J.; Saval, S.; Lopez-Vidal, Y.; Vrvic, M.M.
2001-01-01
Bioremediation is often used for in situ remediation of petroleum-contaminated sites. The primary focus of this study was on understanding the indigenous microbial community which can survive in contaminated environment and is responsible for the degradation. Diesel, toluene and naphthalene-degrading microbial consortia were isolated from diesel-contaminated soil by growing on selective hydrocarbon substrates. The presence and frequency of the catabolic genes responsible for aromatic hydrocarbon biodegradation (xylE, ndoB) within the isolated consortia were screened using polymerase chain reaction PCR and DNA-DNA colony hybridization. The diesel DNA-extract possessed both the xylE catabolic gene for toluene, and the nah catabolic gene for polynuclear aromatic hydrocarbon degradation. The toluene DNA-extract possessed only the xylE catabolic gene, while the naphthalene DNA-extract only the ndoB gene. Restriction enzyme analysis with HaeIII indicated similar restriction patterns for the xylE gene fragment between toluene DNA-extract and a type strain, Pseudomonas putida ATCC 23973. A substantial proportion (74%) of the colonies from the diesel-consortium possessed the xylE gene, and the ndoB gene (78%), while a minority (29%) of the toluene-consortium harbored the xylE gene. 59% of the colonies from the naphthalene-consortium had the ndoB gene, and did not have the xylE gene. These results indicate that the microbial population has been naturally enriched in organisms carrying genes for aromatic hydrocarbon degradation and that significant aromatic biodegradative potential exists at the site. Characterization of the population genotype constitutes a molecular diagnosis which permits the determination of the catabolic potential of the site to degrade the contaminant present. (author)
International Nuclear Information System (INIS)
Cowan, A.K.; Railton, I.D.
1987-01-01
Excised light-grown leaves and etiolated leaves of Hordeum vulgare L. cv Dyan catabolized applied (+/-)-[2- 14 C]abscisic acid ([+/-]-[2- 14 C]ABA) to phaseic acid (PA), dihydrophaseic acid (DPA), and 2'-hydroxymethyl ABA (2'-HMABA). Identification of these catabolites was made by microchemical methods and by combined capillary gas chromatography-mass spectrometry (GC-MS) following high dose feeds of nonlabeled substrate to leaves. Circular dichroism analysis revealed that 2'-HMABA was derived from the (-) enantiomer of ABA. Refeeding studies were used to confirm the catabolic route. The methyl ester of (+/-)-[2 14 C]-ABA was hydrolyzed efficiently by light-grown leaves of H. vulgare. Leaf age played a significant role in (+/-)-ABA catabolism, with younger leaves being less able than their older counterparts to catabolize this compound. The catabolism of (+/-)-ABA was inhibited markedly in water-stressed Hordeum leaves which was characterized by a decreased incorporation of label into 2'-HMABA, DPA, and conjugates. The specific, mixed function oxidase inhibitor, ancymidol, did not inhibit, dramatically (+/-)-ABA catabolism in light-grown leaves of Hordeum whereas the 80s ribosome, translational inhibitor, cycloheximide, inhibited this process markedly. The 70s ribosome translational inhibitors, lincomycin and chloramphenicol, were less effective than cycloheximide in inhibiting (+/-)-ABA catabolism, implying that cytoplasmic protein synthesis is necessary for the catabolism of (+/-)-ABA in Hordeum leaves whereas chloroplast protein synthesis plays only a minor role. This further suggests that the enzymes involved in (+/-)-ABA catabolism in this plant are cytoplasmically synthesized and are turned-over rapidly, although the enzyme responsible for glycosylating (+/-)-ABA itself appeared to be stable
Panzarini, E.; Mariano, S.; Dini, L.
2017-08-01
The effects of glycans-capped AgNPs (30±5 nm average diameter, spherical shape) on biocompatibility and uptake was studied in relation to the glycan capping (glucose AgNPs-G, glucose/sucrose AgNPs-GS, glucose/fructose AgNPs-GF), and to the cell types (HeLa cells, lymphocytes, and HepG2 cells). Glycan capping and type of cells drive morphological changes, viability loss and type and extent of cell death induction; in addition cells response is largely influenced by the AgNPs amount. The MTT photometric method to determine cell metabolism and the analysis of the membrane integrity by Annexin V-Propidium Iodide labelling were used to quantify cell viability and cell death with different concentrations of NPs. It turns out that i) AgNPs-GF are the most toxic, whereas ii) AgNPs-GS are the less toxic NPs, probably due to the stability of glucose/sucrose capping up to 5 days in culture medium; iii) HepG2 cells are the most sensitive to the presence of NPs. A deeper investigation is necessary to explain the interesting PBLs proliferation increase observed in the presence of AgNPs-GS.
Localization of α1-2 Fucose Glycan in the Mouse Olfactory Pathway.
Kondoh, Daisuke; Kamikawa, Akihiro; Sasaki, Motoki; Kitamura, Nobuo
2017-01-01
Glycoconjugates in the olfactory system play critical roles in neuronal formation, and α1-2 fucose (α1-2Fuc) glycan mediates neurite outgrowth and synaptic plasticity. Histochemical findings of α1-2Fuc glycan in the mouse olfactory system detected using Ulex europaeus agglutinin-I (UEA-I) vary. This study histochemically assessed the main olfactory and vomeronasal pathways in male and female ICR and C57BL/6J mice aged 3-4 months using UEA-I. Ulex europaeus agglutinin-I reacted with most receptor cells arranged mainly at the basal region of the olfactory epithelium. The olfactory nerve layer and glomerular layer of the main olfactory bulb were speckled with positive UEA-I staining, and positive fibers were scattered from the glomerular to the internal plexiform layer. The lateral olfactory tract and rostral migratory stream were also positive for UEA-I. We identified superficial short-axon cells, interneurons of the external plexiform layer, external, middle and internal tufted cells, mitral cells and granule cells as the origins of the UEA-I-positive fibers in the main olfactory bulb. The anterior olfactory nucleus, anterior piriform cortex and olfactory tubercle were negative for UEA-I. Most receptor cells in the vomeronasal epithelium and most glomeruli of the accessory olfactory bulb were positive for UEA-I. Our findings indicated that α1-2Fuc glycan is located within the primary and secondary, but not the ternary, pathways of the main olfactory system, in local circuits of the main olfactory bulb and within the primary, but not secondary, pathway of the vomeronasal system. © 2016 S. Karger AG, Basel.
Transcriptional analysis of prebiotic uptake and catabolism by Lactobacillus acidophilus NCFM.
Directory of Open Access Journals (Sweden)
Joakim Mark Andersen
Full Text Available The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in Lactobacillus acidophilus NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and β-linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS, galactoside pentose hexuronide (GPH permease, and ATP-binding cassette (ABC transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides (GOS. The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and β-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6-phospho-β-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of L. acidophilus NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively influence the gastrointestinal microbiota.
Clarkson, Sonya M; Giannone, Richard J; Kridelbaugh, Donna M; Elkins, James G; Guss, Adam M; Michener, Joshua K
2017-09-15
The production of biofuels from lignocellulose yields a substantial lignin by-product stream that currently has few applications. Biological conversion of lignin-derived compounds into chemicals and fuels has the potential to improve the economics of lignocellulose-derived biofuels, but few microbes are able both to catabolize lignin-derived aromatic compounds and to generate valuable products. While Escherichia coli has been engineered to produce a variety of fuels and chemicals, it is incapable of catabolizing most aromatic compounds. Therefore, we engineered E. coli to catabolize protocatechuate, a common intermediate in lignin degradation, as the sole source of carbon and energy via heterologous expression of a nine-gene pathway from Pseudomonas putida KT2440. We next used experimental evolution to select for mutations that increased growth with protocatechuate more than 2-fold. Increasing the strength of a single ribosome binding site in the heterologous pathway was sufficient to recapitulate the increased growth. After optimization of the core pathway, we extended the pathway to enable catabolism of a second model compound, 4-hydroxybenzoate. These engineered strains will be useful platforms to discover, characterize, and optimize pathways for conversions of lignin-derived aromatics. IMPORTANCE Lignin is a challenging substrate for microbial catabolism due to its polymeric and heterogeneous chemical structure. Therefore, engineering microbes for improved catabolism of lignin-derived aromatic compounds will require the assembly of an entire network of catabolic reactions, including pathways from genetically intractable strains. Constructing defined pathways for aromatic compound degradation in a model host would allow rapid identification, characterization, and optimization of novel pathways. We constructed and optimized one such pathway in E. coli to enable catabolism of a model aromatic compound, protocatechuate, and then extended the pathway to a related
Directory of Open Access Journals (Sweden)
Neil Dani
Full Text Available A Drosophila transgenic RNAi screen targeting the glycan genome, including all N/O/GAG-glycan biosynthesis/modification enzymes and glycan-binding lectins, was conducted to discover novel glycan functions in synaptogenesis. As proof-of-product, we characterized functionally paired heparan sulfate (HS 6-O-sulfotransferase (hs6st and sulfatase (sulf1, which bidirectionally control HS proteoglycan (HSPG sulfation. RNAi knockdown of hs6st and sulf1 causes opposite effects on functional synapse development, with decreased (hs6st and increased (sulf1 neurotransmission strength confirmed in null mutants. HSPG co-receptors for WNT and BMP intercellular signaling, Dally-like Protein and Syndecan, are differentially misregulated in the synaptomatrix of these mutants. Consistently, hs6st and sulf1 nulls differentially elevate both WNT (Wingless; Wg and BMP (Glass Bottom Boat; Gbb ligand abundance in the synaptomatrix. Anterograde Wg signaling via Wg receptor dFrizzled2 C-terminus nuclear import and retrograde Gbb signaling via synaptic MAD phosphorylation and nuclear import are differentially activated in hs6st and sulf1 mutants. Consequently, transcriptional control of presynaptic glutamate release machinery and postsynaptic glutamate receptors is bidirectionally altered in hs6st and sulf1 mutants, explaining the bidirectional change in synaptic functional strength. Genetic correction of the altered WNT/BMP signaling restores normal synaptic development in both mutant conditions, proving that altered trans-synaptic signaling causes functional differentiation defects.
Belyakov, Alexei Ye; Burygin, Gennady L; Arbatsky, Nikolai P; Shashkov, Alexander S; Selivanov, Nikolai Yu; Matora, Larisa Yu; Knirel, Yuriy A; Shchyogolev, Sergei Yu
2012-11-01
This is the first report to have identified an O-linked repetitive glycan in bacterial flagellin, a structural protein of the flagellum. Studies by sugar analysis, Smith degradation, (1)H and (13)C NMR spectroscopy, and mass spectrometry showed that the glycan chains of the polar flagellum flagellin of the plant-growth-promoting rhizobacterium Azospirillum brasilense Sp7 are represented by a polysaccharide with a molecular mass of 7.7 kDa, which has a branched tetrasaccharide repeating unit of the following structure: Copyright © 2012. Published by Elsevier Ltd.
CSIR Research Space (South Africa)
Kahari, D
2008-11-01
Full Text Available profiling techniques. The gene encoding Lip2 was cloned as a C-terminally His-tagged protein, expressed in Yarrowia lipolytica (Madzak, C et al;2004) and the glycan composition of the purified protein was analysed by HPLC and MALDITOF. The HPLC techniques...
Solovieva, Elena; Shikanai, Toshihide; Fujita, Noriaki; Narimatsu, Hisashi
2018-04-18
Inherited mutations in glyco-related genes can affect the biosynthesis and degradation of glycans and result in severe genetic diseases and disorders. The Glyco-Disease Genes Database (GDGDB), which provides information about these diseases and disorders as well as their causative genes, has been developed by the Research Center for Medical Glycoscience (RCMG) and released in April 2010. GDGDB currently provides information on about 80 genetic diseases and disorders caused by single-gene mutations in glyco-related genes. Many biomedical resources provide information about genetic disorders and genes involved in their pathogenesis, but resources focused on genetic disorders known to be related to glycan metabolism are lacking. With the aim of providing more comprehensive knowledge on genetic diseases and disorders of glycan biosynthesis and degradation, we enriched the content of the GDGDB database and improved the methods for data representation. We developed the Genetic Glyco-Diseases Ontology (GGDonto) and a RDF/SPARQL-based user interface using Semantic Web technologies. In particular, we represented the GGDonto content using Semantic Web languages, such as RDF, RDFS, SKOS, and OWL, and created an interactive user interface based on SPARQL queries. This user interface provides features to browse the hierarchy of the ontology, view detailed information on diseases and related genes, and find relevant background information. Moreover, it provides the ability to filter and search information by faceted and keyword searches. Focused on the molecular etiology, pathogenesis, and clinical manifestations of genetic diseases and disorders of glycan metabolism and developed as a knowledge-base for this scientific field, GGDonto provides comprehensive information on various topics, including links to aid the integration with other scientific resources. The availability and accessibility of this knowledge will help users better understand how genetic defects impact the
Khan, Sumia; Afzal, Muhammad; Iqbal, Samina; Mirza, Muhammad Sajjad; Khan, Qaiser M
2013-04-01
Plant-bacteria partnership is a promising approach for remediating soil contaminated with organic pollutants. The colonization and metabolic activity of an inoculated microorganism depend not only on environmental conditions but also on the physiological condition of the applied microorganisms. This study assessed the influence of different inoculum pretreatments on survival, gene abundance and catabolic gene expression of an applied strain (Pantoea sp. strain BTRH79) in the rhizosphere of ryegrass vegetated in diesel contaminated soil. Maximum bacterium survival, gene abundance and expression were observed in the soil inoculated with bacterial cells that had been pregrown on complex medium, and hydrocarbon degradation and genotoxicity reduction were also high in this soil. These findings propose that use of complex media for growing plant inocula may enhance bacterial survival and colonization and subsequently the efficiency of pollutant degradation. Copyright © 2013 Elsevier Ltd. All rights reserved.
DEFF Research Database (Denmark)
Blixt, Klas Ola; Kumagai-Braesch, A.; Tibell, A.
2009-01-01
Ten patients with type I diabetes were transplanted with porcine fetal islet-like cell clusters (ICC) between 1990 and 1993. A significant rise in the anti-a-Gal antibody titers was seen posttransplant, but also non-a-Gal-specific antibodies were detected in some patients. We have reanalyzed...... the carbohydrate specificity of antibodies in the sera from seven of these patients taken before transplantation, 1, 6 and 12 months posttransplantation using a glycan array with 200 structurally defined glycans. The main findings were: (i) prepig ICC transplantation patients had antibodies reactive with terminal...... compounds; (ii) the titers of all carbohydrate-specific antibodies detected before transplantation rose after transplantation; (iii) the kinetics of the antibody responses differed between patients; (iv) in some patients antibodies reacting with Gala1,3Lex and several structures terminated with Neu5Gc...
DEFF Research Database (Denmark)
Klisch, Karl; Jeanrond, Evelyne; Pang, Poh-Choo
2008-01-01
assisted laser desorption ionisation-mass spectrometry (MALDI-MS) analysis and linkage analysis, we show that by far, the most abundant N-glycan of PAGs in midpregnancy is a tetraantennary core-fucosylated structure with a bisecting N-acetylglucosamine (GlcNAc). All four antennae consist of the Sd......(a)-antigen (NeuAcalpha2-3[GalNAcbeta1-4]Galbeta1-4GlcNAc-). Immunohistochemistry with the mono- clonal antibody CT1, which recognizes the Sd(a)-antigen, shows that BNC granules contain the Sd(a)-antigen from gestation day (gd) 32 until a few days before parturition. Lectin histochemistry with Maackia amurensis...
Xue, Hui; Veit, Christiane; Abas, Lindy; Tryfona, Theodora; Maresch, Daniel; Ricardi, Martiniano M; Estevez, José Manuel; Strasser, Richard; Seifert, Georg J
2017-08-01
Fasciclin-like arabinogalactan proteins (FLAs) are involved in numerous important functions in plants but the relevance of their complex structure to physiological function and cellular fate is unresolved. Using a fully functional fluorescent version of Arabidopsis thaliana FLA4 we show that this protein is localized at the plasma membrane as well as in endosomes and soluble in the apoplast. FLA4 is likely to be GPI-anchored, is highly N-glycosylated and carries two O-glycan epitopes previously associated with arabinogalactan proteins. The activity of FLA4 was resistant against deletion of the amino-proximal fasciclin 1 domain and was unaffected by removal of the GPI-modification signal, a highly conserved N-glycan or the deletion of predicted O-glycosylation sites. Nonetheless these structural changes dramatically decreased endoplasmic reticulum (ER)-exit and plasma membrane localization of FLA4, with N-glycosylation acting at the level of ER-exit and O-glycosylation influencing post-secretory fate. We show that FLA4 acts predominantly by molecular interactions involving its carboxy-proximal fasciclin 1 domain and that its amino-proximal fasciclin 1 domain is required for stabilization of plasma membrane localization. FLA4 functions as a soluble glycoprotein via its carboxy-proximal Fas1 domain and its normal cellular trafficking depends on N- and O-glycosylation. © 2017 The Authors. The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.
Detection and isolation of novel rhizopine-catabolizing bacteria from the environment
Gardener; de Bruijn FJ
1998-12-01
Microbial rhizopine-catabolizing (Moc) activity was detected in serial dilutions of soil and rhizosphere washes. The activity observed generally ranged between 10(6) and 10(7) catabolic units per g, and the numbers of nonspecific culture-forming units were found to be approximately 10 times higher. A diverse set of 37 isolates was obtained by enrichment on scyllo-inosamine-containing media. However, none of the bacteria that were isolated were found to contain DNA sequences homologous to the known mocA, mocB, and mocC genes of Sinorhizobium meliloti L5-30. Twenty-one of the isolates could utilize an SI preparation as the sole carbon and nitrogen source for growth. Partial sequencing of 16S ribosomal DNAs (rDNAs) amplified from these strains indicated that five distinct bacterial genera (Arthrobacter, Sinorhizobium, Pseudomonas, Aeromonas, and Alcaligenes) were represented in this set. Only 6 of these 21 isolates could catabolize 3-O-methyl-scyllo-inosamine under standard assay conditions. Two of these, strains D1 and R3, were found to have 16S rDNA sequences very similar to those of Sinorhizobium meliloti. However, these strains are not symbiotically effective on Medicago sativa, and DNA sequences homologous to the nodB and nodC genes were not detected in strains D1 and R3 by Southern hybridization analysis.
Increased fat catabolism sustains water balance during fasting in zebra finches.
Rutkowska, Joanna; Sadowska, Edyta T; Cichoń, Mariusz; Bauchinger, Ulf
2016-09-01
Patterns of physiological flexibility in response to fasting are well established, but much less is known about the contribution of water deprivation to the observed effects. We investigated body composition and energy and water budget in three groups of zebra finches: birds with access to food and water, food-deprived birds having access to drinking water and food-and-water-deprived birds. Animals were not stimulated by elevated energy expenditure and they were in thermoneutral conditions; thus, based on previous studies, water balance of fasting birds was expected to be maintained by increased catabolism of proteins. In contrast to this expectation, we found that access to water did not prevent reduction of proteinaceous tissue, but it saved fat reserves of the fasting birds. Thus, water balance of birds fasting without access to water seemed to be maintained by elevated fat catabolism, which generated 6 times more metabolic water compared with that in birds that had access to water. Therefore, we revise currently established views and propose fat to serve as the primary source for metabolic water production. Previously assumed increased protein breakdown for maintenance of water budget would occur if fat stores were depleted or if fat catabolism reached its upper limits due to high energy demands. © 2016. Published by The Company of Biologists Ltd.
Draft Genome Sequences of Three β-Lactam-Catabolizing Soil Proteobacteria
DEFF Research Database (Denmark)
Crofts, Terence S.; Wang, Bin; Spivak, Aaron
2017-01-01
Most antibiotics are derived from the soil, but their catabolism there, which is necessary to close the antibiotic carbon cycle, remains uncharacterized. We report the first draft genome sequences of soil Proteobacteria identified for subsisting solely on β-lactams as their carbon sources...
Directory of Open Access Journals (Sweden)
Denong Wang
2015-03-01
Full Text Available Identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. Owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as HIV-1. However, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. Thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. In this study, we characterized two broadly HIV-neutralizing agents, human monoclonal antibody 2G12 and Galanthus nivalis lectin (GNA, for their viral targeting activities. Although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. The former is HIV-1 specific; the latter is broadly reactive and is able to neutralize viruses of distinct phylogenetic origins, such as HIV-1, severe acute respiratory syndrome coronavirus (SARS-CoV, and human cytomegalovirus (HCMV. In carbohydrate microarray analyses, we explored the molecular basis underlying the striking differences in the spectrum of anti-virus activities of the two probes. Unlike 2G12, which is strictly specific for the high-density Man9GlcNAc2Asn (Man9-clusters, GNA recognizes a number of N-glycan cryptic sugar moieties. These include not only the known oligomannosyl antigens but also previously unrecognized tri-antennary or multi-valent GlcNAc-terminating N-glycan epitopes (Tri/m-Gn. These findings highlight the potential of N-glycan cryptic sugar moieties as conserved targets for broad-spectrum virus neutralization and suggest the GNA-model of glycan-binding warrants focused investigation.
Wang, Wei; Zhang, Fuyang; Xia, Yunlong; Zhao, Shihao; Yan, Wenjun; Wang, Helin; Lee, Yan; Li, Congye; Zhang, Ling; Lian, Kun; Gao, Erhe; Cheng, Hexiang; Tao, Ling
2016-11-01
Cardiac metabolic remodeling is a central event during heart failure (HF) development following myocardial infarction (MI). It is well known that myocardial glucose and fatty acid dysmetabolism contribute to post-MI cardiac dysfunction and remodeling. However, the role of amino acid metabolism in post-MI HF remains elusive. Branched chain amino acids (BCAAs) are an important group of essential amino acids and function as crucial nutrient signaling in mammalian animals. The present study aimed to determine the role of cardiac BCAA metabolism in post-MI HF progression. Utilizing coronary artery ligation-induced murine MI models, we found that myocardial BCAA catabolism was significantly impaired in response to permanent MI, therefore leading to an obvious elevation of myocardial BCAA abundance. In MI-operated mice, oral BCAA administration further increased cardiac BCAA levels, activated the mammalian target of rapamycin (mTOR) signaling, and exacerbated cardiac dysfunction and remodeling. These data demonstrate that BCAAs act as a direct contributor to post-MI cardiac pathologies. Furthermore, these BCAA-mediated deleterious effects were improved by rapamycin cotreatment, revealing an indispensable role of mTOR in BCAA-mediated adverse effects on cardiac function/structure post-MI. Of note, pharmacological inhibition of branched chain ketoacid dehydrogenase kinase (BDK), a negative regulator of myocardial BCAA catabolism, significantly improved cardiac BCAA catabolic disorders, reduced myocardial BCAA levels, and ameliorated post-MI cardiac dysfunction and remodeling. In conclusion, our data provide the evidence that impaired cardiac BCAA catabolism directly contributes to post-MI cardiac dysfunction and remodeling. Moreover, improving cardiac BCAA catabolic defects may be a promising therapeutic strategy against post-MI HF. Copyright © 2016 the American Physiological Society.
Rational Design of a New Trypanosoma rangeli Trans-Sialidase for Efficient Sialylation of Glycans
DEFF Research Database (Denmark)
Jers, Carsten; Michalak, Malwina; Larsen, Dorte Møller
2014-01-01
This paper reports rational engineering of Trypanosoma rangeli sialidase to develop an effective enzyme for a potentially important type of reactivity: production of sialylated prebiotic glycans. The Trypanosoma cruzi trans-sialidase and the homologous T. rangeli sialidase has previously been use...
International Nuclear Information System (INIS)
Petzke, K.J.; Albrecht, V.; Przybilski, H.
1986-01-01
Male albino rats were adapted to isocaloric purified diets that differed mainly in their glycine and casein contents. Controls received a 30% casein diet. In experimental diets gelatin or gelatin hydrolysate was substituted for half of the 30% casein. An additional group was fed a glycine-supplemented diet, which corresponded in glycine level to the gelatin diet but in which the protein level was nearly the same as that of the casein control diet. Another group received a 15% casein diet. Rat liver glycine cleavage system, serine hydroxymethyltransferase and serine dehydratase activities were measured. 14 CO 2 production from the catabolism of 14 C-labeled glycine was measured in vivo and in vitro (from isolated hepatocytes). Serine dehydratase and glycine cleavage system activities were higher in animals fed 30% casein diets than in those fed 15% casein diets. Serine hydroxymethyltransferase activity of the cytosolic and mitochondrial fractions was highest when a high glycine diet (glycine administered as pure, protein bound in gelatin or peptide bound in gelatin hydrolysate) was fed. 14 CO 2 formation from [1- 14 C]- and [2- 14 C]glycine both in vivo and in isolated hepatocytes was higher when a high glycine diet was fed than when a casein diet was fed. These results suggest that glycine catabolism is dependent on and adaptable to the glycine content of the diet. Serine hydroxymethyltransferase appears to play a major role in the regulation of glycine degradation via serine and pyruvate
Development of phenanthrene catabolism in natural and artificial soils
International Nuclear Information System (INIS)
Rhodes, Angela H.; Hofman, Jakub; Semple, Kirk T.
2008-01-01
The characteristics of natural soils often vary from those of artificial soil (e.g. OECD), which may lead to substantial differences in the bioavailability of test substances. The aim of this investigation was to characterise the development of phenanthrene catabolism in both natural and artificial soils with varying total organic carbon (TOC) content after 1, 14, 42 and 84 d soil-phenanthrene contact time. Indigenous catabolic activity was measured via the addition of 14 C-phenanthrene using the respirometric soil slurry assay. Notably, the lag phases, fastest rates and total extents of 14 C-phenanthrene degradation were relatively comparable in soils with similar TOC content after 1 d contact time. However, natural soils generally exhibited significantly shorter lag phases, faster rates and higher extents of mineralisation, than their artificial counterparts after 42 and 84 d contact time. Such findings suggest that the extrapolation of results from artificial soils to real/natural soils may not be straightforward. - Natural and artificial soils display different phenanthrene mineralisation profiles suggesting that the extrapolation of results from artificial soils to real/natural soils may not be straightforward
Sandhoff, Konrad
2016-11-01
Glycosphingolipids and sphingolipids of cellular plasma membranes (PMs) reach luminal intra-lysosomal vesicles (LVs) for degradation mainly by pathways of endocytosis. After a sorting and maturation process (e.g. degradation of sphingomyelin (SM) and secretion of cholesterol), sphingolipids of the LVs are digested by soluble enzymes with the help of activator (lipid binding and transfer) proteins. Inherited defects of lipid-cleaving enzymes and lipid binding and transfer proteins cause manifold and fatal, often neurodegenerative diseases. The review summarizes recent findings on the regulation of sphingolipid catabolism and cholesterol secretion from the endosomal compartment by lipid modifiers, an essential stimulation by anionic membrane lipids and an inhibition of crucial steps by cholesterol and SM. Reconstitution experiments in the presence of all proteins needed, hydrolase and activator proteins, reveal an up to 10-fold increase of ganglioside catabolism just by the incorporation of anionic lipids into the ganglioside carrying membranes, whereas an additional incorporation of cholesterol inhibits GM2 catabolism substantially. It is suggested that lipid and other low molecular modifiers affect the genotype-phenotype relationship observed in patients with lysosomal diseases. Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.
Yang, Weizhun; Ramadan, Sherif; Yang, Bo; Yoshida, Keisuke; Huang, Xuefei
2016-12-02
Among many hurdles in synthesizing proteoglycan glycopeptides, one challenge is the incorporation of aspartic acid in the peptide backbone and acid sensitive O-sulfated glycan chains. To overcome this, a new strategy was developed utilizing homoserine as an aspartic acid precursor. The conversion of homoserine to aspartic acid in the glycopeptide was successfully accomplished by late stage oxidation using (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) and bis(acetoxy)iodobenzene (BAIB). This is the first time that a glycopeptide containing aspartic acid and an O-sulfated glycan was synthesized.
Directory of Open Access Journals (Sweden)
Ying-Ming Wang
Full Text Available Acutobin isolated from Deinagkistrodon acutus venom has been used to prevent or treat stroke in patients. This defibrinogenating serine protease is a 39 kDa glycoprotein containing terminal disialyl-capped N-glycans. After sialidase treatment, the enzyme showed similar catalytic activities toward chromogenic substrate, and cleaved the Aα chain of fibrinogen as efficiently as the native acutobin did. However, the level of fibrinogen degradation products in mice after i.p.-injection of desialylated-acutobin was significantly lower than the level after acutobin injection, suggesting that the disialyl moieties may improve or prolong the half-life of acutobin. Two recombinant enzymes with identical protein structures and similar amidolytic activities to those of native acutobin were expressed from HEK293T and SW1353 cells and designated as HKATB and SWATB, respectively. Mass spectrometric profiling showed that their glycans differed from those of acutobin. In contrast to acutobin, HKATB cleaved not only the Aα chain but also the Bβ and γ chains of human fibrinogens, while SWATB showed a reduced α-fibrinogenase activity. Non-denaturing deglycosylation of these proteases by peptide N-glycosidase F significantly reduced their fibrinogenolytic activities and thermal stabilities. The in vivo defibrinogenating effect of HKATB was inferior to that of acutobin in mice. Taken together, our results suggest that the conjugated glycans of acutobin are involved in its interaction with fibrinogen, and that the selection of cells optimally expressing efficient glycoforms and further glycosylation engineering are desirable before a recombinant product can replace the native enzyme for clinical use.
Date, Kimie; Satoh, Ayano; Iida, Kaoruko; Ogawa, Haruko
2015-01-01
α-Amylase, a major pancreatic protein and starch hydrolase, is essential for energy acquisition. Mammalian pancreatic α-amylase binds specifically to glycoprotein N-glycans in the brush-border membrane to activate starch digestion, whereas it significantly inhibits glucose uptake by Na+/glucose cotransporter 1 (SGLT1) at high concentrations (Asanuma-Date, K., Hirano, Y., Le, N., Sano, K., Kawasaki, N., Hashii, N., Hiruta, Y., Nakayama, K., Umemura, M., Ishikawa, K., Sakagami, H., and Ogawa, H. (2012) Functional regulation of sugar assimilation by N-glycan-specific interaction of pancreatic α-amylase with glycoproteins of duodenal brush border membrane. J. Biol. Chem. 287, 23104–23118). However, how the inhibition is stopped was unknown. Here, we show a new mechanism for the regulation of intestinal glucose absorption. Immunohistochemistry revealed that α-amylase in the duodena of non-fasted, but not fasted, pigs was internalized from the pancreatic fluid and immunostained. We demonstrated that after N-glycan binding, pancreatic α-amylase underwent internalization into lysosomes in a process that was inhibited by α-mannoside. The internalized α-amylase was degraded, showing low enzymatic activity and molecular weight at the basolateral membrane. In a human intestinal Caco-2 cell line, Alexa Fluor 488-labeled pancreatic α-amylase bound to the cytomembrane was transported to lysosomes through the endocytic pathway and then disappeared, suggesting degradation. Our findings indicate that N-glycan recognition by α-amylase protects enterocytes against a sudden increase in glucose concentration and restores glucose uptake by gradual internalization, which homeostatically controls the postprandial blood glucose level. The internalization of α-amylase may also enhance the supply of amino acids required for the high turnover of small intestine epithelial cells. This study provides novel and significant insights into the control of blood sugar during the absorption
Boyce, Kylie J.; McLauchlan, Alisha; Schreider, Lena; Andrianopoulos, Alex
2015-01-01
During infection, pathogens must utilise the available nutrient sources in order to grow while simultaneously evading or tolerating the host’s defence systems. Amino acids are an important nutritional source for pathogenic fungi and can be assimilated from host proteins to provide both carbon and nitrogen. The hpdA gene of the dimorphic fungus Penicillium marneffei, which encodes an enzyme which catalyses the second step of tyrosine catabolism, was identified as up-regulated in pathogenic yeast cells. As well as enabling the fungus to acquire carbon and nitrogen, tyrosine is also a precursor in the formation of two types of protective melanin; DOPA melanin and pyomelanin. Chemical inhibition of HpdA in P. marneffei inhibits ex vivo yeast cell production suggesting that tyrosine is a key nutrient source during infectious growth. The genes required for tyrosine catabolism, including hpdA, are located in a gene cluster and the expression of these genes is induced in the presence of tyrosine. A gene (hmgR) encoding a Zn(II)2-Cys6 binuclear cluster transcription factor is present within the cluster and is required for tyrosine induced expression and repression in the presence of a preferred nitrogen source. AreA, the GATA-type transcription factor which regulates the global response to limiting nitrogen conditions negatively regulates expression of cluster genes in the absence of tyrosine and is required for nitrogen metabolite repression. Deletion of the tyrosine catabolic genes in the cluster affects growth on tyrosine as either a nitrogen or carbon source and affects pyomelanin, but not DOPA melanin, production. In contrast to other genes of the tyrosine catabolic cluster, deletion of hpdA results in no growth within macrophages. This suggests that the ability to catabolise tyrosine is not required for macrophage infection and that HpdA has an additional novel role to that of tyrosine catabolism and pyomelanin production during growth in host cells. PMID:25812137
Directory of Open Access Journals (Sweden)
Hamna Saleem
2016-01-01
Full Text Available Petroleum hydrocarbons are harmful to living organisms when they are exposed in natural environment. Once they come in contact, it is not an easy to remove them because many of their constituents are persistent in nature. To achieve this target, different approaches have been exploited by using plants, bacteria, and plant-bacteria together. Among them, combined use of plants and bacteria has gained tremendous attention as bacteria possess set of catabolic genes which produce catabolic enzymes to decontaminate hydrocarbons. In return, plant ooze out root exudates containing nutrients and necessary metabolites which facilitate the microbial colonization in plant rhizosphere. This results into high gene abundance and gene expression in the rhizosphere and, thus, leads to enhanced degradation. Moreover, high proportions of beneficial bacteria helps plant to gain more biomass due to their plant growth promoting activities and production of phytohromones. This review focuses functioning and mechanisms of catabolic genes responsible for degradation of straight chain and aromatic hydrocarbons with their potential of degradation in bioremediation. With the understanding of expression mechanisms, rate of degradation can be enhanced by adjusting environmental factors and acclimatizing plant associated bacteria in plant rhizosphere.
