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Sample records for 1a glycan determined

  1. The function of the human interferon-beta 1a glycan determined in vivo

    Dissing-Olesen, Lasse; Thaysen-Andersen, Morten; Meldgaard, Michael; Højrup, Peter; Finsen, Bente

    2008-01-01

    counterpart, due to a protein stabilization/solubilization effect of the glycan. It is interesting to note that the terminating sialic acids were essential for these effects. Conclusively, the structure/bioactivity relationship of rhIFN-beta1a was determined in vivo, and it provided a novel insight into the...

  2. Mutations in HNF1A result in marked alterations of plasma glycan profile

    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), and...

  3. Glycan analysis of the chicken synaptic plasma membrane glycoproteins - a major synaptic N-glycan carries the LewisX determinant

    2005-09-01

    Full Text Available The majority of synaptic plasma membrane components are glycosylated. It is now widely accepted that this post-translational modification is crucial during the establishment, maintenance and function of the nervous system. Despite its significance, structural information about the glycosylation of nervous system specific glycoproteins is very limited. In the present study the major glycan structures of the chicken synaptic plasma membrane (SPM associated glycoprotein glycans were determined. N-glycans were released by hydrazinolysis, labelled with 2-aminobenzamide, treated with neuraminidase and subsequently fractionated by size exclusion chromatography. Individual fractions were characterized by the combination of high-pressure liquid chromatography, exoglycosidase treatment or reagent array analysis method (RAAM. In addition to oligomannose-type glycans, core-fucosylated complex glycans with biantennary bisecting glycans carrying the LewisX epitope were most abundant. The overall chicken glycan profile was strikingly similar to the rat brain glycan profile. The presence of the LewisX determinant in relatively large proportions suggests a tissue-specific function for these glycans.

  4. IgG N-glycans as potential biomarkers for determining galactose tolerance in Classical Galactosaemia.

    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.

  5. The essential role of the sialic acid residues for IFN-β1a activity determined in vivo

    Olesen, Lasse Dissing; Andersen, Morten Thaysen

    a protein stabilization/solubilization effect of the glycan. Interestingly, the terminating sialic acids were essential for these effects. Conclusively, the structure/bioactivity relationship of rhIFN-β1a was determined in vivo and provided a novel insight into the role of the rhIFN-β1a glycan and...

  6. Determinants of glycan receptor specificity of H2N2 influenza A virus hemagglutinin.

    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.

  7. Automated motif discovery from glycan array data.

    Cholleti, Sharath R; Agravat, Sanjay; Morris, Tim; Saltz, Joel H; Song, Xuezheng; Cummings, Richard D; Smith, David F

    2012-10-01

    Assessing interactions of a glycan-binding protein (GBP) or lectin with glycans on a microarray generates large datasets, making it difficult to identify a glycan structural motif or determinant associated with the highest apparent binding strength of the GBP. We have developed a computational method, termed GlycanMotifMiner, that uses the relative binding of a GBP with glycans within a glycan microarray to automatically reveal the glycan structural motifs recognized by a GBP. We implemented the software with a web-based graphical interface for users to explore and visualize the discovered motifs. The utility of GlycanMotifMiner was determined using five plant lectins, SNA, HPA, PNA, Con A, and UEA-I. Data from the analyses of the lectins at different protein concentrations were processed to rank the glycans based on their relative binding strengths. The motifs, defined as glycan substructures that exist in a large number of the bound glycans and few non-bound glycans, were then discovered by our algorithm and displayed in a web-based graphical user interface ( http://glycanmotifminer.emory.edu ). The information is used in defining the glycan-binding specificity of GBPs. The results were compared to the known glycan specificities of these lectins generated by manual methods. A more complex analysis was also carried out using glycan microarray data obtained for a recombinant form of human galectin-8. Results for all of these lectins show that GlycanMotifMiner identified the major motifs known in the literature along with some unexpected novel binding motifs. PMID:22877213

  8. Identification of Antigenic Glycans from Schistosoma mansoni by Using a Shotgun Egg Glycan Microarray.

    Mickum, Megan L; Prasanphanich, Nina Salinger; Song, Xuezheng; Dorabawila, Nelum; Mandalasi, Msano; Lasanajak, Yi; Luyai, Anthony; Secor, W Evan; Wilkins, Patricia P; Van Die, Irma; Smith, David F; Nyame, A Kwame; Cummings, Richard D; Rivera-Marrero, Carlos A

    2016-05-01

    Infection of mammals by the parasitic helminth Schistosoma mansoni induces antibodies to glycan antigens in worms and eggs, but the differential nature of the immune response among infected mammals is poorly understood. To better define these responses, we used a shotgun glycomics approach in which N-glycans from schistosome egg glycoproteins were prepared, derivatized, separated, and used to generate an egg shotgun glycan microarray. This array was interrogated with sera from infected mice, rhesus monkeys, and humans and with glycan-binding proteins and antibodies to gather information about the structures of antigenic glycans, which also were analyzed by mass spectrometry. A major glycan antigen targeted by IgG from different infected species is the FLDNF epitope [Fucα3GalNAcβ4(Fucα3)GlcNAc-R], which is also recognized by the IgG monoclonal antibody F2D2. The FLDNF antigen is expressed by all life stages of the parasite in mammalian hosts, and F2D2 can kill schistosomula in vitro in a complement-dependent manner. Different antisera also recognized other glycan determinants, including core β-xylose and highly fucosylated glycans. Thus, the natural shotgun glycan microarray of schistosome eggs is useful in identifying antigenic glycans and in developing new anti-glycan reagents that may have diagnostic applications and contribute to developing new vaccines against schistosomiasis. PMID:26883596

  9. Glycans In The Immune system and The Altered Glycan Theory of Autoimmunity: A Critical Review

    Maverakis, Emanual; Kim, Kyoungmi; Shimoda, Michiko; Gershwin, M. Eric; Patel, Forum; Wilken, Reason; Raychaudhuri, Siba; Ruhaak, L. Renee; Lebrilla, Carlito B.

    2015-01-01

    Herein we will review the role of glycans in determining the functionality and specificity of various components of the immune system. Specific topics covered include: the specific glycosylation sites of IgE, IgM, IgD, IgE, IgA, and IgG; how glycans can encode “self” identity by functioning as either danger associated molecular patterns (DAMPs) or self-associated molecular patterns (SAMPs); the role of glycans as markers of protein integrity and age; how the glycocalyx can dictate the migrati...

  10. Direct glycan structure determination of intact N-linked glycopeptides by low-energy collision-induced dissociation tandem mass spectrometry and predicted spectral library searching.

    Pai, Pei-Jing; Hu, Yingwei; Lam, Henry

    2016-08-31

    Intact glycopeptide MS analysis to reveal site-specific protein glycosylation is an important frontier of proteomics. However, computational tools for analyzing MS/MS spectra of intact glycopeptides are still limited and not well-integrated into existing workflows. In this work, a new computational tool which combines the spectral library building/searching tool, SpectraST (Lam et al. Nat. Methods2008, 5, 873-875), and the glycopeptide fragmentation prediction tool, MassAnalyzer (Zhang et al. Anal. Chem.2010, 82, 10194-10202) for intact glycopeptide analysis has been developed. Specifically, this tool enables the determination of the glycan structure directly from low-energy collision-induced dissociation (CID) spectra of intact glycopeptides. Given a list of possible glycopeptide sequences as input, a sample-specific spectral library of MassAnalyzer-predicted spectra is built using SpectraST. Glycan identification from CID spectra is achieved by spectral library searching against this library, in which both m/z and intensity information of the possible fragmentation ions are taken into consideration for improved accuracy. We validated our method using a standard glycoprotein, human transferrin, and evaluated its potential to be used in site-specific glycosylation profiling of glycoprotein datasets from LC-MS/MS. In addition, we further applied our method to reveal, for the first time, the site-specific N-glycosylation profile of recombinant human acetylcholinesterase expressed in HEK293 cells. For maximum usability, SpectraST is developed as part of the Trans-Proteomic Pipeline (TPP), a freely available and open-source software suite for MS data analysis. PMID:27506355

  11. Improve accuracy and sensibility in glycan structure prediction by matching glycan isotope abundance

    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

  12. Improve accuracy and sensibility in glycan structure prediction by matching glycan isotope abundance

    Xu Guang [College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan (China); National Research Council Canada, Ottawa, Ont., K1A 0R6 (Canada); Liu Xin [College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan (China); Liu Qingyan [National Research Council Canada, Ottawa, Ont., Canada K1A 0R6 (Canada); Zhou Yanhong, E-mail: yhzhou@mail.hust.edu.cn [College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan (China); Li Jianjun, E-mail: Jianjun.Li@nrc-cnrc.gc.ca [National Research Council Canada, Ottawa, Ont., Canada K1A 0R6 (Canada)

    2012-09-19

    Highlights: Black-Right-Pointing-Pointer A glycan isotope pattern recognition strategy for glycomics. Black-Right-Pointing-Pointer A new data preprocessing procedure to detect ion peaks in a giving MS spectrum. Black-Right-Pointing-Pointer 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

  13. The non glycanated endocan polypeptide slows tumor growth by inducing stromal inflammatory reaction

    Yassine, Hanane; De Freitas Caires, Nathalie; Depontieu, Florence; Scherpereel, Arnaud; Awad, Ali,; Tsicopoulos, Anne; Leboeuf, Christophe; Janin, Anne; Duez, Catherine; Grigoriu, Bogdan,; Lassalle, Philippe

    2014-01-01

    Endocan expression is increasingly studied in various human cancers. Experimental evidence showed that human endocan, through its glycan chain, is implicated in various processes of tumor growth. We functionally characterize mouse endocan which is also a chondroitin sulfate proteoglycan but much less glycanated than human endocan. Distant domains from the O-glycanation site, located within exons 1 and 2 determine the glycanation pattern of endocan. In opposite to the human homologue, overexpr...

  14. Sulfated glycans in inflammation.

    Pomin, Vitor H

    2015-03-01

    Sulfated glycans such as glycosaminoglycans on proteoglycans are key players in both molecular and cellular events of inflammation. They participate in leukocyte rolling along the endothelial surface of inflamed sites; chemokine regulation and its consequential functions in leukocyte guidance, migration and activation; leukocyte transendothelial migration; and structural assembly of the subendothelial basement membrane responsible to control tissue entry of cells. Due to these and other functions, exogenous sulfated glycans of various structures and origins can be used to interventionally down-regulate inflammation processes. In this review article, discussion is given primarily on the anti-inflammatory functions of mammalian heparins, heparan sulfate, chondroitin sulfate, dermatan sulfate and related compounds as well as the holothurian fucosylated chondroitin sulfate and the brown algal fucoidans. Understanding the underlying mechanisms of action of these sulfated glycans in inflammation, helps research programs involved in developing new carbohydrate-based drugs aimed to combat acute and chronic inflammatory disorders. PMID:25576741

  15. Salmonella Degrades the Host Glycocalyx Leading to Altered Infection and Glycan Remodeling.

    Arabyan, Narine; Park, Dayoung; Foutouhi, Soraya; Weis, Allison M; Huang, Bihua C; Williams, Cynthia C; Desai, Prerak; Shah, Jigna; Jeannotte, Richard; Kong, Nguyet; Lebrilla, Carlito B; Weimer, Bart C

    2016-01-01

    Complex glycans cover the gut epithelial surface to protect the cell from the environment. Invasive pathogens must breach the glycan layer before initiating infection. While glycan degradation is crucial for infection, this process is inadequately understood. Salmonella contains 47 glycosyl hydrolases (GHs) that may degrade the glycan. We hypothesized that keystone genes from the entire GH complement of Salmonella are required to degrade glycans to change infection. This study determined that GHs recognize the terminal monosaccharides (N-acetylneuraminic acid (Neu5Ac), galactose, mannose, and fucose) and significantly (p < 0.05) alter infection. During infection, Salmonella used its two GHs sialidase nanH and amylase malS for internalization by targeting different glycan structures. The host glycans were altered during Salmonella association via the induction of N-glycan biosynthesis pathways leading to modification of host glycans by increasing fucosylation and mannose content, while decreasing sialylation. Gene expression analysis indicated that the host cell responded by regulating more than 50 genes resulting in remodeled glycans in response to Salmonella treatment. This study established the glycan structures on colonic epithelial cells, determined that Salmonella required two keystone GHs for internalization, and left remodeled host glycans as a result of infection. These data indicate that microbial GHs are undiscovered virulence factors. PMID:27389966

  16. Antigenic glycans in parasitic infections: implications for vaccines and diagnostics.

    Nyame, A Kwame; Kawar, Ziad S; Cummings, Richard D

    2004-06-15

    Infections by parasitic protozoans and helminths are a major world-wide health concern, but no vaccines exist to the major human parasitic diseases, such as malaria, African trypanosomiasis, amebiasis, leishmaniasis, schistosomiasis, and lymphatic filariasis. Recent studies on a number of parasites indicate that immune responses to parasites in infected animals and humans are directed to glycan determinants within cell surface and secreted glycoconjugates and that glycoconjugates are important in host-parasite interactions. Because of the tremendous success achieved recently in generating carbohydrate-protein conjugate vaccines toward microbial infections, such as Haemophilus influenzae type b, there is renewed interest in defining parasite-derived glycans in the prospect of developing conjugate vaccines and new diagnostics for parasitic infections. Parasite-derived glycans are compelling vaccine targets because they have structural features that distinguish them from mammalian glycans. There have been exciting new developments in techniques for glycan analysis and the methods for synthesizing oligosaccharides by chemical or combined chemo-enzymatic approaches that now make it feasible to generate parasite glycans to test as vaccine candidates. Here, we highlight recent progress made in elucidating the immunogenicity of glycans from some of the major human and animal parasites, the potential for developing conjugate vaccines for parasitic infections, and the possible utilization of these novel glycans in diagnostics. PMID:15158669

  17. CROSSWORK for Glycans: Glycan Identificatin Through Mass Spectrometry and Bioinformatics

    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...

  18. Structure and function of the N-linked glycans of HBP/CAP37/azurocidin

    Iversen, L F; Kastrup, Jette Sandholm Jensen; Bjørn, S E;

    1999-01-01

    structure rather than the glycan itself. The biological in vitro activity assay data show that ng-HBP, contrary to glycosylated HBP, mediates only a very limited stimulation of the lipopolysaccharide induced cytokine release from human monocytes. In animal models of fecal peritonitis, glycosylated HBP......The three N-glycosylation sites of human heparin binding protein (HBP) have been mutated to produce a nonglycosylated HBP (ng-HBP) mutant. ng-HBP has been crystallized and tested for biological activity. Complete X-ray data have been collected to 2.1 A resolution, and the structure has been fully......), and only minor local structural differences are observed. Also, the overall stability of the protein seems to be unaffected by glycosylation, as judged by the B-factors derived from the two X-ray structures. The flexibility of a glycan site may be determined by the local polypeptide sequence and...

  19. Quantitation of Permethylated N-Glycans through Multiple-Reaction Monitoring (MRM) LC-MS/MS

    Zhou, Shiyue; Hu, Yunli; DeSantos-Garcia, Janie L.; Mechref, Yehia

    2015-04-01

    The important biological roles of glycans and their implications in disease development and progression have created a demand for the development of sensitive quantitative glycomics methods. Quantitation of glycans existing at low abundance is still analytically challenging. In this study, an N-linked glycans quantitation method using multiple-reaction monitoring (MRM) on a triple quadrupole instrument was developed. Optimum normalized collision energy (CE) for both sialylated and fucosylated N-glycan was determined to be 30%, whereas it was found to be 35% for either fucosylated or sialylated N-glycans. The optimum CE for mannose and complex type N-glycan was determined to be 35%. Additionally, the use of three transitions was shown to facilitate reliable quantitation. A total of 88 N-glycan compositions in human blood serum were quantified using this MRM approach. Reliable detection and quantitation of these glycans was achieved when the equivalence of 0.005 μL of blood serum was analyzed. Accordingly, N-glycans down to the 100th of a μL level can be reliably quantified in pooled human blood serum, spanning a dynamic concentration range of three orders of magnitude. MRM was also effectively utilized to quantitatively compare the expression of N-glycans derived from brain-targeting breast carcinoma cells (MDA-MB-231BR) and metastatic breast cancer cells (MDA-MB-231). Thus, the described MRM method of permethylated N-glycan enables a rapid and reliable identification and quantitation of glycans derived from glycoproteins purified or present in complex biological samples.

  20. Classical galactosaemia: novel insights in IgG N-glycosylation and N-glycan biosynthesis.

    Maratha, Ashwini; Stockmann, Henning; Coss, Karen P; Estela Rubio-Gozalbo, M; Knerr, Ina; Fitzgibbon, Maria; McVeigh, Terri P; Foley, Patricia; Moss, Catherine; Colhoun, Hugh-Owen; van Erven, Britt; Stephens, Kelly; Doran, Peter; Rudd, Pauline; Treacy, Eileen

    2016-07-01

    Classical galactosaemia (OMIM #230400), a rare disorder of carbohydrate metabolism, is caused by a deficient activity of galactose-1-phosphate uridyltransferase (EC 2.7.7.12). The pathophysiology of the long-term complications, mainly cognitive, neurological and female fertility problems remains poorly understood. The lack of validated biomarkers to determine prognosis, monitor disease progression and responses to new therapies, pose a huge challenge. We report the detailed analysis of an automated robotic hydrophilic interaction ultra-performance liquid chromatography N-glycan analytical method of high glycan peak resolution applied to serum IgG. This has revealed specific N-glycan processing defects observed in 40 adult galactosaemia patients (adults and adolescents), in comparison with 81 matched healthy controls. We have identified a significant increase in core fucosylated neutral glycans (Pperipheral blood mononuclear cells from 32 adult galactosaemia patients. We have noted significant dysregulation of two key N-glycan biosynthesis genes: ALG9 upregulated (P<0.001) and MGAT1 downregulated (P<0.01) in galactosaemia patients, which may contribute to its ongoing pathophysiology. Our data suggest that the use of IgG N-glycosylation analysis with matched N-glycan biosynthesis gene profiles may provide useful biomarkers for monitoring response to therapy and interventions. They also indicate potential gene modifying steps in this N-glycan biosynthesis pathway, of relevance to galactosaemia and related N-glycan biosynthesis disorders. PMID:26733289

  1. Comprehensive analysis of the N-glycan biosynthetic pathway using bioinformatics to generate UniCorn: A theoretical N-glycan structure database.

    Akune, Yukie; Lin, Chi-Hung; Abrahams, Jodie L; Zhang, Jingyu; Packer, Nicolle H; Aoki-Kinoshita, Kiyoko F; Campbell, Matthew P

    2016-08-01

    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. PMID:27318307

  2. Exquisite specificity of mitogenic lectin from Cephalosporium curvulum to core fucosylated N-glycans.

    Inamdar, Shashikala R; Eligar, Sachin M; Ballal, Suhas; Belur, Shivakumar; Kalraiya, Rajiv D; Swamy, Bale M

    2016-02-01

    Lectins are carbohydrate binding proteins that are gaining attention as important tools for the identification of specific glycan markers expressed during different stages of the cancer. We earlier reported the purification of a mitogenic lectin from human pathogenic fungus Cephalosporium curvulum (CSL) that has complex sugar specificity when analysed by hapten inhibition assay. In the present study, we report the fine sugar specificity of CSL as determined by glycan array analysis. The results revealed that CSL has exquisite specificity towards core fucosylated N-glycans. Fucosylated trimannosyl core is the basic structure required for the binding of CSL. The presence of fucose in the side chain further enhances the avidity of CSL towards such glycans. The affinity of CSL is drastically reduced towards the non-core fucosylated glycans, in spite of their side chain fucosylation. CSL showed no binding to the tested O-glycans and monosaccharides. These observations suggest the unique specificity of CSL towards core fucosylated N-glycans, which was further validated by binding of CSL to human colon cancer epithelial and hepatocarcinoma cell lines namely HT29 and HepG2, respectively, that are known to express core fucosylated N-glycans, using AOL and LCA as positive controls. LCA and AOL are fucose specific lectins that are currently being used clinically for the diagnosis of hepatocellular carcinomas. Most of the gastrointestinal markers express core fucosylated N-glycans. The high affinity and exclusive specificity of CSL towards α1-6 linkage of core fucosylated glycans compared to other fucose specific lectins, makes it a promising molecule that needs to be further explored for its application in the diagnosis of gastrointestinal cancer. PMID:26514868

  3. Effect of high mannose glycan pairing on IgG antibody clearance.

    Liu, Yaoqing Diana; Flynn, Gregory C

    2016-05-01

    IgG antibodies contain N-linked glycans on the Fc portion of each heavy chain. The glycan on one heavy chain can either match the glycan on the other heavy chain (symmetrical pairing) or be different (asymmetrical pairing). These Fc glycans influence effector functions and can alter clearance rates. Previous studies showing that high mannose forms result in faster mAb clearance in humans were incapable of differentiating the impact of symmetrically vs. asymmetrically paired HM forms, and, therefore, the effect of pairing on clearance was not clear. Traditional analytical methods, which are used to measure glycans in such studies, do not determine the number of HM glycans per antibody. With a sensitive method designed to measure HM pairing, we followed the levels of symmetrically and asymmetrically paired HM on antibodies in human pharmacokinetic serum samples to determine the impact of Fc HM glycan pairing on therapeutic human IgG clearance in humans. The two HM paired forms cleared at the same rate, indicating that the effect on clearance was not proportional to the degree of modification. Since both forms can exist on therapeutic antibodies and the ratio can differ between products, measuring their relative levels is necessary to properly estimate effects on clearance. PMID:26992607

  4. Glycan Engineering for Cell and Developmental Biology

    Griffin, Matthew E.; Hsieh-Wilson, Linda C.

    2016-01-01

    Cell-surface glycans are a diverse class of macromolecules that participate in many key biological processes, including cell-cell communication, development, and disease progression. Thus, the ability to modulate the structures of glycans on cell surfaces provides a powerful means not only to understand fundamental processes but also to direct activity and elicit desired cellular responses. Here, we describe methods to sculpt glycans on cell surfaces and highlight recent successes in which artificially engineered glycans have been employed to control biological outcomes such as the immune response and stem cell fate. PMID:26933739

  5. Sialylated glycans as receptor and inhibitor of enterovirus 71 infection to DLD-1 intestinal cells

    Yang Kuender D

    2009-09-01

    Full Text Available Abstract Background Many viruses recognize specific sugar residues, particularly sulfated or sialylated glycans, as the infection receptors. A change of sialic acid (2-6-linked galactose (SA-α2,6Gal to SA-α2,3Gal determines the receptor for avian flu infection. The receptor for enterovirus 71 (EV71 infection that frequently causes fatal encephalitis in Asian children remains unclear. Currently, there is no effective vaccine or anti-virus agent for EV71 infection. Using DLD-1 intestinal cells, this study investigated whether SA-linked glycan on DLD-1 intestinal cells was a receptor for EV71, and whether natural SA-linked sugars from human milk could block EV71 infection. Results EV71 specifically infected DLD-1 intestinal cells but not K562 myeloid cells. Depletion of O-linked glycans or glycolipids, but not N-linked glycans, significantly decreased EV71 infection of DLD-1 cells. Pretreatment of DLD-1 cells with sialidase (10 mU, 2 hours significantly reduced 20-fold EV71 replication (p Conclusion This is the first in the literature to demonstrate that EV71 uses SA-linked glycans as receptors for infection, and natural SA-linked glycans from human milk can protect intestinal cells from EV71 infection. Further studies will test how a SA-containing glycan can prevent EV71 in the future.

  6. Characterization of Changes in Serum Anti-Glycan Antibodies in Crohn's Disease – a Longitudinal Analysis

    Rieder, Florian; Lopez, Rocio; Franke, Andre; Wolf, Alexandra; Schleder, Stephan; Dirmeier, Andrea; Schirbel, Anja; Rosenstiel, Philip; Dotan, Nir; Schreiber, Stefan; Rogler, Gerhard; Klebl, Frank

    2011-01-01

    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)...

  7. Improved method for drawing of a glycan map, and the first page of glycan atlas, which is a compilation of glycan maps for a whole organism.

    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.

  8. 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,...

  9. Characterization of N-Glycan Structures on the Surface of Mature Dengue 2 Virus Derived from Insect Cells.

    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.

  10. Computational glycoscience: characterizing the spatial and temporal properties of glycans and glycan-protein complexes.

    Woods, Robert J; Tessier, Matthew B

    2010-10-01

    Modern computational methods offer the tools to provide insight into the structural and dynamic properties of carbohydrate-protein complexes, beyond that provided by experimental structural biology. Dynamic properties such as the fluctuation of inter-molecular hydrogen bonds, the residency times of bound water molecules, side chain motions and ligand flexibility may be readily determined computationally. When taken with respect to the unliganded states, these calculations can also provide insight into the entropic and enthalpic changes in free energy associated with glycan binding. In addition, virtual ligand screening may be employed to predict the three dimensional (3D) structures of carbohydrate-protein complexes, given 3D structures for the components. In principle, the 3D structure of the protein may itself be derived by modeling, leading to the exciting--albeit high risk--realm of virtual structure prediction. This latter approach is appealing, given the difficulties associated with generating experimental 3D structures for some classes of glycan binding proteins; however, it is also the least robust. An unexpected outcome of the development of algorithms for modeling carbohydrate-protein interactions has been the discovery of errors in reported experimental 3D structures and a heightened awareness of the need for carbohydrate-specific computational tools for assisting in the refinement and curation of carbohydrate-containing crystal structures. Here we present a summary of the basic strategies associated with employing classical force field based modeling approaches to problems in glycoscience, with a focus on identifying typical pitfalls and limitations. This is not an exhaustive review of the current literature, but hopefully will provide a guide for the glycoscientist interested in modeling carbohydrates and carbohydrate-protein complexes, as well as the computational chemist contemplating such tasks. PMID:20708922

  11. Notable Aspects of Glycan-Protein Interactions

    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.

  12. Glycan ID 2web_01 [Glycoconjugate Data Bank

    Full Text Available last update: 2011/10/26 Glycan information Glycan ID 2web_01 Description O-glycan 1...mer Structure Sugar residue information Residue ID 2web_01_01 Identifier MAN 324 IUPAC nomenclature a-D-mann...opyranose Common name a-D-mannose Anomeric form a Chair conformation 4C1 PDB information (2web) Requests and comments: Taku Nakahara, the author. Glycan ID 2web_01 ...

  13. Glycan ID 2web_02 [Glycoconjugate Data Bank

    Full Text Available last update: 2011/10/26 Glycan information Glycan ID 2web_02 Description O-glycan 1...mer Structure Sugar residue information Residue ID 2web_02_01 Identifier MAN 325 IUPAC nomenclature a-D-mann...opyranose Common name a-D-mannose Anomeric form a Chair conformation 4C1 PDB information (2web) Requests and comments: Taku Nakahara, the author. Glycan ID 2web_02 ...

  14. Glycobiology simplified: diverse roles of glycan recognition in inflammation.

    Schnaar, Ronald L

    2016-06-01

    Glycans and complementary glycan-binding proteins are essential components in the language of cell-cell interactions in immunity. The study of glycan function is the purview of glycobiology, which has often been presented as an unusually complex discipline. In fact, the human glycome, composed of all of its glycans, is built primarily from only 9 building blocks that are combined by enzymes (writers) with specific and limited biosynthetic capabilities into a tractable and increasingly accessible number of potential glycan patterns that are functionally read by several dozen human glycan-binding proteins (readers). Nowhere is the importance of glycan recognition better understood than in infection and immunity, and knowledge in this area has already led to glycan mimetic anti-infective and anti-inflammatory drugs. This review includes a brief tutorial on human glycobiology and a limited number of specific examples of glycan-binding protein-glycan interactions that initiate and regulate inflammation. Examples include representatives from different glycan-binding protein families, including the C-type lectins (E-selectin, P-selectin, dectin-1, and dectin-2), sialic acid-binding immunoglobulin-like lectins (sialic acid-binding immunoglobulin-like lectins 8 and 9), galectins (galectin-1, galectin-3, and galectin-9), as well as hyaluronic acid-binding proteins. As glycoscience technologies advance, opportunities for enhanced understanding of glycans and their roles in leukocyte cell biology provide increasing opportunities for discovery and therapeutic intervention. PMID:27004978

  15. Cell surface glycan alterations in epithelial mesenchymal transition process of Huh7 hepatocellular carcinoma cell.

    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

  16. Kinetic characterization of a novel endo-β-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans.

    Karav, Sercan; Parc, Annabelle Le; de Moura Bell, Juliana Maria Leite Nobrega; Rouquié, Camille; Mills, David A; Barile, Daniela; Block, David E

    2015-09-01

    EndoBI-1 is a recently isolated endo-β-N-acetylglucosaminidase, which cleaves the N-N'-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commercial use, we determined kinetic parameters for EndoBI-1 on the model substrates ribonuclease B and bovine lactoferrin, as well as on concentrated bovine colostrum whey. Km values ranging from 0.25 to 0.49, 0.43 to 1.00 and 0.90 to 3.18 mg/mL and Vmax values ranging from 3.5×10(-3) to 5.09×10(-3), 4.5×10(-3) to 7.75×10(-3) and 1.9×10(-2)to 5.2×10(-2) mg/mL×min were determined for ribonuclease B, lactoferrin and whey, respectively. In general, EndoBI-1 showed the highest apparent affinity for ribonuclease B, while the maximum reaction rate was the highest for concentrated whey. EndoBI-1-released N-glycans were quantified by a phenol-sulphuric total carbohydrate assay and the resultant N-glycan structures monitored by nano-LC-Chip-Q-TOF MS. The kinetic parameters and structural characterization of glycans released suggest EndoBI-1 can facilitate large-scale release of complex, bioactive glycans from a variety of glycoprotein substrates. Moreover, these results suggest that whey, often considered as a waste product, can be used effectively as a source of prebiotic N-glycans. PMID:26138399

  17. Characterizing the release of bioactive N-glycans from dairy products by a novel endo-β-N-acetylglucosaminidase.

    Karav, Sercan; Bell, Juliana Maria Leite Nobrega De Moura; Le Parc, Annabelle; Liu, Yan; Mills, David A; Block, David E; Barile, Daniela

    2015-01-01

    Endo-β-N-acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI-1) is a novel enzyme that cleaves N-N'-diacetyl chitobiose moieties found in the N-glycan core of high mannose, hybrid, and complex N-glycans. These conjugated N-glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifidobacterium longum subsp. Infantis. The effects of pH (4.45-8.45), temperature (27.5-77.5°C), reaction time (15-475 min), and enzyme/protein ratio (1:3,000-1:333) were evaluated on the release of N-glycans from bovine colostrum whey by EndoBI-1. A central composite design was used, including a two-level factorial design (2(4)) with four center points and eight axial points. In general, low pH values, longer reaction times, higher enzyme/protein ratio, and temperatures around 52°C resulted in the highest yield. The results demonstrated that bovine colostrum whey, considered to be a by/waste product, can be used as a glycan source with a yield of 20 mg N-glycan/g total protein under optimal conditions for the ranges investigated. Importantly, these processing conditions are suitable to be incorporated into routine dairy processing activities, opening the door for an entirely new class of products (released bioactive glycans and glycan-free milk). The new enzyme's activity was also compared with a commercially available enzyme, showing that EndoBI-1 is more active on native proteins than PNGase F and can be efficiently used during pasteurization, streamlining its integration into existing processing strategies. PMID:26097235

  18. Specific glycosylation of membrane proteins in epithelial ovarian cancer cell lines: glycan structures reflect gene expression and DNA methylation status.

    Anugraham, Merrina; Jacob, Francis; Nixdorf, Sheri; Everest-Dass, Arun Vijay; Heinzelmann-Schwarz, Viola; Packer, Nicolle H

    2014-09-01

    Epithelial ovarian cancer is the fifth most common cause of cancer in women worldwide bearing the highest mortality rate among all gynecological cancers. Cell membrane glycans mediate various cellular processes such as cell signaling and become altered during carcinogenesis. The extent to which glycosylation changes are influenced by aberrant regulation of gene expression is nearly unknown for ovarian cancer and remains crucial in understanding the development and progression of this disease. To address this effect, we analyzed the membrane glycosylation of non-cancerous ovarian surface epithelial (HOSE 6.3 and HOSE 17.1) and serous ovarian cancer cell lines (SKOV 3, IGROV1, A2780, and OVCAR 3), the most common histotype among epithelial ovarian cancers. N-glycans were released from membrane glycoproteins by PNGase F and analyzed using nano-liquid chromatography on porous graphitized carbon and negative-ion electrospray ionization mass spectrometry (ESI-MS). Glycan structures were characterized based on their molecular masses and tandem MS fragmentation patterns. We identified characteristic glycan features that were unique to the ovarian cancer membrane proteins, namely the "bisecting N-acetyl-glucosamine" type N-glycans, increased levels of α 2-6 sialylated N-glycans and "N,N'-diacetyl-lactosamine" type N-glycans. These N-glycan changes were verified by examining gene transcript levels of the enzymes specific for their synthesis (MGAT3, ST6GAL1, and B4GALNT3) using qRT-PCR. We further evaluated the potential epigenetic influence on MGAT3 expression by treating the cell lines with 5-azacytidine, a DNA methylation inhibitor. For the first time, we provide evidence that MGAT3 expression may be epigenetically regulated by DNA hypomethylation, leading to the synthesis of the unique "bisecting GlcNAc" type N-glycans on the membrane proteins of ovarian cancer cells. Linking the observation of specific N-glycan substructures and their complex association with epigenetic

  19. Sulfated di-, tri- and tetraantennary N-glycans in human Tamm-Horsfall glycoprotein

    Vliegenthart, J.F.G.; Rooijen, J.J.M. van; Kamerling, J.P.

    1998-01-01

    The primary structures of 32 sulfated di-, tri- and tetraantennary N-glycans of human Tamm-Horsfall glycoprotein (THP) have been determined. THP was isolated from the urine of one healthy male donor. The intact carbohydrate chains were released by PNGase-F and fractionated via FPLC on Resource Q, HP

  20. Rational Design of a New Trypanosoma rangeli Trans-Sialidase for Efficient Sialylation of Glycans

    Jers, Carsten; Michalak, Malwina; Larsen, Dorte Møller;

    2014-01-01

    . rangeli sialidase mutant, Tr13. Conditions for efficient trans-sialylation were determined, and Tr13’s acceptor specificity demonstrated promiscuity with respect to the acceptor molecule enabling sialylation of glycans containing terminal galactose and glucose and even monomers of glucose and fucose...

  1. Differential anti-glycan antibody responses in Schistosoma mansoni-infected children and adults studied by shotgun glycan microarray.

    Angela van Diepen

    Full Text Available BACKGROUND: Schistosomiasis (bilharzia is a chronic and potentially deadly parasitic disease that affects millions of people in (subtropical areas. An important partial immunity to Schistosoma infections does develop in disease endemic areas, but this takes many years of exposure and maturation of the immune system. Therefore, children are far more susceptible to re-infection after treatment than older children and adults. This age-dependent immunity or susceptibility to re-infection has been shown to be associated with specific antibody and T cell responses. Many antibodies generated during Schistosoma infection are directed against the numerous glycans expressed by Schistosoma. The nature of glycan epitopes recognized by antibodies in natural schistosomiasis infection serum is largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: The binding of serum antibodies to glycans can be analyzed efficiently and quantitatively using glycan microarray approaches. Very small amounts of a large number of glycans are presented on a solid surface allowing binding properties of various glycan binding proteins to be tested. We have generated a so-called shotgun glycan microarray containing natural N-glycan and lipid-glycan fractions derived from 4 different life stages of S. mansoni and applied this array to the analysis of IgG and IgM antibodies in sera from children and adults living in an endemic area. This resulted in the identification of differential glycan recognition profiles characteristic for the two different age groups, possibly reflecting differences in age or differences in length of exposure or infection. CONCLUSIONS/SIGNIFICANCE: Using the shotgun glycan microarray approach to study antibody response profiles against schistosome-derived glycan elements, we have defined groups of infected individuals as well as glycan element clusters to which antibody responses are directed in S. mansoni infections. These findings are significant for further

  2. A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins

    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.

  3. A Panel of Recombinant Mucins Carrying a Repertoire of Sialylated O-Glycans Based on Different Core Chains for Studies of Glycan Binding Proteins.

    Cherian, Reeja Maria; Jin, Chunsheng; Liu, Jining; Karlsson, Niclas G; Holgersson, Jan

    2015-01-01

    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. PMID:26274979

  4. Mass Spectrometric Screening of Ovarian Cancer with Serum Glycans

    Jae-Han Kim

    2014-01-01

    Full Text Available Changes of glycosylation pattern in serum proteins have been linked to various diseases including cancer, suggesting possible development of novel biomarkers based on the glycomic analysis. In this study, N-linked glycans from human serum were quantitatively profiled by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF mass spectrometry (MS and compared between healthy controls and ovarian cancer patients. A training set consisting of 40 healthy controls and 40 ovarian cancer cases demonstrated an inverse correlation between P value of ANOVA and area under the curve (AUC of each candidate biomarker peak from MALDI-TOF MS, providing standards for the classification. A multibiomarker panel composed of 15 MALDI-TOF MS peaks resulted in AUC of 0.89, 80~90% sensitivity, and 70~83% specificity in the training set. The performance of the biomarker panel was validated in a separate blind test set composed of 23 healthy controls and 37 ovarian cancer patients, leading to 81~84% sensitivity and 83% specificity with cut-off values determined by the training set. Sensitivity of CA-125, the most widely used ovarian cancer marker, was 74% in the training set and 78% in the test set, respectively. These results indicate that MALDI-TOF MS-mediated serum N-glycan analysis could provide critical information for the screening of ovarian cancer.

  5. IBD-associated TL1A gene (TNFSF15 haplotypes determine increased expression of TL1A protein.

    Kathrin S Michelsen

    Full Text Available BACKGROUND: The recently identified member of the TNF superfamily TL1A (TNFSF15 increases IFN-gamma production by T cells in peripheral and mucosal CCR9+ T cells. TL1A and its receptor DR3 are up-regulated during chronic intestinal inflammation in ulcerative colitis and Crohn's disease (CD. TL1A gene haplotypes increase CD susceptibility in Japanese, European, and US cohorts. METHODOLOGY AND PRINCIPAL FINDINGS: Here we report that the presence of TL1A gene haplotype B increases risk in Jewish CD patients with antibody titers for the E. coli outer membrane porin C (OmpC+ (Haplotype B frequency in Jewish CD patients: 24.9% for OmpC negative and 41.9% for OmpC positive patients, respectively, P< or =0.001. CD14+ monocytes isolated from Jewish OmpC+ patients homozygous for TL1A gene haplotype B express higher levels of TL1A in response to FcgammaR stimulation, a known inducing pathway of TL1A, as measured by ELISA. Furthermore, the membrane expression of TL1A is increased on peripheral monocytes from Jewish but not non-Jewish CD patients with the risk haplotype. CONCLUSIONS AND SIGNIFICANCE: These findings suggest that TL1A gene variation exacerbates induction of TL1A in response to FcgammaR stimulation in Jewish CD patients and this may lead to chronic intestinal inflammation via overwhelming T cell responses. Thus, TL1A may provide an important target for therapeutic intervention in this subgroup of IBD patients.

  6. Capture and characterization of influenza A virus from primary samples using glycan bead arrays.

    Cohen, Miriam; Fisher, Christopher J; Huang, Mia L; Lindsay, LeAnn L; Plancarte, Magdalena; Boyce, Walter M; Godula, Kamil; Gagneux, Pascal

    2016-06-01

    Influenza A viruses (IAVs) utilize sialylated host glycans as ligands for binding and infection. The glycan-binding preference of IAV hemagglutinin (HA) is an important determinant of host specificity. Propagation of IAV in embryonated chicken eggs and cultured mammalian cells yields viruses with amino acid substitutions in the HA that can alter the binding specificity. Therefore, it is important to determine the binding specificity of IAV directly in primary samples since it reflects the actual tropism of virus in nature. We developed a novel platform for analysis of IAV binding specificity in samples that contain very low virus titers. This platform consists of a high-density flexible glycan display on magnetic beads, which promotes multivalent interactions with the viral HA. Glycan-bound virus is detected by quantifying the viral neuraminidase activity via a fluorogenic reporter, 2'-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid. This method eliminates the need for labeling the virus and significantly enhances the sensitivity of detection. PMID:27031581

  7. Glycans pattern the phase behaviour of lipid membranes

    Subramaniam, Anand Bala; Guidotti, Guido; Manoharan, Vinothan N.; Stone, Howard A.

    2013-02-01

    Hydrated networks of glycans (polysaccharides)—in the form of cell walls, periplasms or gel-like matrices—are ubiquitously present adjacent to cellular plasma membranes. Yet, despite their abundance, the function of glycans in the extracellular milieu is largely unknown. Here we show that the spatial configuration of glycans controls the phase behaviour of multiphase model lipid membranes: inhomogeneous glycan networks stabilize large lipid domains at the characteristic length scale of the network, whereas homogeneous networks suppress macroscopic lipid phase separation. We also find that glycan-patterned phase separation is thermally reversible—thus indicating that the effect is thermodynamic rather than kinetic—and that phase patterning probably results from a preferential interaction of glycans with ordered lipid phases. These findings have implications for membrane-mediated transport processes, potentially rationalize long-standing observations that differentiate the behaviour of native and model membranes and may indicate an intimate coupling between cellular lipidomes and glycomes.

  8. Enhanced Aromatic Sequons Increase Oligosaccharyltransferase Glycosylation Efficiency and Glycan Homogeneity.

    Murray, Amber N; Chen, Wentao; Antonopoulos, Aristotelis; Hanson, Sarah R; Wiseman, R Luke; Dell, Anne; Haslam, Stuart M; Powers, David L; Powers, Evan T; Kelly, Jeffery W

    2015-08-20

    N-Glycosylation plays an important role in protein folding and function. Previous studies demonstrate that a phenylalanine residue introduced at the n-2 position relative to an Asn-Xxx-Thr/Ser N-glycosylation sequon increases the glycan occupancy of the sequon in insect cells. Here, we show that any aromatic residue at n-2 increases glycan occupancy in human cells and that this effect is dependent upon oligosaccharyltransferase substrate preferences rather than differences in other cellular processing events such as degradation or trafficking. Moreover, aromatic residues at n-2 alter glycan processing in the Golgi, producing proteins with less complex N-glycan structures. These results demonstrate that manipulating the sequence space surrounding N-glycosylation sequons is useful both for controlling glycosylation efficiency, thus enhancing glycan occupancy, and for influencing the N-glycan structures produced. PMID:26190824

  9. Regulation of intracellular signaling by extracellular glycan remodeling

    Parker, Randy B.; Kohler, Jennifer J.

    2010-01-01

    The plasma membrane of eukaryotic cells is coated with carbohydrates. By virtue of their extracellular position and recognizable chemical features, cell surface glycans mediate many receptor-ligand interactions. Recently, mammalian extracellular hydrolytic enzymes have been shown to modify the structure of cell surface glycans and consequently, alter their binding properties. These cell surface glycan remodeling events can cause rapid changes in critical signal transduction phenomena. This re...