Glycosylation at Asn91 of H1N1 haemagglutinin affects binding to glycan receptors.
Jayaraman, Akila; Koh, Xiaoying; Li, Jing; Raman, Rahul; Viswanathan, Karthik; Shriver, Zachary; Sasisekharan, Ram
2012-06-15
The glycoprotein HA (haemagglutinin) on the surface of influenza A virus plays a central role in recognition and binding to specific host cell-surface glycan receptors and in fusion of viral membrane to the host nuclear membrane during viral replication. Given the abundance of HA on the viral surface, this protein is also the primary target for host innate and adaptive immune responses. Although addition of glycosylation sites on HA are a part of viral evolution to evade the host immune responses, there are specific glycosylation sites that are conserved during most of the evolution of the virus. In the present study, it was demonstrated that one such conserved glycosylation site at Asn(91) in H1N1 HA critically governs the glycan receptor-binding specificity and hence would potentially impinge on the host adaptation of the virus.
International Nuclear Information System (INIS)
McDonald, Terence P.; Jeffree, Chris E.; Li, Ping; Rixon, Helen W. McL.; Brown, Gaie; Aitken, James D.; MacLellan, Kirsty; Sugrue, Richard J.
2006-01-01
Glycan heterogeneity of the respiratory syncytial virus (RSV) fusion (F) protein was demonstrated by proteomics. The effect of maturation of the virus glycoproteins-associated glycans on virus infectivity was therefore examined using the α-mannosidase inhibitors deoxymannojirimycin (DMJ) and swainsonine (SW). In the presence of SW the N-linked glycans on the F protein appeared in a partially mature form, whereas in the presence of DMJ no maturation of the glycans was observed. Neither inhibitor had a significant effect on G protein processing or on the formation of progeny virus. Although the level of infectious virus and syncytia formation was not significantly affected by SW-treatment, DMJ-treatment correlated with a one hundred-fold reduction in virus infectivity. Our data suggest that glycan maturation of the RSV glycoproteins, in particular those on the F protein, is an important step in virus maturation and is required for virus infectivity
International Nuclear Information System (INIS)
Daugherty, A.; Thorpe, S.R.; Lange, L.G.; Sobel, B.E.; Schonfeld, G.
1985-01-01
beta-Very low density lipoprotein (beta-VLDL) may be a major atherogenic lipoprotein, and knowledge of the sites of its catabolism should facilitate elucidation of mechanisms important in the regulation of its plasma concentrations. In this study, catabolic sites of beta-VLDL have been delineated in normolipidemic rabbits with a novel, radioiodinated, residualizing label, 125 I-dilactitol tyramine ( 125 I-DLT). Comparative studies of beta-VLDL and low density lipoprotein catabolism were performed with 125 I-DLT conjugated to each lipoprotein and with lipoproteins iodine-labeled conventionally. Conjugation did not alter size distributions or charge characteristics of lipoprotein particles. The overall processing (binding and degradation) of lipoproteins by cultured rabbit skin fibroblasts was not influenced by 125 I-DLT derivatization, suggesting that attachment of the label did not influence cell receptor-lipoprotein interactions. Furthermore, although degradation products of 125 I-lipoproteins leaked out of the cells and into the medium, the degradation products of 125 I-DLT lipoproteins were retained by the cells. The principal catabolic site of beta-VLDL in normolipidemic rabbits was found to be the liver with 54 +/- 4% of injected 125 I retained in this organ 24 h after injection of 125 I-DLT-beta-VLDL. When catabolism was normalized to tissue weight, the liver and adrenals were found to be approximately equally active in the metabolism of beta-VLDL. In agreement with results of other studies with residualizing labels, the principal organ of catabolism of 125 I-DLT-LDL in vivo was the liver. The adrenals were the most highly catabolizing organ when results were normalized for tissue weight
Xu, Minjun; Kitaura, Yasuyuki; Shindo, Daichi; Shimomura, Yoshiharu
2018-03-01
Branched-chain α-keto acid dehydrogenase (BCKDH) kinase (BDK) suppresses the branched-chain amino acid (BCAA) catabolism by inactivation of the BCKDH complex. The muscle-specific BDK-deficient (BDK-mKO) mice showed accelerated BCAA oxidation in muscle and decreased endurance capacity after training (Xu et al. PLoS One. 12 (2017) e0180989). We here report that BCAA supplementation overcompensated endurance capacity in BDK-mKO mice after training.
Energy Technology Data Exchange (ETDEWEB)
Clarkson, Sonya M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Giannone, Richard J. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Chemical Sciences Division; Kridelbaugh, Donna M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Elkins, James G. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Guss, Adam M. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Michener, Joshua K. [Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center; Vieille, Claire [Michigan State Univ., East Lansing, MI (United States)
2017-07-21
The production of biofuels from lignocellulose yields a substantial lignin by-product stream that currently has few applications. Biological conversion of lignin-derived compounds into chemicals and fuels has the potential to improve the economics of lignocellulose-derived biofuels, but few microbes are able both to catabolize lignin-derived aromatic compounds and to generate valuable products. While
Váradi, Csaba; Mittermayr, Stefan; Millán-Martín, Silvia; Bones, Jonathan
2016-12-01
Capillary electrophoresis (CE) offers excellent efficiency and orthogonality to liquid chromatographic (LC) separations for oligosaccharide structural analysis. Combination of CE with high resolution mass spectrometry (MS) for glycan analysis remains a challenging task due to the MS incompatibility of background electrolyte buffers and additives commonly used in offline CE separations. Here, a novel method is presented for the analysis of 2-aminobenzoic acid (2-AA) labelled glycans by capillary electrophoresis coupled to mass spectrometry (CE-MS). To ensure maximum resolution and excellent precision without the requirement for excessive analysis times, CE separation conditions including the concentration and pH of the background electrolyte, the effect of applied pressure on the capillary inlet and the capillary length were evaluated. Using readily available 12/13 C 6 stable isotopologues of 2-AA, the developed method can be applied for quantitative glycan profiling in a twoplex manner based on the generation of extracted ion electropherograms (EIE) for 12 C 6 'light' and 13 C 6 'heavy' 2-AA labelled glycan isotope clusters. The twoplex quantitative CE-MS glycan analysis platform is ideally suited for comparability assessment of biopharmaceuticals, such as monoclonal antibodies, for differential glycomic analysis of clinical material for potential biomarker discovery or for quantitative microheterogeneity analysis of different glycosylation sites within a glycoprotein. Additionally, due to the low injection volume requirements of CE, subsequent LC-MS analysis of the same sample can be performed facilitating the use of orthogonal separation techniques for structural elucidation or verification of quantitative performance.
Glycan Sulfation Modulates Dendritic Cell Biology and Tumor Growth
Directory of Open Access Journals (Sweden)
Roland El Ghazal
2016-05-01
Full Text Available In cancer, proteoglycans have been found to play roles in facilitating the actions of growth factors, and effecting matrix invasion and remodeling. However, little is known regarding the genetic and functional importance of glycan chains displayed by proteoglycans on dendritic cells (DCs in cancer immunity. In lung carcinoma, among other solid tumors, tumor-associated DCs play largely subversive/suppressive roles, promoting tumor growth and progression. Herein, we show that targeting of DC glycan sulfation through mutation in the heparan sulfate biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1 in mice increased DC maturation and inhibited trafficking of DCs to draining lymph nodes. Lymphatic-driven DC migration and chemokine (CCL21-dependent activation of a major signaling pathway required for DC migration (as measured by phospho-Akt were sensitive to Ndst1 mutation in DCs. Lewis lung carcinoma tumors in mice deficient in Ndst1 were reduced in size. Purified CD11c+ cells from the tumors, which contain the tumor-infiltrating DC population, showed a similar phenotype in mutant cells. These features were replicated in mice deficient in syndecan-4, the major heparan sulfate proteoglycan expressed on the DC surface: Tumors were growth-impaired in syndecan-4–deficient mice and were characterized by increased infiltration by mature DCs. Tumors on the mutant background also showed greater infiltration by NK cells and NKT cells. These findings indicate the genetic importance of DC heparan sulfate proteoglycans in tumor growth and may guide therapeutic development of novel strategies to target syndecan-4 and heparan sulfate in cancer.
Accogli, Gianluca; Douet, Cécile; Ambruosi, Barbara; Martino, Nicola Antonio; Uranio, Manuel Filioli; Deleuze, Stefan; Dell'Aquila, Maria Elena; Desantis, Salvatore; Goudet, Ghylène
2014-12-01
Glycoprotein oligosaccharides play major roles during reproduction, yet their function in gamete interactions is not fully elucidated. Identification and comparison of the glycan pattern in cumulus-oocyte complexes (COCs) from species with different efficiencies of in vitro spermatozoa penetration through the zona pellucida (ZP) could help clarify how oligosaccharides affect gamete interactions. We compared the expression and localization of 12 glycosidic residues in equine and porcine in vitro-matured (IVM) and preovulatory COCs by means of lectin histochemistry. The COCs glycan pattern differed between animals and COC source (IVM versus preovulatory). Among the 12 carbohydrate residues investigated, the IVM COCs from these two species shared: (a) sialo- and βN-acetylgalactosamine (GalNAc)-terminating glycans in the ZP; (b) sialylated and fucosylated glycans in cumulus cells; and (c) GalNAc and N-acetylglucosamine (GlcNAc) glycans in the ooplasm. Differences in the preovulatory COCs of the two species included: (a) sialoglycans and GlcNAc terminating glycans in the equine ZP versus terminal GalNAc and internal GlcNAc in the porcine ZP; (b) terminal galactosides in equine cumulus cells versus terminal GlcNAc and fucose in porcine cohorts; and (c) fucose in the mare ooplasm versus lactosamine and internal GlcNAc in porcine oocyte cytoplasm. Furthermore, equine and porcine cumulus cells and oocytes contributed differently to the synthesis of ZP glycoproteins. These results could be attributed to the different in vitro fertilization efficiencies between these two divergent, large-animal models. © 2014 Wiley Periodicals, Inc.
Hesselink, T.; Rouwendal, G.J.A.; Henquet, M.G.L.; Florack, D.E.A.; Helsper, J.P.F.G.; Bosch, H.J.
2014-01-01
b1,4-Galactosylation of plant N-glycans is a prerequisite for commercial production of certain biopharmaceuticals in plants. Two different types of galactosylated N-glycans have initially been reported in plants as the result of expression of human b1,4-galactosyltransferase 1 (GalT). Here we show
Formation of Flavor Compounds by Amino Acid Catabolism in Cheese (Turkish with English Abstract
Directory of Open Access Journals (Sweden)
2015-02-01
Full Text Available Biochemical reactions which contribute flavor formation occur in result of proteolysis during cheese ripening. Casein as the main protein of cheese has a significant effect on the flavor and textural properties of cheeses via its degradation to small peptides and free amino acids by various factors like coagulant enzymes. Specific flavors of cheeses occur as a result of amino acid catabolism by starter and non-starter bacteria. Some flavor compounds are formed by enzymatic transformations as well as by non-enzymatic, chemical changes in cheese. In this paper, formation of flavor compounds by amino acid catabolism during cheese ripening reviewed.
Hirooka, Kazutake; Kodoi, Yusuke; Satomura, Takenori; Fujita, Yasutaro
2015-12-28
The Bacillus subtilis rhaEWRBMA (formerly yuxG-yulBCDE) operon consists of four genes encoding enzymes for l-rhamnose catabolism and the rhaR gene encoding a DeoR-type transcriptional regulator. DNase I footprinting analysis showed that the RhaR protein specifically binds to the regulatory region upstream of the rhaEW gene, in which two imperfect direct repeats are included. Gel retardation analysis revealed that the direct repeat farther upstream is essential for the high-affinity binding of RhaR and that the DNA binding of RhaR was effectively inhibited by L-rhamnulose-1-phosphate, an intermediate of L-rhamnose catabolism. Moreover, it was demonstrated that the CcpA/P-Ser-HPr complex, primarily governing the carbon catabolite control in B. subtilis, binds to the catabolite-responsive element, which overlaps the RhaR binding site. In vivo analysis of the rhaEW promoter-lacZ fusion in the background of ccpA deletion showed that the L-rhamnose-responsive induction of the rhaEW promoter was negated by the disruption of rhaA or rhaB but not rhaEW or rhaM, whereas rhaR disruption resulted in constitutive rhaEW promoter activity. These in vitro and in vivo results clearly indicate that RhaR represses the operon by binding to the operator site, which is detached by L-rhamnulose-1-phosphate formed from L-rhamnose through a sequence of isomerization by RhaA and phosphorylation by RhaB, leading to the derepression of the operon. In addition, the lacZ reporter analysis using the strains with or without the ccpA deletion under the background of rhaR disruption supported the involvement of CcpA in the carbon catabolite repression of the operon. Since L-rhamnose is a component of various plant-derived compounds, it is a potential carbon source for plant-associating bacteria. Moreover, it is suggested that L-rhamnose catabolism plays a significant role in some bacteria-plant interactions, e.g., invasion of plant pathogens and nodulation of rhizobia. Despite the physiological
International Nuclear Information System (INIS)
Giménez, Estela; Balmaña, Meritxell; Figueras, Joan; Fort, Esther; Bolós, Carme de; Sanz-Nebot, Victòria; Peracaula, Rosa; Rizzi, Andreas
2015-01-01
Highlights: • The method enables relative quantitation of hAGP glycans from pathological samples • Pancreatic cancer samples clearly showed an increase of hAGP fucosylated glycans. • Fucosylated glycans could be potential biomarkers for diagnosing pancreatic cancer. • The established method could be extremely useful to find novel glycoprotein biomarkers - Abstract: In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [ 12 C]- and [ 13 C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (μZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α 1 -acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and μZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in hAGP as a
Novel Route for Agmatine Catabolism in Aspergillus niger Involves 4-Guanidinobutyrase.
Kumar, Sunil; Saragadam, Tejaswani; Punekar, Narayan S
2015-08-15
Agmatine, a significant polyamine in bacteria and plants, mostly arises from the decarboxylation of arginine. The functional importance of agmatine in fungi is poorly understood. The metabolism of agmatine and related guanidinium group-containing compounds in Aspergillus niger was explored through growth, metabolite, and enzyme studies. The fungus was able to metabolize and grow on l-arginine, agmatine, or 4-guanidinobutyrate as the sole nitrogen source. Whereas arginase defined the only route for arginine catabolism, biochemical and bioinformatics approaches suggested the absence of arginine decarboxylase in A. niger. Efficient utilization by the parent strain and also by its arginase knockout implied an arginase-independent catabolic route for agmatine. Urea and 4-guanidinobutyrate were detected in the spent medium during growth on agmatine. The agmatine-grown A. niger mycelia contained significant levels of amine oxidase, 4-guanidinobutyraldehyde dehydrogenase, 4-guanidinobutyrase (GBase), and succinic semialdehyde dehydrogenase, but no agmatinase activity was detected. Taken together, the results support a novel route for agmatine utilization in A. niger. The catabolism of agmatine by way of 4-guanidinobutyrate to 4-aminobutyrate into the Krebs cycle is the first report of such a pathway in any organism. A. niger GBase peptide fragments were identified by tandem mass spectrometry analysis. The corresponding open reading frame from the A. niger NCIM 565 genome was located and cloned. Subsequent expression of GBase in both Escherichia coli and A. niger along with its disruption in A. niger functionally defined the GBase locus (gbu) in the A. niger genome. Copyright © 2015, American Society for Microbiology. All Rights Reserved.
Amino acid repletion does not decrease muscle protein catabolism during hemodialysis.
Raj, Dominic S C; Adeniyi, Oladipo; Dominic, Elizabeth A; Boivin, Michel A; McClelland, Sandra; Tzamaloukas, Antonios H; Morgan, Nancy; Gonzales, Lawrence; Wolfe, Robert; Ferrando, Arny
2007-06-01
Intradialytic protein catabolism is attributed to loss of amino acids in the dialysate. We investigated the effect of amino acid infusion during hemodialysis (HD) on muscle protein turnover and amino acid transport kinetics by using stable isotopes of phenylalanine, leucine, and lysine in eight patients with end-stage renal disease (ESRD). Subjects were studied at baseline (pre-HD), 2 h of HD without amino acid infusion (HD-O), and 2 h of HD with amino acid infusion (HD+AA). Amino acid depletion during HD-O augmented the outward transport of amino acids from muscle into the vein. Increased delivery of amino acids to the leg during HD+AA facilitated the transport of amino acids from the artery into the intracellular compartment. Increase in muscle protein breakdown was more than the increase in synthesis during HD-O (46.7 vs. 22.3%, P HD-O compared with pre-HD (-33.7 +/- 1.5 vs. -6.0 +/- 2.3, P acids, the net balance (-16.9 +/- 1.8) did not switch from net release to net uptake. HD+AA induced a proportional increase in muscle protein synthesis and catabolism. Branched chain amino acid catabolism increased significantly from baseline during HD-O and did not decrease during HD+AA. Protein synthesis efficiency, the fraction of amino acid in the intracellular pool that is utilized for muscle protein synthesis decreased from 42.1% pre-HD to 33.7 and 32.6% during HD-O and HD+AA, respectively (P acid repletion during HD increased muscle protein synthesis but did not decrease muscle protein breakdown.
DEFF Research Database (Denmark)
Gallina, Serafina; Saletti, Rosaria; Cunsolo, Vincenzo
2016-01-01
A comprehensive monosaccharide composition of the N-glycans of donkey milk lactoferrin, isolated by ion exchange chromatography from an individual milk sample, was obtained by means of chymotryptic digestion, TiO2 and HILIC enrichment, reversed-phase high-performance liquid chromatography......, electrospray mass spectrometry, and high collision dissociation fragmentation. The results obtained allowed identifying 26 different glycan structures, including high mannose, complex and hybrid N-glycans, linked to the protein backbone via an amide bond to asparagine residues located at the positions 137, 281...... and 476. Altogether, the N-glycan structures determined revealed that most of the N-glycans identified in donkey milk lactoferrin are neutral complex/hybrid. Indeed, 10 neutral non-fucosylated complex/hybrid N-glycans and 4 neutral fucosylated complex/hybrid N-glycans were found. In addition, two high...
Phosphonate biosynthesis and catabolism: a treasure trove of unusual enzymology.
Peck, Spencer C; van der Donk, Wilfred A
2013-08-01
Natural product biosynthesis has proven a fertile ground for the discovery of novel chemistry. Herein we review the progress made in elucidating the biosynthetic pathways of phosphonate and phosphinate natural products such as the antibacterial compounds dehydrophos and fosfomycin, the herbicidal phosphinothricin-containing peptides, and the antimalarial compound FR-900098. In each case, investigation of the pathway has yielded unusual, and often unprecedented, biochemistry. Likewise, recent investigations have uncovered novel ways to cleave the CP bond to yield phosphate under phosphorus starvation conditions. These include the discovery of novel oxidative cleavage of the CP bond catalyzed by PhnY and PhnZ as well as phosphonohydrolases that liberate phosphate from phosphonoacetate. Perhaps the crown jewel of phosphonate catabolism has been the recent resolution of the longstanding problem of the C-P lyase responsible for reductively cleaving the CP bond of a number of different phosphonates to release phosphate. Taken together, the strides made on both metabolic and catabolic fronts illustrate an array of fascinating biochemistry. Copyright © 2013 Elsevier Ltd. All rights reserved.
DEFF Research Database (Denmark)
Gram, G J; Hemming, A; Bolmstedt, A
1994-01-01
Glycosylation is necessary for HIV-1 gp120 to attain a functional conformation, and individual N-linked glycans of gp120 are important, but not essential, for replication of HIV-1 in cell culture. We have constructed a mutant HIV-1 infectious clone lacking a signal for N-linked glycosylation...... in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor...... affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by Con...
Nock, Adam M; Wargo, Matthew J
2016-09-15
Burkholderia thailandensis is a soil-dwelling bacterium that shares many metabolic pathways with the ecologically similar, but evolutionarily distant, Pseudomonas aeruginosa Among the diverse nutrients it can utilize is choline, metabolizable to the osmoprotectant glycine betaine and subsequently catabolized as a source of carbon and nitrogen, similar to P. aeruginosa Orthologs of genes in the choline catabolic pathway in these two bacteria showed distinct differences in gene arrangement as well as an additional orthologous transcriptional regulator in B. thailandensis In this study, we showed that multiple glutamine amidotransferase 1 (GATase 1)-containing AraC family transcription regulators (GATRs) are involved in regulation of the B. thailandensis choline catabolic pathway (gbdR1, gbdR2, and souR). Using genetic analyses and sequencing the transcriptome in the presence and absence of choline, we identified the likely regulons of gbdR1 (BTH_II1869) and gbdR2 (BTH_II0968). We also identified a functional ortholog for P. aeruginosa souR, a GATR that regulates the metabolism of sarcosine to glycine. GbdR1 is absolutely required for expression of the choline catabolic locus, similar to P. aeruginosa GbdR, while GbdR2 is important to increase expression of the catabolic locus. Additionally, the B. thailandensis SouR ortholog (BTH_II0994) is required for catabolism of choline and its metabolites as carbon sources, whereas in P. aeruginosa, SouR function can by bypassed by GbdR. The strategy employed by B. thailandensis represents a distinct regulatory solution to control choline catabolism and thus provides both an evolutionary counterpoint and an experimental system to analyze the acquisition and regulation of this pathway during environmental growth and infection. Many proteobacteria that occupy similar environmental niches have horizontally acquired orthologous genes for metabolism of compounds useful in their shared environment. The arrangement and differential
Directory of Open Access Journals (Sweden)
Elham Peyfoon
2010-01-01
Full Text Available Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L1004-Q1395. Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS, consistent with the tribranched hexasaccharide previously reported in the cytochrome b558/566 of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.
Nagels, Bieke; Van Damme, Els J M; Callewaert, Nico; Zabeau, Lennart; Tavernier, Jan; Delanghe, Joris R; Boets, Annemie; Castilho, Alexandra; Weterings, Koen
2012-08-31
In the past two decades plants have emerged as a valuable alternative for the production of pharmaceutical proteins. Since N-glycosylation influences functionality and stability of therapeutic proteins, the plant N-glycosylation pathway should be humanized. Here, we report the transient magnICON(®) expression of the erythropoietin fusion protein (EPO-Fc) in Nicotiana benthamiana plants that produce multi-antennary N-glycans without the plant-specific β1,2-xylose and α1,3-fucose residues in a stable manner (Nagels et al., 2011). The EPO-Fc fusion protein consists of EPO with a C-terminal-linked IgG-Fc domain and is used for pulmonary delivery of recombinant EPO to patients (Bitonti et al., 2004). Plant expressed EPO-Fc was quantified using a paramagnetic-particle chemiluminescent immunoassay and shown to be active in vitro via receptor binding experiments in HEK293T cells. Mass spectrometry-based N-glycan analysis confirmed the presence of multi-antennary N-glycans on plant-expressed EPO-Fc. The described research is the next step towards the development of a production platform for pharmaceutical proteins in plants. Copyright © 2012 Elsevier B.V. All rights reserved.
Strasser, Richard; Stadlmann, Johannes; Schähs, Matthias; Stiegler, Gabriela; Quendler, Heribert; Mach, Lukas; Glössl, Josef; Weterings, Koen; Pabst, Martin; Steinkellner, Herta
2008-05-01
A common argument against using plants as a production system for therapeutic proteins is their inability to perform authentic human N-glycosylation (i.e. the presence of beta1,2-xylosylation and core alpha1,3-fucosylation). In this study, RNA interference (RNAi) technology was used to obtain a targeted down-regulation of the endogenous beta1,2-xylosyltransferase (XylT) and alpha1,3-fucosyltransferase (FucT) genes in Nicotiana benthamiana, a tobacco-related plant species widely used for recombinant protein expression. Three glyco-engineered lines with significantly reduced xylosylated and/or core alpha1,3-fucosylated glycan structures were generated. The human anti HIV monoclonal antibody 2G12 was transiently expressed in these glycosylation mutants as well as in wild-type plants. Four glycoforms of 2G12 differing in the presence/absence of xylose and core alpha1,3-fucose residues in their N-glycans were produced. Notably, 2G12 produced in XylT/FucT-RNAi plants was found to contain an almost homogeneous N-glycan species without detectable xylose and alpha1,3-fucose residues. Plant-derived glycoforms were indistinguishable from Chinese hamster ovary (CHO)-derived 2G12 with respect to electrophoretic properties, and exhibited functional properties (i.e. antigen binding and HIV neutralization activity) at least equivalent to those of the CHO counterpart. The generated RNAi lines were stable, viable and did not show any obvious phenotype, thus providing a robust tool for the production of therapeutically relevant glycoproteins in plants with a humanized N-glycan structure.
International Nuclear Information System (INIS)
Redfern, J.S.
1988-01-01
Antral and fundic mucosal homogenates obtained from prostaglandin E2-immunized rabbits converted 14C-arachidonic acid to prostaglandin E2, 6-keto prostaglandin F1 alpha, prostaglandin F2 alpha, and prostaglandin D2. Percentage conversion of 14C-arachidonic acid to these prostaglandin products was not significantly different in prostaglandin E2-immunized rabbits compared with control rabbits (thyroglobulin-immunized and unimmunized rabbits combined). Synthesis of 6-keto prostaglandin F1 alpha, prostaglandin E2 and 13,14-dihydro 15-keto prostaglandin E2 from endogenous arachidonic acid after vortex mixing fundic mucosal homogenates was similar in prostaglandin E2 immunized rabbits and control rabbits. Both in prostaglandin E2-immunized rabbits and controls, 3H-prostaglandin E2 was catabolized extensively by the fundic mucosa, whereas 3H-6-keto prostaglandin F1 alpha, 3H-prostaglandin F2 alpha, and 3H-prostaglandin D2 were not catabolized to any appreciable extent. The rate of catabolism of PGs was not significantly different in prostaglandin E2-immunized rabbits and control rabbits, with the exception of prostaglandin F2 alpha which was catabolized slightly more rapidly in prostaglandin E2-immunized rabbits. These results indicate that development of gastric ulcers in prostaglandin E2-immunized rabbits is not associated with an alteration in the capacity of the gastric mucosa to synthesize or catabolize prostaglandins
Arias-Barrau, Elsa; Olivera, Elías R; Luengo, José M; Fernández, Cristina; Galán, Beatriz; García, José L; Díaz, Eduardo; Miñambres, Baltasar
2004-08-01
Pseudomonas putida metabolizes Phe and Tyr through a peripheral pathway involving hydroxylation of Phe to Tyr (PhhAB), conversion of Tyr into 4-hydroxyphenylpyruvate (TyrB), and formation of homogentisate (Hpd) as the central intermediate. Homogentisate is then catabolized by a central catabolic pathway that involves three enzymes, homogentisate dioxygenase (HmgA), fumarylacetoacetate hydrolase (HmgB), and maleylacetoacetate isomerase (HmgC), finally yielding fumarate and acetoacetate. Whereas the phh, tyr, and hpd genes are not linked in the P. putida genome, the hmgABC genes appear to form a single transcriptional unit. Gel retardation assays and lacZ translational fusion experiments have shown that hmgR encodes a specific repressor that controls the inducible expression of the divergently transcribed hmgABC catabolic genes, and homogentisate is the inducer molecule. Footprinting analysis revealed that HmgR protects a region in the Phmg promoter that spans a 17-bp palindromic motif and an external direct repetition from position -16 to position 29 with respect to the transcription start site. The HmgR protein is thus the first IclR-type regulator that acts as a repressor of an aromatic catabolic pathway. We engineered a broad-host-range mobilizable catabolic cassette harboring the hmgABC, hpd, and tyrB genes that allows heterologous bacteria to use Tyr as a unique carbon and energy source. Remarkably, we show here that the catabolism of 3-hydroxyphenylacetate in P. putida U funnels also into the homogentisate central pathway, revealing that the hmg cluster is a key catabolic trait for biodegradation of a small number of aromatic compounds.
Shak, S; Goldstein, I M
1984-08-25
Leukotriene B4 (LTB4), formed by the 5-lipoxygenase pathway in human polymorphonuclear leukocytes (PMN), may be an important mediator of inflammation. Recent studies suggest that human leukocytes can convert LTB4 to products that are less biologically active. To examine the catabolism of LTB4, we developed (using high performance liquid chromatography) a sensitive, reproducible assay for this mediator and its omega-oxidation products (20-OH- and 20-COOH-LTB4). With this assay, we have found that human PMN (but not human monocytes, lymphocytes, or platelets) convert exogenous LTB4 almost exclusively to 20-OH- and 20-COOH-LTB4 (identified by gas chromatography-mass spectrometry). Catabolism of exogenous LTB4 by omega-oxidation is rapid (t1/2 approximately 4 min at 37 degrees C in reaction mixtures containing 1.0 microM LTB4 and 20 X 10(6) PMN/ml), temperature-dependent (negligible at 0 degrees C), and varies with cell number as well as with initial substrate concentration. The pathway for omega-oxidation in PMN is specific for LTB4 and 5(S),12(S)-dihydroxy-6,8,10,14-eicosatetraenoic acid (only small amounts of other dihydroxylated-derivatives of arachidonic acid are converted to omega-oxidation products). Even PMN that are stimulated by phorbol myristate acetate to produce large amounts of superoxide anion radicals catabolize exogenous leukotriene B4 primarily by omega-oxidation. Finally, LTB4 that is generated when PMN are stimulated with the calcium ionophore, A23187, is rapidly catabolized by omega-oxidation. Thus, human PMN not only generate and respond to LTB4, but also rapidly and specifically catabolize this mediator by omega-oxidation.
Decoding the Role of Glycans in Malaria
Directory of Open Access Journals (Sweden)
Pollyanna S. Gomes
2017-06-01
Full Text Available Complications arising from malaria are a concern for public health authorities worldwide, since the annual caseload in humans usually exceeds millions. Of more than 160 species of Plasmodium, only 4 infect humans, with the most severe cases ascribed to Plasmodium falciparum and the most prevalent to Plasmodium vivax. Over the past 70 years, since World War II, when the first antimalarial drugs were widely used, many efforts have been made to combat this disease, including vectorial control, new drug discoveries and genetic and molecular approaches. Molecular approaches, such as glycobiology, may lead to new therapeutic targets (both in the host and the parasites, since all interactions are mediated by carbohydrates or glycan moieties decorating both cellular surfaces from parasite and host cells. In this review, we address the carbohydrate-mediated glycobiology that directly affects Plasmodium survival or host resistance.
The role of polyamine catabolism in anti-tumour drug response.
Casero, R A; Wang, Y; Stewart, T M; Devereux, W; Hacker, A; Wang, Y; Smith, R; Woster, P M
2003-04-01
Interest in polyamine catabolism has increased since it has been directly associated with the cytotoxic response of multiple tumour types to exposure to specific anti-tumour polyamine analogues. Human polyamine catabolism was considered to be a two-step pathway regulated by the rate-limiting enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) that provides substrate for an acetylpolyamine oxidase (APAO). Further, the super-induction of SSAT by several anti-tumour polyamine analogues has been implicated in the cytotoxic response of specific solid-tumour phenotypes to these agents. This high induction of SSAT has been correlated with cellular response to the anti-tumour polyamine analogues in several systems and considerable progress has been made in understanding the molecular mechanisms that regulate the analogue-induced expression of SSAT. A polyamine response element has been identified and the transacting transcription factors that bind and stimulate transcription of SSAT have been cloned and characterized. The link between SSAT activity and cellular toxicity is thought to be based on the production of H(2)O(2) by the activity of the constitutive APAO that uses the SSAT-produced acetylated polyamines. The high induction of SSAT and the subsequent activity of APAO are linked to the cytotoxic response of some tumour cell types to specific polyamine analogues. However, we have recently cloned a variably spliced human polyamine oxidase (PAOh1) that is inducible by specific polyamine analogues, efficiently uses unacetylated spermine as a substrate, and also produces toxic H(2)O(2) as a product. The results of studies with PAOh1 suggest that it is an additional enzyme in polyamine catabolism that has the potential to significantly contribute to polyamine homoeostasis and drug response. Most importantly, PAOh1 is induced by specific polyamine analogues in a tumour-phenotype-specific manner in cell lines representative of the major forms of solid tumours, including
Energy Technology Data Exchange (ETDEWEB)
Giménez, Estela, E-mail: estelagimenez@ub.edu [Department of Analytical Chemistry, University of Barcelona, Diagonal 647, E-08028 Barcelona (Spain); Balmaña, Meritxell [Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi s/n, 17071 Girona (Spain); Figueras, Joan [Department of Surgery, Dr. Josep Trueta University Hospital, IdlBGi, 17007 Girona (Spain); Fort, Esther [Digestive Unit, Dr. Josep Trueta University Hospital, 17007 Girona (Spain); Bolós, Carme de [Gastroesophagic Cancer Research Group, Research Programme in Cancer, Hospital del Mar Medical Research Institute (IMIM), Dr. Aiguader, 88, 08003 Barcelona (Spain); Sanz-Nebot, Victòria [Department of Analytical Chemistry, University of Barcelona, Diagonal 647, E-08028 Barcelona (Spain); Peracaula, Rosa [Biochemistry and Molecular Biology Unit, Department of Biology, University of Girona, Campus Montilivi s/n, 17071 Girona (Spain); Rizzi, Andreas [Institute of Analytical Chemistry, University of Vienna, Währinger Straße 38, A-1090 Vienna (Austria)
2015-03-25
Highlights: • The method enables relative quantitation of hAGP glycans from pathological samples • Pancreatic cancer samples clearly showed an increase of hAGP fucosylated glycans. • Fucosylated glycans could be potential biomarkers for diagnosing pancreatic cancer. • The established method could be extremely useful to find novel glycoprotein biomarkers - Abstract: In this work we demonstrate the potential of glycan reductive isotope labeling (GRIL) using [{sup 12}C]- and [{sup 13}C]-coded aniline and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry (μZIC-HILIC-ESI-MS) for relative quantitation of glycosylation variants in selected glycoproteins present in samples from cancer patients. Human α{sub 1}-acid-glycoprotein (hAGP) is an acute phase serum glycoprotein whose glycosylation has been described to be altered in cancer and chronic inflammation. However, it is not clear yet whether some particular glycans in hAGP can be used as biomarker for differentiating between these two pathologies. In this work, hAGP was isolated by immunoaffinity chromatography (IAC) from serum samples of healthy individuals and from those suffering chronic pancreatitis and different stages of pancreatic cancer, respectively. After de-N-glycosylation, relative quantitation of the hAGP glycans was carried out using stable isotope labeling and μZIC-HILIC-ESI-MS analysis. First, protein denaturing conditions prior to PNGase F digestion were optimized to achieve quantitative digestion yields, and the reproducibility of the established methodology was evaluated with standard hAGP. Then, the proposed method was applied to the analysis of the clinical samples (control vs. pathological). Pancreatic cancer samples clearly showed an increase in the abundance of fucosylated glycans as the stage of the disease increases and this was unlike to samples from chronic pancreatitis. The results gained here indicate the mentioned glycan in h
Satoh, Tadashi; Toshimori, Takayasu; Noda, Masanori; Uchiyama, Susumu; Kato, Koichi
2016-11-01
The glycoside hydrolase family 31 (GH31) α-glucosidases play vital roles in catabolic and regulated degradation, including the α-subunit of glucosidase II (GIIα), which catalyzes trimming of the terminal glucose residues of N-glycan in glycoprotein processing coupled with quality control in the endoplasmic reticulum (ER). Among the known GH31 enzymes, only GIIα functions with its binding partner, regulatory β-subunit (GIIβ), which harbors a lectin domain for substrate recognition. Although the structural data have been reported for GIIα and the GIIβ lectin domain, the interaction mode between GIIα and GIIβ remains unknown. Here, we determined the structure of a complex formed between GIIα and the GIIα-binding domain of GIIβ, thereby providing a structural basis underlying the functional extension of this unique GH31 enzyme. © 2016 The Protein Society.
DEFF Research Database (Denmark)
Hansen, J.-E.S.; Heegaard, Peter M. H.; Jensen, S.P.
1988-01-01
types were different in copper content, and one type could reversibly be changed into the other. The glycan microheterogeneity of ceruloplasmin was analyzed by crossed affinommunoelectrophoresis with free Lens culinaris agglutinin (LCA) and wheat germ agglutinin (WGA). A third of the ceruloplasmin...
Del Puerto, Lucía; Rovetta, Romina; Navatta, Marco; Fontana, Carolina; Lin, Gerardo; Moyna, Guillermo; Dematteis, Sylvia; Brehm, Klaus; Koziol, Uriel; Ferreira, Fernando; Díaz, Alvaro
2016-05-01
The larval stages of the cestodes Echinococcus multilocularis and Echinococcus granulosus cause the important zoonoses known as larval echinococcoses. These larvae are protected by a unique, massive, mucin-based structure known as the laminated layer. The mucin glycans of the E. granulosus laminated layer are core 1- or core 2-based O-glycans in which the core Galpβ1-3 residue can initiate a chain comprising one to three additional Galpβ1-3 residues, a motif not known in mammalian carbohydrates. This chain can be capped with a Galpα1-4 residue, and can be ramified with GlcNAcpβ1-6 residues. These, as well as the GlcNAcpβ1-6 residue in core 2, can be decorated with the Galpα1-4Galpβ1-4 disaccharide. Here we extend our analysis to the laminated layer of E. multilocularis, showing that the non-decorated cores, together with Galpβ1-3(Galpα1-4Galpβ1-4GlcNAcpβ1-6)GalNAc, comprise over 96% of the glycans in molar terms. This simple laminated layer glycome is exhibited by E. multilocularis grown either in vitro or in vivo. Interestingly, all the differences with the complex laminated layer glycome found in E. granulosus may be explained in terms of strongly reduced activity in E. multilocularis of a putative glycosyltransferase catalysing the elongation with Galpβ1-3. Comparative inter-species analysis of available genomic and transcriptomic data suggested a candidate for this enzyme, amongst more than 20 putative (non-core 1) Gal/GlcNAc β1-3 transferases present in each species as a result of a taeniid-specific gene expansion. The candidate gene was experimentally verified to be transcribed at much higher levels in the larva of E. granulosus than that of E. multilocularis. Copyright © 2016 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.
DEFF Research Database (Denmark)
Gram, G J; Hemming, A; Bolmstedt, A
1994-01-01
affecting viral infectivity in cell culture. We found that the mutated virus lacking an N-linked glycan in the V1-loop of gp120 was more resistant to neutralization by monoclonal antibodies to the V3-loop and neutralization by soluble recombinant CD4 (sCD4). Both viruses were equally well neutralized by Con...... in the V1-loop of HIV-1 gp120. Lack of an N-linked glycan was verified by a mobility enhancement of mutant gp120 in SDS-gel electrophoresis. The mutated virus showed no differences in either gp120 content per infectious unit or infectivity, indicating that the N-linked glycan was neither essential nor...
Directory of Open Access Journals (Sweden)
Rajendra P Settem
Full Text Available Alveolar bone (tooth-supporting bone erosion is a hallmark of periodontitis, an inflammatory disease that often leads to tooth loss. Periodontitis is caused by a select group of pathogens that form biofilms in subgingival crevices between the gums and teeth. It is well-recognized that the periodontal pathogen Porphyromonas gingivalis in these biofilms is responsible for modeling a microbial dysbiotic state, which then initiates an inflammatory response destructive to the periodontal tissues and bone. Eradication of this pathogen is thus critical for the treatment of periodontitis. Previous studies have shown that oral inoculation in mice with an attenuated strain of the periodontal pathogen Tannerella forsythia altered in O-glycan surface composition induces a Th17-linked mobilization of neutrophils to the gingival tissues. In this study, we sought to determine if immune priming with such a Th17-biasing strain would elicit a productive neutrophil response against P. gingivalis. Our data show that inoculation with a Th17-biasing T. forsythia strain is effective in blocking P. gingivalis-persistence and associated alveolar bone loss in mice. This work demonstrates the potential of O-glycan modified Tannerella strains or their O-glycan components for harnessing Th17-mediated immunity against periodontal and other mucosal pathogens.
Settem, Rajendra P; Honma, Kiyonobu; Sharma, Ashu
2014-01-01
Alveolar bone (tooth-supporting bone) erosion is a hallmark of periodontitis, an inflammatory disease that often leads to tooth loss. Periodontitis is caused by a select group of pathogens that form biofilms in subgingival crevices between the gums and teeth. It is well-recognized that the periodontal pathogen Porphyromonas gingivalis in these biofilms is responsible for modeling a microbial dysbiotic state, which then initiates an inflammatory response destructive to the periodontal tissues and bone. Eradication of this pathogen is thus critical for the treatment of periodontitis. Previous studies have shown that oral inoculation in mice with an attenuated strain of the periodontal pathogen Tannerella forsythia altered in O-glycan surface composition induces a Th17-linked mobilization of neutrophils to the gingival tissues. In this study, we sought to determine if immune priming with such a Th17-biasing strain would elicit a productive neutrophil response against P. gingivalis. Our data show that inoculation with a Th17-biasing T. forsythia strain is effective in blocking P. gingivalis-persistence and associated alveolar bone loss in mice. This work demonstrates the potential of O-glycan modified Tannerella strains or their O-glycan components for harnessing Th17-mediated immunity against periodontal and other mucosal pathogens.
Natural Variation in Synthesis and Catabolism Genes Influences Dhurrin Content in Sorghum
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Chad M. Hayes
2015-07-01
Full Text Available Cyanogenic glucosides are natural compounds found in more than 1000 species of angiosperms that produce HCN and are deemed undesirable for agricultural use. However, these compounds are important components of the primary defensive mechanisms of many plant species. One of the best-studied cyanogenic glucosides is dhurrin [(--hydroxymandelonitrile-β--glucopyranoside], which is produced primarily in sorghum [ (L. Moench]. The biochemical basis for dhurrin metabolism is well established; however, little information is available on its genetic control. Here, we dissect the genetic control of leaf dhurrin content through a genome-wide association study (GWAS using a panel of 700 diverse converted sorghum lines (conversion panel previously subjected to pre-breeding and selected for short stature (∼1 m in height and photoperiod insensitivity. The conversion panel was grown for 2 yr in three environments. Wide variation for leaf dhurrin content was found in the sorghum conversion panel, with the Caudatum group exhibiting the highest dhurrin content and the Guinea group showing the lowest dhurrin content. A GWAS using a mixed linear model revealed significant associations (a false discovery rate [FDR] < 0.05 close to both UGT 185B1 in the canonical biosynthetic gene cluster on chromosome 1 and close to the catabolic dhurrinase loci on chromosome 8. Dhurrin content was associated consistently with biosynthetic genes in the two N-fertilized environments, while dhurrin content was associated with catabolic loci in the environment without supplemental N. These results suggest that genes for both biosynthesis and catabolism are important in determining natural variation for leaf dhurrin in sorghum in different environments.
Directory of Open Access Journals (Sweden)
Kamyar Zahedi
Full Text Available Cisplatin-induced nephrotoxicity limits its use in many cancer patients. The expression of enzymes involved in polyamine catabolism, spermidine/spermine N1-acetyltransferase (SSAT and spermine oxidase (SMOX increase in the kidneys of mice treated with cisplatin. We hypothesized that enhanced polyamine catabolism contributes to tissue damage in cisplatin acute kidney injury (AKI. Using gene knockout and chemical inhibitors, the role of polyamine catabolism in cisplatin AKI was examined. Deficiency of SSAT, SMOX or neutralization of the toxic products of polyamine degradation, H2O2 and aminopropanal, significantly diminished the severity of cisplatin AKI. In vitro studies demonstrated that the induction of SSAT and elevated polyamine catabolism in cells increases the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α and enhances the expression of binding immunoglobulin protein BiP/GRP78 and CCAAT-enhancer-binding protein homologous protein (CHOP/GADD153. The increased expression of these endoplasmic reticulum stress response (ERSR markers was accompanied by the activation of caspase-3. These results suggest that enhanced polyamine degradation in cisplatin AKI may lead to tubular damage through the induction of ERSR and the consequent onset of apoptosis. In support of the above, we show that the ablation of the SSAT or SMOX gene, as well as the neutralization of polyamine catabolism products modulate the onset of ERSR (e.g. lower BiP and CHOP and apoptosis (e.g. reduced activated caspase-3. These studies indicate that enhanced polyamine catabolism and its toxic products are important mediators of ERSR and critical to the pathogenesis of cisplatin AKI.
DEFF Research Database (Denmark)
Remmers, Neeley; Anderson, Judy M; Linde, Erin M
2013-01-01
Mucin expression is a common feature of most adenocarcinomas and features prominently in current attempts to improve diagnosis and therapy for pancreatic cancer and other adenocarcinomas. We investigated the expression of a number of mucin core proteins and associated O-linked glycans expressed i...
Glycan analysis of recombinant Aspergillus niger endo-polygalacturonase A.
Woosley, Bryan D; Kim, Young Hwan; Kumar Kolli, V S; Wells, Lance; King, Dan; Poe, Ryan; Orlando, Ron; Bergmann, Carl
2006-10-16
The enzyme endo-polygalacturonase A, or PGA, is produced by the fungus, Aspergillus niger, and appears to play a critical role during invasion of plant cell walls. The enzyme has been homologously overexpressed in order to provide sufficient quantities of purified enzyme for structural and biological studies. We have characterized this enzyme in terms of its post-translational modifications (PTMs) and found it to be both N- and O-glycosylated. Additionally, we have characterized the glycosyl moieties using MALDI-TOF and LC-ESI mass spectrometry. The characterization of all PTMs on PGA, along with molecular modeling, allows us to reveal potential roles played by the glycans in modulating the interaction of the enzyme with other macromolecules.
Energy Technology Data Exchange (ETDEWEB)
Oh, Ji-Su [Department of Oral and Maxillofacial Surgery, Chosun University, Gwangju, 61452 (Korea, Republic of); Cho, In-A; Kang, Kyeong-Rok [Department of Dental Bioengineering, Chosun University, Gwangju, 61452 (Korea, Republic of); You, Jae-Seek [Department of Oral and Maxillofacial Surgery, Chosun University, Gwangju, 61452 (Korea, Republic of); Yu, Sang-Joun [Department of Periodontology, Chosun University, Gwangju, 61452 (Korea, Republic of); Lee, Gyeong-Je [Department of Prosthodontics, Chosun University, Gwangju, 61452 (Korea, Republic of); Seo, Yo-Seob [Department of Oral and Maxillofacial Radiology, Chosun University, Gwangju, 61452 (Korea, Republic of); Kim, Chun Sung; Kim, Do Kyung [Pre-Dentistry, School of Dentistry, Chosun University, Gwangju, 61452 (Korea, Republic of); Kim, Su-Gwan [Department of Oral and Maxillofacial Surgery, Chosun University, Gwangju, 61452 (Korea, Republic of); Seo, Young-Woo [Korea Basic Science Institute, Gwangju Center, Chonnam National University, Gwangju, 61186 (Korea, Republic of); Im, Hee-Jeong [Department of Biochemistry, Rush University Medical Center, Chicago, IL, 60612 (United States); Kim, Jae-Sung, E-mail: js_kim@chosun.ac.kr [Pre-Dentistry, School of Dentistry, Chosun University, Gwangju, 61452 (Korea, Republic of)
2016-09-02
Biochanin-A, a phytoestrogen derived from herbal plants, protected from the IL-1β-induced loss of proteoglycans through the suppression of matrix degrading enzymes such as matrix metalloproteinase (MMP)-13, MMP-3, MMP-1, and ADAMTS-5 in primary rat chondrocytes and the knee articular cartilage. It also suppressed the expression of IL-1β-induced catabolic factors such as nitric oxide synthase 2, cyclooxygenase-2, prostaglandin E{sub 2}, and inflammatory cytokines. Furthermore, biochanin-A suppressed the IL-1β-induced phosphorylation of NFκB, and inhibited its nuclear translocation in primary rat chondrocytes. These results indicate that biochanin-A antagonizes the IL-1β-induced catabolic effects through its anti-inflammatory activity that involves the modulation of NFκB signaling. - Highlights: • Biochanin-A is a phytoestrogen derived from medicinal plants. • It suppressed the IL-1β-induced matrix degrading enzymes and catabolic factors. • It inhibited IL-1β-induced proteoglycan loss in chondrocytes and cartilage tissues. • Its anti-catabolic effects were mediated by modulation of NFκB signaling. • It may be used as a potential anti-catabolic biomaterial for osteoarthritis.
D-Allose catabolism of Escherichia coli
DEFF Research Database (Denmark)
Poulsen, Tim S.; Chang, Ying-Ying; Hove-Jensen, Bjarne
1999-01-01
Genes involved in allose utilization of Escherichia coli K-12 are organized in at least two operons, alsRBACE and alsI, located next to each other on the chromosome but divergently transcribed. Mutants defective in alsI (allose 6-phosphate isomerase gene) and alsE (allulose 6-phosphate epimerase...... gene) were Als-. Transcription of the two allose operons, measured as β-galactosidase activity specified by alsI-lacZ+ or alsE-lacZ+ operon fusions, was induced by allose. Ribose also caused derepression of expression of the regulon under conditions in which ribose phosphate catabolism was impaired....
Wetzel, Margaret E.; Kim, Kun-Soo; Miller, Marilyn; Olsen, Gary J.
2014-01-01
The Ti plasmid in Agrobacterium tumefaciens strain 15955 carries two alleles of traR that regulate conjugative transfer. The first is a functional allele, called traR, that is transcriptionally induced by the opine octopine. The second, trlR, is a nonfunctional, dominant-negative mutant located in an operon that is inducible by the opine mannopine (MOP). Based on these findings, we predicted that there exist wild-type agrobacterial strains harboring plasmids in which MOP induces a functional traR and, hence, conjugation. We analyzed 11 MOP-utilizing field isolates and found five where MOP induced transfer of the MOP-catabolic element and increased production of the acyl-homoserine lactone (acyl-HSL) quormone. The transmissible elements in these five strains represent a set of highly related plasmids. Sequence analysis of one such plasmid, pAoF64/95, revealed that the 176-kb element is not a Ti plasmid but carries genes for catabolism of MOP, mannopinic acid (MOA), agropinic acid (AGA), and the agrocinopines. The plasmid additionally carries all of the genes required for conjugative transfer, including the regulatory genes traR, traI, and traM. The traR gene, however, is not located in the MOP catabolism region. The gene, instead, is monocistronic and located within the tra-trb-rep gene cluster. A traR mutant failed to transfer the plasmid and produced little to no quormone even when grown with MOP, indicating that TraRpAoF64/95 is the activator of the tra regulon. A traM mutant was constitutive for transfer and acyl-HSL production, indicating that the anti-activator function of TraM is conserved. PMID:24363349
DEFF Research Database (Denmark)
Duboc, Philippe Jean; von Stockar, U.; Villadsen, John
1998-01-01
The dynamic behavior of a continuous culture of Saccharomyces cerevisiae subjected to a sudden increase in the dilution rate has been successfully modelled for anaerobic growth on glucose, and for aerobic growth on acetate, on ethanol, and on glucose. The catabolism responded by an immediate jump...... identified in steady state continuous cultures or during batch experiments. Only the time constant of biosynthesis regeneration, tau(x), and the time constant of catabolic capacity regeneration, tau(cat), had to be identified during transient experiments. In most experiments 7, was around 3 h, and tau(cat...
Ghosh, Sumit; Meli, Vijaykumar S.; Kumar, Anil; Thakur, Archana; Chakraborty, Niranjan; Chakraborty, Subhra; Datta, Asis
2011-01-01
Excessive softening of fruits during the ripening process leads to deterioration. This is of significant global importance as softening-mediated deterioration leads to huge postharvest losses. N-glycan processing enzymes are reported to play an important role during climacteric fruit softening: however, to date these enzymes have not been characterized in non-climacteric fruit. Two ripening-specific N-glycan processing enzymes, α-mannosidase (α-Man) and β-D-N-acetylhexosaminidase (β-Hex), have been identified and targeted to enhance the shelf life in non-climacteric fruits such as capsicum (Capsicum annuum). The purification, cloning, and functional characterization of α-Man and β-Hex from capsicum, which belong to glycosyl hydrolase (GH) families 38 and 20, respectively, are described here. α-Man and β-Hex are cell wall glycoproteins that are able to cleave terminal α-mannose and β-D-N-acetylglucosamine residues of N-glycans, respectively. α-Man and β-Hex transcripts as well as enzyme activity increase with the ripening and/or softening of capsicum. The function of α-Man and β-Hex in capsicum softening is investigated through RNA interference (RNAi) in fruits. α-Man and β-Hex RNAi fruits were approximately two times firmer compared with the control and fruit deterioration was delayed by approximately 7 d. It is shown that silencing of α-Man and β-Hex enhances fruit shelf life due to the reduced degradation of N-glycoproteins which resulted in delayed softening. Altogether, the results provide evidence for the involvement of N-glycan processing in non-climacteric fruit softening. In conclusion, genetic engineering of N-glycan processing can be a common strategy in both climacteric and non-climacteric species to reduce the post-harvest crop losses. PMID:21030387
Directory of Open Access Journals (Sweden)
Tomasz Walski
2017-12-01
Full Text Available Glycans are involved in many biological phenomena, including signal transduction, cell adhesion, immune response or differentiation. Although a few papers have reported on the role of glycans in the development and proper functioning of the insect midgut, no data are available regarding the localization of the glycan structures on the surface of the cells in the gut of insects. In this paper, we analyzed the spatial distribution of glycans present on the surface of the midgut cells in larvae of the cotton leafworm Spodoptera littoralis, an important agricultural pest insect worldwide. For this purpose, we established primary midgut cell cultures, probed these individual cells that are freely suspended in liquid medium with a selection of seven fluorescently labeled lectins covering a range of different carbohydrate binding specificities [mannose oligomers (GNA and HHA, GalNAc/Gal (RSA and SSA, GlcNAc (WGA and Nictaba and Neu5Ac(α-2,6Gal/GalNAc (SNA-I], and visualized the interaction of these lectins with the different zones of the midgut cells using confocal microscopy. Our analysis focused on the typical differentiated columnar cells with a microvillar brush border at their apical side, which are dominantly present in the Lepidopteran midgut and function in food digestion and absorption, and as well as on the undifferentiated stem cells that are important for midgut development and repair. Confocal microscopy analyses showed that the GalNAc/Gal-binding lectins SSA and RSA and the terminal GlcNAc-recognizing WGA bound preferentially to the apical microvillar zone of the differentiated columnar cells as compared to the basolateral pole. The reverse result was observed for the mannose-binding lectins GNA and HHA, as well as Nictaba that binds preferentially to GlcNAc oligomers. Furthermore, differences in lectin binding to the basal and lateral zones of the cell membranes of the columnar cells were apparent. In the midgut stem cells, GNA and
Liao, Wei-Ju; Tsao, Ku-Chi; Yang, Ruey-Bing
2016-03-01
SCUBE1 (S1), a secreted and membrane-bound glycoprotein, has a modular protein structure composed of an N-terminal signal peptide sequence followed by nine epidermal growth factor (EGF)-like repeats, a spacer region and three cysteine-rich (CR) motifs with multiple potential N-linked glycosylation sites, and one CUB domain at the C-terminus. Soluble S1 is a biomarker of platelet activation but an active participant of thrombosis via its adhesive EGF-like repeats, whereas its membrane-associated form acts as a bone morphogenetic protein (BMP) co-receptor in promoting BMP signal activity. However, the mechanism responsible for the membrane tethering and the biological importance of N-glycosylation of S1 remain largely unknown. In the present study, molecular mapping analysis identified a polycationic segment (amino acids 501-550) in the spacer region required for its membrane tethering via electrostatic interactions possibly with the anionic heparan sulfate proteoglycans. Furthermore, deglycosylation by peptide N-glycosidase F treatment revealed that N-glycans within the CR motif are essential for membrane recruitment through lectin-mediated surface retention. Injection of mRNA encoding zebrafish wild-type but not N-glycan-deficient scube1 restores the expression of haematopoietic and erythroid markers (scl and gata1) in scube1-knockdown embryos. We describe novel mechanisms in targeting S1 to the plasma membrane and demonstrate that N-glycans are required for S1 functions during primitive haematopoiesis in zebrafish. © 2016 Authors; published by Portland Press Limited.
Catabolism of pyrimidines in yeast: A tool to understand degradation of anticancer drugs
DEFF Research Database (Denmark)
Andersen, Gorm; Merico, A.; Bjornberg, O.
2006-01-01
The pyrimidine catabolic pathway is of crucial importance in cancer patients because it is involved in degradation of several chemotherapeutic drugs, such as 5-fluorouracil; it also is important in plants, unicellular eukaryotes, and bacteria for the degradation of pyrimidine-based biocides/antib...
Wargo, Matthew J; Szwergold, Benjamin S; Hogan, Deborah A
2008-04-01
Glycine betaine (GB), which occurs freely in the environment and is an intermediate in the catabolism of choline and carnitine, can serve as a sole source of carbon or nitrogen in Pseudomonas aeruginosa. Twelve mutants defective in growth on GB as the sole carbon source were identified through a genetic screen of a nonredundant PA14 transposon mutant library. Further growth experiments showed that strains with mutations in two genes, gbcA (PA5410) and gbcB (PA5411), were capable of growth on dimethylglycine (DMG), a catabolic product of GB, but not on GB itself. Subsequent nuclear magnetic resonance (NMR) experiments with 1,2-(13)C-labeled choline indicated that these genes are necessary for conversion of GB to DMG. Similar experiments showed that strains with mutations in the dgcAB (PA5398-PA5399) genes, which exhibit homology to genes that encode other enzymes with demethylase activity, are required for the conversion of DMG to sarcosine. Mutant analyses and (13)C NMR studies also confirmed that the soxBDAG genes, predicted to encode a sarcosine oxidase, are required for sarcosine catabolism. Our screen also identified a predicted AraC family transcriptional regulator, encoded by gbdR (PA5380), that is required for growth on GB and DMG and for the induction of gbcA, gbcB, and dgcAB in response to GB or DMG. Mutants defective in the previously described gbt gene (PA3082) grew on GB with kinetics similar to those of the wild type in both the PAO1 and PA14 strain backgrounds. These studies provided important insight into both the mechanism and the regulation of the catabolism of GB in P. aeruginosa.
Heldens, Genoveva T H; Blaney Davidson, Esmeralda N; Vitters, Elly L; Schreurs, B Willem; Piek, Ester; van den Berg, Wim B; van der Kraan, Peter M
2012-01-01
Articular cartilage has a very limited intrinsic repair capacity leading to progressive joint damage. Therapies involving tissue engineering depend on chondrogenic differentiation of progenitor cells. This chondrogenic differentiation will have to survive in a diseased joint. We postulate that catabolic factors in this environment inhibit chondrogenesis of progenitor cells. We investigated the effect of a catabolic environment on chondrogenesis in pellet cultures of human mesenchymal stem cells (hMSCs). We exposed chondrogenically differentiated hMSC pellets, to interleukin (IL)-1α, tumor necrosis factor (TNF)-α or conditioned medium derived from osteoarthritic synovium (CM-OAS). IL-1α and TNF-α in CM-OAS were blocked with IL-1Ra or Enbrel, respectively. Chondrogenesis was determined by chondrogenic markers collagen type II, aggrecan, and the hypertrophy marker collagen type X on mRNA. Proteoglycan deposition was analyzed by safranin o staining on histology. IL-1α and TNF-α dose-dependently inhibited chondrogenesis when added at onset or during progression of differentiation, IL-1α being more potent than TNF-α. CM-OAS inhibited chondrogenesis on mRNA and protein level but varied in extent between patients. Inhibition of IL-1α partially overcame the inhibitory effect of the CM-OAS on chondrogenesis whereas the TNF-α contribution was negligible. We show that hMSC chondrogenesis is blocked by either IL-1α or TNF-α alone, but that there are additional factors present in CM-OAS that contribute to inhibition of chondrogenesis, demonstrating that catabolic factors present in OA joints inhibit chondrogenesis, thereby impairing successful tissue engineering.
Vasseur, Francis; Sendid, Boualem; Broly, Franck; Gower-Rousseau, Corinne; Sarazin, Aurore; Standaert-Vitse, Annie; Colombel, Jean-Frederic; Poulain, Daniel; Jouault, Thierry
2013-03-18
Crohn's disease (CD) is associated with elevated anti-glycans antibody response in 60% of CD patients, and 25% of healthy first-degree relatives (HFDRs), suggesting a genetic influence for this humoral response. In mice, anti-glucan antibody response depends on the NLRP3 inflammasome. Here, we explored the effect of mutated CARD8, a component of the inflammasome, on anti-glycans antibody response in human. The association between p.C10X mutation (rs2043211) of the CARD8 gene and the levels of anti-glycans antibody response was examined in 39 CD families. The family-based QTDT association test was used to test for the genetic association between CARD8 p.C10X mutation and anti-glycan antibodies in the pedigrees. The difference in antibody responses determined by ELISA was tested in a subgroup of CD probands (one per family) and in a subgroup of HFDRs using the Wilcoxon Kruskal Wallis non-parametric test. The QTDT familial transmission tests showed that the p.C10X mutation of CARD8 was significantly associated with lower levels of antibody to mannans and glucans but not chitin (p=0.024, p=0.0028 and p=0.577, for ASCA, ALCA and ACCA, respectively). These associations were independent of NOD2 and NOD1 genetic backgrounds. The p.C10X mutation significantly associated or displayed a trend toward lower ASCA and ALCA levels (p=0.038 and p=0.08, respectively) only in the subgroup of CD probands. Such associations were not significant for ACCA levels in both subgroups of CD probands and of HFDRs. Our results show that ASCA and ALCA but not ACCA levels are under the influence of CARD8 genotype. Alteration of CARD8, a component of inflammasome, is associated with lower levels of antibodies directed to mannans and glucans at least in CD patients.
Czech Academy of Sciences Publication Activity Database
Smejkal, Petr; Szekrényes, A.; Ryvolová, M.; Foret, František; Guttman, A.; Bek, F.; Macka, M.
2010-01-01
Roč. 22, č. 31 (2010), s. 3783-3786 ISSN 0173-0835 R&D Projects: GA MŠk MEB060821 Institutional research plan: CEZ:AV0Z40310501 Keywords : bioanalyzer * chip-based analysis * glycans Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 3.569, year: 2010
International Nuclear Information System (INIS)
Sić, Siniša; Maier, Norbert M.; Rizzi, Andreas M.