  10. Transduction of Glycan-Lectin Binding using Near Infrared Fluorescent Single Walled Carbon Nanotubes for Glycan Profiling

    Reuel, Nigel; Ahn, Jin-Ho; Kim, Jong-Ho; Zhang, Jingqing; Boghossian, Ardemis; Mahal, Lara; Strano, Michael

    2012-02-01

    In this work, we demonstrate a sensor array employing recombinant lectins as glycan recognition sites tethered via Histidine tags to Ni2+ complexes that act as fluorescent quenchers for semi-conducting single walled carbon nanotubes embedded in a chitosan to measure binding kinetics of model glycans. Two higher-affined glycan-lectin pairs are explored: fucose (Fuc) to PA-IIL and N-acetylglucosamine (GlcNAc) to GafD. The dissociation constants (KD) for these pairs as free glycans (106 and 19 μM respectively) and streptavidin-tethered (142 and 50 μM respectively) were found. The absolute detection limit for the current platform was found to be 2 μg of glycosylated protein or 100 ng of free glycan to 20 μg of lectin. Glycan detection is demonstrated at the single nanotube level (GlcNAc to GafD). Over a population of 1000 nanotubes, 289 of the SWNT sensors had signals strong enough to yield kinetic information (KD of 250 ± 10 μM). We are also able to identify the locations of ``strong-transducers'' on the basis of dissociation constant (4 sensors with KD 5% quench response). The ability to pinpoint strong-binding, single sensors is promising to build a nanoarray of glycan-lectin transducers as a method to profile glycans without protein labeling or glycan liberation pretreatment steps.

  11. Multivalent display of minimal Clostridium difficile glycan epitopes mimics antigenic properties of larger glycans

    Broecker, Felix; Hanske, Jonas; Martin, Christopher E.; Baek, Ju Yuel; Wahlbrink, Annette; Wojcik, Felix; Hartmann, Laura; Rademacher, Christoph; Anish, Chakkumkal; Seeberger, Peter H.

    2016-01-01

    Synthetic cell-surface glycans are promising vaccine candidates against Clostridium difficile. The complexity of large, highly antigenic and immunogenic glycans is a synthetic challenge. Less complex antigens providing similar immune responses are desirable for vaccine development. Based on molecular-level glycan–antibody interaction analyses, we here demonstrate that the C. difficile surface polysaccharide-I (PS-I) can be resembled by multivalent display of minimal disaccharide epitopes on a synthetic scaffold that does not participate in binding. We show that antibody avidity as a measure of antigenicity increases by about five orders of magnitude when disaccharides are compared with constructs containing five disaccharides. The synthetic, pentavalent vaccine candidate containing a peptide T-cell epitope elicits weak but highly specific antibody responses to larger PS-I glycans in mice. This study highlights the potential of multivalently displaying small oligosaccharides to achieve antigenicity characteristic of larger glycans. The approach may result in more cost-efficient carbohydrate vaccines with reduced synthetic effort. PMID:27091615

  12. Multistep Fractionation and Mass Spectrometry Reveal Zwitterionic and Anionic Modifications of the N- and O-glycans of a Marine Snail.

    Eckmair, Barbara; Jin, Chunsheng; Abed-Navandi, Daniel; Paschinger, Katharina

    2016-02-01

    Various studies in the past have revealed that molluscs can produce a wide range of rather complex N-glycan structures, which vary from those occurring in other invertebrate animals; particularly methylated glycans have been found in gastropods, and there are some reports of anionic glycans in bivalves. Due to the high variability in terms of previously described structures and methodologies, it is a major challenge to establish glycomic workflows that yield the maximum amount of detailed structural information from relatively low quantities of sample. In this study, we apply differential release with peptide:N-glycosidases F and A followed by solid-phase extraction on graphitized carbon and reversed-phase materials to examine the glycome of Volvarina rubella (C. B. Adams, 1845), a margin snail of the clade Neogastropoda. The resulting four pools of N-glycans were fractionated on a fused core RP-HPLC column and subject to MALDI-TOF MS and MS/MS in conjunction with chemical and enzymatic treatments. In addition, selected N-glycan fractions, as well as O-glycans released by β-elimination, were analyzed by porous graphitized carbon-LC-MS and MS(n). This comprehensive approach enabled us to determine a number of novel modifications of protein-linked glycans, including N-methyl-2-aminoethylphosphonate on mannose and N-acetylhexosamine residues, core β1,3-linked mannose, zwitterionic moieties on core Galβ1,4Fuc motifs, additional mannose residues on oligomannosidic glycans, and bisubstituted antennal fucose; furthermore, typical invertebrate N-glycans with sulfate and core fucose residues are present in this gastropod. PMID:26598642

  13. Predominant Expression of Hybrid N-Glycans Has Distinct Cellular Roles Relative to Complex and Oligomannose N-Glycans

    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.

  14. Comprehensive N-Glycan Profiling of Avian Immunoglobulin Y

    Millán Martín, Silvia; Wormald, Mark R.; Zapatero-Rodríguez, Julia; Conroy, Paul J.; O’Kennedy, Richard J.; Rudd, Pauline M.; Saldova, Radka

    2016-01-01

    Recent exploitation of the avian immune system has highlighted its suitability for the generation of high-quality, high-affinity antibodies to a wide range of antigens for a number of therapeutic and biotechnological applications. The glycosylation profile of potential immunoglobulin therapeutics is species specific and is heavily influenced by the cell-line/culture conditions used for production. Hence, knowledge of the carbohydrate moieties present on immunoglobulins is essential as certain glycan structures can adversely impact their physicochemical and biological properties. This study describes the detailed N-glycan profile of IgY polyclonal antibodies from the serum of leghorn chickens using a fully quantitative high-throughput N-glycan analysis approach, based on ultra-performance liquid chromatography (UPLC) separation of released glycans. Structural assignments revealed serum IgY to contain complex bi-, tri- and tetra-antennary glycans with or without core fucose and bisects, hybrid and high mannose glycans. High sialic acid content was also observed, with the presence of rare sialic acid structures, likely polysialic acids. It is concluded that IgY is heavily decorated with complex glycans; however, no known non-human or immunogenic glycans were identified. Thus, IgY is a potentially promising candidate for immunoglobulin-based therapies for the treatment of various infectious diseases. PMID:27459092

  15. Comprehensive N-Glycan Profiling of Avian Immunoglobulin Y.

    Gilgunn, Sarah; Millán Martín, Silvia; Wormald, Mark R; Zapatero-Rodríguez, Julia; Conroy, Paul J; O'Kennedy, Richard J; Rudd, Pauline M; Saldova, Radka

    2016-01-01

    Recent exploitation of the avian immune system has highlighted its suitability for the generation of high-quality, high-affinity antibodies to a wide range of antigens for a number of therapeutic and biotechnological applications. The glycosylation profile of potential immunoglobulin therapeutics is species specific and is heavily influenced by the cell-line/culture conditions used for production. Hence, knowledge of the carbohydrate moieties present on immunoglobulins is essential as certain glycan structures can adversely impact their physicochemical and biological properties. This study describes the detailed N-glycan profile of IgY polyclonal antibodies from the serum of leghorn chickens using a fully quantitative high-throughput N-glycan analysis approach, based on ultra-performance liquid chromatography (UPLC) separation of released glycans. Structural assignments revealed serum IgY to contain complex bi-, tri- and tetra-antennary glycans with or without core fucose and bisects, hybrid and high mannose glycans. High sialic acid content was also observed, with the presence of rare sialic acid structures, likely polysialic acids. It is concluded that IgY is heavily decorated with complex glycans; however, no known non-human or immunogenic glycans were identified. Thus, IgY is a potentially promising candidate for immunoglobulin-based therapies for the treatment of various infectious diseases. PMID:27459092

  16. O-Glycan analysis of cellobiohydrolase I from Neurospora crassa.

    Tang, Shu-Lun; Bubner, Patricia; Bauer, Stefan; Somerville, Chris R

    2016-06-01

    We describe here the composition of the O-linked glycans on the Neurospora crassa cellobiohydrolase I (CBHI), which accounts for approximately 40% of the protein secreted by cells growing in the presence of cellulose. CBHI is O-glycosylated with six types of linear, and three types of branched, O-glycans containing approximately equal amounts of mannose and galactose. In addition to the classic fungal O-glycans with reducing end mannoses, we also identified reducing end galactoses which suggest the existence of a protein-O-galactosyltransferase in N. crassa Because of the excellent genetic resources available for N. crassa, the knowledge of the CBHI O-glycans may enable the future evaluation of the role of O-glycosylation on cellulase function and the development of directed O-glycan/cellulase engineering. PMID:26762173

  17. Contribution of N-linked glycans on HSV-2 gB to cell–cell fusion and viral entry

    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.

  18. Contribution of N-linked glycans on HSV-2 gB to cell–cell fusion and viral entry

    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

  19. Association analyses of large-scale glycan microarray data reveal novel host-specific substructures in influenza A virus binding glycans

    Zhao, Nan; Martin, Brigitte E.; Yang, Chun-Kai; Luo, Feng; Wan, Xiu-Feng

    2015-10-01

    Influenza A viruses can infect a wide variety of animal species and, occasionally, humans. Infection occurs through the binding formed by viral surface glycoprotein hemagglutinin and certain types of glycan receptors on host cell membranes. Studies have shown that the α2,3-linked sialic acid motif (SA2,3Gal) in avian, equine, and canine species; the α2,6-linked sialic acid motif (SA2,6Gal) in humans; and SA2,3Gal and SA2,6Gal in swine are responsible for the corresponding host tropisms. However, more detailed and refined substructures that determine host tropisms are still not clear. Thus, in this study, we applied association mining on a set of glycan microarray data for 211 influenza viruses from five host groups: humans, swine, canine, migratory waterfowl, and terrestrial birds. The results suggest that besides Neu5Acα2-6Galβ, human-origin viruses could bind glycans with Neu5Acα2-8Neu5Acα2-8Neu5Ac and Neu5Gcα2-6Galβ1-4GlcNAc substructures; Galβ and GlcNAcβ terminal substructures, without sialic acid branches, were associated with the binding of human-, swine-, and avian-origin viruses; sulfated Neu5Acα2-3 substructures were associated with the binding of human- and swine-origin viruses. Finally, through three-dimensional structure characterization, we revealed that the role of glycan chain shapes is more important than that of torsion angles or of overall structural similarities in virus host tropisms.

  20. The GlycanBuilder: a fast, intuitive and flexible software tool for building and displaying glycan structures

    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

  1. Glycan characterization of biopharmaceuticals: Updates and perspectives.

    Planinc, Ana; Bones, Jonathan; Dejaegher, Bieke; Van Antwerpen, Pierre; Delporte, Cédric

    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. PMID:27126786

  2. Development of recombinant Aleuria aurantia lectins with altered binding specificities to fucosylated glycans

    Romano, Patrick R.; Mackay, Andrew; Vong, Minh; deSa, Johann; Lamontagne, Anne; Comunale, Mary Ann; Hafner, Julie; Block, Timothy; Lec, Ryszard; Mehta, Anand

    2011-01-01

    Changes in glycosylation have long been associated with disease. While there are many methods to detect changes in glycosylation, plant derived lectins are often used to determine changes on specific proteins or molecules of interest. One change in glycosylation that has been observed by us and by others is a disease or antigen associated increase in fucosylation on N-linked glycans. To measure this change, the fucose binding Aleuria aurantia lectin (AAL) is often utilized in plate and soluti...

  3. Complex N-Glycans Influence the Spatial Arrangement of Voltage Gated Potassium Channels in Membranes of Neuronal-Derived Cells.

    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

  4. Discovery and Structural Characterization of Fucosylated Oligomannosidic N-Glycans in Mushrooms

    Grass, Josephine; Pabst, Martin; Kolarich, Daniel; Pöltl, Gerald; Léonard, Renaud; Brecker, Lothar; Altmann, Friedrich

    2010-01-01

    l-Fucose is a common constituent of Asn-linked glycans in vertebrates, invertebrates, and plants, but in fungal glycoproteins, fucose has not been found so far. However, by mass spectrometry we detected N-glycans and O-glycans containing one to six deoxyhexose residues in fruit bodies of several basidiomycetes. The N-glycans of chanterelles (Cantharellus cibarius) contained a deoxyhexose chromatographically identical to fucose and sensitive to α-l-fucosidase. Analysis of individual glycan spe...

  5. Integrated Omics and Computational Glycobiology Reveal Structural Basis for Influenza A Virus Glycan Microheterogeneity and Host Interactions.

    Khatri, Kshitij; Klein, Joshua A; White, Mitchell R; Grant, Oliver C; Leymarie, Nancy; Woods, Robert J; Hartshorn, Kevan L; Zaia, Joseph

    2016-06-01

    Despite sustained biomedical research effort, influenza A virus remains an imminent threat to the world population and a major healthcare burden. The challenge in developing vaccines against influenza is the ability of the virus to mutate rapidly in response to selective immune pressure. Hemagglutinin is the predominant surface glycoprotein and the primary determinant of antigenicity, virulence and zoonotic potential. Mutations leading to changes in the number of HA glycosylation sites are often reported. Such genetic sequencing studies predict at best the disruption or creation of sequons for N-linked glycosylation; they do not reflect actual phenotypic changes in HA structure. Therefore, combined analysis of glycan micro and macro-heterogeneity and bioassays will better define the relationships among glycosylation, viral bioactivity and evolution. We present a study that integrates proteomics, glycomics and glycoproteomics of HA before and after adaptation to innate immune system pressure. We combined this information with glycan array and immune lectin binding data to correlate the phenotypic changes with biological activity. Underprocessed glycoforms predominated at the glycosylation sites found to be involved in viral evolution in response to selection pressures and interactions with innate immune-lectins. To understand the structural basis for site-specific glycan microheterogeneity at these sites, we performed structural modeling and molecular dynamics simulations. We observed that the presence of immature, high-mannose type glycans at a particular site correlated with reduced accessibility to glycan remodeling enzymes. Further, the high mannose glycans at sites implicated in immune lectin recognition were predicted to be capable of forming trimeric interactions with the immune-lectin surfactant protein-D. PMID:26984886

  6. Glycotope sharing between snail hemolymph and larval schistosomes: larval transformation products alter shared glycan patterns of plasma proteins.

    Yoshino, Timothy P; Wu, Xiao-Jun; Liu, Hongdi; Gonzalez, Laura A; Deelder, André M; Hokke, Cornelis H

    2012-01-01

    Recent evidence supports the involvement of inducible, highly diverse lectin-like recognition molecules in snail hemocyte-mediated responses to larval Schistosoma mansoni. Because host lectins likely are involved in initial parasite recognition, we sought to identify specific carbohydrate structures (glycans) shared between larval S. mansoni and its host Biomphalaria glabrata to address possible mechanisms of immune avoidance through mimicry of elements associated with the host immunoreactivity. A panel of monoclonal antibodies (mABs) to specific S. mansoni glycans was used to identify the distribution and abundance of shared glycan epitopes (glycotopes) on plasma glycoproteins from B. glabrata strains that differ in their susceptibilities to infection by S. mansoni. In addition, a major aim of this study was to determine if larval transformation products (LTPs) could bind to plasma proteins, and thereby alter the glycotopes exposed on plasma proteins in a snail strain-specific fashion. Plasma fractions ( 100 kDa) from susceptible (NMRI) and resistant (BS-90) snail strains were subjected to SDS-PAGE and immunoblot analyses using mAB to LacdiNAc (LDN), fucosylated LDN variants, Lewis X and trimannosyl core glycans. Results confirmed a high degree of glycan sharing, with NMRI plasma exhibiting a greater distribution/abundance of LDN, F-LDN and F-LDN-F than BS-90 plasma (LTPs significantly altered the reactivity of specific mABs to shared glycotopes on blots, mainly through the binding of LTPs to plasma proteins resulting in either glycotope blocking or increased glycotope attachment to plasma. Many LTP-mediated changes in shared glycans were snail-strain specific, especially those in the 100 kDa fraction. Our data suggest that differential binding of S. mansoni LTPs to plasma proteins of susceptible and resistant B. glabrata strains may significantly impact early anti-larval immune reactivity, and in turn, compatibility, in this parasite-host system. PMID:22448293

  7. Profiling IgG N-glycans as potential biomarker of chronological and biological ages

    Yu, Xinwei; Wang, Youxin; Kristic, Jasminka; Dong, Jing; Chu, Xi; Ge, Siqi; Wang, Hao; Fang, Honghong; Gao, Qing; Liu, Di; Zhao, Zhongyao; Peng, Hongli; Pucic Bakovic, Maja; Wu, Lijuan; Song, Manshu; Rudan, Igor; Campbell, Harry; Lauc, Gordan; Wang, Wei

    2016-01-01

    Abstract As an important post-translation modifying process, glycosylation significantly affects the structure and function of immunoglobulin G (IgG) molecules and is essential in many steps of the inflammatory cascade. Studies have demonstrated the potential of using glycosylation features of IgG as a component of predictive biomarkers for chronological age in several European populations, whereas no study has been reported in Chinese. Herein, we report various patterns of changes in IgG glycosylation associated with age by analyzing IgG glycosylation in 701 community-based Han Chinese (244 males, 457 females; 23–68 years old). Eleven IgG glycans, including FA2B, A2G1, FA2[6]G1, FA2[3]G1, FA2[6]BG1, FA2[3]BG1, A2G2, A2BG2, FA2G2, FA2G2S1, and FA2G2S2, change considerably with age and specific combinations of these glycan features can explain 23.3% to 45.4% of the variance in chronological age in this population. This indicates that these combinations of glycan features provide more predictive information than other single markers of biological age such as telomere length. In addition, the clinical traits such as fasting plasma glucose and aspartate aminotransferase associated with biological age are strongly correlated with the combined glycan features. We conclude that IgG glycosylation appears to correlate with both chronological and biological ages, and thus its possible role in the aging process merits further study. PMID:27428197

  8. Emerging Technologies for Making Glycan-Defined Glycoproteins

    Wang, Lai-Xi; Lomino, Joseph V.

    2011-01-01

    Protein glycosylation is a common and complex posttranslational modification of proteins, which expands functional diversity while boosting structural heterogeneity. Glycoproteins, the end products of such a modification, are typically produced as mixtures of glycoforms possessing the same polypeptide backbone but differ in the site of glycosylation and/or in the structures of pendant glycans, from which single glycoforms are difficult to isolate. The urgent need for glycan-defined glycoprote...

  9. Human DC-SIGN Binds Specific Human Milk Glycans

    Noll, Alexander J.; Yu, Ying; Lasanajak, Yi; Duska-McEwen, Geralyn; Buck, Rachael H.; Smith, David F.; Cummings, Richard D.

    2016-01-01

    Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys, and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBPs) expressed by dendritic cells (DC) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and Siglecs expressed by DC for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-SIGN showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglecs-5 and -9 showed weak binding to a few glycans. By contrast, most hGBPs bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2′-fucosyllactose (2′-FL) and 3-fucosyllactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2′-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2′-FL had an IC50 of ~1 mM for DC-SIGN, which is within the physiological concentration of 2′-FL in human milk. These results demonstrate that DC-SIGN among the many hGBPs expressed by DC binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant. PMID:26976925

  10. Neutral glycans from sandfish skin can reduce friction of polymers.

    Vihar, Boštjan; Hanisch, Franz Georg; Baumgartner, Werner

    2016-03-01

    The lizardScincus 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

  11. 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

  12. Characterization of Changes in Serum Anti-Glycan Antibodies in Crohn's Disease – a Longitudinal Analysis

    Rieder, Florian; Lopez, Rocio; Franke, Andre; Wolf, Alexandra; Schleder, Stephan; Dirmeier, Andrea; Schirbel, Anja; Rosenstiel, Philip; Dotan, Nir; Schreiber, Stefan; Rogler, Gerhard; Klebl, Frank

    2011-01-01

    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. PMID:21573154

  13. Characterization of changes in serum anti-glycan antibodies in Crohn's disease--a longitudinal analysis.

    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.

  14. Discrimination of epimeric glycans and glycopeptides using IM-MS and its potential for carbohydrate sequencing

    Both, P.; Green, A. P.; Gray, C. J.; Šardzík, R.; Voglmeir, J.; Fontana, C.; Austeri, M.; Rejzek, M.; Richardson, D.; Field, R. A.; Widmalm, G.; Flitsch, S. L.; Eyers, C. E.

    2014-01-01

    Mass spectrometry is the primary analytical technique used to characterize the complex oligosaccharides that decorate cell surfaces. Monosaccharide building blocks are often simple epimers, which when combined produce diastereomeric glycoconjugates indistinguishable by mass spectrometry. Structure elucidation frequently relies on assumptions that biosynthetic pathways are highly conserved. Here, we show that biosynthetic enzymes can display unexpected promiscuity, with human glycosyltransferase pp-α-GanT2 able to utilize both uridine diphosphate N-acetylglucosamine and uridine diphosphate N-acetylgalactosamine, leading to the synthesis of epimeric glycopeptides in vitro. Ion-mobility mass spectrometry (IM-MS) was used to separate these structures and, significantly, enabled characterization of the attached glycan based on the drift times of the monosaccharide product ions generated following collision-induced dissociation. Finally, ion-mobility mass spectrometry following fragmentation was used to determine the nature of both the reducing and non-reducing glycans of a series of epimeric disaccharides and the branched pentasaccharide Man3 glycan, demonstrating that this technique may prove useful for the sequencing of complex oligosaccharides.

  15. Multi-Site N-glycan mapping study 1: Capillary electrophoresis - laser induced fluorescence.

    Szekrényes, Ákos; Park, SungAe Suhr; Santos, Marcia; Lew, Clarence; Jones, Aled; Haxo, Ted; Kimzey, Michael; Pourkaveh, Shiva; Szabó, Zoltán; Sosic, Zoran; Feng, Peng; Váradi, Csaba; de l'Escaille, François; Falmagne, Jean-Bernard; Sejwal, Preeti; Niedringhaus, Thomas; Michels, David; Freckleton, Gordon; Hamm, Melissa; Manuilov, Anastasiya; Schwartz, Melissa; Luo, Jiann-Kae; van Dyck, Jonathan; Leung, Pui-King; Olajos, Marcell; Gu, Yingmei; Gao, Kai; Wang, Wenbo; Wegstein, Jo; Tep, Samnang; Guttman, András

    2016-01-01

    An international team that included 20 independent laboratories from biopharmaceutical companies, universities, analytical contract laboratories and national authorities in the United States, Europe and Asia was formed to evaluate the reproducibility of sample preparation and analysis of N-glycans using capillary electrophoresis of 8-aminopyrene-1,3,6-trisulfonic acid (APTS)-labeled glycans with laser induced fluorescence (CE-LIF) detection (16 sites) and ultra high-performance liquid chromatography (UHPLC, 12 sites; results to be reported in a subsequent publication). All participants used the same lot of chemicals, samples, reagents, and columns/capillaries to run their assays. Migration time, peak area and peak area percent values were determined for all peaks with >0.1% peak area. Our results demonstrated low variability and high reproducibility, both, within any given site as well across all sites, which indicates that a standard N-glycan analysis platform appropriate for general use (clone selection, process development, lot release, etc.) within the industry can be established. PMID:26466659

  16. Comprehensive Profiling of Glycosphingolipid Glycans Using a Novel Broad Specificity Endoglycoceramidase in a High-Throughput Workflow.

    Albrecht, Simone; Vainauskas, Saulius; Stöckmann, Henning; McManus, Ciara; Taron, Christopher H; Rudd, Pauline M

    2016-05-01

    The biological function of glycosphingolipids (GSLs) is largely determined by their glycan headgroup moiety. This has placed a renewed emphasis on detailed GSL headgroup structural analysis. Comprehensive profiling of GSL headgroups in biological samples requires the use of endoglycoceramidases with broad substrate specificity and a robust workflow that enables their high-throughput analysis. We present here the first high-throughput glyco-analytical platform for GSL headgroup profiling. The workflow features enzymatic release of GSL glycans with a novel broad-specificity endoglycoceramidase I (EGCase I) from Rhodococcus triatomea, selective glycan capture on hydrazide beads on a robotics platform, 2AB-fluorescent glycan labeling, and analysis by UPLC-HILIC-FLD. R. triatomea EGCase I displayed a wider specificity than known EGCases and was able to efficiently hydrolyze gangliosides, globosides, (n)Lc-type GSLs, and cerebrosides. Our workflow was validated on purified GSL standard lipids and was applied to the characterization of GSLs extracted from several mammalian cell lines and human serum. This study should facilitate the analytical workflow in functional glycomics studies and biomarker discovery. PMID:27033327

  17. Detailed glycan structural characterization by electronic excitation dissociation.

    Yu, Xiang; Jiang, Yan; Chen, Yajie; Huang, Yiqun; Costello, Catherine E; Lin, Cheng

    2013-11-01

    The structural complexity and diversity of glycans parallel their multilateral functions in living systems. To better understand the vital roles glycans play in biological processes, it is imperative to develop analytical tools that can provide detailed glycan structural information. This was conventionally achieved by multistage tandem mass spectrometry (MS(n)) analysis using collision-induced dissociation (CID) as the fragmentation method. However, the MS(n) approach lacks the sensitivity and throughput needed to analyze complex glycan mixtures from biological sources, often available in limited quantities. We define herein the critical parameters for a recently developed fragmentation technique, electronic excitation dissociation (EED), which can yield rich structurally informative fragment ions during liquid chromatographic (LC)-MS/MS analysis of glycans. We further demonstrate that permethylation, reducing end labeling and judicious selection of the metal charge carrier, can greatly facilitate spectral interpretation. With its high sensitivity, throughput, and compatibility with online chromatographic separation techniques, EED appears to hold great promise for large-scale glycomics studies. PMID:24080071

  18. Approaches toward High-Mannose-Type Glycan Libraries.

    Fujikawa, Kohki; Seko, Akira; Takeda, Yoichi; Ito, Yukishige

    2016-02-01

    Asparagine-linked (N-linked) sugar chains are widely found in the rough endoplasmic reticulum (ER), which has attracted renewed attention because of its participation in the glycoprotein quality control process. In the ER, newly formed glycoproteins are properly folded to higher-order structures by the action of a variety of lectin chaperones and processing enzymes and are transported into the Golgi, while terminally misfolded glycoproteins are carried into the cytosol for degradation. A group of proteins related to this system are known to recognize subtle differences in the high-mannose-type oligosaccharide structures of glycoproteins; however, their molecular foundations are still unclear. In order to gain a more precise understanding, our group has established a strategy for the systematic synthesis of high-mannose-type glycans. More recently, we have developed "top-down" chemoenzymatic approaches that allow expeditious access to theoretically all types of high-mannose glycans. This strategy comprehensively delivered 37 high-mannose-type glycans, including G1M9-M3 glycans, and opened up the possibility of the elucidation of structure-function relationships with a series of high-mannose-type glycans. PMID:26493153

  19. Concept, strategy and realization of lectin-based glycan profiling.

    Hirabayashi, Jun

    2008-08-01

    Lectins are a diverse group of carbohydrate-binding proteins. Each lectin has its own specificity profile. It is believed that lectins exist in all living organisms that produce glycans. From a practical viewpoint, lectins have been used extensively in biochemical fields including proteomics due to their usefulness as detection and enrichment tools for specific glycans. Nevertheless, they have often been underestimated as probes, especially compared with antibodies, because of their low affinity and broad specificity. However, together with the concept of glycomics, such properties of lectins are now considered to be suitable for the task of 'profiling' in order to cover a wider range of ligands. Recently there has been rapid movement in the field of proteomics aimed at the investigation of glycan-related biomarkers. This is partly because of limitations of the present approach of simply following changes in protein-level expression, without paying sufficient attention to the fact and effects of glycosylation. The trend is reflected in the frequent use of lectins in the contexts of glycoprotein enrichment and glycan profiling. However, there are many aspects to be considered in using lectins, which differ considerably from antibodies. In this article, the author, as a developer of two unique methodologies, frontal affinity chromatography (FAC) and the lectin microarray, describes critical points concerning the use of lectins, together with the concept, strategy and means to achieve advances in these emerging glycan profiling technologies. PMID:18390573

  20. Use of the U1A Protein to Facilitate Crystallization and Structure Determination of Large RNAs.

    Ferré-D'Amaré, Adrian R

    2016-01-01

    The preparation of well-ordered crystals of RNAs with complex three-dimensional architecture can be facilitated by engineering a binding site for the spliceosomal protein U1A into a functionally and structurally dispensable stem-loop of the RNA of interest. Once suitable crystals are obtained, the U1A protein, of known structure, can be employed to facilitate preparation of heavy atom or anomalously scattering atom derivatives, or as a source of partial model phases for the molecular replacement method. Here, we describe the methods for making U1A preparations suitable for cocrystallization with RNA. As an example, the cocrystallization of the tetracycline aptamer with U1A is also described. PMID:26227038

  1. Ultrasensitive detection of influenza viruses with a glycan-based impedimetric biosensor

    Hushegyi, András; Pihíková, Dominika; Bertók, Tomáš; Adam, Vojtech; Kizek, René; Tkac, Jan

    2015-01-01

    An ultrasensitive impedimetric glycan-based biosensor for reliable and selective detection of inactivated, but intact influenza viruses H3N2 was developed. Such glycan-based approach has a distinct advantage over antibody-based detection of influenza viruses since glycans are natural viral receptors with a possibility to selectively distinguish between potentially pathogenic influenza subtypes by the glycan-based biosensors. Build-up of the biosensor was carefully optimized with atomic force ...

  2. Glycotope sharing between snail hemolymph and larval schistosomes: larval transformation products alter shared glycan patterns of plasma proteins.

    Timothy P Yoshino

    Full Text Available Recent evidence supports the involvement of inducible, highly diverse lectin-like recognition molecules in snail hemocyte-mediated responses to larval Schistosoma mansoni. Because host lectins likely are involved in initial parasite recognition, we sought to identify specific carbohydrate structures (glycans shared between larval S. mansoni and its host Biomphalaria glabrata to address possible mechanisms of immune avoidance through mimicry of elements associated with the host immunoreactivity. A panel of monoclonal antibodies (mABs to specific S. mansoni glycans was used to identify the distribution and abundance of shared glycan epitopes (glycotopes on plasma glycoproteins from B. glabrata strains that differ in their susceptibilities to infection by S. mansoni. In addition, a major aim of this study was to determine if larval transformation products (LTPs could bind to plasma proteins, and thereby alter the glycotopes exposed on plasma proteins in a snail strain-specific fashion. Plasma fractions ( 100 kDa from susceptible (NMRI and resistant (BS-90 snail strains were subjected to SDS-PAGE and immunoblot analyses using mAB to LacdiNAc (LDN, fucosylated LDN variants, Lewis X and trimannosyl core glycans. Results confirmed a high degree of glycan sharing, with NMRI plasma exhibiting a greater distribution/abundance of LDN, F-LDN and F-LDN-F than BS-90 plasma ( 100 kDa fraction. Our data suggest that differential binding of S. mansoni LTPs to plasma proteins of susceptible and resistant B. glabrata strains may significantly impact early anti-larval immune reactivity, and in turn, compatibility, in this parasite-host system.

  3. 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

  4. Glycan bioengineering in immunogen design for tumor T antigen immunotargeting

    Sendra, Victor G; Zlocowski, Natacha; Ditamo, Yanina;

    2009-01-01

    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...... 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...... 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 for...

  5. Using CRISPR-Cas9 to quantify the contributions of O-glycans, N-glycans and Glycosphingolipids to human leukocyte-endothelium adhesion

    Stolfa, Gino; Mondal, Nandini; Zhu, Yuqi; Yu, Xinheng; Buffone, Alexander; Neelamegham, Sriram

    2016-01-01

    There is often interest in dissecting the relative contributions of the N-glycans, O-glycans and glycosphingolipids (GSLs) in regulating complex biological traits like cell signaling, adhesion, development and metastasis. To address this, we developed a CRISPR-Cas9 toolkit to selectively truncate each of these commonly expressed glycan-types. Here, O-glycan biosynthesis was truncated by knocking-out Core 1 β3Gal-T Specific Molecular Chaperone (COSMC), N-glycans by targeting the β1,2 GlcNAc-transferase (MGAT1) and GSLs by deleting UDP-glucose ceramide glucosyltransferase (UGCG). These reagents were applied to reveal the glycoconjugates regulating human myeloid cell adhesion to selectins under physiological shear-flow observed during inflammation. These functional studies show that leukocyte rolling on P- and L-selectin is ablated in cells lacking O-glycans, with N-glycan truncation also increasing cell rolling velocity on L-selectin. All three glycan families contributed to E-selectin dependent cell adhesion with N-glycans contributing to all aspects of the leukocyte adhesion cascade, O-glycans only being important during initial recruitment, and GSLs stabilizing slow cell rolling and the transition to firm arrest. Overall, the genome editing tools developed here may be broadly applied in studies of cellular glycosylation. PMID:27458028

  6. Glycan Analysis by Reversible Reaction to Hydrazide Beads and Mass Spectrometry

    Yang, Shuang J.; Zhang, Hui

    2016-01-01

    Investigation into glycoproteins and their associated glycans is the key to understanding the function of glycoproteins in biological pathways and disease development. Current methods for glycan analysis are generally based on multiple preparation processes to separate glycans from proteins and other molecules prior to analysis. During the multistep purification processes, glycans are continuously lost and the procedure increases the difficulty for accurate quantitative analysis of glycans. Here we describe the development of a novel technique, which uses hydrazide beads to capture glycans. It is based on the conjugation of glycans to hydrazide beads through the formation of reversible hydrazone, washing out unbound nonglycans, then releasing captured glycans by acids. The results showed that the glycans were able to be isolated from concatenate peptides by using hydrazide beads. This technique was also applied to the analysis of glycans from sera sample. The integrated capture-release on the solid-phase simplifies the procedure for glycan preparation from a complex mixture and can be a powerful tool for glycan analysis. PMID:22304307

  7. Glycan Node Analysis: A Bottom-up Approach to Glycomics.

    Zaare, Sahba; Aguilar, Jesús S; Hu, Yueming; Ferdosi, Shadi; Borges, Chad R

    2016-01-01

    Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that

  8. Human DC-SIGN binds specific human milk glycans.

    Noll, Alexander J; Yu, Ying; Lasanajak, Yi; Duska-McEwen, Geralyn; Buck, Rachael H; Smith, David F; Cummings, Richard D

    2016-05-15

    Human milk glycans (HMGs) are prebiotics, pathogen receptor decoys and regulators of host physiology and immune responses. Mechanistically, human lectins (glycan-binding proteins, hGBP) expressed by dendritic cells (DCs) are of major interest, as these cells directly contact HMGs. To explore such interactions, we screened many C-type lectins and sialic acid-binding immunoglobulin-like lectins (Siglecs) expressed by DCs for glycan binding on microarrays presenting over 200 HMGs. Unexpectedly, DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) showed robust binding to many HMGs, whereas other C-type lectins failed to bind, and Siglec-5 and Siglec-9 showed weak binding to a few glycans. By contrast, most hGBP bound to multiple glycans on other microarrays lacking HMGs. An α-linked fucose residue was characteristic of HMGs bound by DC-SIGN. Binding of DC-SIGN to the simple HMGs 2'-fucosyl-lactose (2'-FL) and 3-fucosyl-lactose (3-FL) was confirmed by flow cytometry to beads conjugated with 2'-FL or 3-FL, as well as the ability of the free glycans to inhibit DC-SIGN binding. 2'-FL had an IC50 of ∼1 mM for DC-SIGN, which is within the physiological concentration of 2'-FL in human milk. These results demonstrate that DC-SIGN among the many hGBP expressed by DCs binds to α-fucosylated HMGs, and suggest that such interactions may be important in influencing immune responses in the developing infant. PMID:26976925

  9. Arabidopsis thaliana KORRIGAN1 protein: N-glycan modification, localization, and function in cellulose biosynthesis and osmotic stress responses

    von Schaewen, Antje; Rips, Stephan; Jeong, In Sil; Koiwa, Hisashi

    2015-01-01

    Plant cellulose biosynthesis is a complex process involving cellulose-synthase complexes (CSCs) and various auxiliary factors essential for proper orientation and crystallinity of cellulose microfibrils in the apoplast. Among them is KORRIGAN1 (KOR1), a type-II membrane protein with multiple N-glycans within its C-terminal cellulase domain. N-glycosylation of the cellulase domain was important for KOR1 targeting to and retention within the trans-Golgi network (TGN), and prevented accumulation...

  10. Sialylated glycans as receptor and inhibitor of enterovirus 71 infection to DLD-1 intestinal cells

    Yang Kuender D; Chuang Hau; Yang Betsy

    2009-01-01

    Abstract Background Many viruses recognize specific sugar residues, particularly sulfated or sialylated glycans, as the infection receptors. A change of sialic acid (2-6)-linked galactose (SA-α2,6Gal) to SA-α2,3Gal determines the receptor for avian flu infection. The receptor for enterovirus 71 (EV71) infection that frequently causes fatal encephalitis in Asian children remains unclear. Currently, there is no effective vaccine or anti-virus agent for EV71 infection. Using DLD-1 intestinal cel...

  11. N-glycan transition of the early developmental stage in Oryza sativa.

    Horiuchi, Risa; Hirotsu, Naoki; Miyanishi, Nobumitsu

    2016-08-26

    N-Glycosylation is one of the post-translational modifications. In animals, N-glycans linked to proteins function in cell-cell recognition, sorting, transport, and other biological phenomena. However, in plants, N-glycan-mediated biological functions remain obscure. In a previous study, we showed that the main type of N-glycan transition is from the paucimannosidic to complex type before and after germination in Oryza sativa, suggesting that transitions of N-glycan, including those of glycoproteins and glycosyltransferases, are closely associated with plant growth. To further elucidate the relationship between N-glycan structure and plant growth, we analyzed the structures of N-glycans expressed in O. sativa seedlings grown under light conditions and performed comparative analyses of the structures in the shoot and root. The analyses show that fundamental N-glycan structures are common to the shoot and root, whereas paucimannosidic-type N-glycans dramatically decreased in the root grown under light conditions. Further, to investigate the effects of light on N-glycan structures in O. sativa seedlings, we analyzed N-glycan structures in O. sativa seedlings grown in the dark. Understandably, N-glycan expression in the root was almost unaffected by light. However, despite a marked difference in phenotype, N-glycan expression in the shoot was also unaffected by light. This result suggests that the shoot and root of O. sativa have different glycoproteins and distinct N-glycan synthetic systems. Thus, we propose that the N-glycan synthetic system of the O. sativa shoot is almost unaffected by light conditions and that many photosynthesis-related proteins are not modified by N-glycans. PMID:27320861

  12. Clozapine disposition covaries with CYP1A2 activity determined by a caffeine test.

    Bertilsson, L; Carrillo, J A; Dahl, M L; Llerena, A; Alm, C; Bondesson, U; Lindström, L; Rodriguez de la Rubia, I; Ramos, S; Benitez, J

    1994-01-01

    In a previous study we showed that the disposition of clozapine after a single oral dose is unrelated to either debrisoquine or S-mephenytoin hydroxylation polymorphism. The same 14 healthy subjects studied in that investigation were given 150 mg of caffeine. The reciprocal of plasma clozapine AUC (0,24), was correlated with an index of the N3-demethylation of caffeine (rs = 0.84; P = 0.0024), used as a measure of cytochrome P4501A2 (CYP1A2) activity. N1- and N7-demethylation indices of caffeine also reflect CYP1A2 activity and were also correlated with clozapine clearance (rs = 0.89 and 0.85; P = 0.0013 and 0.0023; respectively). No significant relationships with xanthine oxidase and N-acetyl transferase activity, also assessed by a caffeine test, were found. This study suggests that clozapine is metabolised by CYP1A2 to a major extent. PMID:7893591

  13. Glycan bioengineering in immunogen design for tumor T antigen immunotargeting.

    Sendra, Victor G; Zlocowski, Natacha; Ditamo, Yanina; Copioli, Silvina; Tarp, Mads P; Bennett, Eric P; Clausen, Henrik; Roth, German A; Nores, Gustavo A; Irazoqui, Fernando J

    2009-10-01

    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 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. Antibodies produced by glycan bioengineering recognize HT29, T47D, MCF7, and CT26 epithelial tumor cells. Epithelial tumor cell adhesion to T antigen-binding lectins and endothelial cells was lower in the presence of antibodies raised against the engineered immunogen. The immune response directed 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 for immunotargeting purposes. PMID:19726087

  14. A Bitter Sweet Symphony: Immune Responses to Altered O-glycan Epitopes in Cancer

    Cornelissen, Lenneke A.M.; Van Vliet, Sandra J.

    2016-01-01

    The appearance of aberrant glycans on the tumor cell surface is one of the emerging hallmarks of cancer. Glycosylation is an important post-translation modification of proteins and lipids and is strongly affected by oncogenesis. Tumor-associated glycans have been extensively characterized regarding their composition and tumor-type specific expression patterns. Nevertheless whether and how tumor-associated glycans contribute to the observed immunomodulatory actions by tumors has not been extensively studied. Here, we provide a detailed overview of the current knowledge on how tumor-associated O-glycans affect the anti-tumor immune response, thereby focusing on truncated O-glycans present on epithelial tumors and mucins. These tumor-associated O-glycans and mucins bind a variety of lectin receptors on immune cells to facilitate the subsequently induction of tolerogenic immune responses. We, therefore, postulate that tumor-associated glycans not only support tumor growth, but also actively contribute to immune evasion. PMID:27153100

  15. Structural heterogeneity of terminal glycans in Campylobacter jejuni lipooligosaccharides.

    Semchenko, Evgeny A; Day, Christopher J; Moutin, Marc; Wilson, Jennifer C; Tiralongo, Joe; Korolik, Victoria

    2012-01-01

    Lipooligosaccharides of the gastrointestinal pathogen Campylobacter jejuni are regarded as a major virulence factor and are implicated in the production of cross-reactive antibodies against host gangliosides, which leads to the development of autoimmune neuropathies such as Guillain-Barré and Fisher Syndromes. C. jejuni strains are known to produce diverse LOS structures encoded by more than 19 types of LOS biosynthesis clusters. This study demonstrates that the final C. jejuni LOS structure cannot always be predicted from the genetic composition of the LOS biosynthesis cluster, as determined by novel lectin array analysis of the terminal LOS glycans. The differences were shown to be partially facilitated by the differential on/off status of three genes wlaN, cst and cj1144-45. The on/off status of these genes was also analysed in C. jejuni strains grown in vitro and in vivo, isolated directly from the host animal without passaging, using immunoseparation. Importantly, C. jejuni strains 331, 421 and 520 encoding cluster type C were shown to produce different LOS, mimicking asialo GM(1), asialo GM(2) and a heterogeneous mix of gangliosides and other glycoconjugates respectively. In addition, individual C. jejuni colonies were shown to consistently produce heterogeneous LOS structures, irrespective of the cluster type and the status of phase variable genes. Furthermore we describe C. jejuni strains (351 and 375) with LOS clusters that do not match any of the previously described LOS clusters, yet are able to produce LOS with asialo GM(2)-like mimicries. The LOS biosynthesis clusters of these strains are likely to contain genes that code for LOS biosynthesis machinery previously not identified, yet capable of synthesising LOS mimicking gangliosides. PMID:22815868

  16. N-Glycan profile analysis of transferrin using a microfluidic compact disc and MALDI-MS.

    Quaranta, Alessandro; Sroka-Bartnicka, Anna; Tengstrand, Erik; Thorsén, Gunnar

    2016-07-01

    It has been known for a long time that diseases can be associated with changes to the glycosylation of specific proteins. This has been shown for cancer, immunological disorders, and neurodegenerative diseases. The possibility of using the glycosylation patterns of proteins as biomarkers for disease would be a great asset for clinical research or diagnosis. There is at present a lack of rapid, automated, and cost-efficient analytical techniques for the determination of the glycosylation of specific serum proteins. We have developed a method for determining the glycosylation pattern of proteins based on the affinity capture of a specific serum protein, the enzymatic release of the N-linked glycans, and the analysis of the glycan pattern using MALDI-MS. All sample preparation is performed in a disposable centrifugal microfluidic disc. The sample preparation is miniaturized, requiring only 1 μL of sample per determination, and automated with the possibility of processing 54 samples in parallel in 3.5 h. We have developed a method for the glycosylation pattern analysis of transferrin. The method has been tested on serum samples from chronic alcohol abusers and a control group. Also, a SIMCA model was created and evaluated to discriminate between the two groups. PMID:27137515

  17. Determination of site-specific glycan heterogeneity on glycoproteins

    Kolarich, Daniel; Jensen, Pia Hønnerup; Altmann, Friedrich;

    2012-01-01

    site-specific heterogeneity, showing examples of the analysis of recombinant human erythropoietin (rHuEPO), α1-proteinase inhibitor (A1PI) and immunoglobulin (IgG). Glycoproteins of interest can be proteolytically digested either in solution or in-gel after electrophoretic separation, and the (glyco......The comprehensive analysis of protein glycosylation is a major requirement for understanding glycoprotein function in biological systems, and is a prerequisite for producing recombinant glycoprotein therapeutics. This protocol describes workflows for the characterization of glycopeptides and their...