2016-01-01
The potential and benefits of isotope-coded labeling in the context of MS-based glycan profiling are evaluated focusing on the analysis of O-glycans. For this purpose, a derivatization strategy using d_0/d_5-1-phenyl-3-methyl-5-pyrazolone (PMP) is employed, allowing O-glycan release and derivatization to be achieved in one single step. The paper demonstrates that this release and derivatization reaction can be carried out also in-gel with only marginal loss in sensitivity compared to in-solution derivatization. Such an effective in-gel reaction allows one to extend this release/labeling method also to glycoprotein/glycoform samples pre-separated by gel-electrophoresis without the need of extracting the proteins/digested peptides from the gel. With highly O-glycosylated proteins (e.g. mucins) LODs in the range of 0.4 μg glycoprotein (100 fmol) loaded onto the electrophoresis gel can be attained, with minor glycosylated proteins (like IgAs, FVII, FIX) the LODs were in the range of 80–100 μg (250 pmol–1.5 nmol) glycoprotein loaded onto the gel. As second aspect, the potential of isotope coded labeling as internal standardization strategy for the reliable determination of quantitative glycan profiles via MALDI-MS is investigated. Towards this goal, a number of established and emerging MALDI matrices were tested for PMP-glycan quantitation, and their performance is compared with that of ESI-based measurements. The crystalline matrix 2,6-dihydroxyacetophenone (DHAP) and the ionic liquid matrix N,N-diisopropyl-ethyl-ammonium 2,4,6-trihydroxyacetophenone (DIEA-THAP) showed potential for MALDI-based quantitation of PMP-labeled O-glycans. We also provide a comprehensive overview on the performance of MS-based glycan quantitation approaches by comparing sensitivity, LOD, accuracy and repeatability data obtained with RP-HPLC-ESI-MS, stand-alone nano-ESI-MS with a spray-nozzle chip, and MALDI-MS. Finally, the suitability of the isotope-coded PMP labeling strategy for
Energy Technology Data Exchange (ETDEWEB)
Sić, Siniša; Maier, Norbert M.; Rizzi, Andreas M., E-mail: Andreas.Rizzi@univie.ac.at
2016-09-07
The potential and benefits of isotope-coded labeling in the context of MS-based glycan profiling are evaluated focusing on the analysis of O-glycans. For this purpose, a derivatization strategy using d{sub 0}/d{sub 5}-1-phenyl-3-methyl-5-pyrazolone (PMP) is employed, allowing O-glycan release and derivatization to be achieved in one single step. The paper demonstrates that this release and derivatization reaction can be carried out also in-gel with only marginal loss in sensitivity compared to in-solution derivatization. Such an effective in-gel reaction allows one to extend this release/labeling method also to glycoprotein/glycoform samples pre-separated by gel-electrophoresis without the need of extracting the proteins/digested peptides from the gel. With highly O-glycosylated proteins (e.g. mucins) LODs in the range of 0.4 μg glycoprotein (100 fmol) loaded onto the electrophoresis gel can be attained, with minor glycosylated proteins (like IgAs, FVII, FIX) the LODs were in the range of 80–100 μg (250 pmol–1.5 nmol) glycoprotein loaded onto the gel. As second aspect, the potential of isotope coded labeling as internal standardization strategy for the reliable determination of quantitative glycan profiles via MALDI-MS is investigated. Towards this goal, a number of established and emerging MALDI matrices were tested for PMP-glycan quantitation, and their performance is compared with that of ESI-based measurements. The crystalline matrix 2,6-dihydroxyacetophenone (DHAP) and the ionic liquid matrix N,N-diisopropyl-ethyl-ammonium 2,4,6-trihydroxyacetophenone (DIEA-THAP) showed potential for MALDI-based quantitation of PMP-labeled O-glycans. We also provide a comprehensive overview on the performance of MS-based glycan quantitation approaches by comparing sensitivity, LOD, accuracy and repeatability data obtained with RP-HPLC-ESI-MS, stand-alone nano-ESI-MS with a spray-nozzle chip, and MALDI-MS. Finally, the suitability of the isotope-coded PMP labeling
The function of the human interferon-beta 1a glycan determined in vivo
DEFF Research Database (Denmark)
Dissing-Olesen, Lasse; Thaysen-Andersen, Morten; Meldgaard, Michael
2008-01-01
Recombinant human interferon-beta (rhIFN-beta) is the leading therapeutic intervention shown to change the cause of relapsing-remitting multiple sclerosis, and both a nonglycosylated and a significantly more active glycosylated variant of rhIFN-beta are used in treatment. This study investigates...... into the role of the rhIFN-beta1a glycan and its carbohydrate residues. The possibilities of improving the pharmacological properties of rhIFN-beta1a using glycoengineering are discussed...
DEFF Research Database (Denmark)
Hägglund, Per; Matthiesen, R.; Elortza, F.
2007-01-01
and N-acetyl-β-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides......, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide- N-glycosidase (PNGase) digestion, with concomitant deamidation...... of the released asparagine residue. The reaction is carried out in H218O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-β- N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond...
Insights into the evolution of sialic acid catabolism among bacteria
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Almagro-Moreno Salvador
2009-05-01
Full Text Available Abstract Background Sialic acids comprise a family of nine-carbon amino sugars that are prevalent in mucus rich environments. Sialic acids from the human host are used by a number of pathogens as an energy source. Here we explore the evolution of the genes involved in the catabolism of sialic acid. Results The cluster of genes encoding the enzymes N-acetylneuraminate lyase (NanA, epimerase (NanE, and kinase (NanK, necessary for the catabolism of sialic acid (the Nan cluster, are confined 46 bacterial species, 42 of which colonize mammals, 33 as pathogens and 9 as gut commensals. We found a putative sialic acid transporter associated with the Nan cluster in most species. We reconstructed the phylogenetic history of the NanA, NanE, and NanK proteins from the 46 species and compared them to the species tree based on 16S rRNA. Within the NanA phylogeny, Gram-negative and Gram-positive bacteria do not form distinct clades. NanA from Yersinia and Vibrio species was most closely related to the NanA clade from eukaryotes. To examine this further, we reconstructed the phylogeny of all NanA homologues in the databases. In this analysis of 83 NanA sequences, Bacteroidetes, a human commensal group formed a distinct clade with Verrucomicrobia, and branched with the Eukaryotes and the Yersinia/Vibrio clades. We speculate that pathogens such as V. cholerae may have acquired NanA from a commensal aiding their colonization of the human gut. Both the NanE and NanK phylogenies more closely represented the species tree but numerous incidences of incongruence are noted. We confirmed the predicted function of the sialic acid catabolism cluster in members the major intestinal pathogens Salmonella enterica, Vibrio cholerae, V. vulnificus, Yersinia enterocolitica and Y. pestis. Conclusion The Nan cluster among bacteria is confined to human pathogens and commensals conferring them the ability to utilize a ubiquitous carbon source in mucus rich surfaces of the human body
Energy Technology Data Exchange (ETDEWEB)
Oganesyan, Vaheh, E-mail: oganesyanv@medimmune.com; Mazor, Yariv; Yang, Chunning; Cook, Kimberly E.; Woods, Robert M. [MedImmune LLC, 1 MedImmune Way, Gaithersburg, MD 20878 (United States); Ferguson, Andrew [AstraZeneca Pharmaceuticals, 35 Gatehouse Drive, Mailstop E3, Waltham, MA 02451 (United States); Bowen, Michael A.; Martin, Tom; Zhu, Jie; Wu, Herren; Dall’Acqua, William F., E-mail: oganesyanv@medimmune.com [MedImmune LLC, 1 MedImmune Way, Gaithersburg, MD 20878 (United States)
2015-10-31
In an effort to identify the critical structural features responsible for the high-affinity interaction of IgG1 Fc with FcγRI, the structure of the corresponding complex was solved at a resolution of 2.4 Å. The three-dimensional structure of a human IgG1 Fc fragment bound to wild-type human FcγRI is reported. The structure of the corresponding complex was solved at a resolution of 2.4 Å using molecular replacement; this is the highest resolution achieved for an unmutated FcγRI molecule. This study highlights the critical structural and functional role played by the second extracellular subdomain of FcγRI. It also explains the long-known major energetic contribution of the Fc ‘LLGG’ motif at positions 234–237, and particularly of Leu235, via a ‘lock-and-key’ mechanism. Finally, a previously held belief is corrected and a differing view is offered on the recently proposed direct role of Fc carbohydrates in the corresponding interaction. Structural evidence is provided that such glycan-related effects are strictly indirect.
Praissman, Jeremy L; Willer, Tobias; Sheikh, M Osman; Toi, Ants; Chitayat, David; Lin, Yung-Yao; Lee, Hane; Stalnaker, Stephanie H; Wang, Shuo; Prabhakar, Pradeep Kumar; Nelson, Stanley F; Stemple, Derek L; Moore, Steven A; Moremen, Kelley W; Campbell, Kevin P; Wells, Lance
2016-01-01
Multiple glycosyltransferases are essential for the proper modification of alpha-dystroglycan, as mutations in the encoding genes cause congenital/limb-girdle muscular dystrophies. Here we elucidate further the structure of an O-mannose-initiated glycan on alpha-dystroglycan that is required to generate its extracellular matrix-binding polysaccharide. This functional glycan contains a novel ribitol structure that links a phosphotrisaccharide to xylose. ISPD is a CDP-ribitol (ribose) pyrophosphorylase that generates the reduced sugar nucleotide for the insertion of ribitol in a phosphodiester linkage to the glycoprotein. TMEM5 is a UDP-xylosyl transferase that elaborates the structure. We demonstrate in a zebrafish model as well as in a human patient that defects in TMEM5 result in muscular dystrophy in combination with abnormal brain development. Thus, we propose a novel structure—a ribitol in a phosphodiester linkage—for the moiety on which TMEM5, B4GAT1, and LARGE act to generate the functional receptor for ECM proteins having LG domains. DOI: http://dx.doi.org/10.7554/eLife.14473.001 PMID:27130732
Protein-Glycan Quinary Interactions in Crowding Environment Unveiled by NMR Spectroscopy.
Diniz, Ana; Dias, Jorge S; Jiménez-Barbero, Jesús; Marcelo, Filipa; Cabrita, Eurico J
2017-09-21
Protein-glycan interactions as modulators for quinary structures in crowding environments were explored. The interaction between human galectin 3 (Gal-3) and distinct macromolecular crowders, such as bovine and human serum albumin (BSA and HSA), Ficoll 70 and PEG3350, was scrutinized. The molecular recognition event of the specific ligand, lactose, by Gal-3 in crowding conditions was evaluated. Gal-3 interactions were monitored by NMR analysing chemical shift perturbation (CSP) and line broadening of 1 H 15 N-HSQC signals. The intensity of the Gal-3 1 H 15 N-HSQC signals decreased in the presence of all crowders, due to the increase in the solution viscosity and to the formation of large protein complexes. When glycosylated containing samples of BSA and HSA were used, signal broadening was more severe than that observed in the presence of the more viscous solutions of PEG3350 and Ficoll 70. However, for the samples containing glycoproteins, the signal intensity of 1 H 15 N-HSQC recovered upon addition of lactose. We show that serum proteins interact with Gal-3, through their α2,3-linked sialylgalactose moieties exposed at their surfaces, competing with lactose for the same binding site. The quinary interaction between Gal-3 and serum glycoproteins, could help to co-localize Gal-3 at the cell surface, and may play a role in adhesion and signalling functions of this protein. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
Kim, Jae-Sung; Ellman, Michael B.; Yan, Dongyao; An, Howard S.; Kc, Ranjan; Li, Xin; Chen, Di; Xiao, Guozhi; Cs-Zabo, Gabriella; Hoskin, David W.; Buechter, D.D.; Van Wijnen, Andre J.; Im, Hee-Jeong
2013-01-01
The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. PMID:23460134
Kim, Jae-Sung; Ellman, Michael B; Yan, Dongyao; An, Howard S; Kc, Ranjan; Li, Xin; Chen, Di; Xiao, Guozhi; Cs-Szabo, Gabriella; Hoskin, David W; Buechter, Doug D; Van Wijnen, Andre J; Im, Hee-Jeong
2013-09-01
The catabolic cytokine interleukin-1 (IL-1) and endotoxin lipopolysaccharide (LPS) are well-known inflammatory mediators involved in degenerative disc disease, and inhibitors of IL-1 and LPS may potentially be used to slow or prevent disc degeneration in vivo. Here, we elucidate the striking anti-catabolic and anti-inflammatory effects of bovine lactoferricin (LfcinB) in the intervertebral disc (IVD) via antagonism of both IL-1 and LPS-mediated catabolic activity using in vitro and ex vivo analyses. Specifically, we demonstrate the biological counteraction of LfcinB against IL-1 and LPS-mediated proteoglycan (PG) depletion, matrix-degrading enzyme production, and enzyme activity in long-term (alginate beads) and short-term (monolayer) culture models using bovine and human nucleus pulposus (NP) cells. LfcinB significantly attenuates the IL-1 and LPS-mediated suppression of PG production and synthesis, and thus restores PG accumulation and pericellular matrix formation. Simultaneously, LfcinB antagonizes catabolic factor mediated induction of multiple cartilage-degrading enzymes, including MMP-1, MMP-3, MMP-13, ADAMTS-4, and ADAMTS-5, in bovine NP cells at both mRNA and protein levels. LfcinB also suppresses the catabolic factor-induced stimulation of oxidative and inflammatory factors such as iNOS, IL-6, and toll-like receptor-2 (TLR-2) and TLR-4. Finally, the ability of LfcinB to antagonize IL-1 and LPS-mediated suppression of PG is upheld in an en bloc intradiscal microinjection model followed by ex vivo organ culture using both mouse and rabbit IVD tissue, suggesting a potential therapeutic benefit of LfcinB on degenerative disc disease in the future. Copyright © 2013 Wiley Periodicals, Inc.
Amino acid catabolism and generation of volatiles by lactic acid bacteria
Tavaria, F. K.; Dahl, S.; Carballo, F. J.; Malcata, F. X.
2002-01-01
Twelve isolates of lactic acid bacteria, belonging to the Lactobacillus, Lactococcus, Leuconostoc, and Enterococcus genera, were previously isolated from 180- d-old Serra da Estrela cheese, a traditional Portuguese cheese manufactured from raw milk and coagulated with a plant rennet. These isolates were subsequently tested for their ability to catabolize free amino acids, when incubated independently with each amino acid in free form or with a mixture thereof. Attempts...
Directory of Open Access Journals (Sweden)
Rodrigo W A Souza
Full Text Available Heart failure (HF is associated with cachexia and consequent exercise intolerance. Given the beneficial effects of aerobic exercise training (ET in HF, the aim of this study was to determine if the ET performed during the transition from cardiac dysfunction to HF would alter the expression of anabolic and catabolic factors, thus preventing skeletal muscle wasting.We employed ascending aortic stenosis (AS inducing HF in Wistar male rats. Controls were sham-operated animals. At 18 weeks after surgery, rats with cardiac dysfunction were randomized to 10 weeks of aerobic ET (AS-ET or to an untrained group (AS-UN. At 28 weeks, the AS-UN group presented HF signs in conjunction with high TNF-α serum levels; soleus and plantaris muscle atrophy; and an increase in the expression of TNF-α, NFκB (p65, MAFbx, MuRF1, FoxO1, and myostatin catabolic factors. However, in the AS-ET group, the deterioration of cardiac function was prevented, as well as muscle wasting, and the atrophy promoters were decreased. Interestingly, changes in anabolic factor expression (IGF-I, AKT, and mTOR were not observed. Nevertheless, in the plantaris muscle, ET maintained high PGC1α levels.Thus, the ET capability to attenuate cardiac function during the transition from cardiac dysfunction to HF was accompanied by a prevention of skeletal muscle atrophy that did not occur via an increase in anabolic factors, but through anti-catabolic activity, presumably caused by PGC1α action. These findings indicate the therapeutic potential of aerobic ET to block HF-induced muscle atrophy by counteracting the increased catabolic state.
A Murine Model of Persistent Inflammation, Immune Suppression, and Catabolism Syndrome
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Amanda M. Pugh
2017-08-01
Full Text Available Critically ill patients that survive sepsis can develop a Persistent Inflammation, Immunosuppression, and Catabolism Syndrome (PICS, which often leads to extended recovery periods and multiple complications. Here, we utilized a cecal ligation and puncture (CLP method in mice with the goal of creating a model that concurrently displays all the characteristics of PICS. We observed that, after eight days, mice that survive the CLP develop persistent inflammation with significant myelopoiesis in the bone marrow and spleen. These mice also demonstrate ongoing immune suppression, as evidenced by the decreased total and naïve splenic CD4 and CD8 T cells with a concomitant increase in immature myeloid cells. The mice further display significant weight loss and decreased muscle mass, indicating a state of ongoing catabolism. When PICS mice are challenged with intranasal Pseudomonas aeruginosa, mortality is significantly elevated compared to sham mice. This mortality difference is associated with increased bacterial loads in the lung, as well as impaired neutrophil migration and neutrophil dysfunction in the PICS mice. Altogether, we have created a sepsis model that concurrently exhibits PICS characteristics. We postulate that this will help determine the mechanisms underlying PICS and identify potential therapeutic targets to improve outcomes for this patient population.
Massafra, Vittoria; Milona, Alexandra; Vos, Harmjan R; Ramos, Rúben J J; Gerrits, Johan; Willemsen, Ellen C L; Ramos Pittol, José M; Ijssennagger, Noortje; Houweling, Martin; Prinsen, Hubertus C M T; Verhoeven-Duif, Nanda M; Burgering, Boudewijn M T; van Mil, Saskia W C
2017-01-01
BACKGROUND & AIMS: The nuclear receptor subfamily 1 group H member 4 (NR1H4 or farnesoid X receptor [FXR]) regulates bile acid synthesis, transport, and catabolism. FXR also regulates postprandial lipid and glucose metabolism. We performed quantitative proteomic analyses of liver tissues from mice
DEFF Research Database (Denmark)
Scott, Nichollas E; Parker, Benjamin L; Connolly, Angela M
2011-01-01
by the resistance of the glycan-peptide bond to enzymatic digestion or ss-elimination, and have previously concentrated on soluble glycoproteins compatible with lectin affinity and gel-based approaches. We developed strategies for enriching C. jejuni HB93-13 glycopeptides using zwitterionic hydrophilic interaction......-linked glycosylation sites in C. jejuni and is the first to utilize HCD fragmentation for glycopeptide identification with intact glycan. We also show that hydrophobic integral membrane proteins are significant targets of glycosylation in this organism. Our data demonstrate that peptide-centric approaches coupled...
Metabolism and catabolism in hip fracture patients: nutritional and anabolic intervention--a review.
Hedström, Margareta; Ljungqvist, Olle; Cederholm, Tommy
2006-10-01
Patients suffering from hip fracture are known to be at risk of catabolism and protein-energy malnutrition. In this review we discuss the pathogenesis of hip fracture-related catabolism per- and postoperatively. We also describe the consequences of malnutrition after a hip fracture and summarize studies that have evaluated the effect of nutritional or anabolic treatment of these patients. There has been relatively little published on the effects of nutritional and anabolic pharmacological interventions for improvement of nutritional status and on the role of nutritional status in clinical outcomes. Even so, there have been 19 randomized studies in this field. 12 studies evaluated nutritional supplementation or protein supplementation. 6 found improved clinical outcome with fewer complications, faster recovery and shorter length of hospital stay, whereas the others reported no difference in clinical outcome. For pharmacological interventions, the outcomes have been even less clear. Supplementation studies in general appear to be underpowered or suffer logistic problems. Studies of higher scientific quality are needed, and enteral feeding, anabolic treatment and multimodal approaches need to be evaluated in greater depth.
DEFF Research Database (Denmark)
Andersen, Mathias Christian Franch; Arentoft, Camilla Anna Søholt; Boos, Irene
2017-01-01
Galactans are linear structures mainly found in arabinogalactan glycans and RG-I side chains. As a follow-up to our work on both β-(1→3)-linked and β-(1→4)-linked galactans, we herein report a convergent synthesis of β-(1→6)-galactan using our previously synthesized 4,6-benzylidene protected disa...
Directory of Open Access Journals (Sweden)
Almada Anthony
2004-12-01
Full Text Available Abstract This study examined whether supplementing the diet with a commercial supplement containing zinc magnesium aspartate (ZMA during training affects zinc and magnesium status, anabolic and catabolic hormone profiles, and/or training adaptations. Forty-two resistance trained males (27 ± 9 yrs; 178 ± 8 cm, 85 ± 15 kg, 18.6 ± 6% body fat were matched according to fat free mass and randomly assigned to ingest in a double blind manner either a dextrose placebo (P or ZMA 30–60 minutes prior to going to sleep during 8-weeks of standardized resistance-training. Subjects completed testing sessions at 0, 4, and 8 weeks that included body composition assessment as determined by dual energy X-ray absorptiometry, 1-RM and muscular endurance tests on the bench and leg press, a Wingate anaerobic power test, and blood analysis to assess anabolic/catabolic status as well as markers of health. Data were analyzed using repeated measures ANOVA. Results indicated that ZMA supplementation non-significantly increased serum zinc levels by 11 – 17% (p = 0.12. However, no significant differences were observed between groups in anabolic or catabolic hormone status, body composition, 1-RM bench press and leg press, upper or lower body muscular endurance, or cycling anaerobic capacity. Results indicate that ZMA supplementation during training does not appear to enhance training adaptations in resistance trained populations.
One Choi; Noboru Tomiya; Jung H. Kim; James M. Slavicek; Michael J. Betenbaugh; Yuan C. Lee
2003-01-01
N-glycan structures of recombinant human serum transferrin (hTf) expressed by Lymantria dispar (gypsy moth) 652Y cells were determined. The gene encoding hTf was incorporated into a Lymantria dispar nucleopolyhedrovirus (LdMNPV) under the control of the polyhedrin promoter. This virus was then...
The Emerging Importance of IgG Fab Glycosylation in Immunity
van de Bovenkamp, Fleur S.; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann
2016-01-01
Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans
STEGEMAN, CA; HUISMAN, RM; DEROUW, B; JOOSTEMA, A; DEJONG, PE
We assessed the agreement between different methods of determining protein catabolic rate (PCR) in hemodialysis patients and the possible influence of postdialysis urea rebound and the length of the interdialytic interval on the PCR determination. Protein catabolic rate derived from measured total
Hanneman, Andrew J S; Strand, James; Huang, Chi-Ting
2014-02-01
Glycosylation is a critical parameter used to evaluate protein quality and consistency. N-linked glycan profiling is fundamental to the support of biotherapeutic protein manufacturing from early stage process development through drug product commercialization. Sialylated glycans impact the serum half-life of receptor-Fc fusion proteins (RFPs), making their quality and consistency a concern during the production of fusion proteins. Here, we describe an analytical approach providing both quantitative profiling and in-depth mass spectrometry (MS)-based structural characterization of sialylated RFP N-glycans. Aiming to efficiently link routine comparability studies with detailed structural characterization, an integrated workflow was implemented employing fluorescence detection, online positive and negative ion tandem mass spectrometry (MS/MS), and offline static nanospray ionization-sequential mass spectrometry (NSI-MS(n)). For routine use, high-performance liquid chromatography profiling employs established fluorescence detection of 2-aminobenzoic acid derivatives (2AA) and hydrophilic interaction anion-exchange chromatography (HIAX) charge class separation. Further characterization of HIAX peak fractions is achieved by online (-) ion orbitrap MS/MS, offering the advantages of high mass accuracy and data-dependent MS/MS. As required, additional characterization uses porous graphitized carbon in the second chromatographic dimension to provide orthogonal (+) ion MS/MS spectra and buffer-free liquid chromatography peak eluants that are optimum for offline (+)/(-) NSI-MS(n) investigations to characterize low-abundance species and specific moieties including O-acetylation and sulfation. © 2013 Wiley Periodicals, Inc. and the American Pharmacists Association.
Lourdais, O; Brischoux, F; DeNardo, D; Shine, R
2004-07-01
In many species the high energetic demands of reproduction induce a negative energy balance, and thus females must rely on tissue catabolism to complete the reproductive process. Previous works have shown that both fat and protein are energy resources during prolonged fasting in vertebrates. While many ecological studies on energy costs of reproduction have focused on variations in fat stores, the impact of protein investment on the female has not been thoroughly investigated. Notably, as there is no specialized storage form for proteins, intense catabolism is likely to entail structural (musculature) loss that may compromise maternal physical performance after reproduction. Measurements on captive rainbow boas ( Epicrates cenchria maurus) confirm that reproducing females undergo significant protein catabolism (as indicated by elevated plasma uric acid levels) and show considerable musculature loss during gestation (as detected by reduced width of the epaxial muscles). Protein mobilization entailed a significant functional loss that was illustrated by decrements in tests of strength and constriction after parturition. In wild situations, such effects are likely to decrease the snakes' ability to forage and apprehend prey. Hence, the time period needed to recover from reproduction can be extended not only because the female must compensate losses of both fat stores and functional muscle, but also because the ability to do so may be compromised. Performance alteration is likely to be of equal or greater importance than reduced energy stores in the physiological mediation of elevated post-reproduction mortality rates and infrequent reproductive bouts (e.g. biannual or triannual), two common ecological traits of female snakes.
The mechanisms of haem catabolism
International Nuclear Information System (INIS)
Brown, S.B.; King, R.F.G.J.
1978-01-01
The pathway of haem breakdown in living rats was studied by using 18 0 in the oxygen that the animals consumed. By cannulation of the common bile duct and collection of bile, labelled bilirubin was isolated and its mass spectrum determined. One set of results was obtained for a rat to which haemoglobin had been intravenously administered and another set obtained for a rat that was not given exogenous haem. Isomerization of bilirubin IXα to the XIIIα and IIIα isomers did not occur to any significant extent. The 18 O-labelling pattern obtained in the bilirubin was consistent with a Two-Molecule Mechanism, whereby the terminal lactam oxygen atoms of bilirubin are derived from different oxygen molecules. The consequences of this mechanism are discussed in terms of the possible intermediates of the catabolic pathway. 18 0-labelled bilirubin appeared in the bile in less than 10 min after exposure of the animals to labelled oxygen. This result suggests that all of the chemical transformations involving production of biliverdin, reduction to bilirubin and conjugation of the bilirubin are fast processes. The quantitative recovery of label obtained in the experiments suggests that there is little or no exchange of newly synthesized bilirubin with existing bilirubin pools in the animal. (author)
Mutations Enhancing Amino Acid Catabolism Confer a Growth Advantage in Stationary Phase
Zinser, Erik R.; Kolter, Roberto
1999-01-01
Starved cultures of Escherichia coli undergo successive rounds of population takeovers by mutants of increasing fitness. These mutants express the growth advantage in stationary phase (GASP) phenotype. Previous work identified the rpoS819 allele as a GASP mutation allowing cells to take over stationary-phase cultures after growth in rich media (M. M. Zambrano, D. A. Siegele, M. A. Almirón, A. Tormo, and R. Kolter, Science 259:1757–1760, 1993). Here we have identified three new GASP loci from an aged rpoS819 strain: sgaA, sgaB, and sgaC. Each locus is capable of conferring GASP on the rpoS819 parent, and they can provide successively higher fitnesses for the bacteria in the starved cultures. All four GASP mutations isolated thus far allow for faster growth on both individual and mixtures of amino acids. Each mutation confers a growth advantage on a different subset of amino acids, and these mutations act in concert to increase the overall catabolic capacity of the cell. We present a model whereby this enhanced ability to catabolize amino acids is responsible for the fitness gain during carbon starvation, as it may allow GASP mutants to outcompete the parental cells when growing on the amino acids released by dying cells. PMID:10482523
Wilkie, Michael P; Claude, Jaime F; Cockshutt, Amanda; Holmes, John A; Wang, Yuxiang S; Youson, John H; Walsh, Patrick J
2006-01-01
The jawless fish, the sea lamprey (Petromyzon marinus), spends part of its life as a burrow-dwelling, suspension-feeding larva (ammocoete) before undergoing a metamorphosis into a free swimming, parasitic juvenile that feeds on the blood of fishes. We predicted that animals in this juvenile, parasitic stage have a great capacity for catabolizing amino acids when large quantities of protein-rich blood are ingested. The sixfold to 20-fold greater ammonia excretion rates (J(Amm)) in postmetamorphic (nonfeeding) and parasitic lampreys compared with ammocoetes suggested that basal rates of amino acid catabolism increased following metamorphosis. This was likely due to a greater basal amino acid catabolizing capacity in which there was a sixfold higher hepatic glutamate dehydrogenase (GDH) activity in parasitic lampreys compared with ammocoetes. Immunoblotting also revealed that GDH quantity was 10-fold and threefold greater in parasitic lampreys than in ammocoetes and upstream migrant lampreys, respectively. Higher hepatic alanine and aspartate aminotransferase activities in the parasitic lampreys also suggested an enhanced amino acid catabolizing capacity in this life stage. In contrast to parasitic lampreys, the twofold larger free amino acid pool in the muscle of upstream migrant lampreys confirmed that this period of natural starvation is accompanied by a prominent proteolysis. Carbamoyl phosphate synthetase III was detected at low levels in the liver of parasitic and upstream migrant lampreys, but there was no evidence of extrahepatic (muscle, intestine) urea production via the ornithine urea cycle. However, detection of arginase activity and high concentrations of arginine in the liver at all life stages examined infers that arginine hydrolysis is an important source of urea. We conclude that metamorphosis is accompanied by a metabolic reorganization that increases the capacity of parasitic sea lampreys to catabolize intermittently large amino acid loads arising
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R van der Geize
2011-08-01
Full Text Available Rhodococcus equi causes fatal pyogranulomatous pneumonia in foals and immunocompromised animals and humans. Despite its importance, there is currently no effective vaccine against the disease. The actinobacteria R. equi and the human pathogen Mycobacterium tuberculosis are related, and both cause pulmonary diseases. Recently, we have shown that essential steps in the cholesterol catabolic pathway are involved in the pathogenicity of M. tuberculosis. Bioinformatic analysis revealed the presence of a similar cholesterol catabolic gene cluster in R. equi. Orthologs of predicted M. tuberculosis virulence genes located within this cluster, i.e. ipdA (rv3551, ipdB (rv3552, fadA6 and fadE30, were identified in R. equi RE1 and inactivated. The ipdA and ipdB genes of R. equi RE1 appear to constitute the α-subunit and β-subunit, respectively, of a heterodimeric coenzyme A transferase. Mutant strains RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, were impaired in growth on the steroid catabolic pathway intermediates 4-androstene-3,17-dione (AD and 3aα-H-4α(3'-propionic acid-5α-hydroxy-7aβ-methylhexahydro-1-indanone (5α-hydroxy-methylhexahydro-1-indanone propionate; 5OH-HIP. Interestingly, RE1ΔipdAB and RE1ΔfadE30, but not RE1ΔfadA6, also displayed an attenuated phenotype in a macrophage infection assay. Gene products important for growth on 5OH-HIP, as part of the steroid catabolic pathway, thus appear to act as factors involved in the pathogenicity of R. equi. Challenge experiments showed that RE1ΔipdAB could be safely administered intratracheally to 2 to 5 week-old foals and oral immunization of foals even elicited a substantial protective immunity against a virulent R. equi strain. Our data show that genes involved in steroid catabolism are promising targets for the development of a live-attenuated vaccine against R. equi infections.
Determination of 3-O- and 4-O-methylated monosaccharide constituents in snail glycans.
Stepan, Herwig; Bleckmann, Christina; Geyer, Hildegard; Geyer, Rudolf; Staudacher, Erika
2010-07-02
The N- and O-glycans of Arianta arbustorum, Achatina fulica, Arion lusitanicus and Planorbarius corneus were analysed for their monosaccharide pattern by reversed-phase HPLC after labelling with 2-aminobenzoic acid or 3-methyl-1-phenyl-2-pyrazolin-5-one and by gas chromatography-mass spectrometry. Glucosamine, galactosamine, mannose, galactose, glucose, fucose and xylose were identified. Furthermore, three different methylated sugars were detected: 3-O-methyl-mannose and 3-O-methyl-galactose were confirmed to be a common snail feature; 4-O-methyl-galactose was detected for the first time in snails. Copyright 2010 Elsevier Ltd. All rights reserved.
Insulin signaling regulates fatty acid catabolism at the level of CoA activation.
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Xiaojun Xu
2012-01-01
Full Text Available The insulin/IGF signaling pathway is a highly conserved regulator of metabolism in flies and mammals, regulating multiple physiological functions including lipid metabolism. Although insulin signaling is known to regulate the activity of a number of enzymes in metabolic pathways, a comprehensive understanding of how the insulin signaling pathway regulates metabolic pathways is still lacking. Accepted knowledge suggests the key regulated step in triglyceride (TAG catabolism is the release of fatty acids from TAG via the action of lipases. We show here that an additional, important regulated step is the activation of fatty acids for beta-oxidation via Acyl Co-A synthetases (ACS. We identify pudgy as an ACS that is transcriptionally regulated by direct FOXO action in Drosophila. Increasing or reducing pudgy expression in vivo causes a decrease or increase in organismal TAG levels respectively, indicating that pudgy expression levels are important for proper lipid homeostasis. We show that multiple ACSs are also transcriptionally regulated by insulin signaling in mammalian cells. In sum, we identify fatty acid activation onto CoA as an important, regulated step in triglyceride catabolism, and we identify a mechanistic link through which insulin regulates lipid homeostasis.
The effect of CreA in glucose and xylose catabolism in Aspergillus nidulans
DEFF Research Database (Denmark)
Prathumpai, Wai; Mcintyre, Mhairi; Nielsen, Jens
2004-01-01
The catabolism of glucose and xylose was studied in a wild type and creA deleted (carbon catabolite de-repressed) strain of Aspergillus nidulans. Both strains were cultivated in bioreactors with either glucose or xylose as the sole carbon source, or in the presence of both sugars. In the cultivat......The catabolism of glucose and xylose was studied in a wild type and creA deleted (carbon catabolite de-repressed) strain of Aspergillus nidulans. Both strains were cultivated in bioreactors with either glucose or xylose as the sole carbon source, or in the presence of both sugars...... on the sugar mixture, glucose repression of xylose utilisation was observed; with xylose utilisation occurring only after glucose was depleted. This phenomenon was not seen in the creA deleted strain, where glucose and xylose were catabolised simultaneously. Measurement of key metabolites and the activities...... of key enzymes in the xylose utilisation pathway revealed that xylose metabolism was occurring in the creA deleted strain, even at high glucose concentrations. Conversely, in the wild type strain, activities of the key enzymes for xylose metabolism increased only when the effects of glucose repression...
Iverson, Andrew; Garza, Erin; Manow, Ryan; Wang, Jinhua; Gao, Yuanyuan; Grayburn, Scott; Zhou, Shengde
2016-04-16
Anaerobic rather than aerobic fermentation is preferred for conversion of biomass derived sugars to high value redox-neutral and reduced commodities. This will likely result in a higher yield of substrate to product conversion and decrease production cost since substrate often accounts for a significant portion of the overall cost. To this goal, metabolic pathway engineering has been used to optimize substrate carbon flow to target products. This approach works well for the production of redox neutral products such as lactic acid from redox neutral sugars using the reducing power NADH (nicotinamide adenine dinucleotide, reduced) generated from glycolysis (2 NADH per glucose equivalent). Nevertheless, greater than two NADH per glucose catabolized is needed for the production of reduced products (such as xylitol) from redox neutral sugars by anaerobic fermentation. The Escherichia coli strain AI05 (ΔfrdBC ΔldhA ΔackA Δ(focA-pflB) ΔadhE ΔptsG ΔpdhR::pflBp 6-(aceEF-lpd)), previously engineered for reduction of xylose to xylitol using reducing power (NADH equivalent) of glucose catabolism, was further engineered by 1) deleting xylAB operon (encoding for xylose isomerase and xylulokinase) to prevent xylose from entering the pentose phosphate pathway; 2) anaerobically expressing the sdhCDAB-sucABCD operon (encoding for succinate dehydrogenase, α-ketoglutarate dehydrogenase and succinyl-CoA synthetase) to enable an anaerobically functional tricarboxcylic acid cycle with a theoretical 10 NAD(P)H equivalent per glucose catabolized. These reducing equivalents can be oxidized by synthetic respiration via xylose reduction, producing xylitol. The resulting strain, AI21 (pAI02), achieved a 96 % xylose to xylitol conversion, with a yield of 6 xylitol per glucose catabolized (molar yield of xylitol per glucose consumed (YRPG) = 6). This represents a 33 % improvement in xylose to xylitol conversion, and a 63 % increase in xylitol yield per glucose catabolized over
Energy Technology Data Exchange (ETDEWEB)
Rivas, Blanca de las; Rodríguez, Héctor [Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain); Angulo, Iván [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); Muñoz, Rosario [Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain); Mancheño, José M., E-mail: xjosemi@iqfr.csic.es [Grupo de Cristalografía Macromolecular y Biología Estructural, Instituto Rocasolano, CSIC, Serrano 119, 28006 Madrid (Spain); Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid (Spain)
2007-07-01
The catabolic ornithine transcarbamylase (cOTC) from L. hilgardii has been overexpressed in E. coli, purified and crystallized under two different experimental conditions. The structure has been solved by the molecular-replacement method using the atomic coordinates of catabolic ornithine transcarbamylase from P. aeruginosa as the search model. The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His{sub 6} tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystals obtained from a solution containing 8%(w/v) PEG 4000, 75 mM sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 Å. Conversely, crystals grown in 20%(v/v) 2-methyl-2,4-pentanediol, 7.5%(w/v) PEG 4000, 100 mM HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 Å, β = 100.25°. Diffraction data were collected in-house to 3.00 and 2.91 Å resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 Å{sup 3} Da{sup −1}, respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code) as the search model.
International Nuclear Information System (INIS)
Rivas, Blanca de las; Rodríguez, Héctor; Angulo, Iván; Muñoz, Rosario; Mancheño, José M.
2007-01-01
The catabolic ornithine transcarbamylase (cOTC) from L. hilgardii has been overexpressed in E. coli, purified and crystallized under two different experimental conditions. The structure has been solved by the molecular-replacement method using the atomic coordinates of catabolic ornithine transcarbamylase from P. aeruginosa as the search model. The catabolic ornithine transcarbamylase (cOTC; EC 2.1.3.3) from the lactic acid bacteria Lactobacillus hilgardii is a key protein involved in the degradation of arginine during malolactic fermentation. cOTC containing an N-terminal His 6 tag has been overexpressed in Escherichia coli, purified and crystallized under two different experimental conditions using the hanging-drop vapour-diffusion method. Crystals obtained from a solution containing 8%(w/v) PEG 4000, 75 mM sodium acetate pH 4.6 belong to the trigonal space group P321 and have unit-cell parameters a = b = 157.04, c = 79.28 Å. Conversely, crystals grown in 20%(v/v) 2-methyl-2,4-pentanediol, 7.5%(w/v) PEG 4000, 100 mM HEPES pH 7.8 belong to the monoclinic space group C2 and have unit-cell parameters a = 80.06, b = 148.90, c = 91.67 Å, β = 100.25°. Diffraction data were collected in-house to 3.00 and 2.91 Å resolution for trigonal and monoclinic crystals, respectively. The estimated Matthews coefficient for the crystal forms were 2.36 and 2.24 Å 3 Da −1 , respectively, corresponding to 48% and 45% solvent content. In both cases, the results are consistent with the presence of three protein subunits in the asymmetric unit. The structure of cOTC has been determined by the molecular-replacement method using the atomic coordinates of cOTC from Pseudomonas aeruginosa (PDB code) as the search model
ARA1 regulates not only l-arabinose but also d-galactose catabolism in Trichoderma reesei
Benocci, Tiziano; Aguilar-Pontes, Maria Victoria; Kun, Roland Sándor; Seiboth, Bernhard; de Vries, Ronald P; Daly, Paul
2017-01-01
Trichoderma reesei is used to produce saccharifying enzyme cocktails for biofuels. There is limited understanding of the transcription factors (TFs) that regulate genes involved in release and catabolism of l-arabinose and d-galactose, as the main TF XYR1 is only partially involved. Here, the T.
Energy Technology Data Exchange (ETDEWEB)
Sun, Jing; Todd, Jonathan D.; Thrash, J. Cameron; Qian, Yanping; Qian, Michael C.; Temperton, Ben; Guo, Jiazhen; Fowler, Emily K.; Aldrich, Joshua T.; Nicora, Carrie D.; Lipton, Mary S.; Smith, Richard D.; De Leenheer, Patrick; Payne, Samuel H.; Johnston, Andrew W. B.; Davie-Martin, Cleo L.; Halsey, Kimberly H.; Giovannoni, Stephen J.
2016-05-16
Marine phytoplankton produce ~109 tons of dimethylsulfoniopropionate (DMSP) per year1,2, an estimated 10% of which is catabolized by bacteria through the DMSP cleavage pathway to the climatically active gas dimethyl sulfide (DMS)3,4. SAR11 Alphaproteobacteria (order Pelagibacterales), the most abundant chemoorganotrophic bacteria in the oceans, have been shown to assimilate DMSP into biomass, thereby supplying this cell’s unusual requirement for reduced sulfur5,6. Here we report that Pelagibacter HTCC1062 produces the gas methanethiol (MeSH) and that simultaneously a second DMSP catabolic pathway, mediated by a DMSP lyase, shunts as much as 59% of DMSP uptake to DMS production. We propose a model in which the allocation of DMSP between these pathways is kinetically controlled to release increasing amounts of DMS as the supply of DMSP exceeds cellular sulfur demands for biosynthesis. These findings suggest that DMSP supply and demand relationships in Pelagibacter metabolism are important to determining rates of oceanic DMS production.
Catabolism of lysine by mixed rumen bacteria
International Nuclear Information System (INIS)
Onodera, Ryoji; Kandatsu, Makoto.
1975-01-01
Metabolites arising from the catabolism of lysine by the mixed rumen bacteria were chromatographically examined by using radioactive lysine. After 6 hr incubation, 241 nmole/ml of lysine was decomposed to give ether-soluble substances and CO 2 by the bacteria and 90 nmole/ml of lysine was incorporated unchanged into the bacteria. delta-Aminovalerate, cadaverine or pipecolate did not seem to be produced from lysine even after incubation of the bacteria with addition of those three amino compounds to trap besides lysine and radioactive lysine. Most of the ether-soluble substances produced from radioactive lysine was volatile fatty acids (VFAs). Fractionation of VFAs revealed that the peaks of butyric and acetic acids coincided with the strong radioactive peaks. Small amounts of radioactivities were detected in propionic acid peak and a peak assumed to be caproic acid. The rumen bacteria appeared to decompose much larger amounts of lysine than the rumen ciliate protozoa did. (auth.)
l-Glucitol Catabolism in Stenotrophomonas maltophilia Ac
Brechtel, Elke; Huwig, Alexander; Giffhorn, Friedrich
2002-01-01
The carbohydrate catabolism of the bacterium Stenotrophomonas maltophilia Ac (previously named Pseudomonas sp. strain Ac), which is known to convert the unnatural polyol l-glucitol to d-sorbose during growth on the former as the sole source of carbon and energy, was studied in detail. All enzymes operating in a pathway that channels l-glucitol via d-sorbose into compounds of the intermediary metabolism were demonstrated, and for some prominent reactions the products of conversion were identified. d-Sorbose was converted by C-3 epimerization to d-tagatose, which, in turn, was isomerized to d-galactose. d-Galactose was the initial substrate of the De Ley-Doudoroff pathway, involving reactions of NAD-dependent oxidation of d-galactose to d-galactonate, its dehydration to 2-keto-3-deoxy-d-galactonate, and its phosphorylation to 2-keto-3-deoxy-d-galactonate 6-phosphate. Finally, aldol cleavage yielded pyruvate and d-glycerate 3-phosphate as the central metabolic intermediates. PMID:11823194
Takeshita, Masaru; Kuno, Atsushi; Suzuki, Katsuya; Matsuda, Atsushi; Shimazaki, Hiroko; Nakagawa, Tomomi; Otomo, Yuki; Kabe, Yasuaki; Suematsu, Makoto; Narimatsu, Hisashi; Takeuchi, Tsutomu
2016-05-21
Nearly all secreted proteins are glycosylated, and serum glycoproteins that exhibit disease-associated glycosylation changes have potential to be biomarkers. In rheumatoid arthritis (RA), C-reactive protein (CRP), and matrix metalloproteinase-3 (MMP-3) are widely used as serologic biomarkers, but they lack sufficient specificity or precision. We performed comparative glycosylation profiling of MMP-3 using a recently developed antibody-overlay lectin microarray technology that allows semicomprehensive and quantitative analysis of specific protein glycosylation to develop an RA-specific disease activity biomarker. Serum was taken from patients with RA (n = 24) whose disease activity was scored using composite measures, and MMP-3 was immunoprecipitated and subjected to lectin microarray analysis. A disease activity index (DAI) based on lectin signal was developed and validated using another cohort (n = 60). Synovial fluid MMP-3 in patients with RA and patients with osteoarthritis (OA) was also analyzed. Intense signals were observed on a sialic acid-binding lectin (Agrocybe cylindracea galectin [ACG]) and O-glycan-binding lectins (Jacalin, Agaricus bisporus agglutinin [ABA], and Amaranthus caudatus agglutinin [ACA]) by applying subnanogram levels of serum MMP-3. ACG, ABA, and ACA revealed differences in MMP-3 quantity, and Jacalin revealed differences in MMP-3 quality. The resultant index, ACG/Jacalin, correlated well with disease activity. Further validation using another cohort confirmed that this index correlated well with several DAIs and their components, and reflected DAI changes following RA treatment, with correlations greater than those for MMP-3 and CRP. Furthermore, MMP-3, which generated a high ACG/Jacalin score, accumulated in synovial fluid of patients with RA but not in that of patients with OA. Sialidase digestion revealed that the difference in quality was derived from O-glycan α-2,6-sialylation. This is the first report of a glycoprotein
A mass spectrometric method to determine activities of enzymes involved in polyamine catabolism
International Nuclear Information System (INIS)
Moriya, Shunsuke; Iwasaki, Kaori; Samejima, Keijiro; Takao, Koichi; Kohda, Kohfuku; Hiramatsu, Kyoko; Kawakita, Masao
2012-01-01
Highlights: ► Compounds in polyamine catabolic pathway were determined by a column-free ESI-TOF MS. ► N 1 - and N 8 -acetylspermidine were determined by a column-free ESI-MS/MS. ► The method was applied to determine activities of APAO, SMO, and SSAT in the pathway. ► The assay method contained stable isotope-labeled natural substrates. ► It is applicable to biological samples containing natural substrate and product. - Abstract: An analytical method for the determination of three polyamines (putrescine, spermidine, and spermine) and five acetylpolyamines [N 1 -acetylspermidine (N 1 AcSpd), N 8 -acetylspermidine (N 8 AcSpd), N 1 -acetylspermine, N 1 ,N 8 -diacetylspermidine, and N 1 ,N 12 -diacetylspermine] involved in the polyamine catabolic pathway has been developed using a hybrid tandem mass spectrometer. Heptafluorobutyryl (HFB) derivatives of these compounds and respective internal standards labeled with stable isotopes were analyzed simultaneously by TOF MS, based on peak areas appearing at appropriate m/z values. The isomers, N 1 AcSpd and N 8 AcSpd were determined from their fragment ions, the acetylamidopropyl and acetylamidobutyl groups, respectively, using MS/MS with 13 C 2 -N 1 AcSpd and 13 C 2 -N 8 AcSpd which have the 13 C 2 -acetyl group as an internal standard. The TOF MS method was successfully applied to measure the activity of enzymes involved in polyamine catabolic pathways, namely N 1 -acetylpolyamine oxidase (APAO), spermine oxidase (SMO), and spermidine/spermine N 1 -acetyltransferase (SSAT). The following natural substrates and products labeled with stable isotopes considering the application to biological samples were identified; for APAO, [4,9,12- 15 N 3 ]-N 1 -acetylspermine and [1,4,8- 15 N 3 ]spermidine ( 15 N 3 -Spd), respectively; for SMO, [1,4,8,12- 15 N 4 ]spermine and 15 N 3 -Spd, respectively; and for SSAT, 15 N 3 -Spd and [1,4,8- 15 N 3 ]-N 1 -acetylspermidine, respectively.
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Subtil Thorsten
2012-03-01
Full Text Available Abstract Background In mixed sugar fermentations with recombinant Saccharomyces cerevisiae strains able to ferment D-xylose and L-arabinose the pentose sugars are normally only utilized after depletion of D-glucose. This has been attributed to competitive inhibition of pentose uptake by D-glucose as pentose sugars are taken up into yeast cells by individual members of the yeast hexose transporter family. We wanted to investigate whether D-glucose inhibits pentose utilization only by blocking its uptake or also by interfering with its further metabolism. Results To distinguish between inhibitory effects of D-glucose on pentose uptake and pentose catabolism, maltose was used as an alternative carbon source in maltose-pentose co-consumption experiments. Maltose is taken up by a specific maltose transport system and hydrolyzed only intracellularly into two D-glucose molecules. Pentose consumption decreased by about 20 - 30% during the simultaneous utilization of maltose indicating that hexose catabolism can impede pentose utilization. To test whether intracellular D-glucose might impair pentose utilization, hexo-/glucokinase deletion mutants were constructed. Those mutants are known to accumulate intracellular D-glucose when incubated with maltose. However, pentose utilization was not effected in the presence of maltose. Addition of increasing concentrations of D-glucose to the hexo-/glucokinase mutants finally completely blocked D-xylose as well as L-arabinose consumption, indicating a pronounced inhibitory effect of D-glucose on pentose uptake. Nevertheless, constitutive overexpression of pentose-transporting hexose transporters like Hxt7 and Gal2 could improve pentose consumption in the presence of D-glucose. Conclusion Our results confirm that D-glucose impairs the simultaneous utilization of pentoses mainly due to inhibition of pentose uptake. Whereas intracellular D-glucose does not seem to have an inhibitory effect on pentose utilization
Convergent evolution of Amadori opine catabolic systems in plasmids of Agrobacterium tumefaciens.
Baek, Chang-Ho; Farrand, Stephen K; Lee, Ko-Eun; Park, Dae-Kyun; Lee, Jeong Kug; Kim, Kun-Soo
2003-01-01
Deoxyfructosyl glutamine (DFG, referred to elsewhere as dfg) is a naturally occurring Amadori compound found in rotting fruits and vegetables. DFG also is an opine and is found in tumors induced by chrysopine-type strains of Agrobacterium tumefaciens. Such strains catabolize this opine via a pathway coded for by their plasmids. NT1, a derivative of the nopaline-type A. tumefaciens strain C58 lacking pTiC58, can utilize DFG as the sole carbon source. Genes for utilization of DFG were mapped to the 543-kb accessory plasmid pAtC58. Two cosmid clones of pAtC58 allowed UIA5, a plasmid-free derivative of C58, harboring pSa-C that expresses MocC (mannopine [MOP] oxidoreductase that oxidizes MOP to DFG), to grow by using MOP as the sole carbon source. Genetic analysis of subclones indicated that the genes for utilization of DFG are located in a 6.2-kb BglII (Bg2) region adjacent to repABC-type genes probably responsible for the replication of pAtC58. This region contains five open reading frames organized into at least two transcriptional soc (santhopine catabolism) groups: socR and socABCD. Nucleotide sequence analysis and analyses of transposon-insertion mutations in the region showed that SocR negatively regulates the expression of socR itself and socABCD. SocA and SocB are responsible for transport of DFG and MOP. SocA is a homolog of known periplasmic amino acid binding proteins. The N-terminal half of SocB is a homolog of the transmembrane transporter proteins for several amino acids, and the C-terminal half is a homolog of the transporter-associated ATP-binding proteins. SocC and SocD could be responsible for the enzymatic degradation of DFG, being homologs of sugar oxidoreductases and an amadoriase from Corynebacterium sp., respectively. The protein products of socABCD are not related at the amino acid sequence level to those of the moc and mot genes of Ti plasmids responsible for utilization of DFG and MOP, indicating that these two sets of genes and their
Czech Academy of Sciences Publication Activity Database
Mareš, Vladislav; Brückner, G.
2001-01-01
Roč. 19, č. 3 (2001), s. 297-303 ISSN 0736-5748 Institutional research plan: CEZ:AV0Z5011922 Keywords : fetal rat brain * fucosylated glycans * cerebrospinal fluid Subject RIV: FH - Neurology Impact factor: 2.156, year: 2001
Carvalho, Fernanda Caroline; Soares, Sandro Gomes; Tamarozzi, Mirela Barros; Rego, Eduardo Magalhães; Roque-Barreira, Maria-Cristina
2011-01-01
ArtinM, a d-mannose-binding lectin from Artocarpus heterophyllus (jackfruit), interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6)Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC50 = 10 µg/mL), as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment. PMID:22132163
Directory of Open Access Journals (Sweden)
Fernanda Caroline Carvalho
Full Text Available ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus (jackfruit, interacts with N-glycosylated receptors on the surface of several cells of hematopoietic origin, triggering cell migration, degranulation, and cytokine release. Because malignant transformation is often associated with altered expression of cell surface glycans, we evaluated the interaction of ArtinM with human myelocytic leukemia cells and investigated cellular responses to lectin binding. The intensity of ArtinM binding varied across 3 leukemia cell lines: NB4>K562>U937. The binding, which was directly related to cell growth suppression, was inhibited in the presence of Manα1-3(Manα1-6Manβ1, and was reverted in underglycosylated NB4 cells. ArtinM interaction with NB4 cells induced cell death (IC(50 = 10 µg/mL, as indicated by cell surface exposure of phosphatidylserine and disruption of mitochondrial membrane potential unassociated with caspase activation or DNA fragmentation. Moreover, ArtinM treatment of NB4 cells strongly induced reactive oxygen species generation and autophagy, as indicated by the detection of acidic vesicular organelles in the treated cells. NB4 cell death was attributed to ArtinM recognition of the trimannosyl core of N-glycans containing a ß1,6-GlcNAc branch linked to α1,6-mannose. This modification correlated with higher levels of N-acetylglucosaminyltransferase V transcripts in NB4 cells than in K562 or U937 cells. Our results provide new insights into the potential of N-glycans containing a β1,6-GlcNAc branch linked to α1,6-mannose as a novel target for anti-leukemia treatment.
Smit, C.H.; van Diepen, A.; Nguyen, D.L.; Wuhrer, M.; Hoffmann, K.F.; Deelder, A.M.; Hokke, C.H.
2015-01-01
Glycans present on glycoproteins and glycolipids of the major human parasite Schistosoma mansoni induce innate as well as adaptive immune responses in the host. To be able to study the molecular characteristics of schistosome infections it is therefore required to determine the expression profiles
Objective: Branched-chain alpha-keto acid dehydrogenase complex (BCKDC) regulates branched-chain amino acid (BCAA) metabolism at the level of branched chain alpha-ketoacid (BCKA) catabolism. It has been demonstrated that the activity of hepatic BCKDC is markedly decreased in type 2 diabetic animal...
CLONING AND CHARACTERIZATION OF THE PHTHALATE CATABOLISM REGION OF PRE1 OF ARTHROBACTER KEYSERI 12B
o-Phthalate (benzene-1,2-dicarboxylate) is a central intermediate in the bacterial degradation of phthalate ester plasticizers as well as of a number of fused-ring polycyclic aromatic hydrocarbons found in fossil fuels. In Arthrobacter keyseri 12B, the genes encoding catabolism o...
Czech Academy of Sciences Publication Activity Database
Vancová, Marie; Nebesářová, Jana
2015-01-01
Roč. 10, č. 12 (2015), č. článku e0145034. E-ISSN 1932-6203 R&D Projects: GA TA ČR(CZ) TE01020118 EU Projects: European Commission(XE) 278976 - ANTIGONE Institutional support: RVO:60077344 Keywords : salivary gland * n-glycans * tick * glycosylation * fucose * sheep Subject RIV: EA - Cell Biology Impact factor: 3.057, year: 2015
Sakaguchi, Kouta; Katoh, Toshihiko; Yamamoto, Kenji
2016-11-01
Glycan conversion of glycoprotein via the transglycosylation activity of endo-β-N-acetylglucosaminidase is a promising chemoenzymatic technology for the production of glycoproteins including bio-medicines with a homogeneous glycoform. Although Endo-M is a key enzyme in this process, its product undergoes rehydrolysis, which leads to a lower yield, and limits the practical application of this enzyme. We developed several Endo-M mutant enzymes including N175Q with glycosynthase-like activity and/or transglycosidase-like activity. We found that the Endo-M N175H mutant showed glycosynthase-like activity comparable to N175Q as well as transglycosidase-like activity superior to N175Q. Using a natural sialylglycopeptide as a donor substrate, N175H readily transferred the sialo-glycan onto an N-acetylglucosamine residue attached to bovine ribonuclease B (RNase B), yielding a nonnative sialoglycosylated RNase B. These results demonstrate that use of Endo-M N175H is an alternative glycoengineering technique, which provides a relatively high yield of transglycosylation product and avoids the laborious synthesis of a sugar oxazoline as a donor substrate. © 2015 International Union of Biochemistry and Molecular Biology, Inc.
International Nuclear Information System (INIS)
Sagee, O.; Riov, J.; Goren, J.
1990-01-01
Exogenous [ 14 C]indole-3-acetic acid (IAA) is conjugated in citrus (Citrus sinensis) leaf tissues to one major substance which has been identified as indole-3-acetylaspartic acid (IAAsp). Ethylene pretreatment enhanced the catabolism of [ 14 C]IAA to indole-3-carboxylic acid (ICA), which accumulated as glucose esters (ICGlu). Increased formation of ICGlu by ethylene was accompanied by a concomitant decrease in IAAsp formation. IAAsp and ICGlu were identified by combined gas chromatography-mass spectrometry. Formation of ICGlu was dependent on the concentration of ethylene and the duration of the ethylene pretreatment. It is suggested that the catabolism of IAA to ICA may be one of the mechanisms by which ethylene endogenous IAA levels
International Nuclear Information System (INIS)
Obernikhin, S.S.; Fuks, B.B.
1992-01-01
E1-4 and P-815 murine tumor cells labelled by 125 I-deoxyuridine or 51 Cr were administered in 7-day subcutaneous syngeneic tumors or subcutaneosly. At the same time different groups of mice were treated by immunomodulators and cytostatics. It was shown that cytostatics and immunomodulators significantly delayed catabolism and withdrawing of 125 I-deoxyuridine (that has not been incorporated in DNA) from tumor cells. This delay was correlated with the inhibition of tumor nodes growth rate. It is concluded that influence of cytostatics and immunomodulators on catabolism and withdrawing rate of 125 I-deoxyuridine from tumor cells relates to their cytostatic effect and may be used at the earliest screening step of immunomodulator analysis
Hosomi, Akira; Suzuki, Tadashi
2015-04-01
Endoplasmic reticulum (ER)-associated degradation (ERAD) is a pathway by which misfolded or improperly assembled proteins in the ER are directed to degradation. The cytoplasmic peptide:N-glycanase (PNGase) is a deglycosylating enzyme that cleaves N-glycans from misfolded glycoproteins during the ERAD process. The mutant form of yeast carboxypeptidase Y (CPY*) is an ERAD model substrate that has been extensively studied in yeast. While a delay in the degradation of CPY* in yeast cells lacking the cytoplasmic PNGase (Png1 in yeast) was evident, the in vivo action of PNGase on CPY* has not been detected. We constructed new ERAD substrates derived from CPY*, bearing epitope tags at both N- and C-termini and examined the degradation intermediates observed in yeast cells with compromised proteasome activity. The occurrence of the PNGase-mediated deglycosylation of intact CPY* and its degradation intermediates was evident. A major endoproteolytic reaction on CPY* appears to occur between amino acid 400 and 404. The findings reported herein clearly indicate that PNGase indeed releases N-glycans from CPY* during the ERAD process in vivo. This report implies that the PNGase-mediated deglycosylation during the ERAD process may occur more abundantly than currently envisaged. Copyright © 2014 Elsevier B.V. All rights reserved.
Long, D.L.; Ulici, V.; Chubinskaya, S.; Loeser, R.F.
2015-01-01
Objective We determined if the epidermal growth factor receptor ligand HB-EGF is produced in cartilage and if it regulates chondrocyte anabolic or catabolic activity. Methods HB-EGF expression was measured by quantitative PCR using RNA isolated from mouse knee joint tissues and from normal and OA human chondrocytes. Immunohistochemistry was performed on normal and OA human cartilage and meniscus sections. Cultured chondrocytes were treated with fibronectin fragments (FN-f) as a catabolic stimulus and osteogenic protein 1 (OP-1) as an anabolic stimulus. Effects of HB-EGF on cell signaling were analyzed by immunoblotting of selected signaling proteins. MMP-13 was measured in conditioned media, proteoglycan synthesis was measured by sulfate incorporation, and matrix gene expression by quantitative PCR. Results HB-EGF expression was increased in 12-month old mice at 8 weeks after surgery to induce OA and increased amounts of HB-EGF were noted in human articular cartilage from OA knees. FN-f stimulated chondrocyte HB-EGF expression and HB-EGF stimulated chondrocyte MMP-13 production. However, HB-EGF was not required for FN-f stimulation of MMP-13 production. HB-EGF activated the ERK and p38 MAP kinases and stimulated phosphorylation of Smad1 at an inhibitory serine site which was associated with inhibition of OP-1 mediated proteoglycan synthesis and reduced aggrecan (ACAN) but not COL2A1 expression. Conclusion HB-EGF is a new factor identified in OA cartilage that promotes chondrocyte catabolic activity while inhibiting anabolic activity suggesting it could contribute to the catabolic-anabolic imbalance seen in OA cartilage. PMID:25937027
Glycan dependence of Galectin-3 self-association properties.
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Hubert Halimi
Full Text Available Human Galectin-3 is found in the nucleus, the cytoplasm and at the cell surface. This lectin is constituted of two domains: an unfolded N-terminal domain and a C-terminal Carbohydrate Recognition Domain (CRD. There are still uncertainties about the relationship between the quaternary structure of Galectin-3 and its carbohydrate binding properties. Two types of self-association have been described for this lectin: a C-type self-association and a N-type self-association. Herein, we have analyzed Galectin-3 oligomerization by Dynamic Light Scattering using both the recombinant CRD and the full length lectin. Our results proved that LNnT induces N-type self-association of full length Galectin-3. Moreover, from Nuclear Magnetic Resonance (NMR and Surface Plasmon Resonance experiments, we observed no significant specificity or affinity variations for carbohydrates related to the presence of the N-terminal domain of Galectin-3. NMR mapping clearly established that the N-terminal domain interacts with the CRD. We propose that LNnT induces a release of the N-terminal domain resulting in the glycan-dependent self-association of Galectin-3 through N-terminal domain interactions.
Sato, Kae; Sato, Kiichi; Okubo, Akira; Yamazaki, Sunao
2005-01-01
A capillary electrophoresis method was developed for the analysis of oligosaccharides combined with derivatization with 2-aminobenzoic acid. Glycosaminoglycan delta-disaccharides were effectively resolved on a fused-silica capillary tube using 150 mM borate, pH 8.5, as a running electrolyte solution. This analytical method was applied to the identification of glycosaminoglycan in combination with enzymatic digestion. The separation of N-glycans or glucose-oligomers was performed with a phosphate buffer containing polyethylene glycol or borate as an electrolyte solution. This method is expected to be useful in the determination of oligosaccharide structures in a glycoprotein.
de Sain-van der Velden, M; Kaysen, GA; Barrett, HA; Stellaard, F; Gadellaa, MM; Voorbij, HA; Reijngoud, DJ; Rabelink, TJ
Increased very low density lipoprotein (VLDL) in nephrotic patients results from a decreased catabolism while increased low density lipoprotein (LDL) results from increased synthesis. Hyperlipidemias a hallmark of nephrotic syndrome that has been associated with increased risk for ischemic heart
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Sanja Goč
2013-01-01
Full Text Available This study was aimed at defining molecular species of prostate-specific antigen (PSA in immune complexes with immunoglobulin M (IgM. Having in mind the oligoreactivity of IgM and its preference for carbohydrate antigens, there is the possibility that it can selectively recognize known PSA glycoisoforms. PSA-IgM complexes and free PSA fractions were separated from the sera of subjects with prostate cancer (PCa and benign prostatic hyperplasia (BPH by gel filtration and subjected to on-chip immunoaffinity and ion-exchange chromatography. PSA-immunoreactive species were detected using surface-enhanced laser desorption/ionization time of flight mass spectrometry. The obtained spectra were analyzed for protein and glycan composition. The general pattern of the molecular species of PCa PSA and BPH PSA found in complexes with IgM was similar. It comprised major peaks at 17 kDa and minor peaks at 28 kDa, corresponding to the entire mature glycosylated PSA. The main difference was the presence of incompletely glycosylated 26.8 kDa species, having putative paucimannosidic structures, observed in PCa PSA-IgM, but not in BPH PSA-IgM. Characteristic PCa PSA-IgM glycoforms pose the question of the possible role of glycosylation as a framework for immune surveillance and may be of interest in light of recent data indicating mannose-containing glycans as cancer biomarker.