  18. Glycoengineered Monoclonal Antibodies with Homogeneous Glycan (M3, G0, G2, and A2 Using a Chemoenzymatic Approach Have Different Affinities for FcγRIIIa and Variable Antibody-Dependent Cellular Cytotoxicity Activities.

    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

  19. Evidence for PPC1, a determinant of the pilei-pellis color of Agaricus bisporus fruitbodies.

    Callac, P; Moquet, F; Imbernon, M; Guedes-Lafargue, M R; Mamoun, M; Olivier, J M

    1998-03-01

    In the present study, we investigated the genetic basis of mushroom cap color. In first generation hybrids between a brown isolate and the white commercial hybrid U 1, the white trait was recessive. Color was determined using color meter technology in second generation hybrids obtained by crossing the homokaryotic progeny of a first generation hybrid with a homokaryon from U 1. Statistical analysis revealed a bimodal distribution describing two classes of white and not-white hybrids. We postulate that a recessive allele at a single locus (PPC1) encodes the white pilei-pellis color. Joint segregation analyses indicated that PPC1 was linked to the ADH (alcohol dehydrogenase) locus. Through the analysis of the heterokaryotic progeny of the first generation hybrid, a recombination model is proposed in which PPC1 is located between the centromere and the ADH locus. PMID:9578631

  20. A distinct sodium channel voltage-sensor locus determines insect selectivity of the spider toxin Dc1a

    Bende, Niraj S.; Dziemborowicz, Sławomir; Mobli, Mehdi; Herzig, Volker; Gilchrist, John; Wagner, Jordan; Nicholson, Graham M.; King, Glenn F.; Bosmans, Frank

    2014-07-01

    β-Diguetoxin-Dc1a (Dc1a) is a toxin from the desert bush spider Diguetia canities that incapacitates insects at concentrations that are non-toxic to mammals. Dc1a promotes opening of German cockroach voltage-gated sodium (Nav) channels (BgNav1), whereas human Nav channels are insensitive. Here, by transplanting commonly targeted S3b-S4 paddle motifs within BgNav1 voltage sensors into Kv2.1, we find that Dc1a interacts with the domain II voltage sensor. In contrast, Dc1a has little effect on sodium currents mediated by PaNav1 channels from the American cockroach even though their domain II paddle motifs are identical. When exploring regions responsible for PaNav1 resistance to Dc1a, we identified two residues within the BgNav1 domain II S1-S2 loop that when mutated to their PaNav1 counterparts drastically reduce toxin susceptibility. Overall, our results reveal a distinct region within insect Nav channels that helps determine Dc1a sensitivity, a concept that will be valuable for the design of insect-selective insecticides.

  1. Property Graph vs RDF Triple Store: A Comparison on Glycan Substructure Search.

    Davide Alocci

    Full Text Available Resource description framework (RDF and Property Graph databases are emerging technologies that are used for storing graph-structured data. We compare these technologies through a molecular biology use case: glycan substructure search. Glycans are branched tree-like molecules composed of building blocks linked together by chemical bonds. The molecular structure of a glycan can be encoded into a direct acyclic graph where each node represents a building block and each edge serves as a chemical linkage between two building blocks. In this context, Graph databases are possible software solutions for storing glycan structures and Graph query languages, such as SPARQL and Cypher, can be used to perform a substructure search. Glycan substructure searching is an important feature for querying structure and experimental glycan databases and retrieving biologically meaningful data. This applies for example to identifying a region of the glycan recognised by a glycan binding protein (GBP. In this study, 19,404 glycan structures were selected from GlycomeDB (www.glycome-db.org and modelled for being stored into a RDF triple store and a Property Graph. We then performed two different sets of searches and compared the query response times and the results from both technologies to assess performance and accuracy. The two implementations produced the same results, but interestingly we noted a difference in the query response times. Qualitative measures such as portability were also used to define further criteria for choosing the technology adapted to solving glycan substructure search and other comparable issues.

  2. Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein

    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.

  3. Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein

    Behrens, Anna-Janina; Vasiljevic, Snezana; Pritchard, Laura K.; Harvey, David J.; Andev, Rajinder S.; Krumm, Stefanie A.; Struwe, Weston B.; Cupo, Albert; Kumar, Abhinav; Zitzmann, Nicole; Seabright, Gemma E.; Kramer, Holger B.; Spencer, Daniel I.R.; Royle, Louise; Lee, Jeong Hyun; Klasse, Per J.; Burton, Dennis R.; Wilson, Ian A.; Ward, Andrew B.; Sanders, Rogier W.; Moore, John P.; Doores, Katie J.; Crispin, Max

    2016-01-01

    Summary 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. PMID:26972002

  4. Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies.

    Angelakis, Emmanouil; Bachar, Dipankar; Henrissat, Bernard; Armougom, Fabrice; Audoly, Gilles; Lagier, Jean-Christophe; Robert, Catherine; Raoult, Didier

    2016-01-01

    Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum level, species richness (Chao index, 227 to 2,714) and diversity (non parametric Shannon, 1.37 to 4.4). Moreover DNA was extracted from stools obtained from 83 different individuals by the fastest extraction assay and by an extraction assay that degradated exopolysaccharides. The fastest extraction method was able to detect 68% to 100% genera and 42% to 95% species whereas the glycan degradation extraction method was able to detect 56% to 93% genera and 25% to 87% species. To allow a good liberation of DNA from exopolysaccharides commonly presented in stools, we recommend the mechanical lysis of stools plus glycan degradation, used here for the first time. Caution must be taken in the interpretation of current metagenomic studies, as the efficiency of DNA extraction varies widely among stool samples. PMID:27188959

  5. Sweet complementarity: the functional pairing of glycans with lectins.

    Gabius, H-J; Manning, J C; Kopitz, J; André, S; Kaltner, H

    2016-05-01

    Carbohydrates establish the third alphabet of life. As part of cellular glycoconjugates, the glycans generate a multitude of signals in a minimum of space. The presence of distinct glycotopes and the glycome diversity are mapped by sugar receptors (antibodies and lectins). Endogenous (tissue) lectins can read the sugar-encoded information and translate it into functional aspects of cell sociology. Illustrated by instructive examples, each glycan has its own ligand properties. Lectins with different folds can converge to target the same epitope, while intrafamily diversification enables functional cooperation and antagonism. The emerging evidence for the concept of a network calls for a detailed fingerprinting. Due to the high degree of plasticity and dynamics of the display of genes for lectins the validity of extrapolations between different organisms of the phylogenetic tree yet is inevitably limited. PMID:26956894

  6. Glycan Side Reaction May Compromise ETD-Based Glycopeptide Identification

    Darula, Zsuzsanna; Medzihradszky, Katalin F.

    2014-06-01

    Tris(hydroxymethyl)aminomethane (Tris) is one of the most frequently used buffer ingredients. Among other things, it is recommended and is usually used for lectin-based affinity enrichment of glycopeptides. Here we report that sialic acid, a common `capping' unit in both N- and O-linked glycans may react with this chemical, and this side reaction may compromise glycopeptide identification when ETD spectra are the only MS/MS data used in the database search. We show that the modification may alter N- as well as O-linked glycans, the Tris-derivative is still prone to fragmentation both in `beam-type' CID (HCD) and ETD experiments, at the same time—since the acidic carboxyl group was `neutralized'—it will display a different retention time than its unmodified counterpart. We also suggest solutions that—when incorporated into existing search engines—may significantly improve the reliability of glycopeptide assignments.

  7. Neutrophil mobilization by surface-glycan altered Th17-skewing bacteria mitigates periodontal pathogen persistence and associated alveolar bone loss.

    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.

  8. Marine Non-Glycosaminoglycan Sulfated Glycans as Potential Pharmaceuticals

    Vitor H. Pomin

    2015-01-01

    Sulfated fucans (SFs) and sulfated galactans (SGs) are currently the marine non-glycosaminoglycan (GAG) sulfated glycans most studied in glycomics. These compounds exhibit therapeutic effects in several pathophysiological systems such as blood coagulation, thrombosis, neovascularization, cancer, inflammation, and microbial infections. As analogs of the largely employed GAGs and due to some limitations of the GAG-based therapies, SFs and SGs comprise new carbohydrate-based therapeutics availab...

  9. Green algae Chlamydomonas reinhardtii possess endogenous sialylated N-glycans

    Mamedov, Tarlan; Yusibov, Vidadi

    2011-01-01

    Green algae have a great potential as biofactories for the production of proteins. Chlamydomonas reinhardtii, a representative of eukaryotic microalgae, has been extensively used as a model organism to study light-induced gene expression, chloroplast biogenesis, photosynthesis, light perception, cell–cell recognition, and cell cycle control. However, little is known about the glycosylation machinery and N-linked glycan structures of green algae. In this study, we performed mass spectrometry a...

  10. Biosynthesis and Function of Extracellular Glycans in Cyanobacteria

    Jan-Christoph Kehr; Elke Dittmann

    2015-01-01

    The cell surface of cyanobacteria is covered with glycans that confer versatility and adaptability to a multitude of environmental factors. The complex carbohydrates act as barriers against different types of stress and play a role in intra- as well as inter-species interactions. In this review, we summarize the current knowledge of the chemical composition, biosynthesis and biological function of exo- and lipo-polysaccharides from cyanobacteria and give an overview of sugar-binding lectins c...

  11. DNA display of glycoconjugates to emulate oligomeric interactions of glycans.

    Novoa, Alexandre; Winssinger, Nicolas

    2015-01-01

    Glycans (carbohydrate portion of glycoproteins and glycolipids) frequently exert their function through oligomeric interactions involving multiple carbohydrate units. In efforts to recapitulate the diverse spatial arrangements of the carbohydrate units, assemblies based on hybridization of nucleic acid conjugates have been used to display simplified ligands with tailored interligand distances and valences. The programmability of the assemblies lends itself to a combinatorial display of multiple ligands. Recent efforts in the synthesis and applications of such conjugates are discussed. PMID:26113879

  12. Direct visualization of specifically modified extracellular glycans in living animals

    Attreed, Matthew; Desbois, Muriel; van Kuppevelt, Toin H.; Bülow, Hannes E.

    2012-01-01

    Modification patterns of the extracellular glycan heparan sulfate coordinate protein function in metazoans, yet in vivo imaging of such non-genetically encoded structures has been impossible. Here we report a transgenic method in Caenorhabditis elegans that allows direct live imaging of specific heparan sulfate modification patterns. This experimental approach reveals a dynamic and cell-specific heparan sulfate landscape and could in principle be adapted to visualize and analyze any extracell...

  13. Glycan Arrays: From Basic Biochemical Research to Bioanalytical and Biomedical Applications.

    Geissner, Andreas; Seeberger, Peter H

    2016-06-12

    A major branch of glycobiology and glycan-focused biomedicine studies the interaction between carbohydrates and other biopolymers, most importantly, glycan-binding proteins. Today, this research into glycan-biopolymer interaction is unthinkable without glycan arrays, tools that enable high-throughput analysis of carbohydrate interaction partners. Glycan arrays offer many applications in basic biochemical research, for example, defining the specificity of glycosyltransferases and lectins such as immune receptors. Biomedical applications include the characterization and surveillance of influenza strains, identification of biomarkers for cancer and infection, and profiling of immune responses to vaccines. Here, we review major applications of glycan arrays both in basic and applied research. Given the dynamic nature of this rapidly developing field, we focus on recent findings. PMID:27306309

  14. Targeting host-derived glycans on enveloped viruses for antibody-based vaccine design.

    Crispin, Max; Doores, Katie J

    2015-04-01

    The surface of enveloped viruses can be extensively glycosylated. Unlike the glycans coating pathogens such as bacteria and fungi, glycans on viruses are added and processed by the host-cell during biosynthesis. Glycoproteins are typically subjected to α-mannosidase processing and Golgi-mediated glycosyltransferase extension to form complex-type glycans. In envelope viruses, exceptions to this default pathway are common and lead to the presence of oligomannose-type glycan structures on the virion surface. In one extreme example, HIV-1 utilises a high density of glycans to limit host antibody recognition of protein. However, the high density limits glycan processing and the resulting oligomannose structures can be recognised by broadly neutralising antibodies isolated from HIV-1 infected patients. Here we discuss how divergence from host-cell glycosylation can be targeted for vaccine design. PMID:25747313

  15. Glycan Arrays: From Basic Biochemical Research to Bioanalytical and Biomedical Applications

    Geissner, Andreas; Seeberger, Peter H.

    2016-06-01

    A major branch of glycobiology and glycan-focused biomedicine studies the interaction between carbohydrates and other biopolymers, most importantly, glycan-binding proteins. Today, this research into glycan-biopolymer interaction is unthinkable without glycan arrays, tools that enable high-throughput analysis of carbohydrate interaction partners. Glycan arrays offer many applications in basic biochemical research, for example, defining the specificity of glycosyltransferases and lectins such as immune receptors. Biomedical applications include the characterization and surveillance of influenza strains, identification of biomarkers for cancer and infection, and profiling of immune responses to vaccines. Here, we review major applications of glycan arrays both in basic and applied research. Given the dynamic nature of this rapidly developing field, we focus on recent findings.

  16. 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...

  17. Density Variant Glycan Microarray for Evaluating Cross-Linking of Mucin-like Glycoconjugates by Lectins

    Godula, Kamil; Bertozzi, Carolyn R.

    2012-01-01

    Interactions of mucin glycoproteins with cognate receptors are dictated by the structures and spatial organization of glycans that decorate the mucin polypeptide backbone. The glycan-binding proteins, or lectins, that interact with mucins are often oligomeric receptors with multiple ligand binding domains. In this work, we employed a microarray platform comprising synthetic glycopolymers that emulate natural mucins arrayed at different surface densities to evaluate how glycan valency and spat...

  18. In good company: association between fungal glycans generates molecular complexes with unique functions

    MarcioRodrigues

    2012-01-01

    The biological properties of fungal immunogens have historically utilized testing of isolated molecules. Recent findings, however, indicate that fungal glycans differing in structure and function can interact to form hybrid complexes with unique properties. In the pathogenic yeast Cryptococcus neoformans, chitin-like molecules associate with capsular glucuronoxylomannan to form functionally distinct glycan complexes. Such interactions between glycans that result in the formation of structures...

  19. In good company: association between fungal glycans generates molecular complexes with unique functions

    Rodrigues, Marcio L.; Nimrichter, Leonardo

    2012-01-01

    The biological properties of fungal immunogens have historically utilized testing of isolated molecules. Recent findings, however, indicate that fungal glycans differing in structure and function can interact to form hybrid complexes with unique properties. In the pathogenic yeast Cryptococcus neoformans, chitin-like molecules associate with capsular glucuronoxylomannan (GXM) to form functionally distinct glycan complexes. Such interactions between glycans that result in the formation of stru...

  20. Current Protocols in Chemical Biology Construction and Use of Glycan Microarrays

    Campbell, Christopher T.; Zhang, Yalong; Gildersleeve, Jeffrey C.

    2010-01-01

    Glycosylation is an important post-translational modification that influences many biological processes critical for development, normal physiologic function, and diseases. Unfortunately, progress towards understanding the roles of glycans in biology has been slow due to the challenges of studying glycans and the proteins that interact with them. Glycan microarrays provide a high-throughput approach for the rapid analysis of carbohydrate-macromolecule interactions. Protocols detailed here are...

  1. A Bitter Sweet Symphony: Immune Responses to Altered O-glycan Epitopes in Cancer

    Lenneke A.M. Cornelissen; Van Vliet, Sandra J.

    2016-01-01

    The appearance of aberrant glycans on the tumor cell surface is one of the emerging hallmarks of cancer. Glycosylation is an important post-translation modification of proteins and lipids and is strongly affected by oncogenesis. Tumor-associated glycans have been extensively characterized regarding their composition and tumor-type specific expression patterns. Nevertheless whether and how tumor-associated glycans contribute to the observed immunomodulatory actions by tumors has not been exten...

  2. Circulating Biomphalaria glabrata hemocyte subpopulations possess shared schistosome glycans and receptors capable of binding larval glycoconjugates

    YOSHINO, TIMOTHY P.; Wu, Xiao-Jun; Gonzalez, Laura A.; Cornelis H Hokke

    2012-01-01

    Host lectin-like recognition molecules may play an important role in innate resistance in Biomphalaria glabrata snails to larval schistosome infection, thus implicating parasite-expressed glycans as putative ligands for these lectin receptors. While host lectins may utilize specific glycan structures for parasite recognition, it also has been hypothesized that the parasite may use this system to evade immune detection by mimicking naturally-expressed host glycans, resulting in reduced immunor...

  3. Comparative analysis of N-glycans in the ungerminated and germinated stages of Oryza sativa.

    Horiuchi, Risa; Hirotsu, Naoki; Miyanishi, Nobumitsu

    2015-12-11

    All fundamental information such as signal transduction, metabolic control, infection, cell-to-cell signaling, and cell differentiation related to the growth of plants are preserved in germs. In preserving these information, glycans have a key role and are involved in the development and differentiation of organisms. Glycans which exist in rice germ are expected to have an important role in germination. In this study, we performed structural and correlation analysis of the N-glycans in rice germ before and after germination. Our results confirmed that the N-glycans in the ungerminated stage of the rice germ had low number of N-glycans consisting only of six kinds especially with high-mannose and paucimannose type N-glycans being 16.0% and 76.7%, respectively. On the other hand, after 48 hours germinated germ stage, there was an increase in the complex type N-glycans with the appearance of Lewis a structure, the most complex type and a decrease in paucimannose types. These results suggest that at least six kinds of N-glycans are utilized for long time preservation of rice seed, while the diversification of most complex types of N-glycans is produced an environment dependent for shoot formation of rice. PMID:26513758

  4. High-temperature LC-MS/MS of permethylated glycans derived from glycoproteins.

    Zhou, Shiyue; Hu, Yunli; Mechref, Yehia

    2016-06-01

    Various glycomic analysis methods have been developed due to the essential roles of glycans in biological processes as well as the potential application of glycomics in biomarker discovery in many diseases. Permethylation is currently considered to be one of the most common derivatization methods in MS-based glycomic analysis. Permethylation not only improves ionization efficiency and stability of sialylated glycans in positive mode but also allows for enhanced separation performance on reversed-phase liquid chromatography (RPLC). Recently, RPLC-MS analysis of permethylated glycans exhibited excellent performance in sensitivity and reproducibility and became a widely-applied comprehensive strategy in glycomics. However, separating permethylated glycans by RPLC always suffers from peak broadening for high-molecular-weight branched glycans, which probably due to the low exchange rate between the stationary phase and mobile phase limited by intermolecular interactions of the methyl groups associated with the branching of the glycan structures. In this study, we employed high separation temperature conditions for RPLC of permethylated glycans, thus achieving enhanced peak capacity, improving peak shape, and enhancing separation efficiency. Additionally, partial isomeric separation were observed in RPLC of permethylated glycans at high-temperature. Mathematical processing of the correlation between retention time and molecular weight also revealed the advantage of high-temperature LC method for both manual and automatic glycan identification. PMID:26914157

  5. Determination of U (1 )A restoration from pion and a0 -meson screening masses: Toward the chiral regime

    Ishii, Masahiro; Yonemura, Koji; Takahashi, Junichi; Kouno, Hiroaki; Yahiro, Masanobu

    2016-01-01

    We incorporate the effective restoration of U (1 )A symmetry in the 2 +1 -flavor entanglement Polyakov-loop extended Nambu-Jona-Lasinio (EPNJL) model by introducing a temperature-dependent strength K (T ) to the Kobayashi-Maskawa-'t Hooft determinant interaction. T dependence of K (T ) is well determined from pion and a0-meson screening masses obtained by lattice QCD (LQCD) simulations with improved p4 staggered fermions. The strength is strongly suppressed in the vicinity of the pseudocritical temperature of chiral transition. The EPNJL model with the K (T ) well reproduces meson susceptibilities calculated by LQCD with domain-wall fermions. The model shows that the chiral transition is second order at the "light-quark chiral-limit" point where the light quark mass is zero and the strange quark mass is fixed at the physical value. This indicates that there exists a tricritical point. Hence, the location is estimated.

  6. Global Identification and Differential Distribution Analysis of Glycans in Subcellular Fractions of Bladder Cells

    Yang, Ganglong; Huang, Luyu; Zhang, Jiaxu; Yu, Hanjie; Li, Zheng; Guan, Feng

    2016-01-01

    Compartmentalization of cellular components and their associated biological processes is crucial for cellular function. Protein glycosylation provides a basis for diversity of protein functions. Diversity of glycan composition in animal cells remains poorly understood. We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade nonmuscle invasive bladder cancer KK47 cells and normal bladder epithelia HCV29 cells: microsomal (Mic), mitochondrial (Mito), nuclear (Nuc), and cytosolic (Cyto). An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins LCA, AAL, MPL, WGA and PWM in YTS1 cell, STL, Jacalin, VVA, LCA and WGA in KK47, and ConA, GNA, VVA and ACA in HCV29 cell. Among a total of 40, 32 and 15 N-glycans in four fractions of three cells detected by MS analysis, high-mannose and fucosylated structures were predominant, 10 N-glycans in YTS1, 5 N-glycans in KK47 and 7 N-glycans in HCV29 were present in all four fractions; and 10 N-glycans in YTS1, 16 N-glycans in KK47, and 3 N-glycans in HCV29 were present in only one fraction. Glycans in the latter category are considered potential markers for the corresponding organelles. The integrated strategy described here allows detailed examination of glycomes subcellular fraction with high resolution and sensitivity, and will be useful for elucidation of the functional roles of glycans and corresponding glycosylated proteins in distinct organelles. PMID:27313494

  7. Global Identification and Differential Distribution Analysis of Glycans in Subcellular Fractions of Bladder Cells.

    Yang, Ganglong; Huang, Luyu; Zhang, Jiaxu; Yu, Hanjie; Li, Zheng; Guan, Feng

    2016-01-01

    Compartmentalization of cellular components and their associated biological processes is crucial for cellular function. Protein glycosylation provides a basis for diversity of protein functions. Diversity of glycan composition in animal cells remains poorly understood. We used differential centrifugation techniques to isolate four subcellular protein fractions from homogenate of metastatic bladder YTS1 cells, low grade nonmuscle invasive bladder cancer KK47 cells and normal bladder epithelia HCV29 cells: microsomal (Mic), mitochondrial (Mito), nuclear (Nuc), and cytosolic (Cyto). An integrated strategy combining lectin microarray and mass spectrometry (MS) analysis was then applied to evaluate protein glycosylation of the four fractions. Lectin microarray analysis revealed significant differences among the four fractions in terms of glycan binding to the lectins LCA, AAL, MPL, WGA and PWM in YTS1 cell, STL, Jacalin, VVA, LCA and WGA in KK47, and ConA, GNA, VVA and ACA in HCV29 cell. Among a total of 40, 32 and 15 N-glycans in four fractions of three cells detected by MS analysis, high-mannose and fucosylated structures were predominant, 10 N-glycans in YTS1, 5 N-glycans in KK47 and 7 N-glycans in HCV29 were present in all four fractions; and 10 N-glycans in YTS1, 16 N-glycans in KK47, and 3 N-glycans in HCV29 were present in only one fraction. Glycans in the latter category are considered potential markers for the corresponding organelles. The integrated strategy described here allows detailed examination of glycomes subcellular fraction with high resolution and sensitivity, and will be useful for elucidation of the functional roles of glycans and corresponding glycosylated proteins in distinct organelles. PMID:27313494

  8. Pre-embedding Method of Electron Microscopy for Glycan Localization in Mammalian Tissues and Cells Using Lectin Probes.

    Akimoto, Yoshihiro; Takata, Kuniaki; Kawakami, Hayato

    2016-01-01

    In recent years, the study of glycans is progressing remarkably by the development of glycan analysis systems using mass spectrometry, glycan profiling systems using lectin microarrays, and glycoprotein analysis by the isotope-coded glycosylation site-specific tagging method. With these methodologies, glycan structures and biological functions are being elucidated. In the study of glycan function as well as disease diagnosis, it is important to examine the localization of glycans in tissues and cells. Histochemical methods using lectin probes can localize glycans in the tissues and cells. This chapter describes a pre-embedding electron microscopic method for glycan localization in which tissue sections and cells are incubated with lectin prior to embedding in resin. PMID:27515086

  9. A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

    R. Pejchal; K.J. Doores; L.M. Walker; R. Khayat; P.S. Huang; S.K. Wang; R.L. Stanfield; J.P. Julien; A. Ramos; M. Crispin; R. Depetris; U. Katpally; A. Marozsan; A. Cupo; S. Maloveste; Y. Liu; R. McBride; Y. Ito; R.W. Sanders; C. Ogohara; J.C. Paulson; T. Feizi; C.N. Scanlan; C.H. Wong; J.P. Moore; W.C. Olson; A.B. Ward; P. Poignard; W.R. Schief; D.R. Burton; I.A. Wilson

    2011-01-01

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 a

  10. Malonic acid suppresses mucin-type O-glycan degradation during hydrazine treatment of glycoproteins.

    Goso, Yukinobu

    2016-03-01

    Hydrazine treatment is frequently used for releasing mucin-type O-glycans (O-glycans) from glycoproteins because the method provides O-glycans that retain a reducible GalNAc at their reducing end, which is available for fluorescent labeling. However, many O-glycans are degraded by "peeling" during this treatment. In the current study, it was found that malonic acid suppressed O-glycan degradation during hydrazine treatment of bovine fetuin or porcine gastric mucin in both the gas and liquid phases. This is paradoxical because the release of O-glycans from glycoproteins occurs under alkaline conditions. However, malonic acid seems to prevent the degradation through its acidic property given that other weak acids also prevented the degradation. Accordingly, disodium malonate did not suppress O-glycan degradation. Application of this method to rat gastric mucin demonstrated that the majority of the major O-glycans obtained in the presence of malonic acid were intact, whereas those obtained in the absence of malonic acid were degraded. These results suggest that hydrazine treatment in the presence of malonic acid would allow glycomic analysis of native mucin glycoproteins. PMID:26723492

  11. Glycoengineering of Chinese hamster ovary cells for enhanced erythropoietin N-glycan branching and sialylation

    Yin, Bojiao; Gao, Yuan; Chung, Cheng-yu;

    2015-01-01

    glycans on EPO 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 (Gn...

  12. Role of Glycans in Cancer Cells Undergoing Epithelial–Mesenchymal Transition

    Li, Xiang; Wang, Xin; Tan, Zengqi; Chen, Si; Guan, Feng

    2016-01-01

    The term “cancer” refers to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body. Epithelial–mesenchymal transition (EMT), a process whereby epithelial cells lose their cell polarity and cell–cell adhesion ability, and acquire migratory and invasive properties to gain mesenchymal phenotype, is an important step leading to tumor metastasis. Glycans, such as N-glycans, O-glycans, and glycosphingolipids, are involved in numerous biological processes, including inflammation, virus/bacteria–host interactions, cell–cell interactions, morphogenesis, and cancer development and progression. Aberrant expression of glycans has been observed in several EMT models, and the functional roles of such glycans in cancer development and progression has been investigated. We summarize here recent research progress regarding the functions of glycans in cancer cells undergoing EMT. Better understanding of the mechanisms underlying aberrant glycan patterns in EMT and cancer will facilitate the development of such glycans as cancer biomarkers or as targets in design and synthesis of anti-tumor drugs. PMID:26925388

  13. Ultrasensitive detection of influenza viruses with a glycan-based impedimetric biosensor.

    Hushegyi, András; Pihíková, Dominika; Bertok, Tomas; Adam, Vojtech; Kizek, René; Tkac, Jan

    2016-05-15

    An ultrasensitive impedimetric glycan-based biosensor for reliable and selective detection of inactivated, but intact influenza viruses H3N2 was developed. Such glycan-based approach has a distinct advantage over antibody-based detection of influenza viruses since glycans are natural viral receptors with a possibility to selectively distinguish between potentially pathogenic influenza subtypes by the glycan-based biosensors. Build-up of the biosensor was carefully optimized with atomic force microscopy applied for visualization of the biosensor surface after binding of viruses with the topology of an individual viral particle H3N2 analyzed. The glycan biosensor could detect a glycan binding lectin with a limit of detection (LOD) of 5aM. The biosensor was finally applied for analysis of influenza viruses H3N2 with LOD of 13 viral particles in 1μl, what is the lowest LOD for analysis of influenza viral particles by the glycan-based device achieved so far. The biosensor could detect H3N2 viruses selectively with a sensitivity ratio of 30 over influenza viruses H7N7. The impedimetric biosensor presented here is the most sensitive glycan-based device for detection of influenza viruses and among the most sensitive antibody or aptamer based biosensor devices. PMID:26765527

  14. A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

    Pejchal, Robert; Doores, Katie J.; Walker, Laura M.; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L.; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W.; Ogohara, Cassandra; Paulson, James C.; Feizi, Ten; Scanlan, Christopher N.; Wong, Chi-Huey; Moore, John P.; Olson, William C.; Ward, Andrew B.; Poignard, Pascal; Schief, William R.; Burton, Dennis R.; Wilson, Ian A. (UWASH); (Progenics); (ICL); (Weill-Med); (NIH); (JSTA); (Scripps); (Oxford)

    2015-10-15

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man{sub 9} at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short {beta}-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

  15. Structural analysis of N- and O-glycans released from glycoproteins

    Jensen, Pia Hønnerup; Karlsson, Niclas G; Kolarich, Daniel; Packer, Nicolle H

    2012-01-01

    4. The time for data interpretation depends on the complexity of the samples analyzed. This method can be used in conjunction with the analysis of enriched glycopeptides by capillary/nanoLC-ESI-MS/MS, which together provide detailed information regarding the site heterogeneity of glycosylation.......This protocol shows how to obtain a detailed glycan compositional and structural profile from purified glycoproteins or protein mixtures, and it can be used to distinguish different isobaric glycan isomers. Glycoproteins are immobilized on PVDF membranes before the N-glycans are enzymatically...... released by PNGase F, isolated and reduced. Subsequently, O-glycans are chemically released from the same protein spot by reductive β-elimination. After desalting with cation exchange microcolumns, the glycans are separated and analyzed by porous graphitized carbon liquid chromatography...

  16. Specific N-glycans of Hepatocellular Carcinoma Cell Surface and the Abnormal Increase of Core-α-1, 6-fucosylated Triantennary Glycan via N-acetylglucosaminyltransferases-IVa Regulation

    Huan Nie; Xia Liu; Yubao Zhang; Tingting Li; Chao Zhan; Wenjuan Huo; Anshun He; Yuanfei Yao; Yu Jin; Youpeng Qu; Xue-Long Sun; Yu Li

    2015-01-01

    Glycosylation alterations of cell surface proteins are often observed during the progression of malignancies. The specific cell surface N-glycans were profiled in hepatocellular carcinoma (HCC) with clinical tissues (88 tumor and adjacent normal tissues) and the corresponding serum samples of HCC patients. The level of core-α-1,6-fucosylated triantennary glycan (NA3Fb) increased both on the cell surface and in the serum samples of HCC patients (p 

  17. The multiple roles of epidermal growth factor repeat O-glycans in animal development.

    Haltom, Amanda R; Jafar-Nejad, Hamed

    2015-10-01

    The epidermal growth factor (EGF)-like repeat is a common, evolutionarily conserved motif found in secreted proteins and the extracellular domain of transmembrane proteins. EGF repeats harbor six cysteine residues which form three disulfide bonds and help generate the three-dimensional structure of the EGF repeat. A subset of EGF repeats harbor consensus sequences for the addition of one or more specific O-glycans, which are initiated by O-glucose, O-fucose or O-N-acetylglucosamine. These glycans are relatively rare compared to mucin-type O-glycans. However, genetic experiments in model organisms and cell-based assays indicate that at least some of the glycosyltransferases involved in the addition of O-glycans to EGF repeats play important roles in animal development. These studies, combined with state-of-the-art biochemical and structural biology experiments have started to provide an in-depth picture of how these glycans regulate the function of the proteins to which they are linked. In this review, we will discuss the biological roles assigned to EGF repeat O-glycans and the corresponding glycosyltransferases. Since Notch receptors are the best studied proteins with biologically-relevant O-glycans on EGF repeats, a significant part of this review is devoted to the role of these glycans in the regulation of the Notch signaling pathway. We also discuss recently identified proteins other than Notch which depend on EGF repeat glycans to function properly. Several glycosyltransferases involved in the addition or elongation of O-glycans on EGF repeats are mutated in human diseases. Therefore, mechanistic understanding of the functional roles of these carbohydrate modifications is of interest from both basic science and translational perspectives. PMID:26175457

  18. A simple chromatographic method for determining norfloxacin and enoxacin in pharmacokinetic study assessing CYP1A2 inhibition.

    Kobayashi, Toshimi; Homma, Masato; Momo, Kenji; Kobayashi, Daisuke; Kohda, Yukinao

    2011-04-01

    We developed a simple assay method for the determination of serum and urine norfloxacin and enoxacin using reversed-phase high-performance liquid chromatography and perchloric acid precipitation for sample pre-treatment. Optimized conditions can permit detection of norfloxacin and enoxacin in the same chromatogram, so either compound can be used as an internal standard for another determinant. Supernatants of the precipitated samples were analyzed by the octadecylsilyl silica-gel column under ambient temperature and an ultraviolet wavelength of 272  nm. A mobile phase solvent consisting of 20 mm sodium dihydrogenphosphate (pH 3.0) and acetonitrile (85:15, v/v) was pumped at a flow rate of 1.0 mL/min. The calibration curves for norfloxacin and enoxacin at a concentration of 62.5-1000 ng/mL for serum and 250-4000 ng/mL for urine were linear (r > 0.9997). The recoveries of norfloxacin and enoxacin from serum and urine were >94% with the coefficient of variations (CV) <5%. The CVs for intra- and inter-day assay of norfloxacin and enoxacin were <4.2 and <5.5%, respectively. This method can be applied to the pharmacokinetic study of norfloxacin and enoxacin after repeated administration to assess changes in CYP1A2 activity in healthy subjects. PMID:20662110

  19. Glycan changes: cancer metastasis and anti-cancer vaccines

    Min Li; Lujun Song; Xinyu Qin

    2010-12-01

    Complex carbohydrates, which are major components of the cell membrane, perform important functions in cell–cell and cell–extracellular matrix interactions, as well as in signal transduction. They comprise three kinds of biomolecules: glycoproteins, proteoglycans and glycosphingolipids. Recent studies have also shown that glycan changes in malignant cells take a variety of forms and mediate key pathophysiological events during the various stages of tumour progression. Glycosylation changes are universal hallmarks of malignant transformation and tumour progression in human cancer, which take place on the whole cells or some specific molecules. Accordingly, those changes make them prominent candidates for cancer biomarkers in the meantime. This review mainly focuses on the correlation between glycosylation and the metastasis potential of tumour cells from comprehensive aspects to further address the vital roles of glycans in oncogenesising. Moreover, utilizing these glycosylation changes to ward off tumour metastasis by means of anti-adhesion approach or devising anti-cancer vaccine is one of promising targets of future study.

  20. The interindividual differences in the 3-demthylation of caffeine alias CYP1A2 is determined by both genetic and environmental factors

    Rasmussen, Birgitte B; Brix, Thomas H; Kyvik, Kirsten O;

    2002-01-01

    This study investigated the role of genetic factors (CYP1A2) in caffeine metabolism. The CYP1A2 activity was determined in 378 Danish twins following oral intake of a single dose of 200 mg caffeine and subsequent determination of the caffeine ratio (AFMU+1MU+1MX)/17DMU in a 6-h urine sample. The ...

  1. Schistosoma mansoni-infected mice produce antibodies that cross-react with plant, insect, and mammalian glycoproteins and recognize the truncated biantennaryN-glycan Man3GlcNAc2-R.

    Remoortere, van, A.; Bank, CM; Nyame, AK; Cummings, RD; Deelder, A. M.; Die, de, M.

    2003-01-01

    To reveal the role of cross-reactive carbohydrate determinants in the host immune response in helminth infections and allergenicity, we developed monoclonal antibodies (mAbs) that recognize glycan epitopes present on glycoconjugates from both helminths and plants. An IgM mAb (100-4G11-A) was selected from a panel of anti-glycan mAbs generated from Schistosoma-infected or immunized mice because it recognized both a plant glycoprotein horseradish peroxidase and phospholipase A2 from honeybee ve...

  2. Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans

    De Castro, Cristina; Molinaro, Antonio; Piacente, Francesco; Gurnon, James R.; Sturiale, Luisa; Palmigiano, Angelo; Lanzetta, Rosa; Parrilli, Michelangelo; Garozzo, Domenico; Tonetti, Michela G.; Van Etten, James L.

    2013-01-01

    The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) ...

  3. GlyTouCan 1.0 – The international glycan structure repository

    Aoki-Kinoshita, Kiyoko; Agravat, Sanjay; Aoki, Nobuyuki P.; Arpinar, Sena; Cummings, Richard D.; Fujita, Akihiro; Fujita, Noriaki; Hart, Gerald M.; Haslam, Stuart M.; Kawasaki, Toshisuke; Matsubara, Masaaki; Moreman, Kelley W.; Okuda, Shujiro; Pierce, Michael; Ranzinger, René; Shikanai, Toshihide; Shinmachi, Daisuke; Solovieva, Elena; Suzuki, Yoshinori; Tsuchiya, Shinichiro; Yamada, Issaku; York, William S.; Zaia, Joseph; Narimatsu, Hisashi

    2016-01-01

    Glycans are known as the third major class of biopolymers, next to DNA and proteins. They cover the surfaces of many cells, serving as the ‘face’ of cells, whereby other biomolecules and viruses interact. The structure of glycans, however, differs greatly from DNA and proteins in that they are branched, as opposed to linear sequences of amino acids or nucleotides. Therefore, the storage of glycan information in databases, let alone their curation, has been a difficult problem. This has caused many duplicated efforts when integration is attempted between different databases, making an international repository for glycan structures, where unique accession numbers are assigned to every identified glycan structure, necessary. As such, an international team of developers and glycobiologists have collaborated to develop this repository, called GlyTouCan and is available at http://glytoucan.org/, to provide a centralized resource for depositing glycan structures, compositions and topologies, and to retrieve accession numbers for each of these registered entries. This will thus enable researchers to reference glycan structures simply by accession number, as opposed to by chemical structure, which has been a burden to integrate glycomics databases in the past. PMID:26476458

  4. GlyTouCan 1.0--The international glycan structure repository.

    Aoki-Kinoshita, Kiyoko; Agravat, Sanjay; Aoki, Nobuyuki P; Arpinar, Sena; Cummings, Richard D; Fujita, Akihiro; Fujita, Noriaki; Hart, Gerald M; Haslam, Stuart M; Kawasaki, Toshisuke; Matsubara, Masaaki; Moreman, Kelley W; Okuda, Shujiro; Pierce, Michael; Ranzinger, René; Shikanai, Toshihide; Shinmachi, Daisuke; Solovieva, Elena; Suzuki, Yoshinori; Tsuchiya, Shinichiro; Yamada, Issaku; York, William S; Zaia, Joseph; Narimatsu, Hisashi

    2016-01-01

    Glycans are known as the third major class of biopolymers, next to DNA and proteins. They cover the surfaces of many cells, serving as the 'face' of cells, whereby other biomolecules and viruses interact. The structure of glycans, however, differs greatly from DNA and proteins in that they are branched, as opposed to linear sequences of amino acids or nucleotides. Therefore, the storage of glycan information in databases, let alone their curation, has been a difficult problem. This has caused many duplicated efforts when integration is attempted between different databases, making an international repository for glycan structures, where unique accession numbers are assigned to every identified glycan structure, necessary. As such, an international team of developers and glycobiologists have collaborated to develop this repository, called GlyTouCan and is available at http://glytoucan.org/, to provide a centralized resource for depositing glycan structures, compositions and topologies, and to retrieve accession numbers for each of these registered entries. This will thus enable researchers to reference glycan structures simply by accession number, as opposed to by chemical structure, which has been a burden to integrate glycomics databases in the past. PMID:26476458

  5. Automated Glycan Sequencing from Tandem Mass Spectra of N-Linked Glycopeptides.

    Yu, Chuan-Yih; Mayampurath, Anoop; Zhu, Rui; Zacharias, Lauren; Song, Ehwang; Wang, Lei; Mechref, Yehia; Tang, Haixu

    2016-06-01

    Mass spectrometry has become a routine experimental tool for proteomic biomarker analysis of human blood samples, partly due to the large availability of informatics tools. As one of the most common protein post-translational modifications (PTMs) in mammals, protein glycosylation has been observed to alter in multiple human diseases and thus may potentially be candidate markers of disease progression. While mass spectrometry instrumentation has seen advancements in capabilities, discovering glycosylation-related markers using existing software is currently not straightforward. Complete characterization of protein glycosylation requires the identification of intact glycopeptides in samples, including identification of the modification site as well as the structure of the attached glycans. In this paper, we present GlycoSeq, an open-source software tool that implements a heuristic iterated glycan sequencing algorithm coupled with prior knowledge for automated elucidation of the glycan structure within a glycopeptide from its collision-induced dissociation tandem mass spectrum. GlycoSeq employs rules of glycosidic linkage as defined by glycan synthetic pathways to eliminate improbable glycan structures and build reasonable glycan trees. We tested the tool on two sets of tandem mass spectra of N-linked glycopeptides cell lines acquired from breast cancer patients. After employing enzymatic specificity within the N-linked glycan synthetic pathway, the sequencing results of GlycoSeq were highly consistent with the manually curated glycan structures. Hence, GlycoSeq is ready to be used for the characterization of glycan structures in glycopeptides from MS/MS analysis. GlycoSeq is released as open source software at https://github.com/chpaul/GlycoSeq/ . PMID:27111718

  6. Surface expression patterns of defined glycan antigens change during Schistosoma mansoni cercarial transformation and development of schistosomula.