Goč, Sanja; Janković, Miroslava
2013-01-01
This study was aimed at defining molecular species of prostate-specific antigen (PSA) in immune complexes with immunoglobulin M (IgM). Having in mind the oligoreactivity of IgM and its preference for carbohydrate antigens, there is the possibility that it can selectively recognize known PSA glycoisoforms. PSA-IgM complexes and free PSA fractions were separated from the sera of subjects with prostate cancer (PCa) and benign prostatic hyperplasia (BPH) by gel filtration and subjected to on-chip immunoaffinity and ion-exchange chromatography. PSA-immunoreactive species were detected using surface-enhanced laser desorption/ionization time of flight mass spectrometry. The obtained spectra were analyzed for protein and glycan composition. The general pattern of the molecular species of PCa PSA and BPH PSA found in complexes with IgM was similar. It comprised major peaks at 17 kDa and minor peaks at 28 kDa, corresponding to the entire mature glycosylated PSA. The main difference was the presence of incompletely glycosylated 26.8 kDa species, having putative paucimannosidic structures, observed in PCa PSA-IgM, but not in BPH PSA-IgM. Characteristic PCa PSA-IgM glycoforms pose the question of the possible role of glycosylation as a framework for immune surveillance and may be of interest in light of recent data indicating mannose-containing glycans as cancer biomarker.
Energy Technology Data Exchange (ETDEWEB)
Oliveira, Joao Ezequiel de
2007-07-01
A novel, fast and practical two-step purification strategy, consisting of a classical ion exchange and a reversed-phase high performance liquid chromatography (RP-HPLC), for rapidly obtaining CHO-derived hTSH, was set up providing r-hTSH with 70% yield and > 99% purity. A consistent increase of {approx} 60% in the secretion yields of r-hTSH-IPEN was observed by changing cell culture CO{sub 2} conditions from 5% CO{sub 2} to air environment (0.03% CO{sub 2}). The overall quality of the products obtained under both conditions was evaluated for what concerns N-glycan structure, charge isomers and biological activity in comparison with a well known recombinant biopharmaceutical (Thyrogen{sup R}) and with a pituitary reference preparation (p-hTSH) from National Hormone and Pituitary Program (NIDDK, USA). The N-glycans identified in the recombinant preparations were of the complex type, presenting bi-, tri- and tetra-antennary structures, sometimes fucosylated, 86-88% of the identified structures being sialylated at variable levels. The three most abundant structures were monosialylated glycans, representing {approx} 69% of all identified forms in the three preparations. The main difference was found in terms of antennarity, with 8-10% more bi-antennary structures obtained in the absence of CO{sub 2} and 7-9% more tri-antennary structures in its presence. In the case of p-hTSH, complex, high-mannose and hybrid N-glycan structures were identified, most of them containing sialic acid and/or sulphate terminal residues. The two most abundant structures were shown to contain one or two sulphate residues, one of which unexpectedly bound to galactose. The sialic acid-galactose linkage was also determined, having found that 68 3 {+-} 10% was in the {alpha} 2,6 and 32 {+-} 10% in the {alpha}2,3 conformation. No remarkable difference in charge isomers was observed between the three recombinant preparations, the isoelectric focusing profiles showing six distinct bands in the 5
Carrino, David A; Onnerfjord, Patrik; Sandy, John D; Cs-Szabo, Gabriella; Scott, Paul G; Sorrell, J Michael; Heinegård, Dick; Caplan, Arnold I
2003-05-09
Dramatic changes occur in skin as a function of age, including changes in morphology, physiology, and mechanical properties. Changes in extracellular matrix molecules also occur, and these changes likely contribute to the overall age-related changes in the physical properties of skin. The major proteoglycans detected in extracts of human skin are decorin and versican. In addition, adult human skin contains a truncated form of decorin, whereas fetal skin contains virtually undetectable levels of this truncated decorin. Analysis of this molecule, herein referred to as decorunt, indicates that it is a catabolic fragment of decorin rather than a splice variant. With antibody probes to the core protein, decorunt is found to lack the carboxyl-terminal portion of decorin. Further analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry shows that the carboxyl terminus of decorunt is at Phe(170) of decorin. This result indicates that decorunt represents the amino-terminal 43% of the mature decorin molecule. Such a structure is inconsistent with alternative splicing of decorin and suggests that decorunt is a catabolic fragment of decorin. A neoepitope antiserum, anti-VRKVTF, was generated against the carboxyl terminus of decorunt. This antiserum does not recognize intact decorin in any skin proteoglycan sample tested on immunoblots but recognizes every sample of decorunt tested. The results with anti-VRKVTF confirm the identification of the carboxyl terminus of decorunt. Analysis of collagen binding by surface plasmon resonance indicates that the affinity of decorunt for type I collagen is 100-fold less than that of decorin. This observation correlates with the structural analysis of decorunt, in that it lacks regions of decorin previously shown to be important for interaction with type I collagen. The detection of a catabolic fragment of decorin suggests the existence of a specific catabolic pathway for this proteoglycan. Because of the
Expression of eicosanoid biosynthetic and catabolic enzymes in peritoneal endometriosis.
Lousse, J-C; Defrère, S; Colette, S; Van Langendonckt, A; Donnez, J
2010-03-01
Increased peritoneal eicosanoid concentrations have been reported in endometriosis patients and might be important in disease-associated pain and inflammation. Here, we evaluated the expression of key biosynthetic and catabolic enzymes involved in this abnormal eicosanoid production in peritoneal macrophages and endometriotic lesions. Peritoneal macrophages, endometriotic lesions and matched eutopic endometrium were collected from endometriosis patients (n = 40). Peritoneal macrophages and eutopic endometrium samples were also collected from disease-free women (n = 25). Expression of type IIA secretory phospholipase A(2) (sPLA(2)-IIA), cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), 15-hydroxyprostaglandin dehydrogenase (15-PGDH) and 5-lipoxygenase (5-LO) was quantified by real-time PCR, and these five key enzymes were localized by immunohistochemistry. sPLA(2)-IIA, COX-2 and mPGES-1 mRNA was significantly increased in peritoneal macrophages of endometriosis patients compared with controls (P = 0.006, P = 0.016 and P = 0.025, respectively). In endometriosis patients, sPLA(2)-IIA, mPGES-1 and 15-PGDH mRNA was significantly enhanced in peritoneal lesions compared with matched eutopic endometrium (P endometriosis group compared with controls (P = 0.023). Finally, sPLA(2)-IIA, COX-2, mPGES-1 and 15-PGDH immunostaining was found mainly in endometrial glands, whereas 5-LO was distributed throughout the glands and stroma. Our study highlights an imbalance between eicosanoid biosynthesis and degradation in endometriosis patients. Both peritoneal macrophages and endometriotic lesions may be involved. Research into new molecules inhibiting biosynthetic enzymes (such as sPLA(2)-IIA and mPGES-1) and/or activating catabolic enzymes (such as 15-PGDH) may prove to be a major field of investigation in the development of targeted medical therapies.
Lipid catabolism of invertebrate predator indicates widespread wetland ecosystem degradation
Anteau, Michael J.; Afton, Alan D.
2011-01-01
Animals frequently undergo periods when they accumulate lipid reserves for subsequent energetically expensive activities, such as migration or breeding. During such periods, daily lipid-reserve dynamics (DLD) of sentinel species can quantify how landscape modifications affect function, health, and resilience of ecosystems. Aythya affinis (Eyton 1838; lesser scaup; diving duck) are macroinvertebrate predators; they migrate through an agriculturally dominated landscape in spring where they select wetlands with the greatest food density to refuel and accumulate lipid reserves for subsequent reproduction. We index DLD by measuring plasma-lipid metabolites of female scaup (n = 459) that were refueling at 75 spring migration stopover areas distributed across the upper Midwest, USA. We also indexed DLD for females (n = 44) refueling on a riverine site (Pool 19) south of our upper Midwest study area. We found that mean DLD estimates were significantly (P<0.05) less than zero in all ecophysiographic regions of the upper Midwest, and the greatest negative value was in the Iowa Prairie Pothole region (-31.6). Mean DLD was 16.8 at Pool 19 and was markedly greater than in any region of the upper Midwest. Our results indicate that females catabolized rather than stored lipid reserves throughout the upper Midwest. Moreover, levels of lipid catabolism are alarming, because scaup use the best quality wetlands available within a given stopover area. Accordingly, these results provide evidence of wetland ecosystem degradation across this large agricultural landscape and document affects that are carried-up through several trophic levels. Interestingly, storing of lipids by scaup at Pool 19 likely reflects similar ecosystem perturbations as observed in the upper Midwest because wetland drainage and agricultural runoff nutrifies the riverine habitat that scaup use at Pool 19. Finally, our results underscore how using this novel technique to monitor DLD, of a carefully selected sentinel
Lipid catabolism of invertebrate predator indicates widespread wetland ecosystem degradation.
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Michael J Anteau
Full Text Available Animals frequently undergo periods when they accumulate lipid reserves for subsequent energetically expensive activities, such as migration or breeding. During such periods, daily lipid-reserve dynamics (DLD of sentinel species can quantify how landscape modifications affect function, health, and resilience of ecosystems. Aythya affinis (Eyton 1838; lesser scaup; diving duck are macroinvertebrate predators; they migrate through an agriculturally dominated landscape in spring where they select wetlands with the greatest food density to refuel and accumulate lipid reserves for subsequent reproduction. We index DLD by measuring plasma-lipid metabolites of female scaup (n = 459 that were refueling at 75 spring migration stopover areas distributed across the upper Midwest, USA. We also indexed DLD for females (n = 44 refueling on a riverine site (Pool 19 south of our upper Midwest study area. We found that mean DLD estimates were significantly (P<0.05 less than zero in all ecophysiographic regions of the upper Midwest, and the greatest negative value was in the Iowa Prairie Pothole region (-31.6. Mean DLD was 16.8 at Pool 19 and was markedly greater than in any region of the upper Midwest. Our results indicate that females catabolized rather than stored lipid reserves throughout the upper Midwest. Moreover, levels of lipid catabolism are alarming, because scaup use the best quality wetlands available within a given stopover area. Accordingly, these results provide evidence of wetland ecosystem degradation across this large agricultural landscape and document affects that are carried-up through several trophic levels. Interestingly, storing of lipids by scaup at Pool 19 likely reflects similar ecosystem perturbations as observed in the upper Midwest because wetland drainage and agricultural runoff nutrifies the riverine habitat that scaup use at Pool 19. Finally, our results underscore how using this novel technique to monitor DLD, of a carefully
Tyrosine biosynthesis, metabolism, and catabolism in plants.
Schenck, Craig A; Maeda, Hiroshi A
2018-05-01
L-Tyrosine (Tyr) is an aromatic amino acid (AAA) required for protein synthesis in all organisms, but synthesized de novo only in plants and microorganisms. In plants, Tyr also serves as a precursor of numerous specialized metabolites that have diverse physiological roles as electron carriers, antioxidants, attractants, and defense compounds. Some of these Tyr-derived plant natural products are also used in human medicine and nutrition (e.g. morphine and vitamin E). While the Tyr biosynthesis and catabolic pathways have been extensively studied in microbes and animals, respectively, those of plants have received much less attention until recently. Accumulating evidence suggest that the Tyr biosynthetic pathways differ between microbes and plants and even within the plant kingdom, likely to support the production of lineage-specific plant specialized metabolites derived from Tyr. The interspecies variations of plant Tyr pathway enzymes can now be used to enhance the production of Tyr and Tyr-derived compounds in plants and other synthetic biology platforms. Copyright © 2018 Elsevier Ltd. All rights reserved.
Nass, Sara R; Long, Jonathan Z; Schlosburg, Joel E; Cravatt, Benjamin F; Lichtman, Aron H; Kinsey, Steven G
2015-06-01
Cannabinoid receptor agonists, such as Δ(9)-THC, the primary active constituent of Cannabis sativa, have anti-pyrogenic effects in a variety of assays. Recently, attention has turned to the endogenous cannabinoid system and how endocannabinoids, including 2-arachidonoylglycerol (2-AG) and anandamide, regulate multiple homeostatic processes, including thermoregulation. Inhibiting endocannabinoid catabolic enzymes, monoacylglycerol lipase (MAGL) or fatty acid amide hydrolase (FAAH), elevates levels of 2-AG or anandamide in vivo, respectively. The purpose of this experiment was to test the hypothesis that endocannabinoid catabolic enzymes function to maintain thermal homeostasis in response to hypothermic challenge. In separate experiments, male C57BL/6J mice were administered a MAGL or FAAH inhibitor, and then challenged with the bacterial endotoxin lipopolysaccharide (LPS; 2 mg/kg ip) or a cold (4 °C) ambient environment. Systemic LPS administration caused a significant decrease in core body temperature after 6 h, and this hypothermia persisted for at least 12 h. Similarly, cold environment induced mild hypothermia that resolved within 30 min. JZL184 exacerbated hypothermia induced by either LPS or cold challenge, both of which effects were blocked by rimonabant, but not SR144528, indicating a CB1 cannabinoid receptor mechanism of action. In contrast, the FAAH inhibitor, PF-3845, had no effect on either LPS-induced or cold-induced hypothermia. These data indicate that unlike direct acting cannabinoid receptor agonists, which elicit profound hypothermic responses on their own, neither MAGL nor FAAH inhibitors affect normal body temperature. However, these endocannabinoid catabolic enzymes play distinct roles in thermoregulation following hypothermic challenges.
Pietravalle, Stéphane; Aspray, Thomas J
2013-05-01
Multiple substrate induced respiration (MSIR) assays which assess the response of soils to carbon source amendment are effective approaches to determine catabolic diversity of soils. Many assays are based on a single short term (hydrocarbon contaminated soils using continuous CO2 and O2 respiration measurements. Based on cumulative CO2 and O2 measurements at 4, 24 and 120 h, the soils were found to be distinct in terms of their catabolic diversity. Most noteworthy, however, was the response to the addition of maleic acid which provided strong evidence of abiotic CO2 efflux to be the overriding process, raising questions about the interpretation of CO2 only responses from organic acid addition in MSIR assays. Copyright © 2013 Elsevier Ltd. All rights reserved.
Lackey, Denise E.; Lynch, Christopher J.; Olson, Kristine C.; Mostaedi, Rouzbeh; Ali, Mohamed; Smith, William H.; Karpe, Fredrik; Humphreys, Sandy; Bedinger, Daniel H.; Dunn, Tamara N.; Thomas, Anthony P.; Oort, Pieter J.; Kieffer, Dorothy A.; Amin, Rajesh; Bettaieb, Ahmed; Haj, Fawaz G.; Permana, Paska; Anthony, Tracy G.
2013-01-01
Elevated blood branched-chain amino acids (BCAA) are often associated with insulin resistance and type 2 diabetes, which might result from a reduced cellular utilization and/or incomplete BCAA oxidation. White adipose tissue (WAT) has become appreciated as a potential player in whole body BCAA metabolism. We tested if expression of the mitochondrial BCAA oxidation checkpoint, branched-chain α-ketoacid dehydrogenase (BCKD) complex, is reduced in obese WAT and regulated by metabolic signals. WAT BCKD protein (E1α subunit) was significantly reduced by 35–50% in various obesity models (fa/fa rats, db/db mice, diet-induced obese mice), and BCKD component transcripts significantly lower in subcutaneous (SC) adipocytes from obese vs. lean Pima Indians. Treatment of 3T3-L1 adipocytes or mice with peroxisome proliferator-activated receptor-γ agonists increased WAT BCAA catabolism enzyme mRNAs, whereas the nonmetabolizable glucose analog 2-deoxy-d-glucose had the opposite effect. The results support the hypothesis that suboptimal insulin action and/or perturbed metabolic signals in WAT, as would be seen with insulin resistance/type 2 diabetes, could impair WAT BCAA utilization. However, cross-tissue flux studies comparing lean vs. insulin-sensitive or insulin-resistant obese subjects revealed an unexpected negligible uptake of BCAA from human abdominal SC WAT. This suggests that SC WAT may not be an important contributor to blood BCAA phenotypes associated with insulin resistance in the overnight-fasted state. mRNA abundances for BCAA catabolic enzymes were markedly reduced in omental (but not SC) WAT of obese persons with metabolic syndrome compared with weight-matched healthy obese subjects, raising the possibility that visceral WAT contributes to the BCAA metabolic phenotype of metabolically compromised individuals. PMID:23512805
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Alessandra Ravidà
2016-04-01
Full Text Available Fascioliasis, caused by the liver fluke Fasciola hepatica, is a neglected tropical disease infecting over 1 million individuals annually with 17 million people at risk of infection. Like other helminths, F. hepatica employs mechanisms of immune suppression in order to evade its host immune system. In this study the N-glycosylation of F. hepatica's tegumental coat (FhTeg and its carbohydrate-dependent interactions with bone marrow derived dendritic cells (BMDCs were investigated. Mass spectrometric analysis demonstrated that FhTeg N-glycans comprised mainly of oligomannose and to a lesser extent truncated and complex type glycans, including a phosphorylated subset. The interaction of FhTeg with the mannose receptor (MR was investigated. Binding of FhTeg to MR-transfected CHO cells and BMDCs was blocked when pre-incubated with mannan. We further elucidated the role played by MR in the immunomodulatory mechanism of FhTeg and demonstrated that while FhTeg's binding was significantly reduced in BMDCs generated from MR knockout mice, the absence of MR did not alter FhTeg's ability to induce SOCS3 or suppress cytokine secretion from LPS activated BMDCs. A panel of negatively charged monosaccharides (i.e. GlcNAc-4P, Man-6P and GalNAc-4S were used in an attempt to inhibit the immunoregulatory properties of phosphorylated oligosaccharides. Notably, GalNAc-4S, a known inhibitor of the Cys-domain of MR, efficiently suppressed FhTeg binding to BMDCs and inhibited the expression of suppressor of cytokine signalling (SOCS 3, a negative regulator the TLR and STAT3 pathway. We conclude that F. hepatica contains high levels of mannose residues and phosphorylated glycoproteins that are crucial in modulating its host's immune system, however the role played by MR appears to be limited to the initial binding event suggesting that other C-type lectin receptors are involved in the immunomodulatory mechanism of FhTeg.
Comprehensive glycan analysis of recombinant Aspergillus niger endo-polygalacturonase C.
Woosley, Bryan; Xie, Min; Wells, Lance; Orlando, Ron; Garrison, Derek; King, Daniel; Bergmann, Carl
2006-07-01
The enzyme PGC is produced by the fungus Aspergillus niger during invasion of plant cell walls. The enzyme has been homologously overexpressed to provide sufficient quantities of purified enzyme for biological studies. We have characterized this enzyme in terms of its posttranslational modifications (PTMs) and found it to be both N- and O-glycosylated. The glycosyl moieties have also been characterized. This has involved a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), liquid chromatography (LC)-ion trap, and LC-electrospray ionization (ESI) mass spectrometries in conjunction with trypsin degradation and beta-elimination, followed by Michael addition with dithiothreitol (BEMAD). This is the first demonstration of the ability of BEMAD to map glycosylation sites other than O-GlcNAc sites. The complete characterization of all PTMs on PGC allows us to model them on the peptide backbone, revealing potential roles played by the glycans in modulating the interaction of the enzyme with other macromolecules.
Kudo, Toshiaki; Kobiyama, Atsushi; Rashid, Jonaira; Reza, Shaheed; Yamada, Yuichiro; Ikeda, Yuri; Ikeda, Daisuke; Mizusawa, Nanami; Ikeo, Kazuho; Sato, Shigeru; Ogata, Takehiko; Jimbo, Mitsuru; Kaga, Shinnosuke; Watanabe, Shiho; Naiki, Kimiaki; Kaga, Yoshimasa; Segawa, Satoshi; Mineta, Katsuhiko; Bajic, Vladimir B.; Gojobori, Takashi; Watabe, Shugo
2018-01-01
Ofunato Bay is located in the northeastern Pacific Ocean area of Japan, and it has the highest biodiversity of marine organisms in the world, primarily due to tidal influences from the cold Oyashio and warm Kuroshio currents. Our previous results from performing shotgun metagenomics indicated that Candidatus Pelagibacter ubique and Planktomarina temperata were the dominant bacteria (Reza et al., 2018a, 2018b). These bacteria are reportedly able to catabolize dimethylsulfoniopropionate (DMSP) produced from phytoplankton into dimethyl sulfide (DMS) or methanethiol (MeSH). This study was focused on seasonal changes in the abundances of bacterial genes (dddP, dmdA) related to DMSP catabolism in the seawater of Ofunato Bay by BLAST+ analysis using shotgun metagenomic datasets. We found seasonal changes among the Candidatus Pelagibacter ubique strains, including those of the HTCC1062 type and the Red Sea type. A good correlation was observed between the chlorophyll a concentrations and the abundances of the catabolic genes, suggesting that the bacteria directly interact with phytoplankton in the marine material cycle system and play important roles in producing DMS and MeSH from DMSP as signaling molecules for the possible formation of the scent of the tidewater or as fish attractants.
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Corinne Barbey
Full Text Available The virulence of numerous Gram-negative bacteria is under the control of a quorum sensing process based on synthesis and perception of N-acyl homoserine lactones. Rhodococcus erythropolis, a Gram-positive bacterium, has recently been proposed as a biocontrol agent for plant protection against soft-rot bacteria, including Pectobacterium. Here, we show that the γ-lactone catabolic pathway of R. erythropolis disrupts Pectobacterium communication and prevents plant soft-rot. We report the first characterization and demonstration of N-acyl homoserine lactone quenching in planta. In particular, we describe the transcription of the R. erythropolis lactonase gene, encoding the key enzyme of this pathway, and the subsequent lactone breakdown. The role of this catabolic pathway in biocontrol activity was confirmed by deletion of the lactonase gene from R. erythropolis and also its heterologous expression in Escherichia coli. The γ-lactone catabolic pathway is induced by pathogen communication rather than by pathogen invasion. This is thus a novel and unusual biocontrol pathway, differing from those previously described as protecting plants from phytopathogens. These findings also suggest the existence of an additional pathway contributing to plant protection.
Kudo, Toshiaki
2018-04-26
Ofunato Bay is located in the northeastern Pacific Ocean area of Japan, and it has the highest biodiversity of marine organisms in the world, primarily due to tidal influences from the cold Oyashio and warm Kuroshio currents. Our previous results from performing shotgun metagenomics indicated that Candidatus Pelagibacter ubique and Planktomarina temperata were the dominant bacteria (Reza et al., 2018a, 2018b). These bacteria are reportedly able to catabolize dimethylsulfoniopropionate (DMSP) produced from phytoplankton into dimethyl sulfide (DMS) or methanethiol (MeSH). This study was focused on seasonal changes in the abundances of bacterial genes (dddP, dmdA) related to DMSP catabolism in the seawater of Ofunato Bay by BLAST+ analysis using shotgun metagenomic datasets. We found seasonal changes among the Candidatus Pelagibacter ubique strains, including those of the HTCC1062 type and the Red Sea type. A good correlation was observed between the chlorophyll a concentrations and the abundances of the catabolic genes, suggesting that the bacteria directly interact with phytoplankton in the marine material cycle system and play important roles in producing DMS and MeSH from DMSP as signaling molecules for the possible formation of the scent of the tidewater or as fish attractants.
Energy Technology Data Exchange (ETDEWEB)
Ritzl, F.; Feinendegen, L. E. [Institute of Medicine, Kernforschungsanlage Juelich Gmbh, Juelich, Federal Republic of Germany (Germany)
1971-02-15
Double labelling of a peptide with {sup 51}Cr and {sup 125}({sup 131})I results in an isotopic ratio that changes when and where the molecule in vivo is catabolized. Intracellular hydrolysis of the peptide liberates the iodine into the iodine pool, whereas the chromium by virtue of being a multivalent ion enters a new linkage at the site of breakdown. The isotopic ratio at the site of breakdown alters concomitantly with the hydrolysis rate. Experiments with {sup 51}Cr- and {sup 125}I-labelled insulin in mice in vivo and in vitro showed the liver (not muscle), bone (including marrow) and thyroid gland to be the major site of insulin catabolism with a half-life of approximately 10 min. In eight normal persons and diabetic patients insulin catabolism was analysed by the whole body counter following an iv injection of 0.77-0.95 {mu}g insulin labelled with {sup 51}Cr and {sup 131}I. Counts were taken simultaneously from the area of the liver, thyroid, thigh and posterior pelvis. Again, the.data indicated the liver as the site of insulin catabolism, the normal half-life being approximately 20 min. Iodine- labelled insulin was commercially supplied. {sup 51}Cr-labelled insulin, prepared according to the methods of Kavai and Kesztyues, was analysed by immune precipitation and Sephadex G200 chromatography. In the countercurrent distribution the {sup 51}Cr insulin showed enhanced water solubility. (author)
Turnover of pigment granules: cyclic catabolism and anabolism of ommochromes within epidermal cells.
Insausti, T C; Casas, J
2009-12-01
Ommochromes are end products of the tryptophan metabolism in arthropods. While the anabolism of ommochromes has been well studied, the catabolism is totally unknown. In order to study it, we used the crab-spider Misumena vatia, which is able to change color reversibly in a few days, from yellow to white and back. Ommochromes is the only pigment class responsible for the body coloration in this animal. The aim of this study was to analyze the fine structure of the epidermal cells in bleaching spiders, in an attempt to correlate morphological changes with the fate of the pigment granules. Central to the process of bleaching is the lysis of the ommochrome granules. In the same cell, intact granules and granules in different degradation stages are found. The degradation begins with granule autolysis. Some components are extruded in the extracellular space and others are recycled via autophagy. Abundant glycogen appears associated to granulolysis. In a later stage of bleaching, ommochrome progranules, typical of white spiders, appear in the distal zone of the same epidermal cell. Catabolism and anabolism of pigment granules thus take place simultaneously in spider epidermal cells. A cyclic pathway of pigment granules formation and degradation, throughout a complete cycle of color change is proposed, together with an explanation for this turnover, involving photoprotection against UV by ommochromes metabolites. The presence of this turnover for melanins is discussed.
Acetone Formation in the Vibrio Family: a New Pathway for Bacterial Leucine Catabolism
Nemecek-Marshall, Michele; Wojciechowski, Cheryl; Wagner, William P.; Fall, Ray
1999-01-01
There is current interest in biological sources of acetone, a volatile organic compound that impacts atmospheric chemistry. Here, we determined that leucine-dependent acetone formation is widespread in the Vibrionaceae. Sixteen Vibrio isolates, two Listonella species, and two Photobacterium angustum isolates produced acetone in the presence of l-leucine. Shewanella isolates produced much less acetone. Growth of Vibrio splendidus and P. angustum in a fermentor with controlled aeration revealed that acetone was produced after a lag in late logarithmic or stationary phase of growth, depending on the medium, and was not derived from acetoacetate by nonenzymatic decarboxylation in the medium. l-Leucine, but not d-leucine, was converted to acetone with a stoichiometry of approximately 0.61 mol of acetone per mol of l-leucine. Testing various potential leucine catabolites as precursors of acetone showed that only α-ketoisocaproate was efficiently converted by whole cells to acetone. Acetone production was blocked by a nitrogen atmosphere but not by electron transport inhibitors, suggesting that an oxygen-dependent reaction is required for leucine catabolism. Metabolic labeling with deuterated (isopropyl-d7)-l-leucine revealed that the isopropyl carbons give rise to acetone with full retention of deuterium in each methyl group. These results suggest the operation of a new catabolic pathway for leucine in vibrios that is distinct from the 3-hydroxy-3-methylglutaryl-coenzyme A pathway seen in pseudomonads. PMID:10601206
Directory of Open Access Journals (Sweden)
Markus F Bartels
Full Text Available Protein O-mannosylation is a post-translational modification essential for correct development of mammals. In humans, deficient O-mannosylation results in severe congenital muscular dystrophies often associated with impaired brain and eye development. Although various O-mannosylated proteins have been identified in the recent years, the distribution of O-mannosyl glycans in the mammalian brain and target proteins are still not well defined. In the present study, rabbit monoclonal antibodies directed against the O-mannosylated peptide YAT(α1-ManAV were generated. Detailed characterization of clone RKU-1-3-5 revealed that this monoclonal antibody recognizes O-linked mannose also in different peptide and protein contexts. Using this tool, we observed that mono-O-mannosyl glycans occur ubiquitously throughout the murine brain but are especially enriched at inhibitory GABAergic neurons and at the perineural nets. Using a mass spectrometry-based approach, we further identified glycoproteins from the murine brain that bear single O-mannose residues. Among the candidates identified are members of the cadherin and plexin superfamilies and the perineural net protein neurocan. In addition, we identified neurexin 3, a cell adhesion protein involved in synaptic plasticity, and inter-alpha-trypsin inhibitor 5, a protease inhibitor important in stabilizing the extracellular matrix, as new O-mannosylated glycoproteins.
Bartels, Markus F.; Winterhalter, Patrick R.; Yu, Jin; Liu, Yan; Lommel, Mark; Möhrlen, Frank; Hu, Huaiyu; Feizi, Ten; Westerlind, Ulrika; Ruppert, Thomas; Strahl, Sabine
2016-01-01
Protein O-mannosylation is a post-translational modification essential for correct development of mammals. In humans, deficient O-mannosylation results in severe congenital muscular dystrophies often associated with impaired brain and eye development. Although various O-mannosylated proteins have been identified in the recent years, the distribution of O-mannosyl glycans in the mammalian brain and target proteins are still not well defined. In the present study, rabbit monoclonal antibodies directed against the O-mannosylated peptide YAT(α1-Man)AV were generated. Detailed characterization of clone RKU-1-3-5 revealed that this monoclonal antibody recognizes O-linked mannose also in different peptide and protein contexts. Using this tool, we observed that mono-O-mannosyl glycans occur ubiquitously throughout the murine brain but are especially enriched at inhibitory GABAergic neurons and at the perineural nets. Using a mass spectrometry-based approach, we further identified glycoproteins from the murine brain that bear single O-mannose residues. Among the candidates identified are members of the cadherin and plexin superfamilies and the perineural net protein neurocan. In addition, we identified neurexin 3, a cell adhesion protein involved in synaptic plasticity, and inter-alpha-trypsin inhibitor 5, a protease inhibitor important in stabilizing the extracellular matrix, as new O-mannosylated glycoproteins. PMID:27812179
Novel Insights into the Diversity of Catabolic Metabolism from Ten Haloarchaeal Genomes
Energy Technology Data Exchange (ETDEWEB)
Anderson, Iain; Scheuner, Carmen; Goker, Markus; Mavromatis, Kostas; Hooper, Sean D.; Porat, Iris; Klenk, Hans-Peter; Ivanova, Natalia; Kyrpides, Nikos
2011-05-03
The extremely halophilic archaea are present worldwide in saline environments and have important biotechnological applications. Ten complete genomes of haloarchaea are now available, providing an opportunity for comparative analysis. We report here the comparative analysis of five newly sequenced haloarchaeal genomes with five previously published ones. Whole genome trees based on protein sequences provide strong support for deep relationships between the ten organisms. Using a soft clustering approach, we identified 887 protein clusters present in all halophiles. Of these core clusters, 112 are not found in any other archaea and therefore constitute the haloarchaeal signature. Four of the halophiles were isolated from water, and four were isolated from soil or sediment. Although there are few habitat-specific clusters, the soil/sediment halophiles tend to have greater capacity for polysaccharide degradation, siderophore synthesis, and cell wall modification. Halorhabdus utahensis and Haloterrigena turkmenica encode over forty glycosyl hydrolases each, and may be capable of breaking down naturally occurring complex carbohydrates. H. utahensis is specialized for growth on carbohydrates and has few amino acid degradation pathways. It uses the non-oxidative pentose phosphate pathway instead of the oxidative pathway, giving it more flexibility in the metabolism of pentoses. These new genomes expand our understanding of haloarchaeal catabolic pathways, providing a basis for further experimental analysis, especially with regard to carbohydrate metabolism. Halophilic glycosyl hydrolases for use in biofuel production are more likely to be found in halophiles isolated from soil or sediment.