    Smit, Cornelis H; Homann, Arne; van Hensbergen, Vincent P; Schramm, Gabriele; Haas, Helmut; van Diepen, Angela; Hokke, Cornelis H

    2015-12-01

    During the complex lifecycle of Schistosoma mansoni, a large variety of glycans is expressed. To many of these glycans, antibodies are induced by the infected host and some might be targets for vaccines or diagnostic tests. Spatial changes in glycan expression during schistosome development are largely unexplored. To study the surface-exposed glycans during the important initial stages of infection, we analyzed the binding of a panel of anti-glycan monoclonal antibodies (mAbs) to cercariae and schistosomula up to 72 h after transformation by immunofluorescence microscopy. The mAb specificity toward their natural targets was studied using a microarray containing a wide range of schistosomal N-glycans, O-glycans and glycosphingolipid glycans. With the exception of GalNAcβ1-4(Fucα1-3)GlcNAc (LDN-F), mono- and multifucosylated GalNAcβ1-4GlcNAc (LDN)-motifs were exposed at the surface of all developmental stages studied. Multifucosylated LDN-motifs were present on cercarial glycocalyx-derived O-glycans as well as cercarial glycolipids. In contrast, the Galβ1-4(Fucα1-3)GlcNAc (Lewis X) and LDN-F-motifs, also expressed on cercarial glycolipids, and in addition on a range of cercarial N- and O-glycans, became surface expressed only after transformation of cercariae to schistosomula. In line with the documented shedding of the O-glycan-rich cercarial glycocalyx after transformation these observations suggest that surface accessible multifucosylated LDN-motifs are mostly expressed by O-glycans in cercariae, but principally by glycosphingolipids in schistosomula. We hypothesize that these temporal changes in surface exposure of glycan antigens are relevant to the interaction with the host during the initial stages of infection with schistosomes and discuss the potential of these glycan antigens as intervention targets. PMID:26347524

  7. In good company: association between fungal glycans generates molecular complexes with unique functions

    Marcio eRodrigues

    2012-07-01

    Full Text Available The biological properties of fungal immunogens have historically utilized testing of isolated molecules. Recent findings, however, indicate that fungal glycans differing in structure and function can interact to form hybrid complexes with unique properties. In the pathogenic yeast Cryptococcus neoformans, chitin-like molecules associate with capsular glucuronoxylomannan to form functionally distinct glycan complexes. Such interactions between glycans that result in the formation of structures with different functions strongly suggest that additional molecular complexes with unknown properties may exist in fungal pathogens. Moreover, the identification of these novel complexes has stimulated the search of new immunogens with potential to protect human and animal hosts against systemic mycoses.

  8. N-Glycans in Xenopus laevis testis characterised by lectin histochemistry.

    Valbuena, Galder; Madrid, Juan Francisco; Martínez de Ubago, María; Gómez-Santos, Laura; Alonso, Edurne; Díaz-Flores, Lucio; Sáez, Francisco J

    2016-03-01

    Analysis of glycan chains of glycoconjugates is difficult because of their considerable variety. Despite this, several functional roles for these glycans have been reported. N-Glycans are oligosaccharides linked to asparagine residues of proteins. They are synthesised in the endoplasmic reticulum (ER) in a unique way, and later modified in both the ER and Golgi apparatus, developing different oligosaccharide chains. An essential role for complex N-glycans in mammalian spermatogenesis has been reported. The aim of the present study was to analyse the N-glycans of the Xenopus laevis testis by means of lectin histochemistry. Five lectins were used that specifically recognise mannose-containing and complex glycans, namely Galanthus nivalis agglutinin (GNA) from snowdrops, concanavalin A (Con A) from the Jack bean, Lens culinaris agglutinin (LCA) from lentils and Phaseolus vulgaris erythroagglutinin (PHA-E) and P. vulgaris leukoagglutinin (PHA-L) from the common bean. GNA and Con A labelled the interstitium and most of the germ cell types, whereas LCA and PHA-E showed affinity only for the interstitium. A granular cytoplasmic region was labelled in spermatogonia and spermatocytes by GNA and PHA-L, whereas GNA and LCA labelled a spermatid region that is probably associated with the centriolar basal body of the nascent flagellum. There was no specific labelling in the acrosome. Some unexpected results were found when deglycosylative pretreatments were used: pre-incubation of tissue sections with peptide N glycosidase F, which removes N-linked glycans, reduced or removed labelling with most lectins, as expected. However, after this pretreatment, the intensity of labelling remained or increased for Con A in the follicle (Sertoli) and post-meiotic germ cells. The β-elimination procedure, which removes O-linked glycans, revealed new labelling patterns with GNA, LCA and PHA-L, suggesting that some N-glycans were masked by O-glycans, and thus they became accessible to these

  9. Schistosoma mansoni-infected mice produce antibodies that cross-react with plant, insect, and mammalian glycoproteins and recognize the truncated biantennaryN-glycan Man3GlcNAc2-R.

    Remoortere, van A.; Bank, CM; Nyame, AK; Cummings, RD; Deelder, A.M.; Die, van I.M.

    2003-01-01

    To reveal the role of cross-reactive carbohydrate determinants in the host immune response in helminth infections and allergenicity, we developed monoclonal antibodies (mAbs) that recognize glycan epitopes present on glycoconjugates from both helminths and plants. An IgM mAb (100-4G11-A) was selecte

  10. Biosynthesis and Function of Extracellular Glycans in Cyanobacteria

    Jan-Christoph Kehr

    2015-01-01

    Full Text Available The cell surface of cyanobacteria is covered with glycans that confer versatility and adaptability to a multitude of environmental factors. The complex carbohydrates act as barriers against different types of stress and play a role in intra- as well as inter-species interactions. In this review, we summarize the current knowledge of the chemical composition, biosynthesis and biological function of exo- and lipo-polysaccharides from cyanobacteria and give an overview of sugar-binding lectins characterized from cyanobacteria. We discuss similarities with well-studied enterobacterial systems and highlight the unique features of cyanobacteria. We pay special attention to colony formation and EPS biosynthesis in the bloom-forming cyanobacterium, Microcystis aeruginosa.

  11. Specific N-glycans of Hepatocellular Carcinoma Cell Surface and the Abnormal Increase of Core-α-1, 6-fucosylated Triantennary Glycan via N-acetylglucosaminyltransferases-IVa Regulation.

    Nie, Huan; Liu, Xia; Zhang, Yubao; Li, Tingting; Zhan, Chao; Huo, Wenjuan; He, Anshun; Yao, Yuanfei; Jin, Yu; Qu, Youpeng; Sun, Xue-Long; Li, Yu

    2015-01-01

    Glycosylation alterations of cell surface proteins are often observed during the progression of malignancies. The specific cell surface N-glycans were profiled in hepatocellular carcinoma (HCC) with clinical tissues (88 tumor and adjacent normal tissues) and the corresponding serum samples of HCC patients. The level of core-α-1,6-fucosylated triantennary glycan (NA3Fb) increased both on the cell surface and in the serum samples of HCC patients (p virus (HBV)and cirrhosis. Furthermore, the mRNA and protein expression of N-acetylglucosaminyltransferase IVa (GnT-IVa), which was related to the synthesis of the NA3Fb, was substantially increased in HCC tissues. Knockdown of GnT-IVa leads to a decreased level of NA3Fb and decreased ability of invasion and migration in HCC cells. NA3Fb can be regarded as a specific cell surface N-glycan of HCC. The high expression of GnT-IVa is the cause of the abnormal increase of NA3Fb on the HCC cell surface, which regulates cell migration. This study demonstrated the specific N-glycans of the cell surface and the mechanisms of altered glycoform related with HCC. These findings lead to better understanding of the function of glycan and glycosyltransferase in the tumorigenesis, progression and metastasis of HCC. PMID:26537865

  12. Glycan Sulfation Modulates Dendritic Cell Biology and Tumor Growth

    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.

  13. The GlycanBuilder and GlycoWorkbench glycoinformatics tools: updates and new developments.

    Damerell, David; Ceroni, Alessio; Maass, Kai; Ranzinger, Rene; Dell, Anne; Haslam, Stuart M

    2012-11-01

    During the EUROCarbDB project our group developed the GlycanBuilder and GlycoWorkbench glycoinformatics tools. This short communication summarizes the capabilities of these two tools and updates which have been made since the original publications in 2007 and 2008. GlycanBuilder is a tool that allows for the fast and intuitive drawing of glycan structures; this tool can be used standalone, embedded in web pages and can also be integrated into other programs. GlycoWorkbench has been designed to semi-automatically annotate glycomics data. This tool can be used to annotate mass spectrometry (MS) and MS/MS spectra of free oligosaccharides, N and O-linked glycans, GAGs (glycosaminoglycans) and glycolipids, as well as MS spectra of glycoproteins. PMID:23109548

  14. Enzymes for N-Glycan Branching and Their Genetic and Nongenetic Regulation in Cancer

    Kizuka, Yasuhiko; Taniguchi, Naoyuki

    2016-01-01

    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. PMID:27136596

  15. 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. PMID:26876105

  16. Enzymes for N-Glycan Branching and Their Genetic and Nongenetic Regulation in Cancer

    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.

  17. Enzymes for N-Glycan Branching and Their Genetic and Nongenetic Regulation in Cancer.

    Kizuka, Yasuhiko; Taniguchi, Naoyuki

    2016-01-01

    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. PMID:27136596

  18. Characterization of the Structurally Diverse N-Linked Glycans of Campylobacter Species

    Jervis, Adrian J.; Butler, Jonathan A.; Lawson, Andrew J.; Langdon, Rebecca; Wren, Brendan W.; Linton, Dennis

    2012-01-01

    The Gram-negative bacterium Campylobacter jejuni encodes an extensively characterized N-linked protein glycosylation system that modifies many surface proteins with a heptasaccharide glycan. In C. jejuni, the genes that encode the enzymes required for glycan biosynthesis and transfer to protein are located at a single pgl gene locus. Similar loci are also present in the genome sequences of all other Campylobacter species, although variations in gene content and organization are evident. In th...

  19. Targeted glycomics by selected reaction monitoring for highly sensitive glycan compositional analysis

    Zhang, Hongquan; Wang, Zhaohui; Stupak, Jacek; Ghribi, Othman; Geiger, Jonathan D.; Liu, Qing Yan; Li, Jianjun

    2012-01-01

    The development of glycomics increasingly requires the detection and quantification of large numbers of glycans, which is only partially achieved by current glycomics approaches. Taking advantage of selected reaction monitoring to enhance both sensitivity and selectivity, we report here a strategy termed targeted glycomics that enables highly sensitive and consistent identification and quantification of diverse glycans across multiple samples at the same time. In this proof-of-principle study...

  20. Increased levels of anti-glycan antibodies in patients with cystic fibrosis

    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.

  1. Human Serum Processing and Analysis Methods for Rapid and Reproducible N-Glycan Mass Profiling

    Kronewitter, Scott R.; de Leoz, Maria Lorna A.; Peacock, Kyle S.; McBride, Kelly R.; An, Hyun Joo; Miyamoto, Suzanne; Leiserowitz, Gary S.; Lebrilla, Carlito B.

    2010-01-01

    Glycans constitute a new class of compounds for biomarker discovery. Glycosylation is a common post-translational modification and is often associated with transformation to malignancy. To analyze glycans, they are released from proteins, enriched, and measured with mass spectrometry. For biomarker discovery, repeatability at every step of the process is important. Locating and minimizing the process variability is key to establishing a robust platform stable enough for biomarker discovery. U...

  2. Chemoenzymatic synthesis of GDP-l-fucose and the Lewis X glycan derivatives

    Wang, Wei; Hu, Tianshun; Frantom, Patrick A.; Zheng, Tianqing; Gerwe, Brian; Del Amo, David Soriano; Garret, Sarah; Seidel, Ronald D.; Wu, Peng

    2009-01-01

    Lewis X (Lex)-containing glycans play important roles in numerous cellular processes. However, the absence of robust, facile, and cost-effective methods for the synthesis of Lex and its structurally related analogs has severely hampered the elucidation of the specific functions of these glycan epitopes. Here we demonstrate that chemically defined guanidine 5′-diphosphate-β-l-fucose (GDP-fucose), the universal fucosyl donor, the Lex trisaccharide, and their C-5 substituted derivatives can be s...

  3. Assay for quantitative determination of CYP1A1 enzyme activity using 7-Ethoxyresorufin as standard substrate (EROD assay)

    sprotocols

    2014-01-01

    Authors: Afshin Mohammadi-Bardbori ### Abstract The activity of the enzyme 7-ethoxy-resorufin-O-deethylase (EROD) has been extensively employed in biomonitoring studies of persistent organic pollutants (POPs) for more than a decade. Although the procedure is simple, convenient, sensitive and accurate. The cytochrome P450 monooxygenase 1A (CYP1A) is induced by several planar toxic compounds and endogenous chemicals, and the induction of this protein is often measured in terms of EROD a...

  4. 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]). PMID:27222848

  5. Biological significance of complex N-glycans in plants and their impact on plant physiology

    Richard eStrasser

    2014-07-01

    Full Text Available 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 beta1,2-xylose, core alpha1,3-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 signalling events. By 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.

  6. Emerging Structural Insights into Glycoprotein Quality Control Coupled with N-Glycan Processing in the Endoplasmic Reticulum

    Tadashi Satoh

    2015-01-01

    Full Text Available In the endoplasmic reticulum (ER, the sugar chain is initially introduced onto newly synthesized proteins as a triantennary tetradecasaccharide (Glc3Man9GlcNAc2. The attached oligosaccharide chain is subjected to stepwise trimming by the actions of specific glucosidases and mannosidases. In these processes, the transiently expressed N-glycans, as processing intermediates, function as signals for the determination of glycoprotein fates, i.e., folding, transport, or degradation through interactions of a series of intracellular lectins. The monoglucosylated glycoforms are hallmarks of incompletely folded states of glycoproteins in this system, whereas the outer mannose trimming leads to ER-associated glycoprotein degradation. This review outlines the recently emerging evidence regarding the molecular and structural basis of this glycoprotein quality control system, which is regulated through dynamic interplay among intracellular lectins, glycosidases, and glycosyltransferase. Structural snapshots of carbohydrate-lectin interactions have been provided at the atomic level using X-ray crystallographic analyses. Conformational ensembles of uncomplexed triantennary high-mannose-type oligosaccharides have been characterized in a quantitative manner using molecular dynamics simulation in conjunction with nuclear magnetic resonance spectroscopy. These complementary views provide new insights into glycoprotein recognition in quality control coupled with N-glycan processing.

  7. Influence of the host (Cho) and of the cultivation strategy on glycan structures and molecular properties of human thyrotrophin

    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 CO2 conditions from 5% CO2 to air environment (0.03% CO2). 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 (ThyrogenR) 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 CO2 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 distribution

  8. Effect of Bu-Zhong-Yi-Qi-Tang on deficiency of N-glycan/nitric oxide and islet damage induced by streptozotocin in diabetic rats

    Xiao-Qiu Liu; Ling Wu; Xue-Jun Guo

    2009-01-01

    AIM: To investigate the effect of Bu-Zhong-Yi-Qi-Tang (Decoction for Reinforcing Middle Jiao and Replenishing Qi) on deficiency of N-glycan/nitric oxide (NO) and islet damage induced by injecting two medium doses of streptozotocin (STZ). METHODS: Diabetes was induced by intraperitoneal injection of STZ at 55 mg/kg on day 1 and day 8. Islet damage was evaluated using a scoring system. Nitrite, nitrate, α-mannosidase and amylase activities were measured by colorimetry. N-glycan patterns of amylase were determined with lectin [ConA, pisum sativum agglutinin (PSA), peanut agglutinin (PNA), and lens culinaris agglutinin (LCA)] affinity precipitation method. RESULTS: Severe islet necrosis and mild islet atrophy were observed in diabetic rats. The number and size of islets, the activities of α-mannosidase, amylase and nitrite were decreased, while the binding of PNA and LCA to amylase was increased. All of which were improved after treatment with Bu-Zhong-Yi-Qi-Tang. Islet damage was significantly correlated with nitrite, nitrate, α-mannosidase, amylase and the binding of LCA, PNA, and PSA to amylase. PNA, and PSA to amylase.CONCLUSION: STZ- induced i s let damage i s related to N-glycan def iciency in proteins by blocking α-mannosidase activity and no deficiency, accumulation of unfolded proteins, and endoplasmic reticulum stress and activation of cellular signals, all of which are improved after treatment with Bu-Zhong-Yi- Qi-Tang.

  9. Structure of N-linked oligosaccharides attached to chlorovirus PBCV-1 major capsid protein reveals unusual class of complex N-glycans.

    De Castro, Cristina; Molinaro, Antonio; Piacente, Francesco; Gurnon, James R; Sturiale, Luisa; Palmigiano, Angelo; Lanzetta, Rosa; Parrilli, Michelangelo; Garozzo, Domenico; Tonetti, Michela G; Van Etten, James L

    2013-08-20

    The major capsid protein Vp54 from the prototype chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains four Asn-linked glycans. The structure of the four N-linked oligosaccharides and the type of substitution at each glycosylation site was determined by chemical, spectroscopic, and spectrometric analyses. Vp54 glycosylation is unusual in many ways, including: (i) unlike most viruses, PBCV-1 encodes most, if not all, of the machinery to glycosylate its major capsid protein; (ii) the glycans are attached to the protein by a β-glucose linkage; (iii) the Asn-linked glycans are not located in a typical N-X-(T/S) consensus site; and (iv) the process probably occurs in the cytoplasm. The four glycoforms share a common core structure, and the differences are related to the nonstoichiometric presence of two monosaccharides. The most abundant glycoform consists of nine neutral monosaccharide residues, organized in a highly branched fashion. Among the most distinctive features of the glycoforms are (i) a dimethylated rhamnose as the capping residue of the main chain, (ii) a hyperbranched fucose unit, and (iii) two rhamnose residues with opposite absolute configurations. These glycoforms differ from what has been reported so far in the three domains of life. Considering that chloroviruses and other members of the family Phycodnaviridae may have a long evolutionary history, we suggest that the chlorovirus glycosylation pathway is ancient, possibly existing before the development of the endoplasmic reticulum and Golgi pathway, and involves still unexplored mechanisms. PMID:23918378

  10. Impact of 4 Lactobacillus plantarum capsular polysaccharide clusters on surface glycan composition and host cell signaling

    Remus Daniela M

    2012-11-01

    -J produced decreased levels of surface polysaccharides, whereas the molar mass and the composition of polysaccharides was not affected by these cluster mutations. In the quadruple mutant, the amount of surface polysaccharides was strongly reduced. The impact of the cps cluster mutations on toll-like receptor (TLR-mediated human nuclear factor (NF-κB activation in host cells was evaluated using a TLR2 reporter cell line. In comparison to a L. plantarum wild-type derivative, TLR2 activation remained unaffected by the Δcps1A-I and Δcps3A-J mutants but appeared slightly increased after stimulation with the Δcps2A-J and Δcps4A-J mutants, while the Δcps1A-3J and Δcps1A-3J, Δcps4A-J mutants elicited the strongest responses and clearly displayed enhanced TLR2 signaling. Conclusions Our study reveals that modulation of surface glycan characteristics in L. plantarum highlights the role of these molecules in shielding of cell envelope embedded host receptor ligands. Although the apparently complete cps clusters (cps2A-J and cps4A-J contributed individually to this shielding, the removal of all cps clusters led to the strongest signaling enhancement. Our findings provide new insights into cell surface glycan biosynthesis in L. plantarum, which bears relevance in the context of host-cell signaling by probiotic bacteria.

  11. Upregulation of Glycans Containing 3’ Fucose in a Subset of Pancreatic Cancers Uncovered Using Fusion-Tagged Lectins

    Singh, Sudhir; Pal, Kuntal; Yadav, Jessica; Tang, Huiyuan; Partyka, Katie; Kletter, Doron; Hsueh, Peter; Ensink, Elliot; Birendra, KC; Hostetter, Galen; Xu, H. Eric; Bern, Marshall; Smith, David F.; Mehta, Anand S.; Brand, Randall

    2015-01-01

    The fucose post-translational modification is frequently increased in pancreatic cancer, thus forming the basis for promising biomarkers, but a subset of pancreatic cancer patients does not elevate the known fucose-containing biomarkers. We hypothesized that such patients elevate glycan motifs with fucose in linkages and contexts different from the known fucose-containing biomarkers. We used a database of glycan array data to identify the lectins CCL2 to detect glycan motifs with fucose in a ...

  12. Homology with vesicle fusion mediator syntaxin-1a predicts determinants ofepimorphin/syntaxin-2 function in mammary epithelial morphogenesis

    Chen, Connie S.; Nelson, Celeste M.; Khauv, Davitte; Bennett, Simone; Radisky, Evette S.; Hirai, Yohei; Bissell, Mina J.; Radisky, Derek C.

    2009-06-03

    We have shown that branching morphogenesis of mammary ductal structures requires the action of the morphogen epimorphin/syntaxin-2. Epimorphin, originally identified as an extracellular molecule, is identical to syntaxin-2, an intracellular molecule that is a member of the extensively investigated syntaxin family of proteins that mediate vesicle trafficking. We show here that although epimorphin/syntaxin-2 is highly homologous to syntaxin-1a, only epimorphin/syntaxin-2 can stimulate mammary branching morphogenesis. We construct a homology model of epimorphin/syntaxin-2 based on the published structure of syntaxin-1a, and we use this model to identify the structural motif responsible for the morphogenic activity. We identify four residues located within the cleft between helices B and C that differ between syntaxin-1a and epimorphin/syntaxin-2; through site-directed mutagenesis of these four amino acids, we confer the properties of epimorphin for cell adhesion, gene activation, and branching morphogenesis onto the inactive syntaxin-1a template. These results provide a dramatic demonstration of the use of structural information about one molecule to define a functional motif of a second molecule that is related at the sequence level but highly divergent functionally.

  13. Modular synthesis of N-glycans and arrays for the hetero-ligand binding analysis of HIV antibodies

    Shivatare, Sachin S.; Chang, Shih-Huang; Tsai, Tsung-I.; Tseng, Susan Yu; Shivatare, Vidya S.; Lin, Yih-Shyan; Cheng, Yang-Yu; Ren, Chien-Tai; Lee, Chang-Chun David; Pawar, Sujeet; Tsai, Charng-Sheng; Shih, Hao-Wei; Zeng, Yi-Fang; Liang, Chi-Hui; Kwong, Peter D.; Burton, Dennis R.; Wu, Chung-Yi; Wong, Chi-Huey

    2016-04-01

    A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120—a glycoprotein found on the surface of the virus envelope—thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.

  14. Restricted motion of the conserved immunoglobulin G1 N-glycan is essential for efficient FcγRIIIa binding

    Subedi, Ganesh P.; Hanson, Quinlin M.; Barb, Adam W.

    2014-01-01

    Summary Immunoglobulin G1(IgG1)-based therapies are widespread and many function through interactions with low-affinity Fc γ receptors (FcγR). N-glycosylation of the IgG1 Fc domain is required for FcγR binding, though it is unclear why. Structures of the FcγR:Fc complex fail to explain this because the FcγR polypeptide does not bind the N-glycan. Here we identify a link between motion of the N-glycan and Fc:FcγRIIIa affinity that explains the N-glycan requirement. Fc F241 and F243 mutations decreased the N-glycan/polypeptide interaction and increased N-glycan mobility. The affinity of the Fc mutants for FcγRIIIa was directly proportional to the degree of glycan restriction (R2=0.82). The IgG1 Fc K246F mutation stabilized the N-glycan and enhanced affinity for FcγRIIIa. Allosteric modulation of a protein/protein interaction represents a previously undescribed role for N-glycans in biology. Conserved features suggesting a similar N-glycan/aromatic interaction were also found in IgD, E and M, but not A. PMID:25199692

  15. Comparative analysis to identify determinants of changing life style in Thermosynechococcus elongatus BP-1, a thermophilic cyanobacterium

    Prabha, Ratna; Singh, Dhananjaya P; Gupta, Shailendra K.; de Farias, Sávio Torres; Rai, Anil

    2013-01-01

    A comparative genomics analysis among all forty whole genome sequences available for cyanobacteria (3 thermophiles– Thermosynechococcus elongatus BP-1, Synechococcus sp. JA-2-3B'a (2-13), Synechococcus sp. JA-3-3Ab and 37 mesophiles) was performed to identify genomic and proteomic factors responsible for the behaviour of T. elongatus BP-1, a thermophilic unicellular cyanobacterium with optimum growth temperature [OGT] of 55°C. Majority of genomic and proteomic characteristics for this cyanoba...

  16. Characterization of the N-glycans of female Angiostrongylus cantonensis worms.

    Veríssimo, Carolina M; Morassutti, Alessandra L; von Itzstein, Mark; Sutov, Grigorij; Hartley-Tassell, Lauren; McAtamney, Sarah; Dell, Anne; Haslam, Stuart M; Graeff-Teixeira, Carlos

    2016-07-01

    Glycoconjugates play a crucial role in the host-parasite relationships of helminthic infections, including angiostrongyliasis. It has previously been shown that the antigenicity of proteins from female Angiostrongylus cantonensis worms may depend on their associated glycan moieties. Here, an N-glycan profile of A. cantonensis is reported. A total soluble extract (TE) was prepared from female A. cantonensis worms and was tested by western blot before and after glycan oxidation or N- and O-glycosidase treatment. The importance of N-glycans for the immunogenicity of A. cantonensis was demonstrated when deglycosylation of the TE with PNGase F completely abrogated IgG recognition. The TE was also fractionated using various lectin columns [Ulex europaeus (UEA), concanavalin A (Con A), Arachis hypogaea (PNA), Triticum vulgaris (WGA) and Lycopersicon esculentum (LEA)], and then each fraction was digested with PNGase F. Released N-glycans were analyzed with matrix-assisted laser desorption ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) and MALDI-TOF/TOF-MS/MS. Complex-type, high mannose, and truncated glycan structures were identified in all five fractions. Sequential MALDI-TOF-TOF analysis of the major MS peaks identified complex-type structures, with a α1-6 fucosylated core and truncated antennas. Glycoproteins in the TE were labeled with BodipyAF558-SE dye for a lectin microarray analysis. Fluorescent images were analyzed with ProScanArray imaging software followed by statistical analysis. A total of 29 lectins showed positive binding to the TE. Of these, Bandeiraea simplicifolia (BS-I), PNA, and Wisteria floribunda (WFA), which recognize galactose (Gal) and N-acetylgalactosamine (GalNAc), exhibited high affinity binding. Taken together, our findings demonstrate that female A. cantonensis worms have characteristic helminth N-glycans. PMID:27107931

  17. Neonatal protection by an innate immune system of human milk consisting of oligosaccharides and glycans.

    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

  18. Circulating Biomphalaria glabrata hemocyte subpopulations possess shared schistosome glycans and receptors capable of binding larval glycoconjugates.

    Yoshino, Timothy P; Wu, Xiao-Jun; Gonzalez, Laura A; Hokke, Cornelis H

    2013-01-01

    Host lectin-like recognition molecules may play an important role in innate resistance in Biomphalaria glabrata snails to larval schistosome infection, thus implicating parasite-expressed glycans as putative ligands for these lectin receptors. While host lectins may utilize specific glycan structures for parasite recognition, it also has been hypothesized that the parasite may use this system to evade immune detection by mimicking naturally-expressed host glycans, resulting in reduced immunorecognition capacity. By employing immunocytochemical (ICC) and Western blot assays using schistosome glycan-specific monoclonal antibodies (mABs) we sought to identify specific glycan epitopes (glycotopes) shared in common between larval Schistosoma mansoni and B. glabrata hemocytes, the primary immune effector cells in snails. Results confirmed the presence of selected larval glycotopes on subpopulations of hemocytes by ICC and association with numerous hemocyte proteins by Western blot analyses, including a trimannosyl core N-glycan (TriMan), and two fucosylated lacdiNAc (LDN) variants, F-LDN and F-LDN-F. Snail strain differences were seen in the prevalence of constitutively expressed F-LDN on hemocytes, and in the patterns of protein immunoreactivity with these mABs. In contrast, there was little to no hemocyte reactivity with mABs for Lewis X (LeX), LDN, LDN-F or LDN-DF. When intact hemocytes were exposed to larval transformation products (LTPs), distinct cell subpopulations displayed weak (LeX, LDN-DF) to moderate (LDN, LDN-F) glycotope reactivity by ICC, including snail strain differences in the prevalence of LDN-reactive cellular subsets. Far-Western blot analyses of the hemocytes following exposure to larval transformation proteins (LTPs) also revealed multiple mAB-reactive hemocyte protein bands for LeX, LDN, LDN-F, and LDN-DF. These results demonstrate the existence of complex patterns of shared larval glycan constitutively expressed on hemocytes and their proteins

  19. Mass Spectrometric Quantification of N-Linked Glycans by Reference to Exogenous Standards.

    Mehta, Nickita; Porterfield, Mindy; Struwe, Weston B; Heiss, Christian; Azadi, Parastoo; Rudd, Pauline M; Tiemeyer, Michael; Aoki, Kazuhiro

    2016-09-01

    Environmental and metabolic processes shape the profile of glycoprotein glycans expressed by cells, whether in culture, developing tissues, or mature organisms. Quantitative characterization of glycomic changes associated with these conditions has been achieved historically by reductive coupling of oligosaccharides to various fluorophores following release from glycoprotein and subsequent HPLC or capillary electrophoretic separation. Such labeling-based approaches provide a robust means of quantifying glycan amount based on fluorescence yield. Mass spectrometry, on the other hand, has generally been limited to relative quantification in which the contribution of the signal intensity for an individual glycan is expressed as a percent of the signal intensity summed over the total profile. Relative quantification has been valuable for highlighting changes in glycan expression between samples; sensitivity is high, and structural information can be derived by fragmentation. We have investigated whether MS-based glycomics is amenable to absolute quantification by referencing signal intensities to well-characterized oligosaccharide standards. We report the qualification of a set of N-linked oligosaccharide standards by NMR, HPLC, and MS. We also demonstrate the dynamic range, sensitivity, and recovery from complex biological matrices for these standards in their permethylated form. Our results indicate that absolute quantification for MS-based glycomic analysis is reproducible and robust utilizing currently available glycan standards. PMID:27432553

  20. Density variant glycan microarray for evaluating cross-linking of mucin-like glycoconjugates by lectins.

    Godula, Kamil; Bertozzi, Carolyn R

    2012-09-26

    Interactions of mucin glycoproteins with cognate receptors are dictated by the structures and spatial organization of glycans that decorate the mucin polypeptide backbone. The glycan-binding proteins, or lectins, that interact with mucins are often oligomeric receptors with multiple ligand binding domains. In this work, we employed a microarray platform comprising synthetic glycopolymers that emulate natural mucins arrayed at different surface densities to evaluate how glycan valency and spatial separation affect the preferential binding mode of a particular lectin. We evaluated a panel of four lectins (Soybean agglutinin (SBA), Wisteria floribunda lectin (WFL), Vicia villosa-B-4 agglutinin (VVA), and Helix pomatia agglutin (HPA)) with specificity for α-N-acetylgalactosamine (α-GalNAc), an epitope displayed on mucins overexpressed in many adenocarcinomas. While these lectins possess the ability to agglutinate A(1)-blood cells carrying the α-GalNAc epitope and cross-link low valency glycoconjugates, only SBA showed a tendency to form intermolecular cross-links among the arrayed polyvalent mucin mimetics. These results suggest that glycopolymer microarrays can reveal discrete higher-order binding preferences beyond the recognition of individual glycan epitopes. Our findings indicate that glycan valency can set thresholds for cross-linking by lectins. More broadly, well-defined synthetic glycopolymers enable the integration of glycoconjugate structural and spatial diversity in a single microarray screening platform. PMID:22967056

  1. The N-glycans of Trichomonas vaginalis contain variable core and antennal modifications.

    Paschinger, Katharina; Hykollari, Alba; Razzazi-Fazeli, Ebrahim; Greenwell, Pamela; Leitsch, David; Walochnik, Julia; Wilson, Iain B H

    2012-02-01

    Trichomonad species are widespread unicellular flagellated parasites of vertebrates which interact with their hosts through carbohydrate-lectin interactions. In the past, some data have been accumulated regarding their lipo(phospho)glycans, a major glycoconjugate on their cell surfaces; on the other hand, other than biosynthetic aspects, few details about their N-linked oligosaccharides are known. In this study, we present both mass spectrometric and high-performance liquid chromatography data about the N-glycans of different strains of Trichomonas vaginalis, a parasite of the human reproductive tract. The major structure in all strains examined is a truncated oligomannose form (Man(5)GlcNAc(2)) with α1,2-mannose residues, compatible with a previous bioinformatic examination of the glycogenomic potential of T. vaginalis. In addition, dependent on the strain, N-glycans modified by pentose residues, phosphate or phosphoethanolamine and terminal N-acetyllactosamine (Galβ1,4GlcNAc) units were found. The modification of N-glycans by N-acetyllactosamine in at least some strains is shared with the lipo(phospho)glycan and may represent a further interaction partner for host galectins, thereby playing a role in binding of the parasite to host epithelia. On the other hand, the variation in glycosylation between strains may be the result of genetic diversity within this species. PMID:21983210

  2. Unique, polyfucosylated glycan-receptor interactions are essential for regeneration of Hydra magnipapillata.

    Sahadevan, Sonu; Antonopoulos, Aristotelis; Haslam, Stuart M; Dell, Anne; Ramaswamy, Subramanian; Babu, Ponnusamy

    2014-01-17

    Cell-cell communications, cell-matrix interactions, and cell migrations play a major role in regeneration. However, little is known about the molecular players involved in these critical events, especially cell surface molecules. Here, we demonstrate the role of specific glycan-receptor interactions in the regenerative process using Hydra magnipapillata as a model system. Global characterization of the N- and O-glycans expressed by H. magnipapillata using ultrasensitive mass spectrometry revealed mainly polyfucosylated LacdiNAc antennary structures. Affinity purification showed that a putative C-type lectin (accession number Q6SIX6) is a likely endogenous receptor for the novel polyfucosylated glycans. Disruption of glycan-receptor interactions led to complete shutdown of the regeneration machinery in live Hydra. A time-dependent, lack-of-regeneration phenotype observed upon incubation with exogenous fuco-lectins suggests the involvement of a polyfucose receptor-mediated signaling mechanism during regeneration. Thus, for the first time, the results presented here provide direct evidence for the role of polyfucosylated glycan-receptor interactions in the regeneration of H. magnipapillata. PMID:23972202

  3. Epigenetic Determinants of CYP1A1 Induction by the Aryl Hydrocarbon Receptor Agonist 3,3',4,4',5-Pentachlorobiphenyl (PCB 126

    Sabine U. Vorrink

    2014-08-01

    Full Text Available Many enzymes involved in xenobiotic metabolism, including cytochrome P450 (CYP 1A1, are regulated by the aryl hydrocarbon receptor (AhR. 3,3',4,4',5-Penta chlorobiphenyl (PCB 126 is a potent ligand for AhR and can thus induce the expression of CYP1A1. Interestingly, we observed that human carcinoma cell lines derived from different types of epithelial cells displayed divergent degrees of CYP1A1 induction after exposure to PCB 126. Since epigenetic mechanisms are known to be involved in cell type-specific gene expression, we sought to assess the epigenetic determinants of CYP1A1 induction in these carcinoma cell lines. In contrast to HepG2 hepatocarcinoma cells, HeLa cervical carcinoma cells showed significantly lower levels of CYP1A1 mRNA expression following PCB 126 exposure. Our results show that the two cell lines maintained differences in the chromatin architecture along the CYP1A1 promoter region. Furthermore, treatment with the epigenetic modifiers, trichostatin A (TSA and 5-aza-2'-deoxycytidine (5-Aza-dC, significantly increased the expression of CYP1A1 after PCB 126 treatment in HeLa cells. However, we did not observe apparent differences in methylation levels or specific location of CpG DNA methylation between the two cell lines in the analyzed CYP1A1 promoter region. Taken together, our findings suggest that the differences in CYP1A1 expression between HepG2 and HeLa cells are due to differences in the chromatin architecture of the CYP1A1 promoter and thus establish a role of epigenetic regulation in cell-specific CYP1A1 expression.

  4. LC-MS/MS Peptide Mapping with Automated Data Processing for Routine Profiling of N-Glycans in Immunoglobulins

    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.

  5. N-Linked Glycans of Chloroviruses Sharing a Core Architecture without Precedent.

    De Castro, Cristina; Speciale, Immacolata; Duncan, Garry; Dunigan, David D; Agarkova, Irina; Lanzetta, Rosa; Sturiale, Luisa; Palmigiano, Angelo; Garozzo, Domenico; Molinaro, Antonio; Tonetti, Michela; Van Etten, James L

    2016-01-11

    N-glycosylation is a fundamental modification of proteins and exists in the three domains of life and in some viruses, including the chloroviruses, for which a new type of core N-glycan is herein described. This N-glycan core structure, common to all chloroviruses, is a pentasaccharide with a β-glucose linked to an asparagine residue which is not located in the typical sequon N-X-T/S. The glucose is linked to a terminal xylose unit and a hyperbranched fucose, which is in turn substituted with a terminal galactose and a second xylose residue. The third position of the fucose unit is always linked to a rhamnose, which is a semiconserved element because its absolute configuration is virus-dependent. Additional decorations occur on this core N-glycan and represent a molecular signature for each chlorovirus. PMID:26582281

  6. Metabolic Reprogramming by Hexosamine Biosynthetic and Golgi N-Glycan Branching Pathways.

    Ryczko, Michael C; Pawling, Judy; Chen, Rui; Abdel Rahman, Anas M; Yau, Kevin; Copeland, Julia K; Zhang, Cunjie; Surendra, Anu; Guttman, David S; Figeys, Daniel; Dennis, James W

    2016-01-01

    De novo uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) biosynthesis requires glucose, glutamine, acetyl-CoA and uridine, however GlcNAc salvaged from glycoconjugate turnover and dietary sources also makes a significant contribution to the intracellular pool. Herein we ask whether dietary GlcNAc regulates nutrient transport and intermediate metabolism in C57BL/6 mice by increasing UDP-GlcNAc and in turn Golgi N-glycan branching. GlcNAc added to the drinking water showed a dose-dependent increase in growth of young mice, while in mature adult mice fat and body-weight increased without affecting calorie-intake, activity, energy expenditure, or the microbiome. Oral GlcNAc increased hepatic UDP-GlcNAc and N-glycan branching on hepatic glycoproteins. Glucose homeostasis, hepatic glycogen, lipid metabolism and response to fasting were altered with GlcNAc treatment. In cultured cells GlcNAc enhanced uptake of glucose, glutamine and fatty-acids, and enhanced lipid synthesis, while inhibition of Golgi N-glycan branching blocked GlcNAc-dependent lipid accumulation. The N-acetylglucosaminyltransferase enzymes of the N-glycan branching pathway (Mgat1,2,4,5) display multistep ultrasensitivity to UDP-GlcNAc, as well as branching-dependent compensation. Indeed, oral GlcNAc rescued fat accumulation in lean Mgat5(-/-) mice and in cultured Mgat5(-/-) hepatocytes, consistent with N-glycan branching compensation. Our results suggest GlcNAc reprograms cellular metabolism by enhancing nutrient uptake and lipid storage through the UDP-GlcNAc supply to N-glycan branching pathway. PMID:26972830

  7. Glycan masking of Plasmodium vivax Duffy Binding Protein for probing protein binding function and vaccine development.

    Sowmya Sampath

    Full Text Available Glycan masking is an emerging vaccine design strategy to focus antibody responses to specific epitopes, but it has mostly been evaluated on the already heavily glycosylated HIV gp120 envelope glycoprotein. Here this approach was used to investigate the binding interaction of Plasmodium vivax Duffy Binding Protein (PvDBP and the Duffy Antigen Receptor for Chemokines (DARC and to evaluate if glycan-masked PvDBPII immunogens would focus the antibody response on key interaction surfaces. Four variants of PVDBPII were generated and probed for function and immunogenicity. Whereas two PvDBPII glycosylation variants with increased glycan surface coverage distant from predicted interaction sites had equivalent binding activity to wild-type protein, one of them elicited slightly better DARC-binding-inhibitory activity than wild-type immunogen. Conversely, the addition of an N-glycosylation site adjacent to a predicted PvDBP interaction site both abolished its interaction with DARC and resulted in weaker inhibitory antibody responses. PvDBP is composed of three subdomains and is thought to function as a dimer; a meta-analysis of published PvDBP mutants and the new DBPII glycosylation variants indicates that critical DARC binding residues are concentrated at the dimer interface and along a relatively flat surface spanning portions of two subdomains. Our findings suggest that DARC-binding-inhibitory antibody epitope(s lie close to the predicted DARC interaction site, and that addition of N-glycan sites distant from this site may augment inhibitory antibodies. Thus, glycan resurfacing is an attractive and feasible tool to investigate protein structure-function, and glycan-masked PvDBPII immunogens might contribute to P. vivax vaccine development.

  8. A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins.

    McBride, Ryan; Paulson, James C; de Vries, Robert P

    2016-01-01

    Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a α2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a α2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with α2-3 or α2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency. PMID:27284789

  9. Holes in the Glycan Shield of the Native HIV Envelope Are a Target of Trimer-Elicited Neutralizing Antibodies

    Laura E. McCoy

    2016-08-01

    Full Text Available A major advance in the search for an HIV vaccine has been the development of a near-native Envelope trimer (BG505 SOSIP.664 that can induce robust autologous Tier 2 neutralization. Here, potently neutralizing monoclonal antibodies (nAbs from rabbits immunized with BG505 SOSIP.664 are shown to recognize an immunodominant region of gp120 centered on residue 241. Residue 241 occupies a hole in the glycan defenses of the BG505 isolate, with fewer than 3% of global isolates lacking a glycan site at this position. However, at least one conserved glycan site is missing in 89% of viruses, suggesting the presence of glycan holes in most HIV isolates. Serum evidence is consistent with targeting of holes in natural infection. The immunogenic nature of breaches in the glycan shield has been under-appreciated in previous attempts to understand autologous neutralizing antibody responses and has important potential consequences for HIV vaccine design.

  10. Immunoglobulin-E reactivity to a glycosylated food allergen (peanuts) due to interference with cross-reactive carbohydrate determinants in heavy drinkers

    Vidal, C; Vizcaino, L; Díaz-Peromingo, J A;

    2009-01-01

    BACKGROUND: N-glycans in plant and invertebrate glycoproteins can induce extensive IgE cross-reactivity therefore limiting the specificity of in vitro allergy tests. IgE sensitization to N-glycans (cross-reactive carbohydrate determinants, CCDs) may be increased in heavy drinkers, who therefore...

  11. Further insight into the roles of the glycans attached to human blood protein C inhibitor

    Sun, Wei; Parry, Simon; Ubhayasekera, Wimal;

    2010-01-01

    . Furthermore, we have provided experimental evidence that PCI in both individuals is O-glycosylated on Thr20 with a core type 1 O-glycan, which is mostly NeuAcGalGalNAc. Modeling suggested that the O-glycan attachment site is located in proximity to several ligand-binding sites of the inhibitor.......Protein C inhibitor (PCI) is a 57-kDa glycoprotein that exists in many tissues and secretions in human. As a member of the serpin superfamily of proteins it displays unusually broad protease specificity. PCI is implicated in the regulation of a wide range of processes, including blood coagulation...

  12. TWO TYPES OF GALACTOSYLATED FUCOSE MOTIFS ARE PRESENT ON N-GLYCANS OF HAEMONCHUS CONTORTUS

    Paschinger, Katharina; Wilson, Iain B. H.

    2015-01-01

    N-glycans from the nematode Haemonchus contortus (barber pole worm), a parasite of sheep and cattle, were the first to be described to possess up to three fucose residues associated with the N,N’-diacetylchitobiosyl core, two being on the reducing-terminal proximal GlcNAc and one on the distal core GlcNAc residue. The assumption was that truncated glycans from this organism with three hexose residues have the composition Man3GlcNAc2Fuc1-3. In this study we have performed HPLC and MALDI-TOF MS...

  13. Complexity of the Ruminococcus flavefaciens cellulosome reflects an expansion in glycan recognition

    Venditto, Immacolata; Luis, Ana S.; Rydahl, Maja; Schückel, Julia; Fernandes, Vânia O.; Vidal-Melgosa, Silvia; Bule, Pedro; Goyal, Arun; Pires, Virginia M. R.; Dourado, Catarina G.; Ferreira, Luís M. A.; Coutinho, Pedro M.; Henrissat, Bernard; Knox, J. Paul; Baslé, Arnaud; Najmudin, Shabir; Gilbert, Harry J.; Willats, William G. T.; Fontes, Carlos M. G. A.

    2016-01-01

    The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBM families that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose. PMID:27298375

  14. Characterisation of N-glycans bound to IGFBP-3 in sera from healthy adults.

    Masnikosa, Romana; Baricević, Ivona; Lagundzin, Dragana; Nedić, Olgica

    2010-01-01

    Human IGFBP-3 contains three potential N-linked glycosylation sites. Published data concerning the type and saccharide composition of the N-glycans is scarce. The aim of this study was to characterise N-glycans covalently attached to IGFBP-3 from sera of healthy adults (men and women). In order to do that a panel of eight lectins covering broad saccharide specificity was used: agarose-immobilised SNA (Sambucus nigra agglutinin), Con A (lectin from Canavalia ensiformis), RCA I (Ricinus communis agglutinin I), PHA-E (Phaseolus vulgaris erythroagglutinin), PHA-L (P. vulgaris leukoagglutinin), succinylated WGA (wheat germ agglutinin), ECL (Erythrina cristagalli lectin) and UEA (Ulex europaeus agglutinin). IGFBP-3 interacted with SNA, Con A, RCA I, PHA-E and, to a much lesser extent, with PHA-L. These results indicate that human IGFBP-3 bears mostly biantennary complex type N-glycans with a very high content of alpha-2,6-linked Sia at their termini. Hybrid type and high-mannose type N-glycans are present, as well as a bisecting GlcNAc residue, which may be core fucosylated. N-glycosylation of IGFBP-3 follows the N-glycosylation pattern of major serum proteins. This study represents a ground for the future research of glycosylation pattern of IGFBP-3 from the circulation of men and women diagnosed with different illnesses. PMID:19800385

  15. Nuclear repartitioning of galectin-1 by an extracellular glycan switch regulates mammary morphogenesis.

    Bhat, Ramray; Belardi, Brian; Mori, Hidetoshi; Kuo, Peiwen; Tam, Andrew; Hines, William C; Le, Quynh-Thu; Bertozzi, Carolyn R; Bissell, Mina J

    2016-08-16

    Branching morphogenesis in the mammary gland is achieved by the migration of epithelial cells through a microenvironment consisting of stromal cells and extracellular matrix (ECM). Here we show that galectin-1 (Gal-1), an endogenous lectin that recognizes glycans bearing N-acetyllactosamine (LacNAc) epitopes, induces branching migration of mammary epithelia in vivo, ex vivo, and in 3D organotypic cultures. Surprisingly, Gal-1's effects on mammary patterning were independent of its glycan-binding ability and instead required localization within the nuclei of mammary epithelia. Nuclear translocation of Gal-1, in turn, was regulated by discrete cell-surface glycans restricted to the front of the mammary end buds. Specifically, α2,6-sialylation of terminal LacNAc residues in the end buds masked Gal-1 ligands, thereby liberating the protein for nuclear translocation. Within mammary epithelia, Gal-1 localized within nuclear Gemini bodies and drove epithelial invasiveness. Conversely, unsialylated LacNAc glycans, enriched in the epithelial ducts, sequestered Gal-1 in the extracellular environment, ultimately attenuating invasive potential. We also found that malignant breast cells possess higher levels of nuclear Gal-1 and α2,6-SA and lower levels of LacNAc than nonmalignant cells in culture and in vivo and that nuclear localization of Gal-1 promotes a transformed phenotype. Our findings suggest that differential glycosylation at the level of tissue microanatomy regulates the nuclear function of Gal-1 in the context of mammary gland morphogenesis and in cancer progression. PMID:27496330

  16. Viral hemagglutinin-esterases: Mediators of dynamic virion-glycan interactions

    Langereis, M.A.

    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 chemi

  17. Function and 3D Structure of the N-Glycans on Glycoproteins

    Yoshiki Yamaguchi

    2012-07-01

    Full Text Available Glycosylation is one of the most common post-translational modifications in eukaryotic cells and plays important roles in many biological processes, such as the immune response and protein quality control systems. It has been notoriously difficult to study glycoproteins by X-ray crystallography since the glycan moieties usually have a heterogeneous chemical structure and conformation, and are often mobile. Nonetheless, recent technical advances in glycoprotein crystallography have accelerated the accumulation of 3D structural information. Statistical analysis of “snapshots” of glycoproteins can provide clues to understanding their structural and dynamic aspects. In this review, we provide an overview of crystallographic analyses of glycoproteins, in which electron density of the glycan moiety is clearly observed. These well-defined N-glycan structures are in most cases attributed to carbohydrate-protein and/or carbohydrate-carbohydrate interactions and may function as “molecular glue” to help stabilize inter- and intra-molecular interactions. However, the more mobile N-glycans on cell surface receptors, the electron density of which is usually missing on X-ray crystallography, seem to guide the partner ligand to its binding site and prevent irregular protein aggregation by covering oligomerization sites away from the ligand-binding site.

  18. Chemoenzymatic synthesis of the sialyl Lewis X glycan and its derivatives

    del Amo, David Soriano; Wang, Wei; Besanceney, Christen; Zheng, Tianqing; He, Yizheng; Gerwe, Brian; Seidel, Ronald D; Wu, Peng

    2010-01-01

    A combination of recombinant FKP and α-(1→3)-fucosyltransferase allows the facile synthesis of the sialyl Lewis X tetrasaccharide glycan and its derivatives in excellent yield. In this system, the universal fucosyl donor, guanidine 5′-diphosphate-β-L-fucose (GDP-fucose), or its analogues can be generated in situ by cofactor recycling using pyruvate kinase.

  19. Structural characterization of the dual glycan binding adeno-associated virus serotype 6.

    Ng, Robert; Govindasamy, Lakshmanan; Gurda, Brittney L; McKenna, Robert; Kozyreva, Olga G; Samulski, R Jude; Parent, Kristin N; Baker, Timothy S; Agbandje-McKenna, Mavis

    2010-12-01

    The three-dimensional structure of adeno-associated virus (AAV) serotype 6 (AAV6) was determined using cryo-electron microscopy and image reconstruction and using X-ray crystallography to 9.7- and 3.0-Å resolution, respectively. The AAV6 capsid contains a highly conserved, eight-stranded (βB to βI) β-barrel core and large loop regions between the strands which form the capsid surface, as observed in other AAV structures. The loops show conformational variation compared to other AAVs, consistent with previous reports that amino acids in these loop regions are involved in differentiating AAV receptor binding, transduction efficiency, and antigenicity properties. Toward structure-function annotation of AAV6 with respect to its unique dual glycan receptor (heparan sulfate and sialic acid) utilization for cellular recognition, and its enhanced lung epithelial transduction compared to other AAVs, the capsid structure was compared to that of AAV1, which binds sialic acid and differs from AAV6 in only 6 out of 736 amino acids. Five of these residues are located at or close to the icosahedral 3-fold axis of the capsid, thereby identifying this region as imparting important functions, such as receptor attachment and transduction phenotype. Two of the five observed amino acids are located in the capsid interior, suggesting that differential AAV infection properties are also controlled by postentry intracellular events. Density ordered inside the capsid, under the 3-fold axis in a previously reported, conserved AAV DNA binding pocket, was modeled as a nucleotide and a base, further implicating this capsid region in AAV genome recognition and/or stabilization. PMID:20861247

  20. Kinetics and thermodynamics of glycans and glycoproteins binding to Holothuria scabra lectin: a fluorescence and surface plasmon resonance spectroscopic study.

    Gowda, Nagaraj M; Gaikwad, Sushama M; Khan, M Islam

    2013-11-01

    Holothuria scabra produces a monomeric lectin (HSL) of 182 kDa. HSL showed strong antibacterial activity and induced bacterial agglutination under in vitro conditions, indicating its role in animals' innate immune responses. Very few lectins have been reported from echinoderms and none of these lectins have been explored in detail for their sugar-binding kinetics. Affinity, kinetics and thermodynamic analysis of glycans and glycoproteins binding to HSL were studied by fluorescence and surface plasmon resonance spectroscopy. Lectin binds with higher affinity to O-linked than N-linked asialo glycans, and the affinities were relatively higher than that for sialated glycans and glycoproteins. T-antigen α-methyl glycoside was the most potent ligand having the highest affinity (Ka 8.32 ×10(7) M(-1)). Thermodynamic and kinetic analysis indicated that the binding of galactosyl Tn-antigen and asialo glycans is accompanied by an enthalpic contribution in addition to higher association rate coupled by low activation energy for the association process. Presence of sialic acid or protein matrix inhibits binding. Higher affinity of HSL for O-glycans than N-glycans had biological implications; since HSL specifically recognizes bacteria, which have mucin or O-glycan cognate on their cell surfaces and play a major role in animal innate immunity. Since, HSL had higher affinity to T-antigen, makes it a useful tool for cancer diagnostic purpose. PMID:23736907

  1. Glycan profile of oviductal isthmus epithelium in normal and superovulated ewes.

    Desantis, Salvatore; Accogli, Gianluca; Silvestre, Fabio; Binetti, Francesco; Cox, Sharon Natasha; Roscino, Mariateresa; Caira, Michele; Lacalandra, Giovanni Michele

    2016-04-01

    Glycans of oviductal isthmus are implicated in sperm-isthmus interaction, sperm storage, survival, and capacitation. Isthmus morphology and glycoprotein production are controlled by sex steroids, which could be responsible for alterations of some reproductive events in the superovulated ewes (SE). In this study, the oviductal isthmus epithelium was evaluated in normal and in SE using morphologic and lectin histochemical analysis. The epithelium of normal isthmi was significantly taller in folds than in crypts, whereas it significantly decreased in the folds of SE. Nonciliated cells (NCs) from normal, showed apical blebs revealing apocrine secretory activity, which was missing in SE. The quantitative analysis of lectin staining revealed higher Con A, DBA, and PNA reactivity but lower affinity to KOH-sialidase- (Ks)WGA, GSA II, LTA, UEA I, SBA, GSA I-B4, RCA120, KsPNA, MAL II, SNA in control isthmi compared with superovulated ones. The NCs apical blebs showed terminal fucose (Fuc), N-acetylgalactosamine (GalNAc), galactose (Gal), lactosamine, and O- and N-sialoglycans. In normal isthmi, the luminal surface of NCs and ciliated cells expressed Fuc, highly mannosilated N-glycans terminating with lactosamine as well as O-glycans ending with N-acetylglucosamine (GlcNAc) and GalNAc. Moreover, NCs microvilli contained Gal and α2-3-linked sialic acids. In SE, the luminal surface lacked Gal and GalNAcα1, 3(LFucα1,2)Galβ1,3/4GlcNAcβ1, whereas it was enriched with Fuc in the folds and with α2-3sialo-mucins both in crypts and in folds. The apical surface showed additional O- and N-linked sialoglycans in NCs and αGal in the cilia, which expressed α2-6-linked sialic acid only in the folds. The cytoplasm of control NCs showed highly mannosilated N-glycans throughout the epithelium and GlcNAc in the folds. After superovulation treatment, NCs expressed cytoplasmic terminal Fuc, βGalNAc, lactosamine, α2-3-, and α2-6-linked sialic acids in the folds. The cytoplasm of normal

  2. A lectin from Platypodium elegans with unusual specificity and affinity for asymmetric complex N-glycans.

    Benevides, Raquel Guimarães; Ganne, Géraldine; Simões, Rafael da Conceição; Schubert, Volker; Niemietz, Mathäus; Unverzagt, Carlo; Chazalet, Valérie; Breton, Christelle; Varrot, Annabelle; Cavada, Benildo Sousa; Imberty, Anne

    2012-07-27

    Lectin activity with specificity for mannose and glucose has been detected in the seed of Platypodium elegans, a legume plant from the Dalbergieae tribe. The gene of Platypodium elegans lectin A has been cloned, and the resulting 261-amino acid protein belongs to the legume lectin family with similarity with Pterocarpus angolensis agglutinin from the same tribe. The recombinant lectin has been expressed in Escherichia coli and refolded from inclusion bodies. Analysis of specificity by glycan array evidenced a very unusual preference for complex type N-glycans with asymmetrical branches. A short branch consisting of one mannose residue is preferred on the 6-arm of the N-glycan, whereas extensions by GlcNAc, Gal, and NeuAc are favorable on the 3-arm. Affinities have been obtained by microcalorimetry using symmetrical and asymmetrical Asn-linked heptasaccharides prepared by the semi-synthetic method. Strong affinity with K(d) of 4.5 μm was obtained for both ligands. Crystal structures of Platypodium elegans lectin A complexed with branched trimannose and symmetrical complex-type Asn-linked heptasaccharide have been solved at 2.1 and 1.65 Å resolution, respectively. The lectin adopts the canonical dimeric organization of legume lectins. The trimannose bridges the binding sites of two neighboring dimers, resulting in the formation of infinite chains in the crystal. The Asn-linked heptasaccharide binds with the 6-arm in the primary binding site with extensive additional contacts on both arms. The GlcNAc on the 6-arm is bound in a constrained conformation that may rationalize the higher affinity observed on the glycan array for N-glycans with only a mannose on the 6-arm. PMID:22692206

  3. A Lectin from Platypodium elegans with Unusual Specificity and Affinity for Asymmetric Complex N-Glycans*

    Benevides, Raquel Guimarães; Ganne, Géraldine; Simões, Rafael da Conceição; Schubert, Volker; Niemietz, Mathäus; Unverzagt, Carlo; Chazalet, Valérie; Breton, Christelle; Varrot, Annabelle; Cavada, Benildo Sousa; Imberty, Anne

    2012-01-01

    Lectin activity with specificity for mannose and glucose has been detected in the seed of Platypodium elegans, a legume plant from the Dalbergieae tribe. The gene of Platypodium elegans lectin A has been cloned, and the resulting 261-amino acid protein belongs to the legume lectin family with similarity with Pterocarpus angolensis agglutinin from the same tribe. The recombinant lectin has been expressed in Escherichia coli and refolded from inclusion bodies. Analysis of specificity by glycan array evidenced a very unusual preference for complex type N-glycans with asymmetrical branches. A short branch consisting of one mannose residue is preferred on the 6-arm of the N-glycan, whereas extensions by GlcNAc, Gal, and NeuAc are favorable on the 3-arm. Affinities have been obtained by microcalorimetry using symmetrical and asymmetrical Asn-linked heptasaccharides prepared by the semi-synthetic method. Strong affinity with Kd of 4.5 μm was obtained for both ligands. Crystal structures of Platypodium elegans lectin A complexed with branched trimannose and symmetrical complex-type Asn-linked heptasaccharide have been solved at 2.1 and 1.65 Å resolution, respectively. The lectin adopts the canonical dimeric organization of legume lectins. The trimannose bridges the binding sites of two neighboring dimers, resulting in the formation of infinite chains in the crystal. The Asn-linked heptasaccharide binds with the 6-arm in the primary binding site with extensive additional contacts on both arms. The GlcNAc on the 6-arm is bound in a constrained conformation that may rationalize the higher affinity observed on the glycan array for N-glycans with only a mannose on the 6-arm. PMID:22692206

  4. The fucomic potential of mosquitoes: Fucosylated N-glycan epitopes and their cognate fucosyltransferases.

    Kurz, Simone; King, Jonas G; Dinglasan, Rhoel R; Paschinger, Katharina; Wilson, Iain B H

    2016-01-01

    Fucoconjugates are key mediators of protein-glycan interactions in prokaryotes and eukaryotes. As examples, N-glycans modified with the non-mammalian core α1,3-linked fucose have been detected in various organisms ranging from plants to insects and are immunogenic in mammals. The rabbit polyclonal antibody raised against plant horseradish peroxidase (anti-HRP) is able to recognize the α1,3-linked fucose epitope and is also known to specifically stain neural tissues in the fruit fly Drosophila melanogaster. In this study, we have detected and localized the anti-HRP cross-reactivity in another insect species, the malaria mosquito vector Anopheles gambiae. We were able to identify and structurally elucidate fucosylated N-glycans including core mono- and difucosylated structures (responsible for anti-HRP cross reactivity) as well as a Lewis-type antennal modification on mosquito anionic N-glycans by applying enzymatic and chemical treatments. The three mosquito fucosyltransferase open reading frames (FucT6, FucTA and FucTC) required for the in vivo biosynthesis of the fucosylated N-glycan epitopes were identified in the Anopheles gambiae genome, cloned and recombinantly expressed in Pichia pastoris. Using a robust MALDI-TOF MS approach, we characterised the activity of the three recombinant fucosyltransferases in vitro and demonstrate that they share similar enzymatic properties as compared to their homologues from D. melanogaster and Apis mellifera. Thus, not only do we confirm the neural reactivity of anti-HRP in a mosquito species, but also demonstrate enzymatic activity for all its α1,3- and α1,6-fucosyltransferase homologues, whose specificity matches the results of glycomic analyses. PMID:26617287

  5. A recombinant fungal lectin for labeling truncated glycans on human cancer cells.

    Aymeric Audfray

    Full Text Available Cell surface glycoconjugates present alterations of their structures in chronic diseases and distinct oligosaccharide epitopes have been associated with cancer. Among them, truncated glycans present terminal non-reducing β-N-acetylglucosamine (GlcNAc residues that are rare on healthy tissues. Lectins from unconventional sources such as fungi or algi provide novel markers that bind specifically to such epitopes, but their availability may be challenging. A GlcNAc-binding lectin from the fruiting body of the fungus Psathyrella velutina (PVL has been produced in good yield in bacterial culture. A strong specificity for terminal GlcNAc residues was evidenced by glycan array. Affinity values obtained by microcalorimetry and surface plasmon resonance demonstrated a micromolar affinity for GlcNAcβ1-3Gal epitopes and for biantennary N-glycans with GlcNAcβ1-2Man capped branches. Crystal structure of PVL complexed with GlcNAcβ1-3Gal established the structural basis of the specificity. Labeling of several types of cancer cells and use of inhibitors of glycan metabolism indicated that rPVL binds to terminal GlcNAc but also to sialic acid (Neu5Ac. Analysis of glycosyltransferase expression confirmed the higher amount of GlcNAc present on cancer cells. rPVL binding is specific to cancer tissue and weak or no labeling is observed for healthy ones, except for stomach glands that present unique αGlcNAc-presenting mucins. In lung, breast and colon carcinomas, a clear delineation could be observed between cancer regions and surrounding healthy tissues. PVL is therefore a useful tool for labeling agalacto-glycans in cancer or other diseases.

  6. Characterizing the Release of Bioactive N-Glycans from Dairy Products by a Novel Endo-β-N-Acetylglucosaminidase

    KARAV, Sercan; Bell, Juliana Maria Leite Nobrega De Moura; Le Parc, Annabelle; Liu, Yan; Mills, David A.; Block, David E.; Barile, Daniela

    2015-01-01

    Endo-β-N-acetylglucosaminidase isolated from B. infantis ATCC 15697 (EndoBI-1) is a novel enzyme that cleaves N-N′-diacetyl chitobiose moieties found in the N-glycan core of high mannose, hybrid, and complex N-glycans. These conjugated N-glycans are recently shown as a new prebiotic source that stimulates the growth of a key infant gut microbe, Bifi-dobacterium longum subsp. Infantis. The effects of pH (4.45–8.45), temperature (27.5–77.5°C), reaction time (15–475 min), and enzyme/protein rati...

  7. Kinetic characterization of a novel endo-β-N-acetylglucosaminidase on concentrated bovine colostrum whey to release bioactive glycans

    KARAV, Sercan; Le Parc, Annabelle; de Moura Bell, Juliana Maria Leite Nobrega; Rouquié, Camille; Mills, David A.; Barile, Daniela; Block, David E.

    2015-01-01

    EndoBI-1 is a recently isolated endo-β-N-acetylglucosaminidase, which cleaves the N-N′-diacetyl chitobiose moiety found in the N-glycan core of high mannose, hybrid and complex N-glycans. These N-glycans have selective prebiotic activity for a key infant gut microbe, Bifidobacterium longum subsp. infantis. The broad specificity of EndoBI-1 suggests the enzyme may be useful for many applications, particularly for deglycosylating milk glycoproteins in dairy processing. To facilitate its commerc...

  8. Restricted motion of the conserved immunoglobulin G1 N-glycan is essential for efficient FcγRIIIa binding

    Subedi, Ganesh P.; Hanson, Quinlin M.; Barb, Adam W

    2014-01-01

    Immunoglobulin G1(IgG1)-based therapies are widespread and many function through interactions with low-affinity Fc γ receptors (FcγR). N-glycosylation of the IgG1 Fc domain is required for FcγR binding, though it is unclear why. Structures of the FcγR:Fc complex fail to explain this because the FcγR polypeptide does not bind the N-glycan. Here we identify a link between motion of the N-glycan and Fc:FcγRIIIa affinity that explains the N-glycan requirement. Fc F241 and F243 mutations decreased...

  9. Cell surface N-glycans influence the level of functional E-cadherin at the cell–cell border

    M Kristen Hall; Douglas A Weidner; Sahil Dayal; Ruth A. Schwalbe

    2014-01-01

    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...

  10. Comparative Genome Sequence Analysis Reveals the Extent of Diversity and Conservation for Glycan-Associated Proteins in Burkholderia spp.

    Ong, Hui San; Mohamed, Rahmah; Firdaus-Raih, Mohd

    2012-01-01

    Members of the Burkholderia family occupy diverse ecological niches. In pathogenic family members, glycan-associated proteins are often linked to functions that include virulence, protein conformation maintenance, surface recognition, cell adhesion, and immune system evasion. Comparative analysis of available Burkholderia genomes has revealed a core set of 178 glycan-associated proteins shared by all Burkholderia of which 68 are homologous to known essential genes. The genome sequence compari...

  11. Microfluidic Chip-LC/MS-based Glycomic Analysis Revealed Distinct N-glycan Profile of Rat Serum

    Wei-Na Gao; Lee-Fong Yau; Liang Liu; Xing Zeng; Da-Can Chen; Min Jiang; Ju Liu; Jing-Rong Wang; Zhi-Hong Jiang

    2015-01-01

    The rat is an important alternative for studying human pathology owing to certain similarities to humans. Glycomic studies on rat serum have revealed that variations in the N-glycans of glycoproteins correlated with disease progression, which is consistent with the findings in human serum. Therefore, we comprehensively characterized the rat serum N-glycome using microfluidic chip-LC-ESI-QTOF MS and MS/MS techniques. In total, 282 N-glycans, including isomers, were identified. This study is th...

  12. Comprehensive analysis of protein glycosylation by solid-phase extraction of N-linked glycans and glycosite-containing peptides

    Sun, Shisheng; Shah, Punit; Eshghi, Shadi Toghi; Yang, Weiming; Trikannad, Namita; Yang, Shuang; Chen, Lijun; Aiyetan, Paul; Höti, Naseruddin; Zhang, Zhen; Chan, Daniel W.; Hui ZHANG

    2015-01-01

    Comprehensive characterization of protein glycosylation is critical for understanding the structure and function of glycoproteins. However, due to the complexity and heterogeneity of glycoprotein conformations, current glycoprotein analyses focus mainly on either the de-glycosylated glycosylation site (glycosite)-containing peptides or the released glycans. Here, we describe a chemoenzymatic method called solid phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) for ...

  13. N-glycans of the porcine nematode parasite Ascaris suum are modified with phosphorylcholine and core fucose residues

    Pöltl, Gerald; Kerner, Denise; Paschinger, Katharina; Wilson, Iain B. H.

    2006-01-01

    In recent years, the glycoconjugates of many parasitic nematodes have attracted interest due to their immunogenic and immunomodulatory nature. Previous studies with the porcine roundworm parasite Ascaris suum have focussed on its glycosphingolipids which were found, in part, to be modified by phosphorylcholine. Using mass spectrometry and Western blotting, we have now analysed the PNGase A-released N-glycans of adults of this species. The presence of hybrid, bi- and triantennary N-glycans, so...

  14. Site-specific glycoproteomics confirms that protein structure dictates formation of N-glycan type, core fucosylation and branching.

    Thaysen-Andersen, Morten; Packer, Nicolle H

    2012-11-01

    Growing evidence indicates that the individualized and highly reproducible N-glycan repertoires on each protein glycosylation site modulate function. Relationships between protein structures and the resulting N-glycoforms have previously been observed, but remain to be quantitatively confirmed and examined in detail to define the responsible mechanisms in the conserved mammalian glycosylation machinery. Here, we investigate this relationship by manually extracting and analyzing quantitative and qualitative site-specific glycoprofiling data from 117 research papers. Specifically, N-glycan structural motifs were correlated with the structure of the protein carriers, focusing on the solvent accessibility of the individual glycosylation sites and the physicochemical properties of the surrounding polypeptide chains. In total, 474 glycosylation sites from 169 mammalian N-glycoproteins originating from different tissues/body fluids were investigated. It was confirmed statistically that the N-glycan type, degree of core fucosylation and branching are strongly influenced by the glycosylation site accessibility. For these three N-glycan features, glycosylation sites carrying highly processed glycans were significantly more solvent-accessible than those carrying less processed counterparts. The glycosylation site accessibilities could be linked to molecular signatures at the primary and secondary protein levels, most notably to the glycoprotein size and the proportion of glycosylation sites located in accessible β-turns. In addition, the subcellular location of the glycoproteins influenced the formation of the N-glycan structures. These data confirm that protein structures dictate site-specific formation of several features of N-glycan structures by affecting the biosynthetic pathway. Mammals have, as such, evolved mechanisms enabling proteins to influence the N-glycans they present to the extracellular environment. PMID:22798492

  15. Automated Solution-Phase Synthesis of Insect Glycans to Probe the Binding Affinity of Pea Enation Mosaic Virus.

    Tang, Shu-Lun; Linz, Lucas B; Bonning, Bryony C; Pohl, Nicola L B

    2015-11-01

    Pea enation mosaic virus (PEMV)--a plant RNA virus transmitted exclusively by aphids--causes disease in multiple food crops. However, the aphid-virus interactions required for disease transmission are poorly understood. For virus transmission, PEMV binds to a heavily glycosylated receptor aminopeptidase N in the pea aphid gut and is transcytosed across the gut epithelium into the aphid body cavity prior to release in saliva as the aphid feeds. To investigate the role of glycans in PEMV-aphid interactions and explore the possibility of viral control through blocking a glycan interaction, we synthesized insect N-glycan terminal trimannosides by automated solution-phase synthesis. The route features a mannose building block with C-5 ester enforcing a β-linkage, which also provides a site for subsequent chain extension. The resulting insect N-glycan terminal trimannosides with fluorous tags were used in a fluorous microarray to analyze binding with fluorescein isothiocyanate-labeled PEMV; however, no specific binding between the insect glycan and PEMV was detected. To confirm these microarray results, we removed the fluorous tag from the trimannosides for isothermal titration calorimetry studies with unlabeled PEMV. The ITC studies confirmed the microarray results and suggested that this particular glycan-PEMV interaction is not involved in virus uptake and transport through the aphid. PMID:26457763

  16. Structural basis for diverse N-glycan recognition by HIV-1-neutralizing V1-V2-directed antibody PG16

    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.

  17. Growth phase-dependent expression of proteins with decreased plant-specific N-glycans and immunogenicity in tobacco BY2 cells

    2009-01-01

    Plants possess some desirable characteristics to synthesize recombinant glycoproteins for pharma-ceutical application.However,the mammalian glycoproteins produced in plants are somewhat different from their natural counterparts in terms of N-glycoforms.The immunogenicity of plant-specific glyco-epitopes is the major concern in human therapy.Here,the distribution of N-glycans in different growth phases of tobacco BY2 cells and their immunogenicity in mice were determined.It was ob-served that the percentage of β1,2-xylose and α1,3-fucose in proteins of growing cells increased and the corresponding protein extracts caused accelerated immune response in mice.Based on this observation,the recombinant erythropoietin in BY2 cells was expressed and characterized,and Western blot analysis showed that the recombinant erythropoietin contained a relatively small amount of plant-specific glyco-epitopes in the early phase of culture growth.This study may provide a simple but effective strategy for the production of therapeutic glycoproteins with human-like N-glycan structures in plant hosts to avoid a great allergenic risk.

  18. Structural studies on a non toxic homologue of type II RIPs from bitter gourd: Molecular basis of non toxicity, conformational selection and glycan structure

    Thyageshwar Chandran; Alok Sharma; M Vijayan

    2015-12-01

    The structures of nine independent crystals of bitter gourd seed lectin (BGSL), a non-toxic homologue of type II RIPS, and its sugar complexes have been determined. The four-chain, two-fold symmetric, protein is made up of two identical two-chain modules, each consisting of a catalytic chain and a lectin chain, connected by a disulphide bridge. The lectin chain is made up of two domains. Each domain carries a carbohydrate binding site in type II RIPS of known structure. BGSL has a sugar binding site only on one domain, thus impairing its interaction at the cell surface. The adenine binding site in the catalytic chain is defective. Thus, defects in sugar binding as well as adenine binding appear to contribute to the non-toxicity of the lectin. The plasticity of the molecule is mainly caused by the presence of two possible well defined conformations of a surface loop in the lectin chain. One of them is chosen in the sugar complexes, in a case of conformational selection, as the chosen conformation facilitates an additional interaction with the sugar, involving an arginyl residue in the loop. The -glycosylation of the lectin involves a plant-specific glycan while that in toxic type H RIPS of known structure involves a glycan which is animal as well as plant specific.

  19. Immune recruitment or suppression by glycan engineering of endogenous and therapeutic antibodies.

    Le, Ngoc Phuong Lan; Bowden, Thomas A; Struwe, Weston B; Crispin, Max

    2016-08-01

    Human serum IgG contains multiple glycoforms which exhibit a range of binding properties to effector molecules such as cellular Fc receptors. Emerging knowledge of how the Fc glycans contribute to the antibody structure and effector functions has opened new avenues for the exploitation of defined antibody glycoforms in the treatment of diseases. Here, we review the structure and activity of antibody glycoforms and highlight developments in antibody glycoengineering by both the manipulation of the cellular glycosylation machinery and by chemoenzymatic synthesis. We discuss wide ranging applications of antibody glycoengineering in the treatment of cancer, autoimmunity and inflammation. This article is part of a Special Issue entitled "Glycans in personalised medicine" Guest Editor: Professor Gordan Lauc. PMID:27105835

  20. Mucins and associated glycan signatures in colon adenoma-carcinoma sequence

    Krishn, Shiv Ram; Kaur, Sukhwinder; Smith, Lynette M;

    2016-01-01

    study, we investigated the combined expression of multiple mucins and mucin-associated glycans during the adenoma-carcinoma sequence of colon cancer progression. Further, we evaluated their applicability as markers for differentiating adenomas/adenocarcinomas from hyperplastic polyps....... Immunohistochemical analyses performed on colon disease tissue microarrays revealed downregulation of MUC2 and MUC4 expression (p < 0.0001) while MUC1 and MUC5AC expressions were upregulated (p = 0.01) during adenoma-adenocarcinoma progression. Expression of MUC17 was downregulated in inflamed tissues compared to...... normal tissues, but its increased expression differentiated adenomas (p = 0.0028) and adenocarcinomas (p = 0.025) from inflammation. Glycan epitope-Tn/STn on MUC1 showed higher expression in hyperplastic polyps (p = 0.023), adenomas (p = 0.042) and adenocarcinomas (p = 0.0096) compared to normal tissues...

  1. Effect of Separation Temperature on Structure Specific Glycan Migration in Capillary Electrophoresis.

    Guttman, Andras; Kerekgyarto, Marta; Jarvas, Gabor

    2015-12-01

    Temperature dependent differential migration shifts were studied in capillary electrophoresis between linear (maltooligosaccharides) and branched (sialylated, neutral and core fucosylated biantennary IgG glycans) carbohydrates. Background electrolytes without as well as with low and high molecular weight additives (ethylene glycol, linear polyacrylamide and poly(ethylene oxide)) were investigated for this phenomena in the temperature range of 20-50 °C. Glucose unit (GU) value shifts were observed with increasing temperature for the all IgG glycans both in additive-free and additive-containing background electrolytes, emphasizing the importance of tight temperature control during glycosylation analysis by capillary electrophoresis. The activation energy concept was applied to understand the structure specific electrophoretic migration of the different sugar molecules. Activation energy values were derived from the slopes of the Arrhenius plots of logarithmic mobility vs reciprocal absolute temperature and compared for the linear and branched sugars as well as for the various background electrolyte additives. PMID:26544759

  2. Glycan elongation beyond the mucin associated Tn antigen protects tumor cells from immune-mediated killing

    Madsen, Caroline B; Lavrsen, Kirstine; Steentoft, Catharina; Vester-Christensen, Malene B; Clausen, Henrik; Wandall, Hans H; Pedersen, Anders Elm

    2013-01-01

    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...... 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...

  3. Sialoglycoproteins and N-Glycans from Secreted Exosomes of Ovarian Carcinoma Cells

    Escrevente, Cristina; Grammel, Nicolas; Kandzia, Sebastian; Zeiser, Johannes; Tranfield, Erin M; Conradt, Harald S.; Costa, Júlia

    2013-01-01

    Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abun...

  4. The N-glycan Glycoprotein Deglycosylation Complex (Gpd) from Capnocytophaga canimorsus Deglycosylates Human IgG

    Renzi, Francesco; Manfredi, Pablo; Mally, Manuela; Moes, Suzanne; Jenö, Paul; Cornelis, Guy R

    2011-01-01

    Author Summary Capnocytophaga canimorsus are Gram-negative bacteria from the normal oral flora of dogs and cats. They cause rare but severe infections in humans that have been bitten or simply licked by a dog or cat. Fulminant septicemia and peripheral gangrene with a high mortality are the most common symptoms. A surprising feature of these bacteria is their capacity to feed by foraging the glycan moieties of glycoproteins from animal cells, including phagocytes. Here we show that C. canimor...

  5. Native and modified N-linked glycan analysis by HILIC/MS

    Křenková, Jana; Šesták, Jozef; Moravcová, Dana; Planeta, Josef; Foret, František

    2015. P-14. ISBN N. [International Symposium on Electro- and Liquid Phase-Separation Techniques (ITP2015) /22./ and the Nordic Separation Science (NoSSS) symposium /8./. 30.08.2015-03.09.2015, Helsinki] R&D Projects: GA ČR(CZ) GA14-06319S; GA ČR(CZ) GBP206/12/G014; GA MV VG20112015021 Institutional support: RVO:68081715 Keywords : oligosaccharides * glycans * HILIC * MS Subject RIV: CB - Analytical Chemistry, Separation

  6. N-glycan alterations are associated with drug resistance in human hepatocellular carcinoma

    Nakagawa Takahito

    2007-05-01

    Full Text Available Abstract Background Correlations of disease phenotypes with glycosylation changes have been analysed intensively in the tumor biology field. Glycoforms potentially associated with carcinogenesis, tumor progression and cancer metastasis have been identified. In cancer therapy, drug resistance is a severe problem, reducing therapeutic effect of drugs and adding to patient suffering. Although multiple mechanisms likely underlie resistance of cancer cells to anticancer drugs, including overexpression of transporters, the relationship of glycans to drug resistance is not well understood. Results We established epirubicin (EPI – and mitoxantrone (MIT – resistant cell lines (HLE-EPI and HLE-MIT from the human hepatocellular carcinoma cell line (HLE. HLE-EPI and HLE-MIT overexpressed transporters MDR1/ABCB1 and BCRP/ABCG2, respectively. Here we compared the glycomics of HLE-EPI and HLE-MIT cells with the parental HLE line. Core fucosylated triantennary oligosaccharides were increased in the two resistant lines. We investigated mRNA levels of glycosyltransferases synthesizing this oligosaccharide, namely, N-acetylglucosaminyltransferase (GnT-IVa, GnT-IVb and α1,6-fucosyltransferase (α1,6-FucT, and found that α1,6-FucT was particularly overexpressed in HLE-MIT cells. In HLE-EPI cells, GnT-IVa expression was decreased, while GnT-IVb was increased. Both GnT-IVs were downregulated in HLE-MIT cells. HLE-MIT cells also showed decreases in fucosylated tetraantennary oligosaccharide, the product of GnT-V. GnT-V expression was decreased in both lines, but particularly so in HLE-MIT cells. Thus both N-glycan and glycosyltransferase expression was altered as cells acquired tolerance, suggesting novel mechanisms of drug resistance. Conclusion N-glycan and glycosyltransferase expression in HLE-EPI and HLE-MIT were analysed and presented that glycans altered according with acquired tolerance. These results suggested novel mechanisms of drug resistance.

  7. Bisected, complex N-glycans and galectins in mouse mammary tumor progression and human breast cancer

    Miwa, Hazuki E.; Koba, Wade R; Fine, Eugene J; Giricz, Orsi; Kenny, Paraic A; Stanley, Pamela

    2013-01-01

    Bisected, complex N-glycans on glycoproteins are generated by the glycosyltransferase MGAT3 and cause reduced cell surface binding of galectins. Previously, we showed that MGAT3 reduces growth factor signaling and retards mammary tumor progression driven by the Polyoma middle T antigen (PyMT) expressed in mammary epithelium under the mouse mammary tumor virus (MMTV) promoter. However, the penetrance of the tumor phenotype became variable in mixed FVB/N and C57BL/6 female mice and we therefore...

  8. Oriented immobilization of PNgase F on a porous polymer monolith for rapid N-glycan release

    Szekrényes, A.; Křenková, J. (Jana); Keresztessy, Z.; Foret, F; Guttman, A

    2012-01-01

    We demonstrate a simple and rapid method for the oriented immobilization of peptide-N4-(Nacetyl- glucosaminyl) asparagine amidase F (PNGase F) on a porous polymer monolith. The oriented immobilization is based on the affinity of glutathione-S-transferase (GST) tagged PNGase F towards glutathione modified monolith prepared in the capillary format. This approach allows the oriented and easily replaceable immobilization of PNGase F for rapid and efficient release of N-linked glycans. The react...

  9. A serotonin-induced N-glycan switch regulates platelet aggregation.

    Mercado, Charles P.; Quintero, Maritza V.; Yicong Li; Preeti Singh; Byrd, Alicia K.; Krajang Talabnin; Mayumi Ishihara; Parastoo Azadi; Rusch, Nancy J.; Balagurunathan Kuberan; Luc Maroteaux; Fusun Kilic

    2013-01-01

    Serotonin (5-HT) is a multifunctional signaling molecule that plays different roles in a concentration-dependent manner. We demonstrated that elevated levels of plasma 5-HT accelerate platelet aggregation resulting in a hypercoagulable state in which the platelet surface becomes occupied by several glycoproteins. Here we study the novel hypothesis that an elevated level of plasma 5-HT results in modification of the content of N-glycans on the platelet surface and this abnormality is associate...

  10. Targeting the glycans of gp120: a novel approach aimed at the Achilles heel of HIV

    Balzarini, Jan

    2005-01-01

    The development of drug resistance in HIV compromises the long-term efficacy of current therapies. Furthermore, vaccine development faces huge problems, mainly because of the low antigenicity and immunogenicity of the HIV envelope glycoprotein gp120 and the efficient hiding of highly immunogenic epitopes by its glycans. There is evidence that mutant HIV strains containing glycosylation site deletions trigger the production of specific neutralising antibodies to previously hidden gp120 epitope...

  11. Glycan-Foraging Systems Reveal the Adaptation of Capnocytophaga canimorsus to the Dog Mouth

    Renzi, Francesco; Manfredi, Pablo; Dol, Mélanie; Fu, Jian; Vincent, Stéphane; Cornelis, Guy Richard

    2015-01-01

    ABSTRACT Capnocytophaga canimorsus is known to form two kinds of cells on blood agar plates (coccoid and bacillary), evoking phase variation. When grown in coculture with animal cells these bacteria appeared only as bacilli, but in the presence of vancomycin they were round, indicating that coccoid shapes likely result from weakening of the peptidoglycan layer. Polysaccharide utilization locus 5 (PUL5) and sialidase mutant bacteria, unable to retrieve glycans from glycoproteins, grew less tha...