Hitting the Sweet Spot: Glycans as Targets of Fungal Defense Effector Proteins
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Markus Künzler
2015-05-01
Full Text Available Organisms which rely solely on innate defense systems must combat a large number of antagonists with a comparatively low number of defense effector molecules. As one solution of this problem, these organisms have evolved effector molecules targeting epitopes that are conserved between different antagonists of a specific taxon or, if possible, even of different taxa. In order to restrict the activity of the defense effector molecules to physiologically relevant taxa, these target epitopes should, on the other hand, be taxon-specific and easily accessible. Glycans fulfill all these requirements and are therefore a preferred target of defense effector molecules, in particular defense proteins. Here, we review this defense strategy using the example of the defense system of multicellular (filamentous fungi against microbial competitors and animal predators.
The ygeW encoded protein from Escherichia coli is a knotted ancestral catabolic transcarbamylase
Energy Technology Data Exchange (ETDEWEB)
Li, Yongdong; Jin, Zhongmin; Yu, Xiaolin; Allewell, Norma M.; Tuchman, Mendel; Shi, Dashuang (Maryland); (GWU); (Georgia)
2012-06-28
Purine degradation plays an essential role in nitrogen metabolism in most organisms. Uric acid is the final product of purine catabolism in humans, anthropoid apes, birds, uricotelic reptiles, and almost all insects. Elevated levels of uric acid in blood (hyperuricemia) cause human diseases such as gout, kidney stones, and renal failure. Although no enzyme has been identified that further degrades uric acid in humans, it can be oxidized to produce allantoin by free-radical attack. Indeed, elevated levels of allantoin are found in patients with rheumatoid arthritis, chronic lung disease, bacterial meningitis, and noninsulin-dependent diabetes mellitus. In other mammals, some insects and gastropods, uric acid is enzymatically degraded to the more soluble allantoin through the sequential action of three enzymes: urate oxidase, 5-hydroxyisourate (HIU) hydrolase and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase. Therefore, an elective treatment for acute hyperuricemia is the administration of urate oxidase. Many organisms, including plants, some fungi and several bacteria, are able to catabolize allantoin to release nitrogen, carbon, and energy. In Arabidopsis thaliana and Eschrichia coli, S-allantoin has recently been shown to be degraded to glycolate and urea by four enzymes: allantoinase, allantoate amidohydrolase, ureidoglycine aminohydrolase, and ureidoglycolate amidohydrolase.
Yang, Weizhun; Ramadan, Sherif; Yang, Bo; Yoshida, Keisuke; Huang, Xuefei
2016-01-01
Among many hurdles in synthesizing proteoglycan glycopeptides, one challenge is the incorporation of aspartic acid in the peptide backbone and acid sensitive O-sulfated glycan chains. To overcome this, a new strategy was developed utilizing homoserine as an aspartic acid precursor. The conversion of homoserine to aspartic acid in the glycopeptide was successfully accomplished by late stage oxidation using 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and bis(acetoxy)iodobenzene (BAIB). This is...
DEFF Research Database (Denmark)
Jensen, J B; Egsgaard, H; Van Onckelen, H
1995-01-01
Some strains of Bradyrhizobium japonicum have the ability to catabolize indole-3-acetic acid. Indoleacetic acid (IAA), 4-chloro-IAA (4-Cl-IAA), and 5-Cl-IAA were metabolized to different extents by strains 61A24 and 110. Metabolites were isolated and analyzed by high-performance liquid chromatogr...
van Ree, R.; Cabanes-Macheteau, M.; Akkerdaas, J.; Milazzo, J. P.; Loutelier-Bourhis, C.; Rayon, C.; Villalba, M.; Koppelman, S.; Aalberse, R.; Rodriguez, R.; Faye, L.; Lerouge, P.
2000-01-01
Primary structures of the N-glycans of two major pollen allergens (Lol p 11 and Ole e 1) and a major peanut allergen (Ara h 1) were determined. Ole e 1 and Ara h 1 carried high mannose and complex N-glycans, whereas Lol p 11 carried only the complex. The complex structures all had a beta(1,2)-xylose
Mongkhon, John-Max; Thach, Maryane; Shi, Qin; Fernandes, Julio C; Fahmi, Hassan; Benderdour, Mohamed
2014-08-01
Our study was designed to elucidate the precise molecular mechanisms by which sorbitol-modified hyaluronic acid (HA/sorbitol) exerts beneficial effects in osteoarthritis (OA). Human OA chondrocytes were treated with increasing doses of HA/sorbitol ± anti-CD44 antibody or with sorbitol alone and thereafter with or without interleukin-1beta (IL-1β) or hydrogen peroxide (H2O2). Signal transduction pathways and parameters related to oxidative stress, apoptosis, inflammation, and catabolism were investigated. HA/sorbitol prevented IL-1β-induced oxidative stress, as measured by reactive oxygen species, p47-NADPH oxidase phosphorylation, 4-hydroxynonenal (HNE) production and HNE-metabolizing glutathione-S-transferase A4-4 expression. Moreover, HA/sorbitol stifled IL-1β-induced metalloproteinase-13, nitric oxide (NO) and prostaglandin E2 release as well as inducible NO synthase expression. Study of the apoptosis process revealed that this gel significantly attenuated cell death, caspase-3 activation and DNA fragmentation elicited by exposure to a cytotoxic H2O2 dose. Examination of signaling pathway components disclosed that HA/sorbitol prevented IL-1β-induced p38 mitogen-activated protein kinase and nuclear factor-kappa B activation, but not that of extracellular signal-regulated kinases 1 and 2. Interestingly, the antioxidant as well as the anti-inflammatory and anti-catabolic effects of HA/sorbitol were attributed to sorbitol and HA, respectively. Altogether, our findings support a beneficial effect of HA/sorbitol in OA through the restoration of redox status and reduction of apoptosis, inflammation and catabolism involved in cartilage damage.
Energy Technology Data Exchange (ETDEWEB)
Koivistoinen, O.
2013-11-01
The use of metabolic engineering as a tool for production of biochemicals and biofuels requires profound understanding of cell metabolism. The pathways for the most abundant and most important hexoses have already been studied quite extensively but it is also important to get a more complete picture of sugar catabolism. In this thesis, catabolic pathways of L-rhamnose and D-galactose were studied in fungi. Both of these hexoses are present in plant biomass, such as in hemicellulose and pectin. Galactoglucomannan, a type of hemicellulose that is especially rich in softwood, is an abundant source of D-galactose. As biotechnology is moving from the usage of edible and easily metabolisable carbon sources towards the increased use of lignocellulosic biomass, it is important to understand how the different sugars can be efficiently turned into valuable biobased products. Identification of the first fungal L-rhamnose 1-dehydrogenase gene, which codes for the first enzyme of the fungal catabolic L-rhamnose pathway, showed that the protein belongs to a protein family of short-chain alcohol dehydrogenases. Sugar dehydrogenases oxidising a sugar to a sugar acid are not very common in fungi and thus the identification of the L-rhamnose dehydrogenase gene provides more understanding of oxidative sugar catabolism in eukaryotic microbes. Further studies characterising the L-rhamnose cluster in the yeast Scheffersomyces stipitis including the expression of the L-rhamnonate dehydratase in Saccharomyces cerevisiae finalised the biochemical characterisation of the enzymes acting on the pathway. In addition, more understanding of the regulation and evolution of the pathway was gained. D-Galactose catabolism was studied in the filamentous fungus Aspergillus niger. Two genes coding for the enzymes of the oxido-reductive pathway were identified. Galactitol dehydrogenase is the second enzyme of the pathway converting galactitol to L-xylo-3-hexulose. The galactitol dehydrogenase encoding
Directory of Open Access Journals (Sweden)
Ximena Escalera-Fanjul
2017-06-01
Full Text Available Gene duplication is one of the major evolutionary mechanisms providing raw material for the generation of genes with new or modified functions. The yeast Saccharomyces cerevisiae originated after an allopolyploidization event, which involved mating between two different ancestral yeast species. ScALT1 and ScALT2 codify proteins with 65% identity, which were proposed to be paralogous alanine transaminases. Further analysis of their physiological role showed that while ScALT1 encodes an alanine transaminase which constitutes the main pathway for alanine biosynthesis and the sole pathway for alanine catabolism, ScAlt2 does not display alanine transaminase activity and is not involved in alanine metabolism. Moreover, phylogenetic studies have suggested that ScALT1 and ScALT2 come from each one of the two parental strains which gave rise to the ancestral hybrid. The present work has been aimed to the understanding of the properties of the ancestral type Lacchancea kluyveri LkALT1 and Kluyveromyces lactis KlALT1, alanine transaminases in order to better understand the ScALT1 and ScALT2 evolutionary history. These ancestral -type species were chosen since they harbor ALT1 genes, which are related to ScALT2. Presented results show that, although LkALT1 and KlALT1 constitute ScALT1 orthologous genes, encoding alanine transaminases, both yeasts display LkAlt1 and KlAlt1 independent alanine transaminase activity and additional unidentified alanine biosynthetic and catabolic pathway(s. Furthermore, phenotypic analysis of null mutants uncovered the fact that KlAlt1 and LkAlt1 have an additional role, not related to alanine metabolism but is necessary to achieve wild type growth rate. Our study shows that the ancestral alanine transaminase function has been retained by the ScALT1 encoded enzyme, which has specialized its catabolic character, while losing the alanine independent role observed in the ancestral type enzymes. The fact that ScAlt2 conserves 64
Kolarich, Daniel; Léonard, Renaud; Hemmer, Wolfgang; Altmann, Friedrich
2005-10-01
Hyaluronidase (E.C. 3.2.1.35), one of the three major allergens of yellow jacket venom, is a glycoprotein of 45 kDa that is largely responsible for the cross-reactivity of wasp and bee venoms with sera of allergic patients. The asparagine-linked carbohydrate often appears to constitute the common IgE-binding determinant. Using a combination of MALDI MS and HPLC of 2-aminopyridine-labelled glycans, we found core-difucosylated paucimannosidic glycans to be the major species in the 43-45 kDa band of Vespula vulgaris and also in the corresponding bands of venoms from five other wasp species (V. germanica, V. maculifrons, V. pensylvanica, V. flavopilosa and V. squamosa). Concomitant peptide mapping of the V. vulgaris 43 kDa band identified the known hyaluronidase, Ves v 2 (SwissProt P49370), but only as a minor component. De novo sequencing by tandem MS revealed the predominating peptides to resemble a different, yet homologous, sequence. cDNA cloning retrieved a sequence with 58 and 59% homology to the previously known isoform and to the Dolichovespula maculata and Polistes annularis hyaluronidases. Close homologues of this new, putative hyaluronidase b (Ves v 2b) were also the major isoform in the other wasp venoms.
Directory of Open Access Journals (Sweden)
Omai B Garner
2010-07-01
Full Text Available Nipah virus targets human endothelial cells via NiV-F and NiV-G envelope glycoproteins, resulting in endothelial syncytia formation and vascular compromise. Endothelial cells respond to viral infection by releasing innate immune effectors, including galectins, which are secreted proteins that bind to specific glycan ligands on cell surface glycoproteins. We demonstrate that galectin-1 reduces NiV-F mediated fusion of endothelial cells, and that endogenous galectin-1 in endothelial cells is sufficient to inhibit syncytia formation. Galectin-1 regulates NiV-F mediated cell fusion at three distinct points, including retarding maturation of nascent NiV-F, reducing NiV-F lateral mobility on the plasma membrane, and directly inhibiting the conformational change in NiV-F required for triggering fusion. Characterization of the NiV-F N-glycome showed that the critical site for galectin-1 inhibition is rich in glycan structures known to bind galectin-1. These studies identify a unique set of mechanisms for regulating pathophysiology of NiV infection at the level of the target cell.
Jenabian, Mohammad-Ali; Mehraj, Vikram; Costiniuk, Cecilia T.; Vyboh, Kishanda; Kema, Ido; Rollet, Kathleen; Ramirez, Robert Paulino; Klein, Marina B.; Routy, Jean-Pierre
2016-01-01
Background: We previously reported an association between tryptophan (Trp) catabolism and immune dysfunction in HIV monoinfection. Coinfection with HIV is associated with more rapid evolution of hepatitis C virus (HCV)-associated liver disease despite antiretroviral therapy (ART), possibly due to
Czech Academy of Sciences Publication Activity Database
Nešvera, Jan; Rucká, Lenka; Pátek, Miroslav
2015-01-01
Roč. 93, č. 2015 (2015), s. 107-160 ISSN 0065-2164 R&D Projects: GA TA ČR TA04021212 Institutional support: RVO:61388971 Keywords : Biodegradation * Bioremediation * Phenol catabolism Subject RIV: EE - Microbiology, Virology Impact factor: 4.128, year: 2015
Kaus, Katherine; Lary, Jeffrey W; Cole, James L; Olson, Rich
2014-07-29
Pore-forming toxins (PFTs) are a class of pathogen-secreted molecules that oligomerize to form transmembrane channels in cellular membranes. Determining the mechanism for how PFTs bind membranes is important in understanding their role in disease and for developing possible ways to block their action. Vibrio vulnificus, an aquatic pathogen responsible for severe food poisoning and septicemia in humans, secretes a PFT called V. vulnificus hemolysin (VVH), which contains a single C-terminal targeting domain predicted to resemble a β-trefoil lectin fold. In order to understand the selectivity of the lectin for glycan motifs, we expressed the isolated VVH β-trefoil domain and used glycan-chip screening to identify that VVH displays a preference for terminal galactosyl groups including N-acetyl-d-galactosamine and N-acetyl-d-lactosamine. The X-ray crystal structure of the VVH lectin domain solved to 2.0Å resolution reveals a heptameric ring arrangement similar to the oligomeric form of the related, but inactive, lectin from Vibrio cholerae cytolysin. Structures bound to glycerol, N-acetyl-d-galactosamine, and N-acetyl-d-lactosamine outline a common and versatile mode of recognition allowing VVH to target a wide variety of cell-surface ligands. Sequence analysis in light of our structural and functional data suggests that VVH may represent an earlier step in the evolution of Vibrio PFTs. Copyright © 2014 Elsevier Ltd. All rights reserved.
Automated glycan assembly of a S. pneumoniae serotype 3 CPS antigen
Directory of Open Access Journals (Sweden)
Markus W. Weishaupt
2016-07-01
Full Text Available Vaccines against S. pneumoniae, one of the most prevalent bacterial infections causing severe disease, rely on isolated capsular polysaccharide (CPS that are conjugated to proteins. Such isolates contain a heterogeneous oligosaccharide mixture of different chain lengths and frame shifts. Access to defined synthetic S. pneumoniae CPS structures is desirable. Known syntheses of S. pneumoniae serotype 3 CPS rely on a time-consuming and low-yielding late-stage oxidation step, or use disaccharide building blocks which limits variability. Herein, we report the first iterative automated glycan assembly (AGA of a conjugation-ready S. pneumoniae serotype 3 CPS trisaccharide. This oligosaccharide was assembled using a novel glucuronic acid building block to circumvent the need for a late-stage oxidation. The introduction of a washing step with the activator prior to each glycosylation cycle greatly increased the yields by neutralizing any residual base from deprotection steps in the synthetic cycle. This process improvement is applicable to AGA of many other oligosaccharides.
International Nuclear Information System (INIS)
Yamaguchi, Yoshiki; Kato, Koichi; Shindo, Mitsuru; Aoki, Shin; Furusho, Kumiko; Koga, Kenji; Takahashi, Noriko; Arata, Yoji; Shimada, Ichio
1998-01-01
A systematic method for 13 C labeling of the glycan of immunoglobulin G for NMR study has been developed. A mouse immunoglobulin of subclass IgG2b has been used for the experiment. On the basis of chemical shift and linewidth data, it has been concluded that (1) the mobility of the carbohydrate chain in IgG2b is comparable to that of the backbone polypeptide chain with the exception of the galactose residue at the nonreducing end of the Man α 1-3 branch, which is extremely mobile and (2) agalactosylation does not induce any significant change in the mobility. The results obtained indicate that even in the agalactosyl form the glycans are buried in the protein. Biological significance of the NMR results obtained is also briefly discussed
Yan, Dongyao; Chen, Di; Shen, Jie; Xiao, Guozhi; van Wijnen, Andre J; Im, Hee-Jeong
2013-02-01
Bovine lactoferricin (LfcinB) is a multi-functional peptide derived from proteolytic cleavage of bovine lactoferrin. LfcinB was found to antagonize the biological effects mediated by angiogenic growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF-2) in endothelial cells. However, the effect of LfcinB on human articular cartilage remained unknown. Here, our findings demonstrate that LfcinB restored the proteoglycan loss promoted by catabolic factors (interleukin-1β) IL-1β and FGF-2 in vitro and ex vivo. Mechanistically, LfcinB attenuated the effects of IL-1β and FGF-2 on the expression of cartilage-degrading enzymes (MMP-1, MMP-3, and MMP-13), destructive cytokines (IL-1β and IL-6), and inflammatory mediators (iNOS and TLR2). LfcinB induced protective cytokine expression (IL-4 and IL-10), and downregulated aggrecanase basal expression. LfcinB specifically activated ERK MAPK and Akt signaling pathways, which may account for its anti-inflammatory activity. We also revealed that LfcinB exerted similar protective effects on human synovial fibroblasts challenged by IL-1β, with minimal cytotoxicity. Collectively, our results suggest that LfcinB possesses potent anti-catabolic and anti-inflammatory bioactivities in human articular tissues, and may be utilized for the prevention and/or treatment of OA in the future. Copyright © 2012 Wiley Periodicals, Inc.
Glycan-functionalized diamond nanoparticles as potent E. coli anti-adhesives
Barras, Alexandre; Martin, Fernando Ariel; Bande, Omprakash; Baumann, Jean-Sébastien; Ghigo, Jean-Marc; Boukherroub, Rabah; Beloin, Christophe; Siriwardena, Aloysius; Szunerits, Sabine
2013-02-01
Bacterial attachment and subsequent biofilm formation on biotic surfaces or medical devices is an increasing source of infections in clinical settings. A large proportion of these biofilm-related infections are caused by Escherichia coli, a major nosocomial pathogen, in which the major adhesion factor is the FimH adhesin located at the tip of type 1 fimbriae. Inhibition of FimH-mediated adhesion has been identified as an efficient antibiotic-alternative strategy to potentially reduce E. coli-related infections. In this article we demonstrate that nanodiamond particles, covently modified with mannose moieties by a ``click'' chemistry approach, are able to efficiently inhibit E. coli type 1 fimbriae-mediated adhesion to eukaryotic cells with relative inhibitory potency (RIP) of as high as 9259 (bladder cell adhesion assay), which is unprecedented when compared with RIP values previously reported for alternate multivalent mannose-functionalized nanostructures designed to inhibit E. coli adhesion. Also remarkable is that these novel mannose-modified NDs reduce E. coli biofilm formation, a property previously not observed for multivalent glyco-nanoparticles and rarely demonstrated for other multivalent or monovalent mannose glycans. This work sets the stage for the further evaluation of these novel NDs as an anti-adhesive therapeutic strategy against E. coli-derived infections.Bacterial attachment and subsequent biofilm formation on biotic surfaces or medical devices is an increasing source of infections in clinical settings. A large proportion of these biofilm-related infections are caused by Escherichia coli, a major nosocomial pathogen, in which the major adhesion factor is the FimH adhesin located at the tip of type 1 fimbriae. Inhibition of FimH-mediated adhesion has been identified as an efficient antibiotic-alternative strategy to potentially reduce E. coli-related infections. In this article we demonstrate that nanodiamond particles, covently modified with
Lu, Gang; Sun, Haipeng; She, Pengxiang; Youn, Ji-Youn; Warburton, Sarah; Ping, Peipei; Vondriska, Thomas M; Cai, Hua; Lynch, Christopher J; Wang, Yibin
2009-06-01
The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. In individuals with deficiencies in BCAA, these amino acids can be preserved through inhibition of the branched-chain-alpha-ketoacid dehydrogenase (BCKD) complex, the rate-limiting step in their metabolism. BCKD is inhibited by phosphorylation of its E1alpha subunit at Ser293, which is catalyzed by BCKD kinase. During BCAA excess, phosphorylated Ser293 (pSer293) becomes dephosphorylated through the concerted inhibition of BCKD kinase and the activity of an unknown intramitochondrial phosphatase. Using unbiased, proteomic approaches, we have found that a mitochondrial-targeted phosphatase, PP2Cm, specifically binds the BCKD complex and induces dephosphorylation of Ser293 in the presence of BCKD substrates. Loss of PP2Cm completely abolished substrate-induced E1alpha dephosphorylation both in vitro and in vivo. PP2Cm-deficient mice exhibited BCAA catabolic defects and a metabolic phenotype similar to the intermittent or intermediate types of human maple syrup urine disease (MSUD), a hereditary disorder caused by defects in BCKD activity. These results indicate that PP2Cm is the endogenous BCKD phosphatase required for nutrient-mediated regulation of BCKD activity and suggest that defects in PP2Cm may be responsible for a subset of human MSUD.
Cell wall O-glycoproteins and N-glycoproteins: biosynthesis and some functional aspects.
Directory of Open Access Journals (Sweden)
Eric eNguema-Ona
2014-10-01
Full Text Available Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensin, the O-glycan chains of arabinogalactan proteins are highly heterogeneous consisting mostly of (i a short oligo-arabinoside chain of three to four residues, and (ii a larger -1,3-linked galactan backbone with -1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core 1,2-xylose, core 1,3-fucose residues and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins only extensin and arabinogalactan proteins are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review.
The proteinogenic branched-chain amino acids (BCAAs) leucine, isoleucine, and valine are essential nutrients for mammals. In plants, they double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates...
DEFF Research Database (Denmark)
Astronomo, Rena D; Lee, Hing-Ken; Scanlan, Christopher N
2008-01-01
The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G......12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man(4)) that corresponds to the D1 arm...
Zuo, Hui; Ueland, Per M; Midttun, Øivind; Vollset, Stein E; Tell, Grethe S; Theofylaktopoulou, Despoina; Travis, Ruth C; Boutron-Ruault, Marie-Christine; Fournier, Agnès; Severi, Gianluca; Kvaskoff, Marina; Boeing, Heiner; Bergmann, Manuela M; Fortner, Renée T; Kaaks, Rudolf; Trichopoulou, Antonia; Kotanidou, Anastasia; Lagiou, Pagona; Palli, Domenico; Sieri, Sabina; Panico, Salvatore; Bueno-de-Mesquita, H Bas; Peeters, Petra H; Grankvist, Kjell; Johansson, Mikael; Agudo, Antonio; Garcia, Jose Ramon Quiros; Larranaga, Nerea; Sanchez, Maria-Jose; Chirlaque, Maria Dolores; Ardanaz, Eva; Chuang, Shu-Chun; Gallo, Valentina; Brennan, Paul; Johansson, Mattias; Ulvik, Arve
2018-01-01
Circulating pyridoxal-5'-phosphate (PLP) has been linked to lung cancer risk. The PAr index, defined as the ratio 4-pyridoxic acid/(pyridoxal + PLP), reflects increased vitamin B6 catabolism during inflammation. PAr has been defined as a marker of lung cancer risk in a prospective cohort study, but
Grandys, Marcin; Majerczak, Joanna; Karasinski, Janusz; Kulpa, Jan; Zoladz, Jerzy A
2012-12-01
Gonadal hormones and anabolic-catabolic hormone balance have potent influence on skeletal muscle tissue, but little is known about their action with regard to myosin heavy chain (MHC) transformation in humans. We investigated the relationship between skeletal muscle MHC isoform content in the vastus lateralis muscle and basal testosterone (T) concentration in 3 groups of subjects: endurance trained (E), sprint/strength trained (S), and untrained (U) young men. We have also determined basal sex hormone-binding globulin and cortisol (C) concentrations in untrained subjects to examine the relationship between MHC composition and the anabolic-catabolic hormone balance. Moreover, basal free testosterone (fT) and bioavailable testosterone (bio-T) concentrations were calculated for this subgroup. Despite significant differences in MHC isoform content (69.4 ± 2.39%, 61.4 ± 8.04%, and 37.5 ± 13.80% of MHC-2 for groups S, U, and E, respectively, Kruskal-Wallis: H = 18.58, p 0.5). We have also found that in the U group, type 2 MHC in the vastus lateralis muscle is positively correlated with basal fT:C ratio (r = 0.63, p = 0.01). It is concluded that the differences in the training history and training specificity can be distinguished with regard to the MHC composition but not with regard to the basal T concentration. Simultaneously, it has been shown that MHC isoform content in human vastus lateralis muscle may be related to basal anabolic-catabolic hormone balance, and this hypothesis needs further investigation.
Zuo, Hui; Ueland, Per Magne; Midttun, Øivind; Vollset, Stein Emil; Tell, Grethe S.; Theofylaktopoulou, Despoina; Travis, Ruth C.; Boutron-Ruault, Marie Christine; Fournier, Agnès; Severi, Gianluca; Kvaskoff, Marina; Boeing, Heiner; Bergmann, Manuela M.; Turzanski-Fortner, Renée; Kaaks, Rudolf; Trichopoulou, Antonia; Kotanidou, Anastasia; Lagiou, Pagona; Palli, Domenico; Sieri, Sabina; Panico, Salvatore; Bueno-De-Mesquita, H. Bas; Peeters, Petra H.; Grankvist, Kjell; Johansson, Mikael; Agudo, Antonio; Garcia, Jose Ramon Quiros; Larranaga, Nerea; Sanchez, Maria-Jose; Chirlaque, Maria-Dolores; Ardanaz, Eva; Chuang, Shu Chun; Gallo, Valentina; Brennan, Paul; Johansson, Mattias; Ulvik, Arve
2018-01-01
Circulating pyridoxal-5′-phosphate (PLP) has been linked to lung cancer risk. The PAr index, defined as the ratio 4-pyridoxic acid/(pyridoxal + PLP), reflects increased vitamin B6 catabolism during inflammation. PAr has been defined as a marker of lung cancer risk in a prospective cohort study, but
Long, Aaron; Klimova, Nina; Kristian, Tibor
2017-10-01
NAD + catabolism and mitochondrial dynamics are important parts of normal mitochondrial function and are both reported to be disrupted in aging, neurodegenerative diseases, and acute brain injury. While both processes have been extensively studied there has been little reported on how the mechanisms of these two processes are linked. This review focuses on how downstream NAD + catabolism via NUDIX hydrolases affects mitochondrial dynamics under pathologic conditions. Additionally, several potential targets in mitochondrial dysfunction and fragmentation are discussed, including the roles of mitochondrial poly(ADP-ribose) polymerase 1(mtPARP1), AMPK, AMP, and intra-mitochondrial GTP metabolism. Mitochondrial and cytosolic NUDIX hydrolases (NUDT9α and NUDT9β) can affect mitochondrial and cellular AMP levels by hydrolyzing ADP- ribose (ADPr) and subsequently altering the levels of GTP and ATP. Poly (ADP-ribose) polymerase 1 (PARP1) is activated after DNA damage, which depletes NAD + pools and results in the PARylation of nuclear and mitochondrial proteins. In the mitochondria, ADP-ribosyl hydrolase-3 (ARH3) hydrolyzes PAR to ADPr, while NUDT9α metabolizes ADPr to AMP. Elevated AMP levels have been reported to reduce mitochondrial ATP production by inhibiting the adenine nucleotide translocase (ANT), allosterically activating AMPK by altering the cellular AMP: ATP ratio, and by depleting mitochondrial GTP pools by being phosphorylated by adenylate kinase 3 (AK3), which uses GTP as a phosphate donor. Recently, activated AMPK was reported to phosphorylate mitochondria fission factor (MFF), which increases Drp1 localization to the mitochondria and promotes mitochondrial fission. Moreover, the increased AK3 activity could deplete mitochondrial GTP pools and possibly inhibit normal activity of GTP-dependent fusion enzymes, thus altering mitochondrial dynamics. Published by Elsevier Ltd.
Metabolic profiling of hypoxic cells revealed a catabolic signature required for cell survival.
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Christian Frezza
Full Text Available Hypoxia is one of the features of poorly vascularised areas of solid tumours but cancer cells can survive in these areas despite the low oxygen tension. The adaptation to hypoxia requires both biochemical and genetic responses that culminate in a metabolic rearrangement to counter-balance the decrease in energy supply from mitochondrial respiration. The understanding of metabolic adaptations under hypoxia could reveal novel pathways that, if targeted, would lead to specific death of hypoxic regions. In this study, we developed biochemical and metabolomic analyses to assess the effects of hypoxia on cellular metabolism of HCT116 cancer cell line. We utilized an oxygen fluorescent probe in anaerobic cuvettes to study oxygen consumption rates under hypoxic conditions without the need to re-oxygenate the cells and demonstrated that hypoxic cells can maintain active, though diminished, oxidative phosphorylation even at 1% oxygen. These results were further supported by in situ microscopy analysis of mitochondrial NADH oxidation under hypoxia. We then used metabolomic methodologies, utilizing liquid chromatography-mass spectrometry (LC-MS, to determine the metabolic profile of hypoxic cells. This approach revealed the importance of synchronized and regulated catabolism as a mechanism of adaptation to bioenergetic stress. We then confirmed the presence of autophagy under hypoxic conditions and demonstrated that the inhibition of this catabolic process dramatically reduced the ATP levels in hypoxic cells and stimulated hypoxia-induced cell death. These results suggest that under hypoxia, autophagy is required to support ATP production, in addition to glycolysis, and that the inhibition of autophagy might be used to selectively target hypoxic regions of tumours, the most notoriously resistant areas of solid tumours.
Czech Academy of Sciences Publication Activity Database
Jenčová, V.; Strnad, Hynek; Chodora, Zdeněk; Ulbrich, Pavel; Vlček, Čestmír; Hickey, W. J.; Pačes, Václav
2008-01-01
Roč. 159, č. 2 (2008), s. 118-127 ISSN 0923-2508 R&D Projects: GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z50520514 Keywords : megaplasmid * haloaromatic acid * catabolism Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.055, year: 2008
Hepatic Fatty Acid Oxidation Restrains Systemic Catabolism during Starvation
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Jieun Lee
2016-06-01
Full Text Available The liver is critical for maintaining systemic energy balance during starvation. To understand the role of hepatic fatty acid β-oxidation on this process, we generated mice with a liver-specific knockout of carnitine palmitoyltransferase 2 (Cpt2L−/−, an obligate step in mitochondrial long-chain fatty acid β-oxidation. Fasting induced hepatic steatosis and serum dyslipidemia with an absence of circulating ketones, while blood glucose remained normal. Systemic energy homeostasis was largely maintained in fasting Cpt2L−/− mice by adaptations in hepatic and systemic oxidative gene expression mediated in part by Pparα target genes including procatabolic hepatokines Fgf21, Gdf15, and Igfbp1. Feeding a ketogenic diet to Cpt2L−/− mice resulted in severe hepatomegaly, liver damage, and death with a complete absence of adipose triglyceride stores. These data show that hepatic fatty acid oxidation is not required for survival during acute food deprivation but essential for constraining adipocyte lipolysis and regulating systemic catabolism when glucose is limiting.
Nielsen, Tue Kjærgaard; Rasmussen, Morten; Demanèche, Sandrine; Cecillon, Sébastien; Vogel, Timothy M; Hansen, Lars Hestbjerg
2017-09-01
Bacterial degraders of chlorophenoxy herbicides have been isolated from various ecosystems, including pristine environments. Among these degraders, the sphingomonads constitute a prominent group that displays versatile xenobiotic-degradation capabilities. Four separate sequencing strategies were required to provide the complete sequence of the complex and plastic genome of the canonical chlorophenoxy herbicide-degrading Sphingobium herbicidovorans MH. The genome has an intricate organization of the chlorophenoxy-herbicide catabolic genes sdpA, rdpA, and cadABCD that encode the (R)- and (S)-enantiomer-specific 2,4-dichlorophenoxypropionate dioxygenases and four subunits of a Rieske non-heme iron oxygenase involved in 2-methyl-chlorophenoxyacetic acid degradation, respectively. Several major genomic rearrangements are proposed to help understand the evolution and mobility of these important genes and their genetic context. Single-strain mobilomic sequence analysis uncovered plasmids and insertion sequence-associated circular intermediates in this environmentally important bacterium and enabled the description of evolutionary models for pesticide degradation in strain MH and related organisms. The mobilome presented a complex mosaic of mobile genetic elements including four plasmids and several circular intermediate DNA molecules of insertion-sequence elements and transposons that are central to the evolution of xenobiotics degradation. Furthermore, two individual chromosomally integrated prophages were shown to excise and form free circular DNA molecules. This approach holds great potential for improving the understanding of genome plasticity, evolution, and microbial ecology. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.