  12. Travelling-wave ion mobility and negative ion fragmentation of high-mannose N-glycans.

    Harvey, David J; Scarff, Charlotte A; Edgeworth, Matthew; Struwe, Weston B; Pagel, Kevin; Thalassinos, Konstantinos; Crispin, Max; Scrivens, Jim

    2016-03-01

    The isomeric structure of high-mannose N-glycans can significantly impact biological recognition events. Here, the utility of travelling-wave ion mobility mass spectrometry for isomer separation of high-mannose N-glycans is investigated. Negative ion fragmentation using collision-induced dissociation gave more informative spectra than positive ion spectra with mass-different fragment ions characterizing many of the isomers. Isomer separation by ion mobility in both ionization modes was generally limited, with the arrival time distributions (ATD) often showing little sign of isomers. However, isomers could be partially resolved by plotting extracted fragment ATDs of the diagnostic fragment ions from the negative ion spectra, and the fragmentation spectra of the isomers could be extracted by using ions from limited areas of the ATD peak. In some cases, asymmetric ATDs were observed, but no isomers could be detected by fragmentation. In these cases, it was assumed that conformers or anomers were being separated. Collision cross sections of the isomers in positive and negative fragmentation mode were estimated from travelling-wave ion mobility mass spectrometry data using dextran glycans as calibrant. More complete collision cross section data were achieved in negative ion mode by utilizing the diagnostic fragment ions. Examples of isomer separations are shown for N-glycans released from the well-characterized glycoproteins chicken ovalbumin, porcine thyroglobulin and gp120 from the human immunodeficiency virus. In addition to the cross-sectional data, details of the negative ion collision-induced dissociation spectra of all resolved isomers are discussed. PMID:26956389

  13. Lectin-Glycan Interaction Network-Based Identification of Host Receptors of Microbial Pathogenic Adhesins

    Ielasi, Francesco S.; Alioscha-Perez, Mitchel; Donohue, Dagmara; Claes, Sandra; Sahli, Hichem; Schols, Dominique

    2016-01-01

    ABSTRACT The first step in the infection of humans by microbial pathogens is their adherence to host tissue cells, which is frequently based on the binding of carbohydrate-binding proteins (lectin-like adhesins) to human cell receptors that expose glycans. In only a few cases have the human receptors of pathogenic adhesins been described. A novel strategy—based on the construction of a lectin-glycan interaction (LGI) network—to identify the potential human binding receptors for pathogenic adhesins with lectin activity was developed. The new approach is based on linking glycan array screening results of these adhesins to a human glycoprotein database via the construction of an LGI network. This strategy was used to detect human receptors for virulent Escherichia coli (FimH adhesin), and the fungal pathogens Candida albicans (Als1p and Als3p adhesins) and C. glabrata (Epa1, Epa6, and Epa7 adhesins), which cause candidiasis. This LGI network strategy allows the profiling of potential adhesin binding receptors in the host with prioritization, based on experimental binding data, of the most relevant interactions. New potential targets for the selected adhesins were predicted and experimentally confirmed. This methodology was also used to predict lectin interactions with envelope glycoproteins of human-pathogenic viruses. It was shown that this strategy was successful in revealing that the FimH adhesin has anti-HIV activity. PMID:27406561

  14. Glycan heterogeneity on gold nanoparticles increases lectin discrimination capacity in label-free multiplexed bioassays.

    Otten, Lucienne; Vlachou, Denise; Richards, Sarah-Jane; Gibson, Matthew I

    2016-07-21

    The development of new analytical tools as point-of-care biosensors is crucial to combat the spread of infectious diseases, especially in the context of drug-resistant organisms, or to detect biological warfare agents. Glycan/lectin interactions drive a wide range of recognition and signal transduction processes within nature and are often the first site of adhesion/recognition during infection making them appealing targets for biosensors. Glycosylated gold nanoparticles have been developed that change colour from red to blue upon interaction with carbohydrate-binding proteins and may find use as biosensors, but are limited by the inherent promiscuity of some of these interactions. Here we mimic the natural heterogeneity of cell-surface glycans by displaying mixed monolayers of glycans on the surface of gold nanoparticles. These are then used in a multiplexed, label-free bioassay to create 'barcodes' which describe the lectin based on its binding profile. The increased information content encoded by using complex mixtures of a few sugars, rather than increased numbers of different sugars makes this approach both scalable and accessible. These nanoparticles show increased lectin identification power at a range of lectin concentrations, relative to single-channel sensors. It was also found that some information about the concentration of the lectins can be extracted, all from just a simple colour change, taking this technology closer to being a realistic biosensor. PMID:27181289

  15. Glycan heterogeneity on gold nanoparticles increases lectin discrimination capacity in label-free multiplexed bioassays†

    Otten, Lucienne; Vlachou, Denise; Richards, Sarah-Jane; Gibson, Matthew I.

    2016-01-01

    The development of new analytical tools as point-of-care biosensors is crucial to combat the spread of infectious diseases, especially in the context of drug-resistant organisms, or to detect biological warfare agents. Glycan/lectin interactions drive a wide range of recognition and signal transduction processes within nature and are often the first site of adhesion/recognition during infection making them appealing targets for biosensors. Glycosylated gold nanoparticles have been developed that change colour from red to blue upon interaction with carbohydrate-binding proteins and may find use as biosensors, but are limited by the inherent promiscuity of some of these interactions. Here we mimic the natural heterogeneity of cell-surface glycans by displaying mixed monolayers of glycans on the surface of gold nanoparticles. These are then used in a multiplexed, label-free bioassay to create ‘barcodes’ which describe the lectin based on its binding profile. The increased information content encoded by using complex mixtures of a few sugars, rather than increased numbers of different sugars makes this approach both scalable and accessible. These nanoparticles show increased lectin identification power at a range of lectin concentrations, relative to single-channel sensors. It was also found that some information about the concentration of the lectins can be extracted, all from just a simple colour change, taking this technology closer to being a realistic biosensor. PMID:27181289

  16. Enzymatic passaging of human embryonic stem cells alters central carbon metabolism and glycan abundance

    Badur, Mehmet G.; Zhang, Hui; Metallo, Christian M.

    2016-01-01

    To realize the potential of human embryonic stem cells (hESCs) in regenerative medicine and drug discovery applications, large numbers of cells that accurately recapitulate cell and tissue function must be robustly produced. Previous studies have suggested that genetic instability and epigenetic changes occur as a consequence of enzymatic passaging. However, the potential impacts of such passaging methods on the metabolism of hESCs have not been described. Using stable isotope tracing and mass spectrometry-based metabolomics, we have explored how different passaging reagents impact hESC metabolism. Enzymatic passaging caused significant decreases in glucose utilization throughout central carbon metabolism along with attenuated de novo lipogenesis. In addition, we developed and validated a method for rapidly quantifying glycan abundance and isotopic labeling in hydrolyzed biomass. Enzymatic passaging reagents significantly altered levels of glycans immediately after digestion but surprisingly glucose contribution to glycans was not affected. These results demonstrate that there is an immediate effect on hESC metabolism after enzymatic passaging in both central carbon metabolism and biosynthesis. HESCs subjected to enzymatic passaging are routinely placed in a state requiring re-synthesis of biomass components, subtly influencing their metabolic needs in a manner that may impact cell performance in regenerative medicine applications. PMID:26289220

  17. An improved lectin-based method for the detection of mucin-type O-glycans in biological samples.

    Lee, Cheng-Siang; Muthusamy, Arivalagan; Abdul-Rahman, Puteri Shafinaz; Bhavanandan, Veer P; Hashim, Onn Haji

    2013-06-21

    Mucins and mucin-type glycoproteins, collectively referred to as mucin-type O-glycans, are implicated in many important biological functions and pathological conditions, including malignancy. Presently, there is no reliable method to measure the total mucin-type O-glycans of a sample, which may contain one or more of these macromolecules of unknown structures. We report the development of an improved microassay that is based on the binding of lectins to the unique and constant GalNAc-Ser/Thr structural feature of mucin-type O-glycans. Since the sugar-amino acid linkage in the mucin-type O-glycans is invariably cryptic, we first chemically removed the heterogeneous peripheral and core saccharides of model glycoconjugates before examining for their interactions using an enzyme-linked lectin assay (ELLA). Desialylation of the model glycoconjugates led to maximal binding of the lectins but additional treatments such as Smith degradation did not result in increased binding. Of the lectins tested for their ability to probe the desialylated O-glycans, jacalin showed the highest sensitivity followed by champedak galactose binding (CGB) lectin and Vicia villosa agglutinin. Further improvement in the sensitivity of ELLA was achieved by using microtiter plates that were pre-coated with the CGB lectin, which increased the specificity of the assay to mucin-type O-glycans. Finally, the applicability of the developed sandwich ELLA to crude samples was demonstrated by estimating trace quantities of the mucin-type O-glycans in the human serum. PMID:23665615

  18. Turning-Off Signaling by Siglecs, Selectins, and Galectins: Chemical Inhibition of Glycan-Dependent Interactions in Cancer

    Cagnoni, Alejandro J.; Pérez Sáez, Juan M.; Rabinovich, Gabriel A.; Mariño, Karina V.

    2016-01-01

    Aberrant glycosylation, a common feature associated with malignancy, has been implicated in important events during cancer progression. Our understanding of the role of glycans in cancer has grown exponentially in the last few years, concurrent with important advances in glycomics and glycoproteomic technologies, paving the way for the validation of a number of glycan structures as potential glycobiomarkers. However, the molecular bases underlying cancer-associated glycan modifications are still far from understood. Glycans exhibit a natural heterogeneity, crucial for their diverse functional roles as specific carriers of biologically relevant information. This information is decoded by families of proteins named lectins, including sialic acid-binding immunoglobulin (Ig)-like lectins (siglecs), C-type lectin receptors (CLRs), and galectins. Siglecs are primarily expressed on the surface of immune cells and differentially control innate and adaptive immune responses. Among CLRs, selectins are a family of cell adhesion molecules that mediate interactions between cancer cells and platelets, leukocytes, and endothelial cells, thus facilitating tumor cell invasion and metastasis. Galectins, a family of soluble proteins that bind β-galactoside-containing glycans, have been implicated in diverse events associated with cancer biology such as apoptosis, homotypic cell aggregation, angiogenesis, cell migration, and tumor-immune escape. Consequently, individual members of these lectin families have become promising targets for the design of novel anticancer therapies. During the past decade, a number of inhibitors of lectin–glycan interactions have been developed including small-molecule inhibitors, multivalent saccharide ligands, and more recently peptides and peptidomimetics have offered alternatives for tackling tumor progression. In this article, we review the current status of the discovery and development of chemical lectin inhibitors and discuss novel strategies to

  19. Structure of a SusD Homologue, BT1043, Involved in Mucin O-Glycan Utilization in a Prominent Human Gut Symbiont

    Koropatkin, Nicole; Martens, Eric C.; Gordon, Jeffrey I.; Smith, Thomas J.; (Danforth); (WU-MED)

    2009-05-21

    Mammalian distal gut bacteria have an expanded capacity to utilize glycans. In the absence of dietary sources, some species rely on host-derived mucosal glycans. The ability of Bacteroides thetaiotaomicron, a prominent human gut symbiont, to forage host glycans contributes to both its ability to persist within an individual host and its ability to be transmitted naturally to new hosts at birth. The molecular basis of host glycan recognition by this species is still unknown but likely occurs through an expanded suite of outermembrane glycan-binding proteins that are the primary interface between B. thetaiotaomicron and its environment. Presented here is the atomic structure of the B. thetaiotaomicron protein BT1043, an outer membrane lipoprotein involved in host glycan metabolism. Despite a lack of detectable amino acid sequence similarity, BT1043 is a structural homologue of the B. thetaiotaomicron starch-binding protein SusD. Both structures are dominated by tetratrico peptide repeats that may facilitate association with outer membrane {beta}-barrel transporters required for glycan uptake. The structure of BT1043 complexed with N-acetyllactosamine reveals that recognition is mediated via hydrogen bonding interactions with the reducing end of {beta}-N-acetylglucosamine, suggesting a role in binding glycans liberated from the mucin polypeptide. This is in contrast to CBM 32 family members that target the terminal nonreducing galactose residue of mucin glycans. The highly articulated glycan-binding pocket of BT1043 suggests that binding of ligands to BT1043 relies more upon interactions with the composite sugar residues than upon overall ligand conformation as previously observed for SusD. The diversity in amino acid sequence level likely reflects early divergence from a common ancestor, while the unique and conserved {alpha}-helical fold the SusD family suggests a similar function in glycan uptake.

  20. An Integrated Solution-Based Rapid Sample Preparation Procedure for the Analysis of N-Glycans From Therapeutic Monoclonal Antibodies.

    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. PMID:26886304

  1. Application of highly sensitive UPLC-MS to determine biodistribution at tracer doses: validation with the 5-HT1A ligand [18F]FPWAY

    High-sensitivity and high-resolution LC/MS instrumentation has been applied in positron emission tomography (PET) radiopharmaceutical development to provide quantitative measurement of the mass of radiotracers extracted from tissues of rats. We employed the highly sensitive Waters Q-TOF premier MS coupled with an Acquity UPLC system to demonstrate that LC-MS can generate ex vivo biodistribution data for PET 5-HT1A ligand FPWAY without the need to radiolabel. For the biodistribution studies, we injected rats with [18F]FPWAY containing various amounts of nonradioactive FPWAY. At the end of the allotted time, the animals were killed and six regions of brain and plasma from each animal were processed for quantitative measurement of parent compound concentration by LC-MS. These data were then converted to the differential uptake ratio DUR (%ID/g*body weight/100) and the brain tissue-specific binding ratio to allow direct comparison with data obtained by gamma counting of the coinjected radioactive [18F]FPWAY. The DUR and the brain tissue-specific binding ratio calculated using the LC-MS method were highly correlated to the values obtained by standard radioactivity measurements of [18F]FPWAY. In conclusion, there was significant concordance between the LC/MS and radioactivity method in determination of DUR and the specific binding ratio in the rat brain. This concordance indicated that high-sensitivity LC/MS is an indispensable tool in evaluating the quantity of administered chemical in tissue as part of the development of new molecular imaging probes.

  2. Correlation between the glycan variations and defibrinogenating activities of acutobin and its recombinant glycoforms.

    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.

  3. Incorporation of a non-human glycan mediates human susceptibility to a bacterial toxin

    Byres, Emma; Paton, Adrienne W.; Paton, James C.; Löfling, Jonas C.; Smith, David F.; Wilce, Matthew C.J.; Talbot, Ursula M.; Chong, Damien C.; Yu, Hai; Huang, Shengshu; Chen, Xi; Varki, Nissi M.; Varki, Ajit; Rossjohn, Jamie; Beddoe, Travis (Emory-MED); (UCD); (Adelaide); (Monash)

    2009-01-30

    AB{sub 5} toxins comprise an A subunit that corrupts essential eukaryotic cell functions, and pentameric B subunits that direct target-cell uptake after binding surface glycans. Subtilase cytotoxin (SubAB) is an AB{sub 5} toxin secreted by Shiga toxigenic Escherichia coli (STEC), which causes serious gastrointestinal disease in humans. SubAB causes haemolytic uraemic syndrome-like pathology in mice through SubA-mediated cleavage of BiP/GRP78, an essential endoplasmic reticulum chaperone. Here we show that SubB has a strong preference for glycans terminating in the sialic acid N-glycolylneuraminic acid (Neu5Gc), a monosaccharide not synthesized in humans. Structures of SubB-Neu5Gc complexes revealed the basis for this specificity, and mutagenesis of key SubB residues abrogated in vitro glycan recognition, cell binding and cytotoxicity. SubAB specificity for Neu5Gc was confirmed using mouse tissues with a human-like deficiency of Neu5Gc and human cell lines fed with Neu5Gc. Despite lack of Neu5Gc biosynthesis in humans, assimilation of dietary Neu5Gc creates high-affinity receptors on human gut epithelia and kidney vasculature. This, and the lack of Neu5Gc-containing body fluid competitors in humans, confers susceptibility to the gastrointestinal and systemic toxicities of SubAB. Ironically, foods rich in Neu5Gc are the most common source of STEC contamination. Thus a bacterial toxin's receptor is generated by metabolic incorporation of an exogenous factor derived from food.

  4. Differential expression of glycans in the hippocampus of rats trained on an inhibitory learning paradigm.

    Hidalgo, Alejandra; Burgos, Valeria; Viola, Haydée; Medina, Jorge; Argibay, Pablo

    2006-12-01

    The glycan chains of glycoconjugates play important roles in cell-cell and cell-matrix interactions. In the CNS, previous studies on learning and memory suggest the importance of oligosaccharides attached to glycoconjugates in the modulation of synaptic connections. We studied the hippocampal glycan distribution of rats subject to an inhibitory avoidance task. The expression of glycans was examined by lectin-histochemistry using Vicia villosa lectin (VVL) for terminal alpha/beta N-acetylgalactosamine (alpha/beta GalNAc); Galanthus nivalus lectin (GNL) for terminal mannose alpha-1,3 (Man alpha-1,3); Peanut agglutinin (PNA) for galactose beta-1,3N-acetylgalactosamine (Gal beta-1,3 GalNAc); Erythrina cristagalli lectin (ECL) for galactose beta-1,4 N-acetylglucosamine (Gal beta-1,4 GlcNAc); Sambucus nigra lectin (SNA) for sialic acid alpha-2.6 galactose (SA alpha-2,6 Gal); Maackia amurensis lectin II (MAL II) for sialic acid alpha-2,3 (SA alpha-2,3); Wheat germ agglutinin (WGA) for terminal N-acetylglucosamine with/ without sialic acid (GlcNAc wo SA); succynilated WGA (sWGA) for terminal N-acetylglucosamine without sialic acid (terminal GlcNAc without SA); Griffonia simplicifolia lectin II (GSL II) for terminal alpha/beta N-acetylglucosamine (alpha/beta GlcNAc terminal); and Lotus tetragonolobus lectin (LTL) alpha-fucose. Two groups of 10 animals were examined: non-trained (Control) and Trained rats. ECL, sWGA and GSL II were negative for both groups in all the hippocampal subfields studied. For both groups, VVL was negative in CA4 and granular cells of the Dentate Gyrus (DG) and LTL was negative in the CA4 subfield. Expression of alpha/beta GalNAc, alpha-fucose and GlcNAc in other hippocampal subflields was positive, with no differences between groups. However, expression of Man alpha-1,3 was significantly higher in the CA1, CA2, CA3, and CA4 subfields in the Trained group. On the other hand, expression of Gal beta-1,3 GalNAc was significantly low in CA4 and DG in the

  5. Synthesis, processing, and function of N-glycans in N-glycoproteins

    Bieberich, Erhard

    2014-01-01

    Many membrane-resident and secrected proteins, including growth factors and their receptors are N-glycosylated. The initial N-glycan structure consists of 14 sugar residues (Glc3Man9GlcNAc2) that are first synthesized in the endoplasmic reticulum (ER) as a branched structure on a lipid anchor (dolicholpyrophosphate) and then co-translationally, “en bloc” transferred and linked via N-acetylglucosamine (GlcNAc) to asparagine within a specific N-glycosylation acceptor sequence (Asn-X-Ser/Thr) of...

  6. Branch Specific Sialylation of IgG-Fc Glycans by ST6Gal-I

    Barb, Adam W; Brady, Evan K.; Prestegard, James H.

    2009-01-01

    Sialylated forms of the Fc fragment of immunoglobulin G, produced by the human α2–6 sialyltransferase ST6Gal-I, were identified as potent anti-inflammatory mediators in a mouse model of rheumatoid arthritis and are potentially the active components in IVIG therapies. The activities and specificities of hST6Gal-I are, however, poorly characterized. Here MS and NMR methodology demonstrates glycan modification occurs in a branch-specific manner with the α1–3Man branch of the complex, biantennary...

  7. Concurrent automated sequencing of the glycan and peptide portions of O-linked glycopeptide anions by ultraviolet photodissociation mass spectrometry.

    Madsen, James A; Ko, Byoung Joon; Xu, Hua; Iwashkiw, Jeremy A; Robotham, Scott A; Shaw, Jared B; Feldman, Mario F; Brodbelt, Jennifer S

    2013-10-01

    O-Glycopeptides are often acidic owing to the frequent occurrence of acidic saccharides in the glycan, rendering traditional proteomic workflows that rely on positive mode tandem mass spectrometry (MS/MS) less effective. In this report, we demonstrate the utility of negative mode ultraviolet photodissociation (UVPD) MS for the characterization of acidic O-linked glycopeptide anions. This method was evaluated for a series of singly and multiply deprotonated glycopeptides from the model glycoprotein kappa casein, resulting in production of both peptide and glycan product ions that afforded 100% sequence coverage of the peptide and glycan moieties from a single MS/MS event. The most abundant and frequent peptide sequence ions were a/x-type products which, importantly, were found to retain the labile glycan modifications. The glycan-specific ions mainly arose from glycosidic bond cleavages (B, Y, C, and Z ions) in addition to some less common cross-ring cleavages. On the basis of the UVPD fragmentation patterns, an automated database searching strategy (based on the MassMatrix algorithm) was designed that is specific for the analysis of glycopeptide anions by UVPD. This algorithm was used to identify glycopeptides from mixtures of glycosylated and nonglycosylated peptides, sequence both glycan and peptide moieties simultaneously, and pinpoint the correct site(s) of glycosylation. This methodology was applied to uncover novel site-specificity of the O-linked glycosylated OmpA/MotB from the "superbug" A. baumannii to help aid in the elucidation of the functional role that protein glycosylation plays in pathogenesis. PMID:24006841

  8. Disruption of O-GlcNAc cycling in C. elegans perturbs Nucleotide Sugar pools and Complex Glycans

    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.

  9. Dual Roles of O-Glucose Glycans Redundant with Monosaccharide O-Fucose on Notch in Notch Trafficking.

    Matsumoto, Kenjiroo; Ayukawa, Tomonori; Ishio, Akira; Sasamura, Takeshi; Yamakawa, Tomoko; Matsuno, Kenji

    2016-06-24

    Notch is a transmembrane receptor that mediates cell-cell interactions and controls various cell-fate specifications in metazoans. The extracellular domain of Notch contains multiple epidermal growth factor (EGF)-like repeats. At least five different glycans are found in distinct sites within these EGF-like repeats. The function of these individual glycans in Notch signaling has been investigated, primarily by disrupting their individual glycosyltransferases. However, we are just beginning to understand the potential functional interactions between these glycans. Monosaccharide O-fucose and O-glucose trisaccharide (O-glucose-xylose-xylose) are added to many of the Notch EGF-like repeats. In Drosophila, Shams adds a xylose specifically to the monosaccharide O-glucose. We found that loss of the terminal dixylose of O-glucose-linked saccharides had little effect on Notch signaling. However, our analyses of double mutants of shams and other genes required for glycan modifications revealed that both the monosaccharide O-glucose and the terminal dixylose of O-glucose-linked saccharides function redundantly with the monosaccharide O-fucose in Notch activation and trafficking. The terminal dixylose of O-glucose-linked saccharides and the monosaccharide O-glucose were required in distinct Notch trafficking processes: Notch transport from the apical plasma membrane to adherens junctions, and Notch export from the endoplasmic reticulum, respectively. Therefore, the monosaccharide O-glucose and terminal dixylose of O-glucose-linked saccharides have distinct activities in Notch trafficking, although a loss of these activities is compensated for by the presence of monosaccharide O-fucose. Given that various glycans attached to a protein motif may have redundant functions, our results suggest that these potential redundancies may lead to a serious underestimation of glycan functions. PMID:27129198

  10. Arsenite Regulates Prolongation of Glycan Residues of Membrane Glycoprotein: A Pivotal Study via Wax Physisorption Kinetics and FTIR Imaging

    Chih-Hung Lee

    2016-03-01

    Full Text Available Arsenic exposure results in several human cancers, including those of the skin, lung, and bladder. As skin cancers are the most common form, epidermal keratinocytes (KC are the main target of arsenic exposure. The mechanisms by which arsenic induces carcinogenesis remains unclear, but aberrant cell proliferation and dysregulated energy homeostasis play a significant role. Protein glycosylation is involved in many key physiological processes, including cell proliferation and differentiation. To evaluate whether arsenite exposure affected protein glycosylation, the alteration of chain length of glycan residues in arsenite treated skin cells was estimated. Herein we demonstrated that the protein glycosylation was adenosine triphosphate (ATP-dependent and regulated by arsenite exposure by using Fourier transform infrared (FTIR reflectance spectroscopy, synchrotron-radiation-based FTIR (SR-FTIR microspectroscopy, and wax physisorption kinetics coupled with focal-plane-array-based FTIR (WPK-FPA-FTIR imaging. We were able to estimate the relative length of surface protein-linked glycan residues on arsenite-treated skin cells, including primary KC and two skin cancer cell lines, HSC-1 and HaCaT cells. Differential physisorption of wax adsorbents adhered to long-chain (elongated type and short-chain (regular type glycan residues of glycoprotein of skin cell samples treated with various concentration of arsenite was measured. The physisorption ratio of beeswax remain/n-pentacosane remain for KC cells was increased during arsenite exposure. Interestingly, this increase was reversed after oligomycin (an ATP synthase inhibitor pretreatment, suggesting the chain length of protein-linked glycan residues is likely ATP-dependent. This is the first study to demonstrate the elongation and termination of surface protein-linked glycan residues using WPK-FPA-FTIR imaging in eukaryotes. Herein the result may provide a scientific basis to target surface protein

  11. Fluorinated carbon tag derivatization combined with fluorous solid-phase extraction: a new method for the highly sensitive and selective mass spectrometric analysis of glycans.

    Li, Lulu; Jiao, Jing; Cai, Yan; Zhang, Ying; Lu, Haojie

    2015-01-01

    The sensitive and specific detection of glycans via mass spectrometry (MS) remains a significant challenge due to their low abundance in complex biological mixtures, inherent lack of hydrophobicity, and suppression by other, more abundant biological molecules (proteins/peptides) or contaminants. A new strategy for the sensitive and selective MS analysis of glycans based on fluorous chemistry is reported. Glycan reducing ends were derivatized with a hydrophobic fluorinated carbon tag, increasing glycan ionization efficiency during MS by more than an order of magnitude. More importantly, the fluorinated carbon tag enabled efficient fluorous solid-phase extraction (FSPE) to specifically enrich the glycans from contaminated solutions and protein mixtures. Finally, we successfully analyzed the N-glycome in human serum using this new method. PMID:25884104

  12. N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin

    Lai, Ying-Jung J.; Chang, Hsiang-Hua D.; Lai, Hongyu; Xu, Yuan; Shiao, Frank; Huang, Nanxi; Li, Linpei; Lee, Ming-Shyue; Johnson, Michael D.; Wang, Jehng-Kang; Lin, Chen-Yong

    2015-01-01

    The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB), is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa) due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with ext...

  13. Structural Insights into Polymorphic ABO Glycan Binding by Helicobacter pylori.

    Moonens, Kristof; Gideonsson, Pär; Subedi, Suresh; Bugaytsova, Jeanna; Romaõ, Ema; Mendez, Melissa; Nordén, Jenny; Fallah, Mahsa; Rakhimova, Lena; Shevtsova, Anna; Lahmann, Martina; Castaldo, Gaetano; Brännström, Kristoffer; Coppens, Fanny; Lo, Alvin W; Ny, Tor; Solnick, Jay V; Vandenbussche, Guy; Oscarson, Stefan; Hammarström, Lennart; Arnqvist, Anna; Berg, Douglas E; Muyldermans, Serge; Borén, Thomas; Remaut, Han

    2016-01-13

    The Helicobacter pylori adhesin BabA binds mucosal ABO/Le(b) blood group (bg) carbohydrates. BabA facilitates bacterial attachment to gastric surfaces, increasing strain virulence and forming a recognized risk factor for peptic ulcers and gastric cancer. High sequence variation causes BabA functional diversity, but the underlying structural-molecular determinants are unknown. We generated X-ray structures of representative BabA isoforms that reveal a polymorphic, three-pronged Le(b) binding site. Two diversity loops, DL1 and DL2, provide adaptive control to binding affinity, notably ABO versus O bg preference. H. pylori strains can switch bg preference with single DL1 amino acid substitutions, and can coexpress functionally divergent BabA isoforms. The anchor point for receptor binding is the embrace of an ABO fucose residue by a disulfide-clasped loop, which is inactivated by reduction. Treatment with the redox-active pharmaceutic N-acetylcysteine lowers gastric mucosal neutrophil infiltration in H. pylori-infected Le(b)-expressing mice, providing perspectives on possible H. pylori eradication therapies. PMID:26764597

  14. Polysialylated N-Glycans Identified in Human Serum Through Combined Developments in Sample Preparation, Separations and Electrospray ionization-mass spectrometry

    Kronewitter, Scott R.; Marginean, Ioan; Cox, Jonathan T.; Zhao, Rui; Hagler, Clay D.; Shukla, Anil K.; Carlson, Timothy S.; Adkins, Joshua N.; Camp, David G.; Moore, Ronald J.; Rodland, Karin D.; Smith, Richard D.

    2014-09-02

    The N-glycan diversity of human serum glycoproteins, i.e. the human blood serum N-glycome, is complex due to the range of glycan structures potentially synthesizable by human glycosylation enzymes. The reported glycome, however, is limited by methods of sample preparation, available analytical platforms, e.g., based upon electrospray ionization-mass spectrometry (ESI-MS), and software tools for data analysis. In this report, several improvements have been implemented in sample preparation and analysis to extend ESI-MS glycan characterization and to provide an improved view of glycan diversity. Sample preparation improvements include acidified, microwave-accelerated, PNGase F N-glycan release, and sodium borohydride reduction were optimized to improve quantitative yields and conserve the number of glycoforms detected. Two-stage desalting (during solid phase extraction and on the analytical column) increased the sensitivity by reducing analyte signal division between multiple reducing-end-forms or cation adducts. On-line separations were improved by using extended length graphitized carbon columns and adding TFA as an acid modifier to a formic acid/reversed phase gradient which provides additional resolving power and significantly improved desorption of both large and heavily sialylated glycans. To improve MS sensitivity and provide gentler ionization conditions at the source-MS interface, subambient pressure ionization with nanoelectrospray (SPIN) has been utilized. When method improvements are combined together with the Glycomics Quintavariate Informed Quantification (GlyQ-IQ) recently described1 these technologies demonstrate the ability to significantly extend glycan detection sensitivity and provide expanded glycan coverage. We demonstrate application of these advances in the context of the human serum glycome, and for which our initial observations include detection of a new class of heavily sialylated N-glycans, including polysialylated N-glycans.

  15. Engineering Yarrowia lipolytica to produce glycoproteins homogeneously modified with the universal Man3GlcNAc2 N-glycan core.

    Karen De Pourcq

    Full Text Available Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as "generally recognized as safe." Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man(3GlcNAc(2 structures on its glycoproteins. We obtained unprecedented levels of homogeneity of this glycanstructure. This is the ideal starting point for building human-like sugars. Disruption of the ALG3 gene resulted in modification of proteins mainly with Man(5GlcNAc(2 and GlcMan(5GlcNAc(2 glycans, and to a lesser extent with Glc(2Man(5GlcNAc(2 glycans. To avoid underoccupancy of glycosylation sites, we concomitantly overexpressed ALG6. We also explored several approaches to remove the terminal glucose residues, which hamper further humanization of N-glycosylation; overexpression of the heterodimeric Apergillus niger glucosidase II proved to be the most effective approach. Finally, we overexpressed an α-1,2-mannosidase to obtain Man(3GlcNAc(2 structures, which are substrates for the synthesis of complex-type glycans. The final Yarrowia lipolytica strain produces proteins glycosylated with the trimannosyl core N-glycan (Man(3GlcNAc(2, which is the common core of all complex-type N-glycans. All these glycans can be constructed on the obtained trimannosyl N-glycan using either in vivo or in vitro modification with the appropriate glycosyltransferases. The results demonstrate the high potential of Yarrowia lipolytica to be developed as an efficient expression system for the production of glycoproteins with humanized glycans.

  16. Multiplexed detection of lectins using integrated glycan-coated microring resonators.

    Ghasemi, Farshid; Hosseini, Ehsan Shah; Song, Xuezheng; Gottfried, David S; Chamanzar, Maysamreza; Raeiszadeh, Mehrsa; Cummings, Richard D; Eftekhar, Ali A; Adibi, Ali

    2016-06-15

    We present the systematic design, fabrication, and characterization of a multiplexed label-free lab-on-a-chip biosensor using silicon nitride (SiN) microring resonators. Sensor design is addressed through a systematic approach that enables optimizing the sensor according to the specific noise characteristics of the setup. We find that an optimal 6 dB undercoupled resonator consumes 40% less power in our platform to achieve the same limit-of-detection as the conventional designs using critically coupled resonators that have the maximum light-matter interaction. We lay out an optimization framework that enables the generalization of our method for any type of optical resonator and noise characteristics. The device is fabricated using a CMOS-compatible process, and an efficient swabbing lift-off technique is introduced for the deposition of the protective oxide layer. This technique increases the lift-off quality and yield compared to common lift-off methods based on agitation. The complete sensor system, including microfluidic flow cell and surface functionalization with glycan receptors, is tested for the multiplexed detection of Aleuria Aurantia Lectin (AAL) and Sambucus Nigra Lectin (SNA). Further analysis shows that the sensor limit of detection is 2 × 10(-6) RIU for bulk refractive index, 1 pg/mm(2) for surface-adsorbed mass, and ∼ 10 pM for the glycan/lectins studied here. PMID:26826877

  17. Engineering the Campylobacter jejuni N-glycan to create an effective chicken vaccine

    Nothaft, Harald; Davis, Brandi; Lock, Yee Ying; Perez-Munoz, Maria Elisa; Vinogradov, Evgeny; Walter, Jens; Coros, Colin; Szymanski, Christine M.

    2016-01-01

    Campylobacter jejuni is a predominant cause of human gastroenteritis worldwide. Source-attribution studies indicate that chickens are the main reservoir for infection, thus elimination of C. jejuni from poultry would significantly reduce the burden of human disease. We constructed glycoconjugate vaccines combining the conserved C. jejuni N-glycan with a protein carrier, GlycoTag, or fused to the Escherichia coli lipopolysaccharide-core. Vaccination of chickens with the protein-based or E. coli-displayed glycoconjugate showed up to 10-log reduction in C. jejuni colonization and induced N-glycan-specific IgY responses. Moreover, the live E. coli vaccine was cleared prior to C. jejuni challenge and no selection for resistant campylobacter variants was observed. Analyses of the chicken gut communities revealed that the live vaccine did not alter the composition or complexity of the microbiome, thus representing an effective and low-cost strategy to reduce C. jejuni in chickens and its subsequent entry into the food chain. PMID:27221144

  18. Structural and immunological characterization of the N-glycans from the major yellow jacket allergen Ves v 2: The N-glycan structures are needed for the human antibody recognition

    Seppälä, Ulla; Selby, David; Monsalve, Rafael;

    2009-01-01

    127 of Ves v 2.01, and residues 66 and 81 of Ves v 2.02. Structural analysis of the glycopeptides showed that the majority of the N-glycans contained at least one alpha1,3-fucose and/or alpha1,6-fucose residues in a structure. Interestingly, serum IgE antibodies from vespid allergic patients...

  19. A Targeted Glycan-Related Gene Screen Reveals Heparan Sulfate Proteoglycan Sulfation Regulates WNT and BMP Trans-Synaptic Signaling

    Dani, Neil; Nahm, Minyeop; Lee, Seungbok; Broadie, Kendal

    2012-01-01

    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. PMID:23144627

  20. Cross-presentation through langerin and DC-SIGN targeting requires different formulations of glycan-modified antigens

    Fehres, Cynthia M.; Kalay, Hakan; Bruijns, Sven C M; Musaafir, Sara A M; Ambrosini, Martino; Van Bloois, Louis; Van Vliet, Sandra J.; Storm, Gert; Garcia-Vallejo, Juan J.; Van Kooyk, Yvette

    2015-01-01

    Dendritic cells (DCs) and Langerhans cells (LC) are professional antigen presenting cells (APCs) that initiate humoral and cellular immune responses. Targeted delivery of antigen towards DC- or LC-specific receptors enhances vaccine efficacy. In this study, we compared the efficiency of glycan-based

  1. Cross-presentation through langerin and DC-SIGN targeting requires different formulation of glycan-modified antigens

    Fehres, Cynthia M.; Kalay, Hakan; Bruijns, Sven C.M.; Musaafir, Sara A.M.; Ambrosini, Martino; Bloois, van Louis; Vliet, van Sandra J.; Storm, Gert; Garcia-Vallejo, Juan J.; Kooyk, van Yvette

    2015-01-01

    Dendritic cells (DCs) and Langerhans cells (LC) are professional antigen presenting cells (APCs) that initiate humoral and cellular immune responses. Targeted delivery of antigen towards DC- or LC-specific receptors enhances vaccine efficacy. In this study, we compared the efficiency of glycan-based

  2. A targeted glycan-related gene screen reveals heparan sulfate proteoglycan sulfation regulates WNT and BMP trans-synaptic signaling.

    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.

  3. Structural Analysis of N- and O-glycans Using ZIC-HILIC/Dialysis Coupled to NMR Detection

    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.

  4. Determination of optimal acquisition time of [18F]FCWAY PET for imaging serotonin 1A receptors in the healthy male subjects

    The purpose of this research is to find optimal acquisition time point of [18F]FCWAY PET for the assessment of serotonin 1A receptor (5-HT1A) density. To achieve this goal, we examined the specific-to-nonspecific ratios in various brain regions. The cerebellum has very few 5-HT1A receptors in the brain, so we set this region as the reference tissue. As a result, specific-to-nonspecific binding ratios in the frontal, temporal cortex and the hippocampus were steadily increased at 90 min after injection and remained stable at 120 min. In addition, the binding ratio of the late time was significantly higher than that of the previous time points. From these results, we recommend that 90 min p.i. is a better single time point for the analysis rather than previous time points for assessing [18F]FCWAY binding to 5-HT1A receptors. - Highlights: • For routine clinical study, PET protocol should be conducted on a single time point with short imaging acquisition. • The specific-to-nonspecific ratios in the various brain regions were calculated. • Optimal [18F]FCWAY PET acquisition time point was proposed

  5. Mass spectrometric analysis of neutral and anionic N-glycans from a Dictyostelium discoideum model for human congenital disorder of glycosylation CDG IL.

    Hykollari, Alba; Balog, Crina I A; Rendić, Dubravko; Braulke, Thomas; Wilson, Iain B H; Paschinger, Katharina

    2013-03-01

    The HL241 mutant strain of the cellular slime mold Dictyostelium discoideum is a potential model for human congenital disorder of glycosylation type IL (ALG9-CDG) and has been previously predicted to possess a lower degree of modification of its N-glycans with anionic moieties than the parental wild-type. In this study, we first showed that this strain has a premature stop codon in its alg9 mannosyltransferase gene compatible with the occurrence of truncated N-glycans. These were subject to an optimized analytical workflow, considering that the mass spectrometry of acidic glycans often presents challenges due to neutral loss and suppression effects. Therefore, the protein-bound N-glycans were first fractionated, after serial enzymatic release, by solid phase extraction. Then primarily single glycan species were isolated by mixed hydrophilic-interaction/anion-exchange or reversed-phase HPLC and analyzed using chemical and enzymatic treatments and MS/MS. We show that protein-linked N-glycans of the mutant are of reduced size as compared to those of wild-type AX3, but still contain core α1,3-fucose, intersecting N-acetylglucosamine, bisecting N-acetylglucosamine, methylphosphate, phosphate, and sulfate residues. We observe that a single N-glycan can carry up to four of these six possible modifications. Due to the improved analytical procedures, we reveal fuller details regarding the N-glycomic potential of this fascinating model organism. PMID:23320427

  6. 1H and 13C NMR assignments for the glycans in glycoproteins by using 2H/13C-labeled glucose as a metabolic precursor

    In order to understand the role of the glycans in glycoproteins in solution, structural information obtained by NMR spectroscopy is obviously required. However, the assignment of the NMR signals from the glycans in larger glycoproteins is still difficult, mainly due to the lack of appropriate methods for the assignment of the resonances originating from the glycans. By using [U-13C6,2H7]glucose as a metabolic precursor, we have successfully prepared a glycoprotein whose glycan is uniformly labeled with 13C and partially with D at the sugar residues. The D to H exchange ratios at the C1-C6 positions of the sugar residues have been proven to provide useful information for the spectral assignments of the glycan in the glycoprotein. This is the first report on the residue-specific assignment of the anomeric resonances originating from a glycan attached to a glycoprotein by using the metabolic incorporation of hydrogen from the medium into a glycan labeled with [U-13C6,2H7]glucose

  7. Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans

    Garcia-Campos, Andres; Cwiklinski, Krystyna; Dalton, John P.; Hokke, Cornelis H.; O’Neill, Sandra; Mulcahy, Grace

    2016-01-01

    Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F

  8. Tegument Glycoproteins and Cathepsins of Newly Excysted Juvenile Fasciola hepatica Carry Mannosidic and Paucimannosidic N-glycans.

    Garcia-Campos, Andres; Ravidà, Alessandra; Nguyen, D Linh; Cwiklinski, Krystyna; Dalton, John P; Hokke, Cornelis H; O'Neill, Sandra; Mulcahy, Grace

    2016-05-01

    Recently, the prevalence of Fasciola hepatica in some areas has increased considerably and the availability of a vaccine to protect livestock from infection would represent a major advance in tools available for controlling this disease. To date, most vaccine-target discovery research on this parasite has concentrated on proteomic and transcriptomic approaches whereas little work has been carried out on glycosylation. As the F. hepatica tegument (Teg) may contain glycans potentially relevant to vaccine development and the Newly Excysted Juvenile (NEJ) is the first lifecycle stage in contact with the definitive host, our work has focused on assessing the glycosylation of the NEJTeg and identifying the NEJTeg glycoprotein repertoire. After in vitro excystation, NEJ were fixed and NEJTeg was extracted. Matrix-assisted laser desorption ionisation-time of flight-mass spectrometry (MALDI-TOF-MS) analysis of released N-glycans revealed that oligomannose and core-fucosylated truncated N-glycans were the most dominant glycan types. By lectin binding studies these glycans were identified mainly on the NEJ surface, together with the oral and ventral suckers. NEJTeg glycoproteins were affinity purified after targeted biotinylation of the glycans and identified using liquid chromatography and tandem mass spectrometry (LC-MS/MS). From the total set of proteins previously identified in NEJTeg, eighteen were also detected in the glycosylated fraction, including the F. hepatica Cathepsin B3 (FhCB3) and two of the Cathepsin L3 (FhCL3) proteins, among others. To confirm glycosylation of cathepsins, analysis at the glycopeptide level by LC-ESI-ion-trap-MS/MS with collision-induced dissociation (CID) and electron-transfer dissociation (ETD) was carried out. We established that cathepsin B1 (FhCB1) on position N80, and FhCL3 (BN1106_s10139B000014, scaffold10139) on position N153, carry unusual paucimannosidic Man2GlcNAc2 glycans. To our knowledge, this is the first description of F

  9. CSF N-glycan profile reveals sialylation deficiency in a patient with GM2 gangliosidosis presenting as childhood disintegrative disorder.

    Barone, Rita; Sturiale, Luisella; Fiumara, Agata; Palmigiano, Angelo; Bua, Rosaria O; Rizzo, Renata; Zappia, Mario; Garozzo, Domenico

    2016-04-01

    Protein N-glycosylation consists in the synthesis and processing of the oligosaccharide moiety (N-glycan) linked to a protein and it serves several functions for the proper central nervous system (CNS) development and function. Previous experimental and clinical studies have shown the importance of proper glycoprotein sialylation for the synaptic function and the occurrence of autism spectrum disorders (ASD) in the presence of sialylation deficiency in the CNS. Late-onset Tay Sachs disease (LOTSD) is a lysosomal disorder caused by mutations in the HEXA gene resulting in GM2-ganglioside storage in the CNS. It is characterized by progressive neurological impairment and high co-occurrence of psychiatric disturbances. We studied the N-glycome profile of the cerebrospinal fluid (CSF) in a 14 year-old patient with GM2-gangliosidosis (LOTSD). At the age of 4, the patient presented regressive autism fulfilling criteria for childhood disintegrative disorder (CDD). A CSF sample was obtained in the course of diagnostic work-up for the suspicion of an underlying neurodegenerative disorder. We found definite changes of CSF N-glycans due to a dramatic decrease of sialylated biantennary and triantennary structures and an increase of asialo-core fucosylated bisected N-glycans. No changes of total plasma N-glycans were found. Herein findings highlight possible relationships between the early onset psychiatric disturbance featuring CDD in the patient and defective protein sialylation in the CNS. In conclusion, the study first shows aberrant N-glycan structures of CSF proteins in LOTSD; unveils possible pathomechanisms of GM2-gangliosidosis; supports existing relationships between neuropsychiatric disorders and unproper protein glycosylation in the CNS. Autism Res 2016, 9: 423-428. © 2015 International Society for Autism Research, Wiley Periodicals, Inc. PMID:26286102

  10. A dual approach for improving homogeneity of a human-type N-glycan structure in Saccharomyces cerevisiae.

    Piirainen, Mari A; Boer, Harry; de Ruijter, Jorg C; Frey, Alexander D

    2016-04-01

    N-glycosylation is an important feature of therapeutic and other industrially relevant proteins, and engineering of the N-glycosylation pathway provides opportunities for developing alternative, non-mammalian glycoprotein expression systems. Among yeasts, Saccharomyces cerevisiae is the most established host organism used in therapeutic protein production and therefore an interesting host for glycoengineering. In this work, we present further improvements in the humanization of the N-glycans in a recently developed S. cerevisiae strain. In this strain, a tailored trimannosyl lipid-linked oligosaccharide is formed and transferred to the protein, followed by complex-type glycan formation by Golgi apparatus-targeted human N-acetylglucosamine transferases. We improved the glycan pattern of the glycoengineered strain both in terms of glycoform homogeneity and the efficiency of complex-type glycosylation. Most of the interfering structures present in the glycoengineered strain were eliminated by deletion of the MNN1 gene. The relative abundance of the complex-type target glycan was increased by the expression of a UDP-N-acetylglucosamine transporter from Kluyveromyces lactis, indicating that the import of UDP-N-acetylglucosamine into the Golgi apparatus is a limiting factor for efficient complex-type N-glycosylation in S. cerevisiae. By a combination of the MNN1 deletion and the expression of a UDP-N-acetylglucosamine transporter, a strain forming complex-type glycans with a significantly improved homogeneity was obtained. Our results represent a further step towards obtaining humanized glycoproteins with a high homogeneity in S. cerevisiae. PMID:26983412

  11. EndoE from Enterococcus faecalis hydrolyzes the glycans of the biofilm inhibiting protein lactoferrin and mediates growth.

    Julia Garbe

    Full Text Available Glycosidases are widespread among bacteria. The opportunistic human pathogen Enterococcus faecalis encodes several putative glycosidases but little is known about their functions. The identified endo-β-N-acetylglucosaminidase EndoE has activity on the N-linked glycans of the human immunoglobulin G (IgG. In this report we identified the human glycoprotein lactoferrin (hLF as a new substrate for EndoE. Hydrolysis of the N-glycans from hLF was investigated using lectin blot, UHPLC and mass spectrometry, showing that EndoE releases major glycoforms from this protein. hLF was shown to inhibit biofilm formation of E. faecalis in vitro. Glycans of hLF influence the binding to E. faecalis, and EndoE-hydrolyzed hLF inhibits biofilm formation to lesser extent than intact hLF indicating that EndoE prevents the inhibition of biofilm. In addition, hLF binds to a surface-associated enolase of E. faecalis. Culture experiments showed that the activity of EndoE enables E. faecalis to use the glycans derived from lactoferrin as a carbon source indicating that they could be used as nutrients in vivo when no other preferred carbon source is available. This report adds important information about the enzymatic activity of EndoE from the commensal and opportunist E. faecalis. The activity on the human glycoprotein hLF, and the functional consequences with reduced inhibition of biofilm formation highlights both innate immunity functions of hLF and a bacterial mechanism to evade this innate immunity function. Taken together, our results underline the importance of glycans in the interplay between bacteria and the human host, with possible implications for both commensalism and opportunism.

  12. Regioselectivity of Human UDP-Glucuronsyltransferase 1A1 in the Synthesis of Flavonoid Glucuronides Determined by Metal Complexation and Tandem Mass Spectrometry

    Davis, Barry D.; Brodbelt, Jennifer S.

    2007-01-01

    A three-part tandem mass spectrometric strategy that entails MSn analysis and a post-column LC-MS cobalt complexation method is developed to identify flavonoid monoglucuronide metabolites synthesized using the 1A1 isozyme of human UDP-glucuronosyltransferase (UGT). Ten flavonoid aglycons were used as substrates, spanning the subclasses of flavones, flavonols and flavanones. The products were characterized by LC-MS and LC-MSn, with post-column cobalt complexation employed to pinpoint the speci...

  13. Glycans and glycosylation of platelets: current concepts and implications for transfusion

    Sørensen, Anne Louise; Hoffmeister, Karin M; Wandall, Hans H

    2008-01-01

    galactosylation is not sufficient to restore circulation of long-term refrigerated platelets. Additional data indicate that differential carbohydrate-mediated mechanisms may exist for clearance of short-term and long-term cold-stored platelets. SUMMARY: Room temperature storage of platelet products increases the...... addresses the current knowledge of platelet glycans and the potential of glycosylation for improving platelet storage. RECENT FINDINGS: Removal of refrigerated platelets from the circulation is partly mediated by recognition of clustered beta-N-acetylglucosamine on platelet surface glycoproteins by the...... alphaMbeta2 hepatic lectin receptor. Capping the exposed beta-N-acetylglucosamine residues by enzymatic galactosylation restored the circulation of short-term chilled murine platelets, introducing a novel method that allows for cold storage of platelet. Recent studies have, however, shown that...

  14. Characterization of the phosphatidylinositol-glycan membrane anchor of human placental alkaline phosphatase

    Placental alkaline phosphatase [orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] is a member of a diverse group of membrane proteins whose attachment to the lipid bilayer is mediated by a phosphatidylinositol-glycan. To investigate structural aspects of the glycolipid anchor, cultured WISH cells were used because, they produce the enzyme in abundant quantities. When cell suspensions were incubated with purified phosphatidylinositol-specific phospholipase C, most of the placental alkaline phosphatase was released from membranes in a hydrophilic form. On incubation of the cells with [14C]ethanolamine, [14C]myristic acid, or myo[3H]inositol, each was incorporated into the phosphatase near the carboxyl terminus, showing that these components, which are found in other phosphatidylinositol membrane-linked proteins, are also present in placental alkaline phosphatase

  15. Glycan profiling of gel forming mucus layer from the scleractinian symbiotic coral Oculina arbuscula.

    Coddeville, Bernadette; Maes, Emmanuel; Ferrier-Pages, Christine; Guerardel, Yann

    2011-06-13

    The gel forming mucus layer surrounding scleractinian corals play fundamental functions in the maintenance of a favorable microenvironment required for the survival of these organisms. In particular, it harbors a rich partially species-specific symbiotic community through yet poorly understood molecular interactions. However, removal or contamination of this community by exogenous bacteria is closely linked to the worldwide bleaching events that are presently devastating coral colonies. The present study investigates the structure of major high molecular weight glycoconjugates that are responsible for both rheological properties of mucus and sugar-protein interactions with microbial communities. We demonstrated that it is composed by two distinct types of sulfated macromolecules: mucin type glycoproteins densely substituted by short unusual O-linked glycans and repetitive polysaccharides. PMID:21517058

  16. Combining polysaccharide biosynthesis and transport in a single enzyme: dual-function cell wall glycan synthases.

    Jonathan Kent Davis

    2012-06-01

    Full Text Available Extracellular polysaccharides are synthesized by a wide variety of species, from unicellular bacteria and Archaea to the largest multicellular plants and animals in the biosphere. In every case, the biosynthesis of these polymers requires transport across a membrane, from the cytosol to either the lumen of secretory pathway organelles or directly into the extracellular space. Although some polysaccharide biosynthetic substrates are moved across the membrane to sites of polysaccharide synthesis by separate transporter proteins before being incorporated into polymers by glycosyltransferase proteins, many polysaccharide biosynthetic enzymes appear to have both transporter and transferase activities. In these cases, the biosynthetic enzymes utilize substrate on one side of the membrane and deposit the polymer product on the other side. This review discusses structural characteristics of plant cell wall glycan synthases that couple synthesis with transport, drawing on what is known about such dual-function enzymes in other species.

  17. Hitting the Sweet Spot: Glycans as Targets of Fungal Defense Effector Proteins

    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.

  18. Antigen presenting cell-selective drug delivery by glycan-decorated nanocarriers.

    Frenz, Theresa; Grabski, Elena; Durán, Verónica; Hozsa, Constantin; Stępczyńska, Anna; Furch, Marcus; Gieseler, Robert K; Kalinke, Ulrich

    2015-09-01

    Targeted drug delivery systems hold promise for selective provision of active compounds to distinct tissues or cell subsets. Thus, locally enhanced drug concentrations are obtained that would confer improved efficacy. As a consequence adverse effects should be diminished, as innocent bystander cells are less affected. Currently, several controlled drug delivery systems based on diverse materials are being developed. Some systems exhibit material-associated toxic effects and/or show low drug loading capacity. In contrast, liposomal nanocarriers are particularly favorable because they are well tolerated, poorly immunogenic, can be produced in defined sizes, and offer a reasonable payload capacity. Compared with other immune cells, professional antigen-presenting cells (APCs) demonstrate enhanced liposome uptake mediated by macropinocytosis, phagocytosis and presumably also by clathrin- and caveolae-mediated endocytosis. In order to further enhance the targeting efficacy toward APCs, receptor-mediated uptake appears advisable. Since APC subsets generally do not express single linage-specific receptors, members of the C-type lectin receptor (CLR) family are compelling targets. Examples of CLR expressed by APCs include DEC-205 (CD205) expressed by myeloid dendritic cells (DC) and monocytes, the mannose receptor C type 1 (MR, CD206) expressed by DC, monocytes and macrophages, DC-SIGN (CD209) expressed by DC, and several others. These receptors bind glycans, which are typically displayed by pathogens and thus support pathogen uptake and endocytosis. Further research will elucidate whether glycan-decorated liposomes will not only enhance APCs targeting but also enable preferential delivery of their payload to discrete subcellular compartments. PMID:25701806

  19. Small-scale, high-throughput method for plant N-glycan preparation for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis.

    Matsuo, Kouki

    2011-06-15

    A simple, small-scale, and high-throughput method for preparation of plant N-glycans for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described. This method entailed the extraction of soluble proteins, pepsin digestion, release of N-glycans by glycopeptidase A, and a three-step chromatographic purification process using cation exchange, anion exchange, and graphitized carbon. Homemade minicolumns using commercially available filter unit devices were used for N-glycan purification steps. All purification steps were designed to be easy. Using this method, N-glycans from 10-mg leaf samples of different plant species and only 2 μg of pure horseradish peroxidase were successfully purified. PMID:21320463

  20. O- and N-glycosylation of the Leishmania mexicana-secreted acid phosphatase. Characterization of a new class of phosphoserine-linked glycans.

    Ilg, T; Overath, P; Ferguson, M A; Rutherford, T; Campbell, D G; McConville, M J

    1994-09-30

    The protozoan parasite Leishmania mexicana secretes a heavily glycosylated 100-kDa acid phosphatase (sAP) which is associated with one or more polydisperse proteophosphoglycans. Most of the glycans in this complex were released using mild acid hydrolysis conditions that preferentially cleave phosphodiester linkages. The released saccharides were shown to consist of monomeric mannose and a series of neutral and phosphorylated glycans by Dionex high performance liquid chromatography, methylation analysis, exoglycosidase digestions, and one-dimensional 1H NMR spectroscopy. The neutral species comprised a linear series of oligosaccharides with the structures [Man alpha 1-2]1-5Man. The phosphorylated oligosaccharides were characterized as PO4-6Gal beta 1-4Man and PO4-6[Glc beta 1-3]Gal beta 1-4Man. The attachment of these glycans to the polypeptide backbone via the linkage, Man alpha 1-PO4-Ser, is suggested by: 1) the finding that more than 60% of the serine residues in the polypeptide are phosphorylated and 2) the resistance of the phosphoserine residues to alkaline phosphatase digestion unless the sAP was first treated with either mild acid (to release all glycans) or jack bean alpha-mannosidase (to release neutral mannose glycans). Analysis of the partially resolved components of the complex indicated that the most of the O-linked glycans on the 100-kDa phosphoglycoprotein comprised mannose and the mannose-oligosaccharides. In contrast the major O-linked glycans on the proteophosphoglycan were short phosphoglycan chains, containing on average two repeat units per chain. In addition to the O-linked glycans, both components in the sAP complex contained N-linked glycans. The N-glycanase F-released glycans were characterized by Bio-Gel P4 chromatography and exoglycosidase digestions to be the biantennary oligomannose type with the structures Glc1Man6GlcNAc2 and Man6GlcNAc2. The O-linked glycans of the sAP complex are similar to those found in the phosphoglycan chains of

  1. Distinct N-Glycan Glycosylation of P-glycoprotein Isolated from the Human Uterine Sarcoma Cell Line MES-SA/D×5

    Greer, D.A.; Ivey, S.

    2007-01-01

    The uterine sarcoma human cell line MES-SA/D×5 overexpresses the MDR1 gene product, P-glycoprotein (Pgp). Pgp is a heavily glycosylated, ATP-dependent drug efflux pump expressed in many human cancers. There are more than 50 known isoforms of Pgp, which complicates the characterization of Pgp glycans because each isoform could present a different glycome. The contribution of these oligosaccharides to the structure and function of Pgp remains unclear. We identified distinct Pgp glycans recogniz...

  2. Engineering Yarrowia lipolytica to Produce Glycoproteins Homogeneously Modified with the Universal Man3GlcNAc2 N-Glycan Core

    De Pourcq, Karen; Tiels, Petra; Van Hecke, Annelies; Geysens, Steven; Vervecken, Wouter; Callewaert, Nico

    2012-01-01

    Yarrowia lipolytica is a dimorphic yeast that efficiently secretes various heterologous proteins and is classified as "generally recognized as safe." Therefore, it is an attractive protein production host. However, yeasts modify glycoproteins with non-human high mannose-type N-glycans. These structures reduce the protein half-life in vivo and can be immunogenic in man. Here, we describe how we genetically engineered N-glycan biosynthesis in Yarrowia lipolytica so that it produces Man(3)GlcNAc...

  3. Structural features of free N-glycans occurring in plants and functional features of de-N-glycosylation enzymes, ENGase, and PNGase: the presence of unusual plant complex type N-glycans

    Maeda, Megumi; Kimura, Yoshinobu

    2014-01-01

    Free N-glycans (FNGs) are present at micromolar concentrations in plant cells during their differentiation, growth, and maturation stages. It has been postulated that these FNGs are signaling molecules involved in plant development or fruit ripening. However, the hypothetical biochemical and molecular function of FNGs has not been yet established. The structure of FNGs found ubiquitously in plant tissues such as hypocotyls, leaves, roots, developing seeds, or fruits can be classified into two...

  4. A Prominent Site of Antibody Vulnerability on HIV Envelope Incorporates a Motif Associated with CCR5 Binding and Its Camouflaging Glycans.

    Sok, Devin; Pauthner, Matthias; Briney, Bryan; Lee, Jeong Hyun; Saye-Francisco, Karen L; Hsueh, Jessica; Ramos, Alejandra; Le, Khoa M; Jones, Meaghan; Jardine, Joseph G; Bastidas, Raiza; Sarkar, Anita; Liang, Chi-Hui; Shivatare, Sachin S; Wu, Chung-Yi; Schief, William R; Wong, Chi-Huey; Wilson, Ian A; Ward, Andrew B; Zhu, Jiang; Poignard, Pascal; Burton, Dennis R

    2016-07-19

    The dense patch of high-mannose-type glycans surrounding the N332 glycan on the HIV envelope glycoprotein (Env) is targeted by multiple broadly neutralizing antibodies (bnAbs). This region is relatively conserved, implying functional importance, the origins of which are not well understood. Here we describe the isolation of new bnAbs targeting this region. Examination of these and previously described antibodies to Env revealed that four different bnAb families targeted the (324)GDIR(327) peptide stretch at the base of the gp120 V3 loop and its nearby glycans. We found that this peptide stretch constitutes part of the CCR5 co-receptor binding site, with the high-mannose patch glycans serving to camouflage it from most antibodies. GDIR-glycan bnAbs, in contrast, bound both (324)GDIR(327) peptide residues and high-mannose patch glycans, which enabled broad reactivity against diverse HIV isolates. Thus, as for the CD4 binding site, bnAb effectiveness relies on circumventing the defenses of a critical functional region on Env. PMID:27438765

  5. TSOAK-M1: a computer code to determine tritium reaction/adsorption/release parameters from experimental results of air-detritiation tests

    Land, R.H.; Maroni, V.A.; Minkoff, M.

    1979-01-01

    A computer code has been developed which permits the determination of tritium reaction (T/sub 2/ to HTO)/adsorption/release and instrument correction parameters from enclosure (building) - detritiation test data. The code is based on a simplified model which treats each parameter as a normalized time-independent constant throughout the data-unfolding steps. Because of the complicated four-dimensional mathematical surface generated by the resulting differential equation system, occasional local-minima effects are observed, but these effects can be overcome in most instances by selecting a series of trial guesses for the initial parameter values and observing the reproducibility of final parameter values for cases where the best overall fit to experimental data is achieved. The code was then used to analyze existing small-cubicle test data with good success, and the resulting normalized parameters were employed to evaluate hypothetical reactor-building detritiation scenarios. It was concluded from the latter evaluation that the complications associated with moisture formation, adsorption, and release, particularly in terms of extended cleanup times, may not be as great as was previously thought. It is recommended that the validity of the TSOAK-M1 model be tested using data from detritiation tests conducted on large experimental enclosures (5 to 10 cm/sup 3/) and, if possible, actual facility buildings.

  6. TSOAK-M1: a computer code to determine tritium reaction/adsorption/release parameters from experimental results of air-detritiation tests

    A computer code has been developed which permits the determination of tritium reaction (T2 to HTO)/adsorption/release and instrument correction parameters from enclosure (building) - detritiation test data. The code is based on a simplified model which treats each parameter as a normalized time-independent constant throughout the data-unfolding steps. Because of the complicated four-dimensional mathematical surface generated by the resulting differential equation system, occasional local-minima effects are observed, but these effects can be overcome in most instances by selecting a series of trial guesses for the initial parameter values and observing the reproducibility of final parameter values for cases where the best overall fit to experimental data is achieved. The code was then used to analyze existing small-cubicle test data with good success, and the resulting normalized parameters were employed to evaluate hypothetical reactor-building detritiation scenarios. It was concluded from the latter evaluation that the complications associated with moisture formation, adsorption, and release, particularly in terms of extended cleanup times, may not be as great as was previously thought. It is recommended that the validity of the TSOAK-M1 model be tested using data from detritiation tests conducted on large experimental enclosures (5 to 10 cm3) and, if possible, actual facility buildings

  7. Influence of the host (Cho) and of the cultivation strategy on glycan structures and molecular properties of human thyrotrophin; Influencia do hospedeiro (Cho) e da estrategia de cultivo nas estruturas glicidicas e propriedades moleculares da tireotrofina humana

    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

  8. Mining the “glycocode”—exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy

    Gunning, A. Patrick; Kirby, Andrew R.; Fuell, Christine; Pin, Carmen; Tailford, Louise E.; Juge, Nathalie

    2013-01-01

    Mucins are the main components of the gastrointestinal mucus layer. Mucin glycosylation is critical to most intermolecular and intercellular interactions. However, due to the highly complex and heterogeneous mucin glycan structures, the encoded biological information remains largely encrypted. Here we have developed a methodology based on force spectroscopy to identify biologically accessible glycoepitopes in purified porcine gastric mucin (pPGM) and purified porcine jejunal mucin (pPJM). The...

  9. A clique-based method for the edit distance between unordered trees and its application to analysis of glycan structures

    2011-01-01

    Background Measuring similarities between tree structured data is important for analysis of RNA secondary structures, phylogenetic trees, glycan structures, and vascular trees. The edit distance is one of the most widely used measures for comparison of tree structured data. However, it is known that computation of the edit distance for rooted unordered trees is NP-hard. Furthermore, there is almost no available software tool that can compute the exact edit distance for unordered trees. Result...

  10. Concurrent Automated Sequencing of the Glycan and Peptide Portions of O-Linked Glycopeptide Anions by Ultraviolet Photodissociation Mass Spectrometry

    Madsen, James A.; Ko, Byoung Joon; Xu, Hua; Iwashkiw, Jeremy A; Robotham, Scott A.; Shaw, Jared B.; Feldman, Mario F.; Brodbelt, Jennifer S.

    2013-01-01

    O -glycopeptides are often acidic owing to the frequent occurrence of acidic saccharides in the glycan, rendering traditional proteomic workflows that rely on positive mode tandem mass spectrometry (MS/MS) less effective. In this report, we demonstrate the utility of negative mode ultraviolet photodissociation (UVPD) MS for the characterization of acidic O-linked glycopeptide anions. This method was evaluated for a series of singly- and multiply-deprotonated glycopeptides from the model glyco...

  11. The complexity of membrane-bound glycans in health and disease and the beneficial properties of glyconutrients

    Wagner, Lynn

    2013-01-01

    The surface of all free living cells and all multicellular cell types are covered with a dense and complex array of sugars mostly attached to proteins and lipids. These specific sugars are referred to as glycans and the biological role of these sugars includes cell-cell, cell-matrix, cell-molecule interactions, and interactions between other organisms. The chemistry of carbohydrates has been studied well since the first part of the 20th century without understanding the complexity of the glyc...

  12. Development of a polyimide based liquid junction nanoelectrospray interface for CE/MS analysis of peptides and glycans

    Křenková, Jana; Foret, František

    Geneve , 2015. s. 196-197. ISBN N. [HPLC 2015. International Symposium on High Performance Liquid Phase Separations and Related Techniques /42./. 21.06.2015-25.06.2015, Geneva] R&D Projects: GA ČR(CZ) GA14-06319S; GA ČR(CZ) GBP206/12/G014 Institutional support: RVO:68081715 Keywords : CE/ESI/MS * liquid junction * polyimide * pepties * glycans Subject RIV: CB - Analytical Chemistry, Separation

  13. Glycotope Sharing between Snail Hemolymph and Larval Schistosomes: Larval Transformation Products Alter Shared Glycan Patterns of Plasma Proteins

    YOSHINO, TIMOTHY P.; Wu, Xiao-Jun; Liu, Hongdi; Gonzalez, Laura A.; Deelder, André M; Cornelis H Hokke

    2012-01-01

    Recent evidence supports the involvement of inducible, highly diverse lectin-like recognition molecules in snail hemocyte-mediated responses to larval Schistosoma mansoni. Because host lectins likely are involved in initial parasite recognition, we sought to identify specific carbohydrate structures (glycans) shared between larval S. mansoni and its host Biomphalaria glabrata to address possible mechanisms of immune avoidance through mimicry of elements associated with the host immunoreactivi...

  14. Catalytic and glycan-binding abilities of ppGalNAc-T2 are regulated by acetylation

    Zlocowski, Natacha; Sendra, Victor G; Lorenz, Virginia; Villarreal, Marcos A; Jorge, Alberto; Núñez, Yolanda; Bennett, Eric P; Clausen, Henrik; Nores, Gustavo A; Irazoqui, Fernando J

    2011-01-01

    ); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of pp...... activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation....

  15. 5-HT1A receptor gene silencers Freud-1 and Freud-2 are differently expressed in the brain of rats with genetically determined high level of fear-induced aggression or its absence.

    Kondaurova, Elena M; Ilchibaeva, Tatiana V; Tsybko, Anton S; Kozhemyakina, Rimma V; Popova, Nina K; Naumenko, Vladimir S

    2016-09-01

    Serotonin 5-HT1A receptor is known to play a crucial role in the mechanisms of genetically defined aggression. In its turn, 5-HT1A receptor functional state is under control of multiple factors. Among others, transcriptional factors Freud-1 and Freud-2 are known to be involved in the repression of 5-HT1A receptor gene expression. However, implication of these factors in the regulation of behavior is unclear. Here, we investigated the expression of 5-HT1A receptor and silencers Freud-1 and Freud-2 in the brain of rats selectively bred for 85 generations for either high level of fear-induced aggression or its absence. It was shown that Freud-1 and Freud-2 levels were different in aggressive and nonaggressive animals. Freud-1 protein level was decreased in the hippocampus, whereas Freud-2 protein level was increased in the frontal cortex of highly aggressive rats. There no differences in 5-HT1A receptor gene expression were found in the brains of highly aggressive and nonaggressive rats. However, 5-HT1A receptor protein level was decreased in the midbrain and increased in the hippocampus of highly aggressive rats. These data showed the involvement of Freud-1 and Freud-2 in the regulation of genetically defined fear-induced aggression. However, these silencers do not affect transcription of the 5-HT1A receptor gene in the investigated rats. Our data indicate the implication of posttranscriptional rather than transcriptional regulation of 5-HT1A receptor functional state in the mechanisms of genetically determined aggressive behavior. On the other hand, the implication of other transcriptional regulators for 5-HT1A receptor gene in the mechanisms of genetically defined aggression could be suggested. PMID:27150226

  16. Evaluation of a di-O-methylated glycan as a potential antigenic target for the serodiagnosis of human toxocariasis.

    Elefant, G R; Roldán, W H; Seeböck, A; Kosma, P

    2016-04-01

    Serodiagnosis of human toxocariasis is based on the detection of specific IgG antibodies by the enzyme-linked immunosorbent assay (ELISA) using Toxocara larvae excretory-secretory (TES) antigens, but its production is a laborious and time-consuming process being also limited by the availability of adult females of T. canis as source for ova to obtain larvae. Chemical synthesis of the di-O-methylated (DiM) glycan structure found in the TES antigens has provided material for studying the antibody reactivity in a range of mammalian hosts, showing reactivity with human IgM and IgG. In this study, we have evaluated the performance of the DiM glycan against a panel of sera including patients with toxocariasis (n = 60), patients with other helminth infections (n = 75) and healthy individuals (n = 94), showing that DiM is able to detect IgG antibodies with a sensitivity and specificity of 91·7% and 94·7%, respectively, with a very good agreement with the TES antigens (kappa = 0·825). However, cross-reactivity was observed in some sera from patients with ascariasis, hymenolepiasis and fascioliasis. These results show that the DiM glycan could be a promising antigenic tool for the serodiagnosis of human toxocariasis. PMID:26896376

  17. μ-Theraphotoxin-An1a: primary structure determination and assessment of the pharmacological activity of a promiscuous anti-insect toxin from the venom of the tarantula Acanthoscurria natalensis (Mygalomorphae, Theraphosidae).

    Rates, Breno; Prates, Maura V; Verano-Braga, Thiago; da Rocha, Angela P; Roepstorff, Peter; Borges, Carlos L; Lapied, Bruno; Murillo, Laurence; Pimenta, Adriano M C; Biondi, Ilka; De Lima, Maria Elena

    2013-08-01

    Tarantulas are included in the mygalomorph spider family Theraphosidae. Although the pharmacological diversity of theraphosid toxins (theraphotoxins) is broad, studies dedicated to the characterization of biologically active molecules from the theraphosid genus Acanthoscurria have been restricted to the investigation of antimicrobial peptides and polyamines produced by the hemocytes of Acanthoscurria gomesiana. The present study reports the purification, primary structure determination and electrophysiological effects of an anti-insect toxin, named μ-theraphotoxin-An1a (μ-TRTX-An1a), from the venom of Acanthoscurria natalensis - a tarantula species occurring in the Brazilian biomes caatinga and cerrado. The analysis of the primary structure of μ-TRTX-An1a revealed the similarity of this toxin to theraphosid toxins bearing a huwentoxin-II-like fold. Electrophysiological experiments showed that μ-TRTX-An1a (100 nM) induces membrane depolarization, increases the spontaneous firing frequency and reduces spike amplitude of cockroach dorsal unpaired median (DUM) neurons. In addition, under voltage-clamp conditions, μ-TRTX-An1a (100 nM) only partially blocks voltage-dependent sodium current amplitudes in DUM neurons without any effect on their voltage dependence. This effect correlates well with the reduction of the spontaneous action potential amplitudes. Altogether, these last results suggest that μ-TRTX-An1a affects insect neuronal voltage-dependent sodium channels, which are among possible channels targeted by this promiscuous toxin. PMID:23651762

  18. N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin.

    Ying-Jung J Lai

    Full Text Available The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB, is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N-glycosylation with the 25-kDa likely a precursor of the 30-40-kDa HAI-2, with the ratio of their levels roughly similar among a variety of cells. In breast cancer cells, a significant amount of the 30-40-kDa HAI-2 can translocate to and inhibit matriptase on the cell surface, followed by shedding of the matriptase-HAI-2 complex. The 25-kDa HAI-2 appears to have also exited the ER/Golgi, being localized at the cytoplasmic face of the plasma membrane of breast cancer cells. While the 25-kDa HAI-2 was also detected at the extracellular face of plasma membrane at very low levels it appears to have no role in matriptase inhibition probably due to its paucity on the cell surface. Our study reveals that N-glycan branching regulates HAI-2 through different subcellular distribution and subsequently access to different target proteases.

  19. The N-glycan glycoprotein deglycosylation complex (Gpd from Capnocytophaga canimorsus deglycosylates human IgG.

    Francesco Renzi

    2011-06-01

    Full Text Available C. canimorsus 5 has the capacity to grow at the expenses of glycan moieties from host cells N-glycoproteins. Here, we show that C. canimorsus 5 also has the capacity to deglycosylate human IgG and we analyze the deglycosylation mechanism. We show that deglycosylation is achieved by a large complex spanning the outer membrane and consisting of the Gpd proteins and sialidase SiaC. GpdD, -G, -E and -F are surface-exposed outer membrane lipoproteins. GpdDEF could contribute to the binding of glycoproteins at the bacterial surface while GpdG is a endo-β-N-acetylglucosaminidase cleaving the N-linked oligosaccharide after the first N-linked GlcNAc residue. GpdC, resembling a TonB-dependent OM transporter is presumed to import the oligosaccharide into the periplasm after its cleavage from the glycoprotein. The terminal sialic acid residue of the oligosaccharide is then removed by SiaC, a periplasm-exposed lipoprotein in direct contact with GpdC. Finally, most likely degradation of the oligosaccharide proceeds sequentially from the desialylated non reducing end by the action of periplasmic exoglycosidases, including β-galactosidases, β-N-Acetylhexosaminidases and α-mannosidases.

  20. IgG glycan hydrolysis by EndoS inhibits experimental autoimmune encephalomyelitis

    Benkhoucha Mahdia

    2012-09-01

    Full Text Available Abstract Studies in experimental autoimmune encephalomyelitis (EAE, a mouse model of multiple sclerosis, have shown that B cells markedly influence the course of the disease, although whether their effects are protective or pathological is a matter of debate. EndoS hydrolysis of the IgG glycan has profound effects on IgG effector functions, such as complement activation and Fc receptor binding, suggesting that the enzyme could be used as an immunomodulatory therapeutic agent against IgG-mediated diseases. We demonstrate here that EndoS has a protective effect in myelin oligodendrocyte glycoprotein peptide amino acid 35–55 (MOG35-55-induced EAE, a chronic neuroinflammatory demyelinating disorder of the central nervous system (CNS in which humoral immune responses are thought to play only a minor role. EndoS treatment in chronic MOG35-55-EAE did not impair encephalitogenic T cell priming and recruitment into the CNS of mice, consistent with a primary role of EndoS in controlling IgG effector functions. In contrast, reduced EAE severity coincided with poor serum complement activation and deposition within the spinal cord, suggesting that EndoS treatment impairs B cell effector function. These results identify EndoS as a potential therapeutic agent against antibody-mediated CNS autoimmune disorders.

  1. Some non-cellulosic B-D-glycans from plant sources

    The structures of some non-cellulosic β-D-Glycans from three plant sources have been investigated and each was found to be characterised by a main chain consisting of β-(1→4)- linked D-pyranosyl sugars. The polysaccharides were isolated and purified using standard fractionation methods including chromatographic techniques and selective precipitation methods. Structural information was obtained by employing techniques such as methylation analysis, optical rotation measurements, mass spectrometry, 13C n.m.r. and 1H n.m.r. spectroscopy on the original polysaccharides and on degraded products obtained by methods such as acid- or enzyme-catalysed hydrolysis and Smith degradation. The hemicellulosic components from Agave sisalana leaves have been shown to be xylan and xyloglucan. Finally, the major polysaccharide component from the tubers of Satyrium corrifolium was characterised as a glucommanoglycan consisting of D-mannopyranosyl and D-glucopyranosyl residues in a linear chain of β-(1→4)- linkages. Electron microscopy and x-ray diffraction methods were used in this characterization

  2. The Hepatitis C Virus Glycan Shield and Evasion of the Humoral Immune Response

    Jean Dubuisson

    2011-10-01

    Full Text Available Despite the induction of effective immune responses, 80% of hepatitis C virus (HCV-infected individuals progress from acute to chronic hepatitis. In contrast to the cellular immune response, the role of the humoral immune response in HCV clearance is still subject to debate. Indeed, HCV escapes neutralizing antibodies in chronically infected patients and reinfection has been described in human and chimpanzee. Studies of antibody-mediated HCV neutralization have long been hampered by the lack of cell-culture-derived virus and the absence of a small animal model. However, the development of surrogate models and recent progress in HCV propagation in vitro now enable robust neutralization assays to be performed. These advances are beginning to shed some light on the mechanisms of HCV neutralization. This review summarizes the current state of knowledge of the viral targets of anti-HCV-neutralizing antibodies and the mechanisms that enable HCV to evade the humoral immune response. The recent description of the HCV glycan shield that reduces the immunogenicity of envelope proteins and masks conserved neutralizing epitopes at their surface constitutes the major focus of this review.

  3. Sequential processing of mannose-containing glycans by two α-mannosidases from Solitalea canadensis.

    Liu, Fang F; Kulinich, Anna; Du, Ya M; Liu, Li; Voglmeir, Josef

    2016-04-01

    Two putative α-mannosidase genes isolated from the rather unexplored soil bacterium Solitalea canadensis were cloned and biochemically characterised. Both recombinant enzymes were highly selective in releasing α-linked mannose but no other sugars. The α-mannosidases were designated Sca2/3Man2693 and Sca6Man4191, and showed the following biochemical properties: the temperature optimum for both enzymes was 37 °C, and their pH optima lay at 5.0 and 5.5, respectively. The activity of Sca2/3Man2693 was found to be dependent on Ca(2+) ions, whereas Cu(2+) and Zn(2+) ions almost completely inhibited both α-mannosidases. Specificity screens with various substrates revealed that Sca2/3Man2693 could release both α1-2- and α1-3-linked mannose, whereas Sca6Man4191 only released α1-6-linked mannose. The combined enzymatic action of both recombinant α-mannosidases allowed the sequential degradation of high-mannose-type N-glycans. The facile expression and purification procedures in combination with strict substrate specificities make α-mannosidases from S. canadensis promising candidates for bioanalytical applications. PMID:26864077

  4. Blocking Pseudomonas Aeruginosa, Chromobacterium Violaceum, and Ralstonia Solanacearum Adhesion by Fruit Glycans

    Nechama Gilboa-Garber

    2014-05-01

    Full Text Available The soil-borne pathogens Pseudomonas aeruginosa, Chromobacterium violaceum, and Ralstonia solanacearum, possess the lectins PA-IL, PA-IIL, CV-IIL, RSL, and RS-IIL, which may mediate their adhesion onto animal and plant target cells, enabling infections. Such infections may be prevented by surrounding the sensitive cells with competing glycans, which act as glycodecoys that block patholectins and capture pathogens that bear them. The above-mentioned five lectins have been used by us as probes to reveal progeny-protecting glycodecoys in avian eggs, milk, royal jelly, and seeds. Herein we describe their usage as probes for fruit and onion glycodecoys. They revealed lectin-blocking galactosides, fructose, oligo/polysaccharides, and glycoproteins in most of the examined fruits. Galactose/arabinose- bearing compounds were detected by PA-IL in banana, carob, pineapple, pomegranate, kiwifruit, and dates. Diverse mannose/fucose-bearing compounds were detected by PA-IIL in banana, onion, and pomegranate; by CV-IIL in pineapple; by RSL in banana, carob, date, onion, and pineapple, and by RS-IIL in date and fig. The results show the high efficiency of these lectins as probes for natural infection-preventing glycodecoys. Usage of fruit and seed embryo-protecting glycodecoys, unless allergenic, is advantageous for preventing animal intestinal and external and plant wilting infections since they are natural, harmless, inexpensive, and widely available.

  5. High-Sensitivity and Low-Toxicity Fucose Probe for Glycan Imaging and Biomarker Discovery.

    Kizuka, Yasuhiko; Funayama, Sho; Shogomori, Hidehiko; Nakano, Miyako; Nakajima, Kazuki; Oka, Ritsuko; Kitazume, Shinobu; Yamaguchi, Yoshiki; Sano, Masahiro; Korekane, Hiroaki; Hsu, Tsui-Ling; Lee, Hsiu-Yu; Wong, Chi-Huey; Taniguchi, Naoyuki

    2016-07-21

    Fucose, a terminal sugar in glycoconjugates, critically regulates various physiological and pathological phenomena, including cancer development and inflammation. However, there are currently no probes for efficient labeling and detection of this sugar. We chemically synthesized a novel series of alkynyl-fucose analogs as probe candidates and found that 7-alkynyl-fucose gave the highest labeling efficiency and low cytotoxicity. Among the fucose analogs, 7-alkynyl-fucose was the best substrate against all five fucosyltransferases examined. We confirmed its conversion to the corresponding guanosine diphosphate derivative in cells and found that cellular glycoproteins were labeled much more efficiently with 7-alkynyl-fucose than with an existing probe. 7-Alkynyl-fucose was detected in the N-glycan core by mass spectrometry, and 7-alkynyl-fucose-modified proteins mostly disappeared in core-fucose-deficient mouse embryonic fibroblasts, suggesting that this analog mainly labeled core fucose in these cells. These results indicate that 7-alkynyl-fucose is a highly sensitive and powerful tool for basic glycobiology research and clinical application for biomarker discovery. PMID:27447047

  6. Mutations in four glycosyl hydrolases reveal a highly coordinated pathway for rhodopsin biosynthesis and N-glycan trimming in Drosophila melanogaster.

    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

  7. Heterogeneity and glycan masking of cell wall microstructures in the stems of Miscanthus x giganteus, and its parents M. sinensis and M. sacchariflorus.

    Jie Xue

    Full Text Available Plant cell walls, being repositories of fixed carbon, are important sources of biomass and renewable energy. Miscanthus species are fast growing grasses with a high biomass yield and they have been identified as potential bioenergy crops. Miscanthus x giganteus is the sterile hybrid between M. sinensis and M. sacchariflorus, with a faster and taller growth than its parents. In this study, the occurrence of cell wall polysaccharides in stems of Miscanthus species has been determined using fluorescence imaging with sets of cell wall directed monoclonal antibodies. Heteroxylan and mixed linkage-glucan (MLG epitopes are abundant in stem cell walls of Miscanthus species, but their distributions are different in relation to the interfascicular parenchyma and these epitopes also display different developmental dynamics. Detection of pectic homogalacturonan (HG epitopes was often restricted to intercellular spaces of parenchyma regions and, notably, the high methyl ester LM20 HG epitope was specifically abundant in the pith parenchyma cell walls of M. x giganteus. Some cell wall probes cannot access their target glycan epitopes because of masking by other polysaccharides. In the case of Miscanthus stems, masking of xyloglucan by heteroxylan and masking of pectic galactan by heteroxylan and MLG was detected in certain cell wall regions. Knowledge of tissue level heterogeneity of polysaccharide distributions and molecular architectures in Miscanthus cell wall structures will be important for both understanding growth mechanisms and also for the development of potential strategies for the efficient deconstruction of Miscanthus biomass.

  8. Fluorous modified magnetic mesoporous silica composites-incorporated fluorous solid-phase extraction for the specific enrichment of N-linked glycans with simultaneous exclusion of proteins.