Ye, Nenghui; Li, Haoxuan; Zhu, Guohui; Liu, Yinggao; Liu, Rui; Xu, Weifeng; Jing, Yu; Peng, Xinxiang; Zhang, Jianhua
2014-11-01
Although copper (Cu) is an essential micronutrient for plants, a slight excess of Cu in soil can be harmful to plants. Unfortunately, Cu contamination is a growing problem all over the world due to human activities, and poses a soil stress to plant development. As one of the most important biological processes, seed germination is sensitive to Cu stress. However, little is known about the mechanism of Cu-induced inhibition of seed germination. In the present study, we investigated the relationship between Cu and ABA which is the predominant regulator of seed germination. Cu at a concentration of 30 µM effectively inhibited germination of rice caryopsis. ABA content in germinating seeds under copper stress was also higher than that under control conditions. Quantitative real-time PCR (qRT-PCR) revealed that Cu treatment reduced the expression of OsABA8ox2, a key gene of ABA catabolism in rice seeds. In addition, both malondialdehyde (MDA) and H2O2 contents were increased by Cu stress in the germinating seeds. Antioxidant enzyme assays revealed that only catalase activity was reduced by excess Cu, which was consistent with the mRNA profile of OsCATa during seed germination under Cu stress. Together, our results demonstrate that suppression of ABA catabolism and catalase (CAT) activity by excess Cu leads to the inhibition of seed germination of rice. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.
Araman, Can; Thompson, Robert E; Wang, Siyao; Hackl, Stefanie; Payne, Richard J; Becker, Christian F W
2017-09-01
The prion protein (PrP) is an N -glycosylated protein attached to the outer leaflet of eukaryotic cell membranes via a glycosylphosphatidylinositol (GPI) anchor. Different prion strains have distinct glycosylation patterns and the extent of glycosylation of potentially pathogenic misfolded prion protein (PrP Sc ) has a major impact on several prion-related diseases (transmissible spongiform encephalopathies, TSEs). Based on these findings it is hypothesized that posttranslational modifications (PTMs) of PrP influence conversion of cellular prion protein (PrP C ) into PrP Sc and, as such, modified PrP variants are critical tools needed to investigate the impact of PTMs on the pathogenesis of TSEs. Here we report a semisynthetic approach to generate PrP variants modified with monodisperse polyethyleneglycol (PEG) units as mimics of N-glycans. Incorporating PEG at glycosylation sites 181 and 197 in PrP induced only small changes to the secondary structure when compared to unmodified, wildtype PrP. More importantly, in vitro aggregation was abrogated for all PEGylated PrP variants under conditions at which wildtype PrP aggregated. Furthermore, the addition of PEGylated PrP as low as 10 mol% to wildtype PrP completely blocked aggregation. A similar effect was observed for synthetic PEGylated PrP segments comprising amino acids 179-231 alone if these were added to wildtype PrP in aggregation assays. This behavior raises the question if large N-glycans interfere with aggregation in vivo and if PEGylated PrP peptides could serve as potential therapeutics.
Catabolism of 6-ketoprostaglandin F1alpha by the rat kidney cortex.
Pace-Asciak, C R; Domazet, Z; Carrara, M
1977-05-25
Homogenates of the rat kidney cortex converted 5,8,9,11,12,14,15-hepta-tritiated 6-ketoprostaglandin F 1alpha into one major product identified by gas chromatography-mass spectrometry of the methoxime-methyl ester trimethylsilyl ether derivative as 6,15-diketo-9,11-dihydroxyprost-13-enoic acid. The sequence of derivatisation i.e. methoximation prior to methylation, was crucial as methylation of 15-keto catabolites of the E, F and 6-keto-F series affords degradation products. The corresponding 15-keto-13,14-dihydro catabolite was formed in much smaller quantities. Time course studies indicated that 6-keto-prostaglandin F1alpha was catabolised at a slower rate (about 2-5 fold) than prostaglandin F1alpha. The catabolic activity was blocked by NADH.
Energy Technology Data Exchange (ETDEWEB)
Bruehlmeier, Matthias E-mail: peter.blaeuenstein@psi.ch; Garayoa, Elisa Garcia; Blanc, Alain; Holzer, Barbara; Gergely, Suzanne; Tourwe, Dirk; Schubiger, Pius August; Blaeuenstein, Peter
2002-04-01
Neurotensin (NT) receptors in pancreatic and other neuroendocrine tumors are promising targets for imaging and therapeutic purposes. Here, we report on the effect of distinct changes in the peptide chain on catabolism in vitro for five radiolabeled [{sup 99m}Tc] neurotensin analogues having high affinity for neurotensin receptors. Substitution of NT(1-7) by (N{alpha}His)Ac--the Tc-binding moiety--combined with a reduced bond 8-9 (CH{sub 2}NH), N-methylation of peptide bonds or replacement of Ile(12) by tertiary leucin (Tle) led to peptide stabilization of various degrees. Biodistribution studies in nude mice bearing HT29 xenografts showed higher tumor uptake with more stable peptides, yielding high tumor to blood ratios of up to 70.
Miah, M Fahad; Conseil, Gwenaëlle; Cole, Susan P C
2016-01-22
Multidrug resistance protein 4 (MRP4) is a member of subfamily C of the ATP-binding cassette superfamily of membrane transport proteins. MRP4 mediates the ATP-dependent efflux of many endogenous and exogenous solutes across the plasma membrane, and in polarized cells, it localizes to the apical or basolateral plasma membrane depending on the tissue type. MRP4 is a 170 kDa glycoprotein and here we show that MRP4 is simultaneously N-glycosylated at Asn746 and Asn754. Furthermore, confocal immunofluorescence studies showed that N-glycans do not affect MRP4's apical membrane localization in polarized LLC-PK1 cells or basolateral membrane localization in polarized MDCKI cells. However, vesicular transport assays showed that N-glycans differentially affect MRP4's ability to transport prostaglandin E2, but not estradiol glucuronide. Together these data indicate that N-glycosylation at Asn746 and Asn754 is not essential for plasma membrane localization of MRP4 but cause substrate-selective effects on its transport activity. Copyright © 2015 Elsevier Inc. All rights reserved.
Sugimori, Daisuke; Watanabe, Mika; Utsue, Tomohiro
2013-01-01
The lipids (fats and oils) degradation capabilities of soil microorganisms were investigated for possible application in treatment of lipids-contaminated wastewater. We isolated a strain of the bacterium Raoultella planticola strain 232-2 that is capable of efficiently catabolizing lipids under acidic conditions such as in grease traps in restaurants and food processing plants. The strain 232-2 efficiently catabolized a mixture (mixed lipids) of commercial vegetable oil, lard, and beef tallow (1:1:1, w/w/w) at 20-35 °C, pH 3-9, and 1,000-5,000 ppm lipid content. Highly effective degradation rate was observed at 35 °C and pH 4.0, and the 24-h degradation rate was 62.5 ± 10.5 % for 3,000 ppm mixed lipids. The 24-h degradation rate for 3,000 ppm commercial vegetable oil, lard, beef tallow, mixed lipids, and oleic acid was 71.8 %, 58.7 %, 56.1 %, 55.3 ± 8.5 %, and 91.9 % at pH 4 and 30 °C, respectively. R. planticola NBRC14939 (type strain) was also able to efficiently catabolize the lipids after repeated subculturing. The composition of the culture medium strongly influenced the degradation efficiency, with yeast extract supporting more complete dissimilation than BactoPeptone or beef extract. The acid tolerance of strain 232-2 is proposed to result from neutralization of the culture medium by urease-mediated decomposition of urea to NH(3). The rate of lipids degradation increased with the rates of neutralization and cell growth. Efficient lipids degradation using strain 232-2 has been achieved in the batch treatment of a restaurant wastewater.
International Nuclear Information System (INIS)
Pometto, A.L. III; Crawford, D.L.
1986-01-01
Degradation of ground and hot-water-extracted corn stover (Zea mays) lignocellulose by Streptomyces viridosporus T7A generates a water-soluble lignin degradation intermediate termed acid-precipitable polymeric lignin (APPL). The further catabolism of T7A-APPL by S. viridosporus T7A, S. badius 252, and S. setonii75Vi2 was followed for 3 weeks. APPL catabolism by Phanerochaete chrysosporium was followed in stationary cultures in a low-nitrogen medium containing 1% (wt/vol) glucose and 0.05% (wt/vol) T7A-APPL. Metabolism of the APPL was followed by turbidometric assay (600 nm) and by direct measurement of APPL recoverable from the medium. Accumulation and disappearance of soluble low-molecular-weight products of APPL catabolism were followed by gas-liquid chromatography and by high-pressure liquid chromatography, utilizing a diode array detector. Mineralization of a [ 14 C-lignin]APPL was also followed. The percent 14 C recovered as 14 CO 2 , 14 C-APPL, 14 C-labeled water-soluble products, and cell mass-associated radioactivity, were determined for each microorganism after 1 and 3 weeks of incubation in bubbler tube cultures at 37 0 C. P. chrysosporium evolved the most 14 CO 2 , and S. viridosporus gave the greatest decrease in recoverable 14 C-APPL. The results show that S. badius was not able to significantly degrade the APPL, while the other microorganisms demonstrated various APPL-degrading abilities
He, Amanda; Penix, Stephanie R; Basting, Preston J; Griffith, Jessie M; Creamer, Kaitlin E; Camperchioli, Dominic; Clark, Michelle W; Gonzales, Alexandra S; Chávez Erazo, Jorge Sebastian; George, Nadja S; Bhagwat, Arvind A; Slonczewski, Joan L
2017-06-15
Acid-adapted strains of Escherichia coli K-12 W3110 were obtained by serial culture in medium buffered at pH 4.6 (M. M. Harden, A. He, K. Creamer, M. W. Clark, I. Hamdallah, K. A. Martinez, R. L. Kresslein, S. P. Bush, and J. L. Slonczewski, Appl Environ Microbiol 81:1932-1941, 2015, https://doi.org/10.1128/AEM.03494-14). Revised genomic analysis of these strains revealed insertion sequence (IS)-driven insertions and deletions that knocked out regulators CadC (acid induction of lysine decarboxylase), GadX (acid induction of glutamate decarboxylase), and FNR (anaerobic regulator). Each acid-evolved strain showed loss of one or more amino acid decarboxylase systems, which normally help neutralize external acid (pH 5 to 6) and increase survival in extreme acid (pH 2). Strains from populations B11, H9, and F11 had an IS 5 insertion or IS-mediated deletion in cadC , while population B11 had a point mutation affecting the arginine activator adiY The cadC and adiY mutants failed to neutralize acid in the presence of exogenous lysine or arginine. In strain B11-1, reversion of an rpoC (RNA polymerase) mutation partly restored arginine-dependent neutralization. All eight strains showed deletion or downregulation of the Gad acid fitness island. Strains with the Gad deletion lost the ability to produce GABA (gamma-aminobutyric acid) and failed to survive extreme acid. Transcriptome sequencing (RNA-seq) of strain B11-1 showed upregulated genes for catabolism of diverse substrates but downregulated acid stress genes (the biofilm regulator ariR , yhiM , and Gad). Other strains showed downregulation of H 2 consumption mediated by hydrogenases ( hya and hyb ) which release acid. Strains F9-2 and F9-3 had a deletion of fnr and showed downregulation of FNR-dependent genes ( dmsABC , frdABCD , hybABO , nikABCDE , and nrfAC ). Overall, strains that had evolved in buffered acid showed loss or downregulation of systems that neutralize unbuffered acid and showed altered regulation of
Ghosh, Swagata; Rao, K Hanumantha; Sengupta, Manjistha; Bhattacharya, Sujit K; Datta, Asis
2011-06-01
Pathogenic microorganisms like Vibrio cholerae are capable of adapting to diverse living conditions, especially when they transit from their environmental reservoirs to human host. V. cholerae attaches to N-acetylglucosamine (GlcNAc) residues in glycoproteins and lipids present in the intestinal epithelium and chitinous surface of zoo-phytoplanktons in the aquatic environment for its survival and colonization. GlcNAc utilization thus appears to be important for the pathogen to reach sufficient titres in the intestine for producing clinical symptoms of cholera. We report here the involvement of a second cluster of genes working in combination with the classical genes of GlcNAc catabolism, suggesting the occurrence of a novel variant of the process of biochemical conversion of GlcNAc to Fructose-6-phosphate as has been described in other organisms. Colonization was severely attenuated in mutants that were incapable of utilizing GlcNAc. It was also shown that N-acetylglucosamine specific repressor (NagC) performs a dual role - while the classical GlcNAc catabolic genes are under its negative control, the genes belonging to the second cluster are positively regulated by it. Further application of tandem affinity purification to NagC revealed its interaction with a novel partner. Our results provide a genetic program that probably enables V. cholerae to successfully utilize amino - sugars and also highlights a new mode of transcriptional regulation, not described in this organism. © 2011 Blackwell Publishing Ltd.
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Aurélia Revel
Full Text Available Muscle atrophy has been explained by an anabolic resistance following food intake and an increase of dietary protein intake is recommended. To be optimal, a dietary protein has to be effective not only to initiate but also to prolong a muscle anabolic response in a catabolic state. To our knowledge, whether or not a dairy or a dairy/plant protein blend fulfills these criterions is unknown in a muscle wasting situation.Our aim was, in a control and a catabolic state, to measure continuously muscle anabolism in term of intensity and duration in response to a meal containing casein (CAS, whey (WHEY or a whey/ plant protein blend (BLEND and to evaluate the best protein source to elicit the best post prandial anabolism according to the physio-pathological state.Adult male Yucatan mini pigs were infused with U-13C-Phenylalanine and fed either CAS, WHEY or BLEND. A catabolic state was induced by a glucocorticoid treatment for 8 days (DEX. Muscle protein synthesis, proteolysis and balance were measured with the hind limb arterio-venous differences technique. Repeated time variance analysis were used to assess significant differences.In a catabolic situation, whey proteins were able to initiate muscle anabolism which remained transient in contrast to the stimulated muscle protein accretion with WHEY, CAS or BLEND in healthy conditions. Despite the same leucine intake compared to WHEY, BLEND did not restore a positive protein balance in DEX animals.Even with WHEY, the duration of the anabolic response was not optimal and has to be improved in a catabolic state. The use of BLEND remained of lower efficiency even at same leucine intake than whey.
Tallman, John F.; Johnson, William G.; Brady, Roscoe O.
1972-01-01
The catabolism of Tay-Sachs ganglioside, N-acetylgalactosaminyl- (N-acetylneuraminosyl) -galactosylglucosylceramide, has been studied in lysosomal preparations from normal human brain and brain obtained at biopsy from Tay-Sachs patients. Utilizing Tay-Sachs ganglioside labeled with 14C in the N-acetylgalactosaminyl portion or 3H in the N-acetylneuraminosyl portion, the catabolism of Tay-Sachs ganglioside may be initiated by either the removal of the molecule of N-acetylgalactosamine or N-acetylneuraminic acid. The activity of the N-acetylgalactosamine-cleaving enzyme (hexosaminidase) is drastically diminished in such preparations from Tay-Sachs brain whereas the activity of the N-acetylneuraminic acid-cleaving enzyme (neuraminidase) is at a normal level. Total hexosaminidase activity as measured with an artificial fluorogenic substrate is increased in tissues obtained from patients with the B variant form of Tay-Sachs disease and it is virtually absent in the O-variant patients. The addition of purified neuraminidase and various purified hexosaminidases exerted only a minimal synergistic effect on the hydrolysis of Tay-Sachs ganglioside in the lysosomal preparations from the control or patient with the O variant of Tay-Sachs disease. Images PMID:4639018
Zhang, Yun; Shang, Xiuling; Lai, Shujuan; Zhang, Yu; Hu, Qitiao; Chai, Xin; Wang, Bo; Liu, Shuwen; Wen, Tingyi
2018-02-16
l-Serine, the principal one-carbon source for DNA biosynthesis, is difficult for microorganisms to accumulate due to the coupling of l-serine catabolism and microbial growth. Here, we reprogrammed the one-carbon unit metabolic pathways in Corynebacterium glutamicum to decouple l-serine catabolism from cell growth. In silico model-based simulation showed a negative influence on glyA-encoding serine hydroxymethyltransferase flux with l-serine productivity. Attenuation of glyA transcription resulted in increased l-serine accumulation, and a decrease in purine pools, poor growth and longer cell shapes. The gcvTHP-encoded glycine cleavage (Gcv) system from Escherichia coli was introduced into C. glutamicum, allowing glycine-derived 13 CH 2 to be assimilated into intracellular purine synthesis, which resulted in an increased amount of one-carbon units. Gcv introduction not only restored cell viability and morphology but also increased l-serine accumulation. Moreover, comparative proteomic analysis indicated that abundance changes of the enzymes involved in one-carbon unit cycles might be responsible for maintaining one-carbon unit homeostasis. Reprogramming of the one-carbon metabolic pathways allowed cells to reach a comparable growth rate to accumulate 13.21 g/L l-serine by fed-batch fermentation in minimal medium. This novel strategy provides new insights into the regulation of cellular properties and essential metabolite accumulation by introducing an extrinsic pathway.
Complex Interplay of Hormonal Signals during Grape Berry Ripening
Directory of Open Access Journals (Sweden)
Ana Margarida Fortes
2015-05-01
Full Text Available Grape and wine production and quality is extremely dependent on the fruit ripening process. Sensory and nutritional characteristics are important aspects for consumers and their development during fruit ripening involves complex hormonal control. In this review, we explored data already published on grape ripening and compared it with the hormonal regulation of ripening of other climacteric and non-climacteric fruits. The roles of abscisic acid, ethylene, and brassinosteroids as promoters of ripening are discussed, as well as the role of auxins, cytokinins, gibberellins, jasmonates, and polyamines as inhibitors of ripening. In particular, the recently described role of polyamine catabolism in grape ripening is discussed, together with its putative interaction with other hormones. Furthermore, other recent examples of cross-talk among the different hormones are presented, revealing a complex interplay of signals during grape development and ripening.
Bowman, Kole; Rose, Jack
2017-01-01
Glycogen synthesis by mink uterine glandular and luminal epithelia (GE and LE) is stimulated by estradiol (E 2 ) during estrus. Subsequently, the glycogen deposits are mobilized to near completion to meet the energy requirements of pre-embryonic development and implantation by as yet undetermined mechanisms. We hypothesized that progesterone (P 4 ) was responsible for catabolism of uterine glycogen reserves as one of its actions to ensure reproductive success. Mink were treated with E 2 , P 4 or vehicle (controls) for 3 days and uteri collected 24 h (E 2 , P 4 and vehicle) and 96 h (E 2 ) later. To evaluate E 2 priming, mink were treated with E 2 for 3 days, then P 4 for an additional 3 days (E 2 →P 4 ) and uteri collected 24 h later. Percent glycogen content of uterine epithelia was greater at E 2 + 96 h (GE = 5.71 ± 0.55; LE = 11.54 ± 2.32) than E 2 +24 h (GE = 3.63 ± 0.71; LE = 2.82 ± 1.03), and both were higher than controls (GE = 0.27 ± 0.15; LE = 0.54 ± 0.30; P glycogen content (GE = 0.61 ± 0.16; LE = 0.51 ± 0.13), to levels not different from controls, while concomitantly increasing catabolic enzyme (glycogen phosphorylase m and glucose-6-phosphatase) gene expression and amount of phospho-glycogen synthase protein (inactive) in uterine homogenates. Interestingly, E 2 →P 4 increased glycogen synthase 1 messenger RNA (mRNA) and hexokinase 1mRNA and protein. Our findings suggest to us that while E 2 promotes glycogen accumulation by the mink uterus during estrus and pregnancy, it is P 4 that induces uterine glycogen catabolism, releasing the glucose that is essential to support pre-embryonic survival and implantation. © 2016 Japanese Society of Animal Science.
Jia, Gaozhen; Dong, Zhenyang; Sun, Chenxia; Wen, Fuping; Wang, Haifeng; Guo, Huaizu; Gao, Xu; Xu, Chuanliang; Xu, Chuanliang; Yang, Chenghua; Sun, Yinghao
2017-09-29
The prostate specific antigen (PSA) test is widely used for early diagnosis of prostate cancer (PCa). However, its limited sensitivity has led to over-diagnosis and over-treatment of PCa. Glycosylation alteration is a common phenomenon in cancer development. Different PSA glycan subforms have been proposed as diagnostic markers to better differentiate PCa from benign prostate hyperplasia (BPH). In this study, we purified PSA from expressed prostate secretions (EPS)-urine samples from 32 BPH and 30 PCa patients and provided detailed PSA glycan profiles in Chinese population. We found that most of the PSA glycans from EPS-urine were complex type biantennary glycans. We observed two major patterns in PSA glycan profiles. Overall there was no distinct separation of PSA glycan profiles between BPH and PCa patients. However, we detected a significant increase of glycan FA2 and FM5A2G2S1 in PCa when compared with BPH patients. Furthermore, we observed that the composition of FA2 glycan increased significantly in advanced PCa with Gleason score ≥8, which potentially could be translated to clinic as a marker for aggressive PCa.
Crystal structure of reovirus attachment protein σ1 in complex with sialylated oligosaccharides.
Reiter, Dirk M; Frierson, Johnna M; Halvorson, Elizabeth E; Kobayashi, Takeshi; Dermody, Terence S; Stehle, Thilo
2011-08-01
Many viruses attach to target cells by binding to cell-surface glycans. To gain a better understanding of strategies used by viruses to engage carbohydrate receptors, we determined the crystal structures of reovirus attachment protein σ1 in complex with α-2,3-sialyllactose, α-2,6-sialyllactose, and α-2,8-di-siallylactose. All three oligosaccharides terminate in sialic acid, which serves as a receptor for the reovirus serotype studied here. The overall structure of σ1 resembles an elongated, filamentous trimer. It contains a globular head featuring a compact β-barrel, and a fibrous extension formed by seven repeating units of a triple β-spiral that is interrupted near its midpoint by a short α-helical coiled coil. The carbohydrate-binding site is located between β-spiral repeats two and three, distal from the head. In all three complexes, the terminal sialic acid forms almost all of the contacts with σ1 in an identical manner, while the remaining components of the oligosaccharides make little or no contacts. We used this structural information to guide mutagenesis studies to identify residues in σ1 that functionally engage sialic acid by assessing hemagglutination capacity and growth in murine erythroleukemia cells, which require sialic acid binding for productive infection. Our studies using σ1 mutant viruses reveal that residues 198, 202, 203, 204, and 205 are required for functional binding to sialic acid by reovirus. These findings provide insight into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to endow other trimeric proteins with carbohydrate-binding properties.
Crystal structure of reovirus attachment protein σ1 in complex with sialylated oligosaccharides.
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Dirk M Reiter
2011-08-01
Full Text Available Many viruses attach to target cells by binding to cell-surface glycans. To gain a better understanding of strategies used by viruses to engage carbohydrate receptors, we determined the crystal structures of reovirus attachment protein σ1 in complex with α-2,3-sialyllactose, α-2,6-sialyllactose, and α-2,8-di-siallylactose. All three oligosaccharides terminate in sialic acid, which serves as a receptor for the reovirus serotype studied here. The overall structure of σ1 resembles an elongated, filamentous trimer. It contains a globular head featuring a compact β-barrel, and a fibrous extension formed by seven repeating units of a triple β-spiral that is interrupted near its midpoint by a short α-helical coiled coil. The carbohydrate-binding site is located between β-spiral repeats two and three, distal from the head. In all three complexes, the terminal sialic acid forms almost all of the contacts with σ1 in an identical manner, while the remaining components of the oligosaccharides make little or no contacts. We used this structural information to guide mutagenesis studies to identify residues in σ1 that functionally engage sialic acid by assessing hemagglutination capacity and growth in murine erythroleukemia cells, which require sialic acid binding for productive infection. Our studies using σ1 mutant viruses reveal that residues 198, 202, 203, 204, and 205 are required for functional binding to sialic acid by reovirus. These findings provide insight into mechanisms of reovirus attachment to cell-surface glycans and contribute to an understanding of carbohydrate binding by viruses. They also establish a filamentous, trimeric carbohydrate-binding module that could potentially be used to endow other trimeric proteins with carbohydrate-binding properties.
Eliakim, Alon; Portal, Shawn; Zadik, Zvi; Meckel, Yoav; Nemet, Dan
2013-11-01
We examined the effect of training on hormonal and inflammatory response to a single volleyball practice in elite adolescent players. Thirteen female, national team level, Israeli volleyball players (age 16.0 ± 1.4 years, Tanner stage 4-5) participated in the study. Blood samples were collected before and immediately after a typical 60 minutes of volleyball practice, before and after 7 weeks of training during the initial phase of the season. Training involved tactic and technical drills (20% of time), power and speed drills (25% of time), interval sessions (25% of time), endurance-type training (15% of time), and resistance training (15% of time). To achieve greater training responses, the study was performed during the early phase (first 7 weeks) of the volleyball season. Hormonal measurements included the anabolic hormones growth hormone (GH), insulin-like growth factor-I (IGF-I) and IGF-binding protein-3, the catabolic hormone cortisol, the proinflammatory marker interleukin-6 (IL-6), and the anti-inflammatory marker IL-1 receptor antagonist. Training led to a significant improvement of vertical jump, anaerobic properties (peak and mean power by the Wingate Anaerobic Test), and predicted VO2max (by the 20-m shuttle run). Volleyball practice, both before and after the training intervention, was associated with a significant increase of serum lactate, GH, and IL-6. Training resulted in a significantly reduced cortisol response ([INCREMENT]cortisol: 4.2 ± 13.7 vs. -4.4 ± 12.3 ng · ml, before and after training, respectively; p volleyball practice. The results suggest that along with the improvement of power and anaerobic and aerobic characteristics, training reduces the catabolic and inflammatory response to exercise.
Latimer, Scott; Li, Yubing; Nguyen, Thuong T H; Soubeyrand, Eric; Fatihi, Abdelhak; Elowsky, Christian G; Block, Anna; Pichersky, Eran; Basset, Gilles J
2018-05-09
The proteinogenic branched-chain amino acids (BCAAs) leucine, isoleucine and valine are essential nutrients for mammals. In plants, BCAAs double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates to the tricarboxylic acid cycle. Yet, the actual architecture of the degradation pathways of BCAAs is not well understood. In this study, gene network modeling in Arabidopsis and rice, and plant-prokaryote comparative genomics detected candidates for 3-methylglutaconyl-CoA hydratase (4.2.1.18), one of the missing plant enzymes of leucine catabolism. Alignments of these protein candidates sampled from various spermatophytes revealed non-homologous N-terminal extensions that are lacking in their bacterial counterparts, and green fluorescent protein-fusion experiments demonstrated that the Arabidopsis protein, product of gene At4g16800, is targeted to mitochondria. Recombinant At4g16800 catalyzed the dehydration of 3-hydroxymethylglutaryl-CoA into 3-methylglutaconyl-CoA, and displayed kinetic features similar to those of its prokaryotic homolog. When at4g16800 knockout plants were subjected to dark-induced carbon starvation, their rosette leaves displayed accelerated senescence as compared to control plants, and this phenotype was paralleled by a marked increase in the accumulation of free and total leucine, isoleucine and valine. The seeds of the at4g16800 mutant showed a similar accumulation of free BCAAs. These data suggest that 3-methylglutaconyl-CoA hydratase is not solely involved in the degradation of leucine, but is also a significant contributor to that of isoleucine and valine. Furthermore, evidence is shown that unlike the situation observed in Trypanosomatidae, leucine catabolism does not contribute to the formation of the terpenoid precursor mevalonate. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights
Miyazaki, Takatsugu; Kato, Tatsuya; Park, Enoch Y
2018-02-03
β-1,2-N-Acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi-localized type II transmembrane enzyme that catalyzes the transfer of N-acetylglucosamine to the 6-arm of the trimanosyl core of N-glycans, an essential step in the conversion of oligomannose-type to complex-type N-glycans. Despite its physiological importance, there have been only a few reports on the heterologous expression and structure-function relationship of this enzyme. Here, we constructed a silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system and expressed human GnTII (hGnTII) lacking the N-terminal cytosolic tail and transmembrane region. The recombinant hGnTII was purified from silkworm larval hemolymph in two steps by using tandem affinity purification tags, with a yield of approximately 120 μg from 10 mL hemolymph, and exhibited glycosyltransferase activity and strict substrate specificity. The enzyme was found to be N-glycosylated by the enzymatic cleavage of glycans, while hGnTII expressed in insect cells had not been reported to be glycosylated. Although insects typically produce pauci-mannosidic-type glycans, the structure of N-glycans in the recombinant hGnTII was suggested to be of the complex type, and the removal of the glycans did not affect the enzymatic activity. Copyright © 2018 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
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Teet Seene
2016-05-01
Full Text Available Muscle weakness in corticosteroid myopathy is mainly the result of the destruction and atrophy of the myofibrillar compartment of fast-twitch muscle fibers. Decrease of titin and myosin, and the ratio of nebulin and MyHC in myopathic muscle, shows that these changes of contractile and elastic proteins are the result of increased catabolism of the abovementioned proteins in skeletal muscle. Slow regeneration of skeletal muscle is in good correlation with a decreased number of satellite cells under the basal lamina of muscle fibers. Aging causes a reduction of AMP-activated protein kinase (AMPK activity as the result of the reduced function of the mitochondrial compartment. AMPK activity increases as a result of increased functional activity. Resistance exercise causes anabolic and anticatabolic effects in skeletal muscle: muscle fibers experience hypertrophy while higher myofibrillar proteins turn over. These changes are leading to the qualitative remodeling of muscle fibers. As a result of these changes, possible maximal muscle strength is increasing. Endurance exercise improves capillary blood supply, increases mitochondrial biogenesis and muscle oxidative capacity, and causes a faster turnover rate of sarcoplasmic proteins as well as qualitative remodeling of type I and IIA muscle fibers. The combination of resistance and endurance exercise may be the fastest way to prevent or decelerate muscle atrophy due to the anabolic and anticatabolic effects of exercise combined with an increase in oxidative capacity. The aim of the present short review is to assess the role of myofibrillar protein catabolism in the development of glucocorticoid-caused myopathy from aging and physical activity aspects.
Mielgo Ayuso, Juan; Zourdos, Michael C; Urdampilleta, Aritz; Calleja González, Julio; Seco, Jesús; Córdova, Alfredo
2017-10-24
Specific macronutrient distribution and training can alter acute and chronic hormone behavior and, subsequently, sport performance. The main aim was to examine relationships between dietary intake and anabolic/catabolic hormone response in elite female volleyball players during a 29-week season. Twenty-two elite female volleyballers (26.4 ± 5.6 years; 178 ± 9 cm; 67.1 ± 7.5 kg) had dietary intake (seven-day dietary recall and food frequency questionnaire), blood concentration of anabolic/catabolic hormones concentration, physical performance, and body composition assessed at four time points: a) T1: baseline/pre-testing; b) T2: eleven weeks after T1; c) T3: ten weeks after T2; and d) T4: eight weeks after T3. Hormones evaluated were: total testosterone (TT), free testosterone (FT) adrenocorticotropic hormone (ACTH), and cortisol (C), along with hormone ratios. Positive correlations were observed between carbohydrate/protein ratio with ΔFT (r = 0.955; p 0.05) in body mass or body mass index at any time point, and the sum of six skinfolds improved (p < 0.05) from T1 (86.5 ± 6.9 mm) to T4 (75.2 ± 5.6 mm) as did muscle mass (T1: 28.9 ± 0.7 kg vsT4: 30.1 ± 0.8 kg). Vertical jump, spike-jump and speed improved (p < 0.05) from T1 to T4. A high carbohydrate/protein ratio was associated with positive changes in anabolism, while high protein and low carbohydrates (CHO) were associated with an attenuated anabolic response.