    Zhao, Man; Deng, Chunhui

    2016-10-01

    Taking advantage of fluorine-fluorine interactions, fluorous solid-phase extraction (FSPE) is emerging as a novel approach in proteomics research. Notably, silica gel bound with perfluoroalkyl groups was applied to the FSPE of N-linked glycans. Based on previous studies, mesoporous silica coated magnetic nanoparticles bound with perfluoroalkyl groups were synthesized for the specific enrichment of N-linked glycans in this study. The magnetic nanoparticles-incorporated FSPE strategy successfully identified 22 N-linked glycans from the OVA digest with a concentration of 0.5μg/μL, and achieved a detection limit of 5ng/μL (with 16 N-linked glycans identified). It also showed good day-to-day reproducibility. Its selectivity towards BSA protein is 1:200 (molar ratio), showing excellent size-exclusion effect. In addition, the present method proved to be effective for the analysis of the human serum digest, opening up new prospect for the identification of glycans and proteins with other post-translational modifications in biological environment. PMID:27474286

  9. Mass spectrometric analysis of products of metabolic glycan engineering with azido-modification of sialic acids.

    Bodnar, Edward; Raymond, Céline; Lopez, Paul G; Villacrés, Carina; Butler, Michael; Schoenhofen, Ian C; Durocher, Yves; Perreault, Hélène

    2015-12-01

    Metabolic engineering of glycans present on antibodies and other glycoproteins is becoming an interesting research area for improving our understanding of the glycome. With knowledge of the sialic acid biosynthetic pathways, the experiments described in this report are based on a published procedure involving the addition of a synthesized azido-mannosamine sugar into cell culture media and evaluation of downstream expression as azido-sialic acid. This unique bioorthogonal sugar has the potential for a variety of "click chemistry" reactions through the azide linkage, which allow for it to be isolated and quantified given the choice of label. In this report, mass spectrometry was used to investigate and optimize the cellular absorption of peracetylated N-azidoacetylmannosamine (Ac4ManNAz) to form N-azidoacetylneuraminic acid (SiaNAz) in a Chinese hamster ovary (CHO) cell line transiently expressing a double mutant trastuzumab (TZMm2), human galactosyltransferase 1 (GT), and human α-2,6-sialyltransferase (ST6). This in vivo approach is compared to in vitro enzymatic addition SiaNAz onto TZMm2 using soluble β-galactosamide α-2,6-sialyltransferase 1 and CMP-SiaNAz as donor. The in vivo results suggest that for this mAb, concentrations above 100 μM of Ac4ManNAz are necessary to allow for observation of terminal SiaNAz on tryptic peptides of TZMm2 by matrix-assisted laser desorption ionization (MALDI) mass spectrometry. This is further confirmed by a parallel study on the production of EG2-hFc monoclonal antibody (Zhang J et al. Prot Expr Purific 65(1); 77-82, 2009) in the presence of increasing concentrations of Ac4ManNAz. PMID:26362153

  10. Carbohydrate/glycan-binding specificity of legume lectins in respect to their proposed biological functions

    Márcio Viana Ramos

    2000-01-01

    Full Text Available The lectins, proteins which specifically recognize carbohydrate moieties, have been extensively studied in many biochemical and structural aspects in order to establish the molecular basis of this non-catalytic event. On the other hand, their clinical and agricultural potentials have been growing fast. Although lectins, mainly those from legume plants, had been investigated for biological properties, studies about the physiological functions of lectins are scarce in literature. Therefore, despite the accumulated data on lectins (as proteins, the role played by these signalizing molecules is poorly discussed. In the light of our accumulated results on legume lectins, specially those obtained from plants belonging to the Diocleinae sub-tribe and available data in literature, we discuss here the main hypothesis of their functions according to their carbohydrate/glycan-binding specificity.As lectinas, proteinas que especificamente reconhecem estruturas que contém carboidratos, têm sido extensivamente estudadas em muitos aspectos bioquímicos e estruturais, objetivando estabelecer as bases moleculares deste evento não-catalítico. Por outro lado, os potenciais clínicos e agriculturais destas proteínas têm crescido rapidamente. Embora as lectinas, principalmente aquelas de legumes tenham sido bastante investigadas em suas propriedades biológicas, estudos sobre as funcões fisiológicas de lectinas são escassos na literatura. Além disto, a despeito da quantidade de dados acumulados sobre lectinas (como proteínas, o papel desempenhado por estas moléculas de sinalização é pobremente discutido. Valendo-se de nossos estudos sobre lectinas de leguminosas, principalmente da sub-tribo Diocleinae, e outros dados presentes na literatura, discutimos aqui, as principais hipóteses de suas funções com base na especificidade por carboidratos e glicanos complexos.

  11. Cross-presentation through langerin and DC-SIGN targeting requires different formulations of glycan-modified antigens.

    Fehres, Cynthia M; Kalay, Hakan; Bruijns, Sven C M; Musaafir, Sara A M; Ambrosini, Martino; van Bloois, Louis; van Vliet, Sandra J; Storm, Gert; Garcia-Vallejo, Juan J; van Kooyk, Yvette

    2015-04-10

    Dendritic cells (DCs) and Langerhans cells (LC) are professional antigen presenting cells (APCs) that initiate humoral and cellular immune responses. Targeted delivery of antigen towards DC- or LC-specific receptors enhances vaccine efficacy. In this study, we compared the efficiency of glycan-based antigen targeting to both the human DC-specific C-type lectin receptor (CLR) DC-SIGN and the LC-specific CLR langerin. Since DC-SIGN and langerin are able to recognize the difucosylated oligosaccharide Lewis Y (Le(Y)), we prepared neoglycoconjugates bearing this glycan epitope to allow targeting of both lectins. Le(Y)-modified liposomes, with an approximate diameter of 200nm, were significantly endocytosed by DC-SIGN(+) DCs and mediated efficient antigen presentation to CD4(+) and CD8(+) T cells. Surprisingly, although langerin bound to Le(Y)-modified liposomes, LCs exposed to Le(Y)-modified liposomes could not endocytose liposomes nor mediate antigen presentation to T cells. However, LCs mediated an enhanced cross-presentation when antigen was delivered through langerin using Le(Y)-modified synthetic long peptides. In contrast, Le(Y)-modified synthetic long peptides were recognized by DC-SIGN, but did not trigger antigen internalization nor antigen cross-presentation. These data demonstrate that langerin and DC-SIGN have different size requirements for antigen uptake. Although using glycans remains an interesting option in the design of anti-cancer vaccines targeting multiple CLRs, aspects such as molecule size and conformation need to be taken in consideration. PMID:25656175

  12. Glycans from Fasciola hepatica Modulate the Host Immune Response and TLR-Induced Maturation of Dendritic Cells.

    Rodríguez, Ernesto; Noya, Verónica; Cervi, Laura; Chiribao, María Laura; Brossard, Natalie; Chiale, Carolina; Carmona, Carlos; Giacomini, Cecilia; Freire, Teresa

    2015-12-01

    Helminths express various carbohydrate-containing glycoconjugates on their surface, and they release glycan-rich excretion/secretion products that can be very important in their life cycles, infection and pathology. Recent evidence suggests that parasite glycoconjugates could play a role in the evasion of the immune response, leading to a modified Th2-polarized immune response that favors parasite survival in the host. Nevertheless, there is limited information about the nature or function of glycans produced by the trematode Fasciola hepatica, the causative agent of fasciolosis. In this paper, we investigate whether glycosylated molecules from F. hepatica participate in the modulation of host immunity. We also focus on dendritic cells, since they are an important target of immune-modulation by helminths, affecting their activity or function. Our results indicate that glycans from F. hepatica promote the production of IL-4 and IL-10, suppressing IFNγ production. During infection, this parasite is able to induce a semi-mature phenotype of DCs expressing low levels of MHCII and secrete IL-10. Furthermore, we show that parasite glycoconjugates mediate the modulation of LPS-induced maturation of DCs since their oxidation restores the capacity of LPS-treated DCs to secrete high levels of the pro-inflammatory cytokines IL-6 and IL-12/23p40 and low levels of the anti-inflammatory cytokine IL-10. Inhibition assays using carbohydrates suggest that the immune-modulation is mediated, at least in part, by the recognition of a mannose specific-CLR that signals by recruiting the phosphatase Php2. The results presented here contribute to the understanding of the role of parasite glycosylated molecules in the modulation of the host immunity and might be useful in the design of vaccines against fasciolosis. PMID:26720149

  13. Designed synthesis of carbon-functional magnetic graphene mesoporous silica materials using polydopamine as carbon precursor for the selective enrichment of N-linked glycan.

    Sun, Nianrong; Yao, Jizong; Deng, Chunhui

    2016-02-01

    Glycosylation, which has been confirmed to be associated with many diseases, is an important protein post-translation modification. Taking into account the low abundant of glycan, the purification of complex biological samples is considered to be very significant before mass spectrometry detection. In this work, carbon-functionalized magnetic graphene /mesoporous silica materials (C-Mag G@mSiO2 materials) with high content of carbon were designed and synthesized by using polydopamine as carbon precursor. Taking advantage of the special interaction between carbon and glycan, C-Mag G@mSiO2 materials were successfully applied to enrich N-linked glycans in different complex samples, such as standard glycoprotein digestion, the mixture of standard glycoprotein digestion, glycoprotein and non-glycoprotein, and human serum. PMID:26653470

  14. Aberrant expression of mucin core proteins and o-linked glycans associated with progression of pancreatic cancer

    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 in...... pancreatic adenocarcinoma-sialyl Tn (STn), Tn, T antigen, sialyl Lewis A (CA19-9), sialyl Lewis C (SLeC), Lewis X (LeX), and sialyl LeX (SLeX)-during the progression of pancreatic cancer from early stages to metastatic disease....

  15. Targeting N-Glycan Cryptic Sugar Moieties for Broad-Spectrum Virus Neutralization: Progress in Identifying Conserved Molecular Targets in Viruses of Distinct Phylogenetic Origins

    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.

  16. Neofunctionalization of the Sec1 α1,2fucosyltransferase paralogue in leporids contributes to glycan polymorphism and resistance to rabbit hemorrhagic disease virus.

    Kristina Nyström; Joana Abrantes; Ana Margarida Lopes; Béatrice Le Moullac-Vaidye; Stéphane Marchandeau; Jézabel Rocher; Nathalie Ruvoën-Clouet; Esteves, Pedro J.; Jacques Le Pendu

    2015-01-01

    International audience RHDV (rabbit hemorrhagic disease virus), a virulent calicivirus, causes high mortalities in European rabbit populations (Oryctolagus cuniculus). It uses α1,2fucosylated glycans, histo-blood group antigens (HBGAs), as attachment factors, with their absence or low expression generating resistance to the disease. Synthesis of these glycans requires an α1,2fucosyltransferase. In mammals, there are three closely located α1,2fucosyltransferase genes rSec1, rFut2 and rFut1 ...

  17. The functional O-mannose glycan on α-dystroglycan contains a phospho-ribitol primed for matriglycan addition

    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

  18. ABH-Glycan Microarray Characterizes ABO Subtype Antibodies: Fine Specificity of Immune Tolerance After ABO-Incompatible Transplantation.

    Jeyakanthan, M; Meloncelli, P J; Zou, L; Lowary, T L; Larsen, I; Maier, S; Tao, K; Rusch, J; Chinnock, R; Shaw, N; Burch, M; Beddows, K; Addonizio, L; Zuckerman, W; Pahl, E; Rutledge, J; Kanter, K R; Cairo, C W; Buriak, J M; Ross, D; Rebeyka, I; West, L J

    2016-05-01

    Organ transplantation from ABO blood group-incompatible (ABOi) donors requires accurate detection, effective removal and subsequent surveillance of antidonor antibodies. Because ABH antigen subtypes are expressed differently in various cells and organs, measurement of antibodies specific for the antigen subtypes in the graft is essential. Erythrocyte agglutination, the century-old assay used clinically, does not discriminate subtype-specific ABO antibodies and provides limited information on antibody isotypes. We designed and created an ABO-glycan microarray and demonstrated the precise assessment of both the presence and, importantly, the absence of donor-specific antibodies in an international study of pediatric heart transplant patients. Specific IgM, IgG, and IgA isotype antibodies to nonself ABH subtypes were detected in control participants and recipients of ABO-compatible transplants. Conversely, in children who received ABOi transplants, antibodies specific for A subtype II and/or B subtype II antigens-the only ABH antigen subtypes expressed in heart tissue-were absent, demonstrating the fine specificity of B cell tolerance to donor/graft blood group antigens. In contrast to the hemagglutination assay, the ABO-glycan microarray allows detailed characterization of donor-specific antibodies necessary for effective transplant management, representing a major step forward in precise ABO antibody detection. PMID:26602221

  19. HIV-1 clade C escapes broadly neutralizing autologous antibodies with N332 glycan specificity by distinct mechanisms.

    Deshpande, Suprit; Patil, Shilpa; Kumar, Rajesh; Hermanus, Tandile; Murugavel, Kailapuri G; Srikrishnan, Aylur K; Solomon, Suniti; Morris, Lynn; Bhattacharya, Jayanta

    2016-01-01

    The glycan supersite centered on N332 in the V3 base of the HIV-1 envelope (Env) is a target for broadly neutralizing antibodies (bnAbs) such as PGT121 and PGT128. In this study, we examined the basis of resistance of HIV-1 clade C Envs obtained from broadly cross neutralizing (BCN) plasma of an Indian donor with N332 specificity. Pseudotyped viruses expressing autologous envs were found to be resistant to autologous BCN plasma as well as to PGT121 and PGT128 mAbs despite the majority of Envs containing an intact N332 residue. While resistance of one of the Envs to neutralization by autologous plasma antibodies with shorter V1 loop length was found to be correlated with a N332S mutation, resistance to neutralization of rest of the Envs was found to be associated with longer V1 loop length and acquisition of protective N-glycans. In summary, we show evidence of escape of circulating HIV-1 clade C in an individual from autologous BCN antibodies by three distinct mechanisms. PMID:27576440

  20. Glycan-mediated uptake in urothelial primary cells: Perspectives for improved intravesical drug delivery in urinary tract infections.

    Pichl, Clara Maria; Feilhauer, Sophie; Schwaigerlehner, Rose-Marie; Gabor, Franz; Wirth, Michael; Neutsch, Lukas

    2015-11-30

    Urinary tract infections (UTIs) are among the most common bacterial infections. Despite a wide range of therapeutic options, treatment success is compromised by multiresistance and the efficient mechanism of tissue colonization of uropathogenic Escherichia coli (UPEC). In advanced drug delivery systems, a similar, glycan-mediated targeting mechanism may be realized by conjugating the drug to a plant lectin. This may lead to the drug being more efficiently accumulated at the desired site of action, the bacterial reservoirs. In this study, we aimed at elucidating the potential of this biorecognitive approach. Glycan-triggered interaction cascades and uptake processes of several plant lectins with distinct carbohydrate specificities were characterized using single cells and monolayer culture. Due to pronounced cytoadhesive and cytoinvasive properties, wheat germ agglutinin (WGA) emerged as a promising targeter in porcine urothelial primary cells. The lectin-cell interaction proved highly stabile in artificial urine, simulating the conditions in actual application. Colocalisation studies with internalized WGA and lens culinaris agglutinin (LCA) revealed that intracellular accumulation sites were largely identical for GlcNAc- and Mannose-specific lectins. This indicates that WGA-mediated delivery may indeed constitute a potent tool to reach bacteria taken up via a FimH-triggered invasion process. Existing pitfalls in intravesical treatment schedules may soon be overcome. PMID:26383837

  1. Caenorhabditis elegans N-glycan core beta-galactoside confers sensitivity towards nematotoxic fungal galectin CGL2.

    Alex Butschi

    2010-01-01

    Full Text Available The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galbeta1,4Fucalpha1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galbeta1,4Fucalpha1,6GlcNAc trisaccharide at 1.5 A resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.

  2. The functional O-mannose glycan on α-dystroglycan contains a phospho-ribitol primed for matriglycan addition.

    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. PMID:27130732

  3. Specifically Binding of L-ficolin to N-glycans of HCV Envelope Glycoproteins E1 and E2 Leads to Complement Activation

    Jun Liu; Mohammed A.M.Ali; Yinghua Shi; Yinglan Zhao; Fenglin Luo; Jin Yu; Tian Xiang; Jie Tang; Dongqing Li; Quan Hu; Wenzhe Ho; Xiaolian Zhang

    2009-01-01

    L-ficolin and HCV E1 and E2 glycoproteins was attributed to the N-glycans of E1 and E2.These findings provide new insights into the biological functions of L-ficolin in clinically important hepatic viral diseases.

  4. Fucosylated glycans in the periventricular structures and the cerebrospinal fluid of the fetal rat forebrain. An autoradiographic and lectin binding histiotopic study

    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

  5. Ultrasensitive detection of cancer cells and glycan expression profiling based on a multivalent recognition and alkaline phosphatase-responsive electrogenerated chemiluminescence biosensor

    Chen, Xiaojiao; He, Yao; Zhang, Youyu; Liu, Meiling; Liu, Yang; Li, Jinghong

    2014-09-01

    A multivalent recognition and alkaline phosphatase (ALP)-responsive electrogenerated chemiluminescence (ECL) biosensor for cancer cell detection and in situ evaluation of cell surface glycan expression was developed on a poly(amidoamine) (PAMAM) dendrimer-conjugated, chemically reduced graphene oxide (rGO) electrode interface. In this strategy, the multivalency and high affinity of the cell-targeted aptamers on rGO provided a highly efficient cell recognition platform on the electrode. The ALP and concanavalin A (Con A) coated gold nanoparticles (Au NPs) nanoprobes allowed the ALP enzyme-catalyzed production of phenols that inhibited the ECL reaction of Ru(bpy)32+ on the rGO electrode interface, affording fast and highly sensitive ECL cytosensing and cell surface glycan evaluation. Combining the multivalent aptamer interface and ALP nanoprobes, the ECL cytosensor showed a detection limit of 38 CCRF-CEM cells per mL in human serum samples, broad dynamic range and excellent selectivity. In addition, the proposed biosensor provided a valuable insight into dynamic profiling of the expression of different glycans on cell surfaces, based on the carbohydrates recognized by lectins applied to the nanoprobes. This biosensor exhibits great promise in clinical diagnosis and drug screening.A multivalent recognition and alkaline phosphatase (ALP)-responsive electrogenerated chemiluminescence (ECL) biosensor for cancer cell detection and in situ evaluation of cell surface glycan expression was developed on a poly(amidoamine) (PAMAM) dendrimer-conjugated, chemically reduced graphene oxide (rGO) electrode interface. In this strategy, the multivalency and high affinity of the cell-targeted aptamers on rGO provided a highly efficient cell recognition platform on the electrode. The ALP and concanavalin A (Con A) coated gold nanoparticles (Au NPs) nanoprobes allowed the ALP enzyme-catalyzed production of phenols that inhibited the ECL reaction of Ru(bpy)32+ on the rGO electrode

  6. Comparative evaluation of T11 target structure and its deglycosylated derivative nullifies the importance of glycan moieties in immunotherapeutic efficacy

    Sirshendu Chatterjee; Sagar Acharya; Pankaj Kumar; Ananya Chatterjee; Suhnrita Chaudhuri; Anirban Ghosh; Swapna Chaudhuri

    2012-01-01

    Sheep red blood cell (SRBC),a non-specific biological response modifier that has long been used as a classical antigen,has been shown to exert an immunomodulatory and anti-tumor activities in experimental animals.The active component of SRBC,which is responsible for such effects,was found to be a cell surface acidic glycoprotein molecule,known as T11 target structure (T11TS).In the present study,T11TS was isolated and purified to homogeneity using a five-step protocol involving isolation of sheep erythrocyte membrane from packed cell volume,20% ammonium sulfate cut of the crude membrane proteins mixture,immunoaffinity purification using mouse anti-sheep CD58 mAb (L180/1) tagged matrix,preparative gel electrophoresis,and gel electroelution process.Finally,the purity and identity of the proteins were confirmed by the matrix-assisted laser desorption/ionization (MALDI) mass spectrometric analysis.The in silico glycosylation site analysis showed that the extracellular domain contained three N-glycosylation sites (N-12,N-62,and N-111) and one O-glycosylation site (T-107).However,the experimental analysis negated the presence of O-linked glycan moieties on T11TS.To investigate the role of glycan moieties in the current immunotherapeutic regime,T11TS and its deglycosylated form (dT11TS) were administered intraperitoneally (i.p.) in N-ethyl-N-nitrosoureainduced immune-compromised mice at 0.4 mg/kg body weight.It was observed that both the forms of T11TS could activate the compromised immune status of mice by augmenting immune receptor expression (CD2,CD25,CD8,and CD11b),T-helper 1 shift of cytokine network,enhanced cytotoxicity,and phagocytosis activity.Therefore,the results nullify the active involvement of the N-linked glycan moieties in immunotherapeutic efficacy of T11TS.

  7. Phase-Variable Heptose I Glycan Extensions Modulate Efficacy of 2C7 Vaccine Antibody Directed against Neisseria gonorrhoeae Lipooligosaccharide.

    Chakraborti, Srinjoy; Lewis, Lisa A; Cox, Andrew D; St Michael, Frank; Li, Jianjun; Rice, Peter A; Ram, Sanjay

    2016-06-01

    Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection, gonorrhea, has developed resistance to most conventional antibiotics. Safe and effective vaccines against gonorrhea are needed urgently. A candidate vaccine that targets a lipooligosaccharide (LOS) epitope recognized mAb 2C7 attenuates gonococcal burden in the mouse vaginal colonization model. Glycan extensions from the LOS core heptoses (HepI and HepII) are controlled by phase-variable LOS glycosyltransferase (lgt) genes; we sought to define how HepI glycan extensions affect mAb 2C7 function. Isogenic gonococcal mutants in which the lgt required for mAb 2C7 reactivity (lgtG) was genetically locked on and the lgt loci required for HepI variation (lgtA, lgtC, and lgtD) were genetically locked on or off in different combinations were created. We observed 100% complement-dependent killing by mAb 2C7 of a mutant that expressed lactose (Gal-Glc) from HepI, whereas a mutant that expressed Gal-Gal-Glc-HepI fully resisted killing (>100% survival). Mutants that elaborated 4- (Gal-GlcNAc-Gal-Glc-HepI) and 5-glycan (GalNAc-Gal-GlcNAc-Gal-Glc-HepI) structures displayed intermediate phenotypes (95% killing with 4 μg/ml mAb 2C7). The contrasting phenotypes of the lactose-HepI and the Gal-Gal-Glc-HepI LOS structures were recapitulated with phase variants of a recently isolated clinical strain. Despite lack of killing of the Gal-Gal-Glc-HepI mutants, mAb 2C7 deposited sufficient C3 on these bacteria for opsonophagocytic killing by human neutrophils. In conclusion, mAb 2C7 showed functional activity against all gonococcal HepI LOS structures defined by various lgtA/C/D on/off combinations, thereby providing further impetus for use of the 2C7 epitope in a gonococcal vaccine. PMID:27183633

  8. Increased mannosylphosphorylation of N-glycans by heterologous expression of YlMPO1 in glyco-engineered Saccharomyces cerevisiae for mannose-6-phosphate modification.

    Gil, Jin Young; Park, Jeong-Nam; Lee, Kyung Jin; Kang, Ji-Yeon; Kim, Yeong Hun; Kim, Seonghun; Kim, Sang-Yoon; Kwon, Ohsuk; Lim, Yong Taik; Kang, Hyun Ah; Oh, Doo-Byoung

    2015-07-20

    Mannosylphosphorylated N-glycans found in yeasts can be converted to those containing mannose-6-phosphate, which is a key factor for lysosomal targeting. In the traditional yeast Saccharomyces cerevisiae, both ScMNN4 and ScMNN6 genes are required for efficient mannosylphosphorylation. ScMnn4 protein has been known to be a positive regulator of ScMnn6p, a real enzyme for mannosylphosphorylation. On the other hand, YlMpo1p, a ScMnn4p homologue, mediates mannosylphosphorylation in Yarrowia lypolytica without the involvement of ScMnn6p homologues. In this study, we show that heterologous expression of YlMpo1p can perform and enhance mannosylphosphorylation in S. cerevisiae in the absence of ScMnn4p and ScMnn6p. Moreover, the level of mannosylphosphorylation of N-glycans enhanced by YlMpo1p overexpression is much higher than that with ScMnn4p overexpression, and this is highlighted further in Scmnn4- and Scmnn6-disrupted mutants. When YlMpo1p overexpression is applied to glyco-engineered S. cerevisiae in which the synthesis of immunogenic glycans is abolished, a great increase of bi-mannosylphosphorylated glycan is observed. Through an in vitro process involving the uncapping of the outer mannose residue, this bi-mannosylphosphorylated structure is changed to a bi-phosphorylated structure with high affinity for mannose-6-phosphate receptor. The superior ability of YlMpo1p to increase bi-mannosylphosphorylated glycan in yeast shows promise for the production of therapeutic enzymes with improved lysosomal targeting capability. PMID:25907834

  9. A tetraantennary glycan with bisecting N-acetylglucosamine and the Sda antigen is the predominant N-glycan on bovine pregnancy-associated glycoproteins

    Klisch, Karl; Jeanrond, Evelyne; Pang, Poh-Choo; Pich, Andreas; Schuler, Gerhard; Dantzer, Vibeke; Kowalewski, Mariusz P.; Dell, Anne

    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...

  10. Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4

    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...

  11. Fluorographic detection of tritiated glycopeptides and oligosaccharides separated on polyacrylamide gels: analysis of glycans from Dictyostelium discoideum glycoproteins

    Previous workers have shown that oligosaccharides and glycopeptides can be separated by electrophoresis in buffers containing borate ions. However, normal fluorography of tritium-labeled structures cannot be performed because the glycans are soluble and can diffuse during equilibration with scintillants. This problem has been circumvented by equilibration of the gel with 2,5-diphenyloxazole (PPO) prior to electrophoresis. The presence of PPO in the gel during electrophoresis does not alter mobility of the glycopeptides and oligosaccharides. After electrophoresis, the gel is simply dried and fluorography performed. This allows sensitive and precise comparisons of labeled samples in parallel lanes of a slab gel and, since mobilities are highly reproducible, between different gels. The procedure is preparative in that after fluorography the gel bands can be quantitatively eluted for further study, without any apparent modification by the procedure. In this report, the procedure is illustrated by fractionation of both neutral and anionic glycopeptides produced by the cellular slime mold Dictyostelium discoideum

  12. The Relationship between Glycan Binding and Direct Membrane Interactions in Vibrio cholerae Cytolysin, a Channel-forming Toxin.

    De, Swastik; Bubnys, Adele; Alonzo, Francis; Hyun, Jinsol; Lary, Jeffrey W; Cole, James L; Torres, Victor J; Olson, Rich

    2015-11-20

    Bacterial pore-forming toxins (PFTs) are structurally diverse pathogen-secreted proteins that form cell-damaging channels in the membranes of host cells. Most PFTs are released as water-soluble monomers that first oligomerize on the membrane before inserting a transmembrane channel. To modulate specificity and increase potency, many PFTs recognize specific cell surface receptors that increase the local toxin concentration on cell membranes, thereby facilitating channel formation. Vibrio cholerae cytolysin (VCC) is a toxin secreted by the human pathogen responsible for pandemic cholera disease and acts as a defensive agent against the host immune system. Although it has been shown that VCC utilizes specific glycan receptors on the cell surface, additional direct contacts with the membrane must also play a role in toxin binding. To better understand the nature of these interactions, we conducted a systematic investigation of the membrane-binding surface of VCC to identify additional membrane interactions important in cell targeting. Through cell-based assays on several human-derived cell lines, we show that VCC is unlikely to utilize high affinity protein receptors as do structurally similar toxins from Staphylococcus aureus. Next, we identified a number of specific amino acid residues that greatly diminish the VCC potency against cells and investigated the interplay between glycan binding and these direct lipid contacts. Finally, we used model membranes to parse the importance of these key residues in lipid and cholesterol binding. Our study provides a complete functional map of the VCC membrane-binding surface and insights into the integration of sugar, lipid, and cholesterol binding interactions. PMID:26416894

  13. Identification of an N-linked glycan in the V1-loop of HIV-1 gp120 influencing neutralization by anti-V3 antibodies and soluble CD4

    Gram, G J; Hemming, A; Bolmstedt, A; Jansson, B; Olofsson, S; Akerblom, L; Nielsen, Jens Ole; Hansen, J E

    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......A and a conformation dependent human antibody IAM-2G12. This suggests that the N-linked glycan in the V1-loop modulates the three-dimensional conformation of gp120, without changing the overall functional integrity of the molecule....

  14. Role of Site-Specific N-Glycans Expressed on GluA2 in the Regulation of Cell Surface Expression of AMPA-Type Glutamate Receptors.

    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.

  15. Propriedades funcionais (tecnológicas da parede celular de leveduras da fermentação alcoólica e das frações glicana, manana e glicoproteína Functional (technological properties of yeast cellular wall of alcoholic fermentation and its glycan, mannan, and glycoprotein fractions

    Saula Goulart Chaud

    2006-06-01

    glycan, mannan and glycoprotein. Fractionation was realized by the physico-chemical processes of extraction, centrifugation and spray drying; chemical characterization, by determination of centesimal composition and the functional properties through well-known techniques. In the cellular wall (CW, predominated protein (19% and soluble fiber (74%. Glycoprotein presented 35.5% protein and 56% soluble fiber. In the mannnan (M and soluble glycan (SG, the soluble fiber (70% was predominant, whereas in the insoluble glycan (IG predominated the insoluble fiber (70.7%. Solubility of the various fractions in aqueous media ranged from 40% to 100% and was not pH dependent. Soluble glycan showed the highest water retention capacity (14.4 g H2O/g sample and the higher water solubility indices (WSI were found for mannan (60% and for glycoprotein (83.8%. The glycans (soluble and insoluble presented good gelling properties at 12 and 14% solid concentrations. Glycoprotein, mannan and soluble glycan presented excellent emulsifying capacity (1,500 to 2,000 ml oil/g sample. The glycoprotein and soluble glycan emulsitions were the most stable. Addition of ovalbumin (0.2% in the emulsifying media increased significantly the cellular wall and soluble glycan emulsifying capacity. It also contributed for the stabilization of the mannan (M and glycans (IG and SG emultions.

  16. Antibody-based screening of cell wall matrix glycans in ferns reveals taxon, tissue and cell-type specific distribution patterns

    Leroux, Olivier; Sørensen, Iben; Marcus, Susan E.;

    2015-01-01

    Background: While it is kno3wn that complex tissues with specialized functions emerged during land plant evolution, it is not clear how cell wall polymers and their structural variants are associated with specific tissues or cell types. Moreover, due to the economic importance of many flowering...... plants, ferns have been largely neglected in cell wall comparative studies. Results: To explore fern cell wall diversity sets of monoclonal antibodies directed to matrix glycans of angiosperm cell walls have been used in glycan microarray and in situ analyses with 76 fern species and four species of...... across the ferns and specifically associated with phloem cell walls and similarly the LM11 xylan epitope was associated with xylem cell walls. The LM5 galactan and LM6 arabinan epitopes, linked to pectic supramolecules in angiosperms, were associated with vascular structures with only limited detection...

  17. Dynamics of the carbohydrate chains attached to the Fc portion of immunoglobulin G as studied by NMR spectroscopy assisted by selective 13C labeling of the glycans

    A systematic method for 13C 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 Manα1-3 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

  18. Sialylation by β-galactoside α-2,6-sialyltransferase and N-glycans regulate cell adhesion and invasion in human anaplastic large cell lymphoma

    Suzuki, Osamu; Abe, Masafumi; HASHIMOTO, YUKO

    2015-01-01

    The interaction between cell surface glycans and extracellular matrix (ECM) including galectins is known to be closely associated with tumor cell adhesion, invasion and metastasis. We analyzed the roles of cell surface sialylation or glycosylation in galectin or ECM-mediated cell adhesion and invasion of human malignant lymphoma cells. Neuraminidase from Arthrobacter ureafaciens (AU) treatment resulted in reduction of cell adhesion to galectin-8 in human anaplastic large cell lymphoma (H-ALCL...

  19. Differential chemical derivatization integrated with chromatographic separation for analysis of isomeric sialylated N-glycans: a nano-hydrophilic interaction liquid chromatography-MS platform.

    Tousi, Fateme; Bones, Jonathan; Hancock, William S; Hincapie, Marina

    2013-09-01

    MS analysis of sialylated glycans is challenging due to their low ionization efficiency in positive ion mode as well as the possibility of in-source fragmentation. Chemical derivatization strategies have been developed to address this issue focused on removal of the labile acidic proton prior to MS analysis. Highly sialylated negatively charged glycans also exhibit high retention and unsatisfactory separation efficiency when analyzed by hydrophilic interaction liquid chromatography (HILIC) due to their high polarity. Here, we combined linkage specific derivatization of sialic acids by reaction with the condensation reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM) in methanol with nanoscale liquid chromatographic separation prior to accurate mass Orbitrap MS analysis. Coupling DMT-MM charge neutralization of sialic acids with nano-HILIC-Orbitrap-MS not only allows for linkage specific characterization of sialylated glycans directly from the precursor mass but also improves the preceding HILIC separation by increasing the hydrophobicity and altering the selectivity of the oligosaccharide analytes. We focused on the trisialylated N-glycan fraction from haptoglobin and human plasma, enriched using weak anion exchange chromatography, as this trisialylated fraction has been linked with cancer associated changes in the serum N-glycome. The developed methodology was applied to investigate whether structural alterations in this oligosaccharide pool, enriched from the sera of pathological stage and sex matched patients bearing lung, breast, ovarian, pancreatic, or gastric cancer, demonstrate any degree of cancer specificity or whether changes in expression levels are purely cancer associated. The results of this pilot study indicate limited degrees of cancer specificity, particularly for pancreatic cancer, based on alterations in the relative abundance of specific trisialylated isomers. PMID:23901877

  20. Glycan masking of hemagglutinin for adenovirus vector and recombinant protein immunizations elicits broadly neutralizing antibodies against H5N1 avian influenza viruses.

    Lin, Shih-Chang; Liu, Wen-Chun; Jan, Jia-Tsrong; Wu, Suh-Chin

    2014-01-01

    The highly pathogenic avian influenza (HPAI) H5N1 virus, a known trigger of diseases in poultry and humans, is perceived as a serious threat to public health. There is a clear need for a broadly protective H5N1 vaccine or vaccines for inducing neutralizing antibodies against multiple clades/subclades. We constructed single, double, and triple mutants of glycan-masked hemagglutiinin (HA) antigens at residues 83, 127 and 138 (i.e., g83, g127, g138, g83+g127, g127+g138, g83+g138 and g83+g127+g138), and then obtained their corresponding HA-expressing adenovirus vectors and recombinant HA proteins using a prime-boost immunization strategy. Our results indicate that the glycan-masked g127+g138 double mutant induced more potent HA-inhibition, virus neutralization antibodies, cross-clade protection against heterologous H5N1 clades, correlated with the enhanced bindings to the receptor binding sites and the highly conserved stem region of HA. The immune refocusing stem-specific antibodies elicited by the glycan-masked H5HA g127+g138 and g83+g127+g138 mutants overlapped with broadly neutralizing epitopes of the CR6261 monoclonal antibody that neutralizes most group 1 subtypes. These findings may provide useful information in the development of a broadly protective H5N1 influenza vaccine. PMID:24671139

  1. Removal of either N-glycan site from the envelope receptor binding domain of Moloney and Friend but not AKV mouse ecotropic gammaretroviruses alters receptor usage

    Three N-linked glycosylation sites were removed from the envelope glycoproteins of Friend, Moloney, and AKV mouse ecotropic gammaretroviruses: gs1 and gs2, in the receptor binding domain; and gs8, in a region implicated in post-binding cell fusion. Mutants were tested for their ability to infect rodent cells expressing 4 CAT-1 receptor variants. Three mutants (Mo-gs1, Mo-gs2, and Fr-gs1) infect NIH 3T3 and rat XC cells, but are severely restricted in Mus dunni cells and Lec8, a Chinese hamster cell line susceptible to ecotropic virus. This restriction is reproduced in ferret cells expressing M. dunni dCAT-1, but not in cells expressing NIH 3T3 mCAT-1. Virus binding assays, pseudotype assays, and the use of glycosylation inhibitors further suggest that restriction is primarily due to receptor polymorphism and, in M. dunni cells, to glycosylation of cellular proteins. Virus envelope glycan size or type does not affect infectivity. Thus, host range variation due to N-glycan deletion is receptor variant-specific, cell-specific, virus type-specific, and glycan site-specific.

  2. State-of-the-art technologies for rapid and high-throughput sample preparation and analysis of N-glycans from antibodies.

    Aich, Udayanath; Lakbub, Jude; Liu, Aston

    2016-06-01

    Glycosylation is a PTM that occurs during production of many protein-based biologic drugs and can have a profound impact on their biological, clinical, and pharmacological properties. Quality by design, process optimization, and advance in manufacturing technology create a demand for robust, sensitive, and accurate profiling and quantification of antibody glycosylation. Potential drawbacks in antibody glycosylation profiling include the high hands-on time required for sample preparation and several hours for data acquisition and analysis. Rapid and high-throughput (HTP) N-glycan profiling and characterization along with automation for sample preparation and analysis are essential for extensive antibody glycosylation analysis due to the substantial improvement of turnaround time. The first part of this review article will focus on the recent progress in rapid and HTP sample preparation and analysis of antibody glycosylation. Subsequently, the article will cover a brief overview of various separation and mass spectrometric methods for the rapid and HTP analysis of N-glycans in antibodies. Finally, we will discuss the recent developments in process analytical technologies for the screening and quantification of N-glycans in antibodies. PMID:26829758

  3. N-glycan containing a core α1,3-fucose residue is required for basipetal auxin transport and gravitropic response in rice (Oryza sativa).

    Harmoko, Rikno; Yoo, Jae Yong; Ko, Ki Seong; Ramasamy, Nirmal Kumar; Hwang, Bo Young; Lee, Eun Ji; Kim, Ho Soo; Lee, Kyung Jin; Oh, Doo-Byoung; Kim, Dool-Yi; Lee, Sanghun; Li, Yang; Lee, Sang Yeol; Lee, Kyun Oh

    2016-10-01

    In plants, α1,3-fucosyltransferase (FucT) catalyzes the transfer of fucose from GDP-fucose to asparagine-linked GlcNAc of the N-glycan core in the medial Golgi. To explore the physiological significance of this processing, we isolated two Oryza sativa (rice) mutants (fuct-1 and fuct-2) with loss of FucT function. Biochemical analyses of the N-glycan structure confirmed that α1,3-fucose is missing from the N-glycans of allelic fuct-1 and fuct-2. Compared with the wild-type cv Kitaake, fuct-1 displayed a larger tiller angle, shorter internode and panicle lengths, and decreased grain filling as well as an increase in chalky grains with abnormal shape. The mutant allele fuct-2 gave rise to similar developmental abnormalities, although they were milder than those of fuct-1. Restoration of a normal tiller angle in fuct-1 by complementation demonstrated that the phenotype is caused by the loss of FucT function. Both fuct-1 and fuct-2 plants exhibited reduced gravitropic responses. Expression of the genes involved in tiller and leaf angle control was also affected in the mutants. We demonstrate that reduced basipetal auxin transport and low auxin accumulation at the base of the shoot in fuct-1 account for both the reduced gravitropic response and the increased tiller angle. PMID:27241276

  4. Versatile metal-organic framework-functionalized magnetic graphene nanoporous composites: As deft matrix for high-effective extraction and purification of the N-linked glycans.

    Wang, Jiaxi; Wang, Yanan; Gao, Mingxia; Zhang, Xiangmin; Yang, Pengyuan

    2016-08-17

    The highly selective enrichment of N-linked glycans from complex biological sample is still very important but challenging task due to the ultra-low abundance, complicated structures and strong ion suppress effect caused by distractors such as proteins, peptides and salts. Here, we firstly present a novel metal-organic frameworks (MOFs)-functionalized magnetic nanoporous carbon-graphene composites (C-magG@ZIF-8) synthesized through a smart process. The obtained materials enjoy the unique properties including strong magnetic responsiveness, a large sum of graphitized carbon pore, remarkable biocompatibility and large specific surface area. By virtue of these unique properties, the C-magG@ZIF-8 composites displayed excellent selectivity and sensitivity, good recyclability and incredible size exclusion ability (roughly 2000 times) in the N-linked glycans analysis. Furthermore, 48 N-linked glycans were clearly identified from the normal human serum treated with the C-magG@ZIF-8. There is reason to believe that our smart strategy offers new possibilities for preparing the MOFs-functionalized composites for large-scale characterization of glycoproteomics by mass spectrometry analysis. PMID:27286768

  5. Comparison of analytical methods for profiling N- and O-linked glycans from cultured cell lines : HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.

    Ito, Hiromi; Kaji, Hiroyuki; Togayachi, Akira; Azadi, Parastoo; Ishihara, Mayumi; Geyer, Rudolf; Galuska, Christina; Geyer, Hildegard; Kakehi, Kazuaki; Kinoshita, Mitsuhiro; Karlsson, Niclas G; Jin, Chunsheng; Kato, Koichi; Yagi, Hirokazu; Kondo, Sachiko; Kawasaki, Nana; Hashii, Noritaka; Kolarich, Daniel; Stavenhagen, Kathrin; Packer, Nicolle H; Thaysen-Andersen, Morten; Nakano, Miyako; Taniguchi, Naoyuki; Kurimoto, Ayako; Wada, Yoshinao; Tajiri, Michiko; Yang, Pengyuan; Cao, Weiqian; Li, Hong; Rudd, Pauline M; Narimatsu, Hisashi

    2016-06-01

    The Human Disease Glycomics/Proteome Initiative (HGPI) is an activity in the Human Proteome Organization (HUPO) supported by leading researchers from international institutes and aims at development of disease-related glycomics/glycoproteomics analysis techniques. Since 2004, the initiative has conducted three pilot studies. The first two were N- and O-glycan analyses of purified transferrin and immunoglobulin-G and assessed the most appropriate analytical approach employed at the time. This paper describes the third study, which was conducted to compare different approaches for quantitation of N- and O-linked glycans attached to proteins in crude biological samples. The preliminary analysis on cell pellets resulted in wildly varied glycan profiles, which was probably the consequence of variations in the pre-processing sample preparation methodologies. However, the reproducibility of the data was not improved dramatically in the subsequent analysis on cell lysate fractions prepared in a specified method by one lab. The study demonstrated the difficulty of carrying out a complete analysis of the glycome in crude samples by any single technology and the importance of rigorous optimization of the course of analysis from preprocessing to data interpretation. It suggests that another collaborative study employing the latest technologies in this rapidly evolving field will help to realize the requirements of carrying out the large-scale analysis of glycoproteins in complex cell samples. PMID:26511985

  6. The cattle tick antigen, Bm95, expressed in Pichia pastoris contains short chains of N- and O-glycans.

    González, Luis J; Cremata, José A; Guanche, Yazmín; Ramos, Yassel; Triguero, Ada; Cabrera, Gleysin; Montesino, Raquel; Huerta, Vivian; Pons, Tirso; Boué, Oscar; Farnós, Omar; Rodríguez, Manuel

    2004-12-15

    Bm95 is an antigen isolated from Boophilus microplus strains with low susceptibility to antibodies developed in cattle vaccinated with the recombinant Bm86 antigen (Gavac, HeberBiotec S.A., Cuba). It is a Bm86-like surface protein, which by similarity contains seven EGF-like domains and a lipid-binding GPI-anchor site at the C-terminal region. The primary structure of the recombinant (rBm95) protein expressed in Pichia pastoris was completely verified by LC/MS. The four potential glycosylation sites (Asn 122, 163, 329, and 363) are glycosylated partially with short N-glycans, from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) of which, Man(8-9)GlcNAc(2) were the most abundant. O-Glycopeptides are distributed mostly towards the protein N-terminus. While the first N-glycosylated site (Asn(122)) is located between EGF-like domains 2 and 3, where the O-glycopeptides were found, two other N-glycosylated sites (Asn(329) and Asn(363)) are located between EGF-like domains 5 and 6, a region devoid of O-glycosylated Ser or Thr. PMID:15542059

  7. Host-derived glycans serve as selected nutrients for the gut microbe: human milk oligosaccharides and bifidobacteria.

    Katayama, Takane

    2016-04-01

    Lactation is a common feeding strategy of eutherian mammals, but its functions go beyond feeding the neonates. Ever since Tissier isolated bifidobacteria from the stool of breast-fed infants, human milk has been postulated to contain compounds that selectively stimulate the growth of bifidobacteria in intestines. However, until relatively recently, there have been no reports to link human milk compound(s) with bifidobacterial physiology. Over the past decade, successive studies have demonstrated that infant-gut-associated bifidobacteria are equipped with genetic and enzymatic toolsets dedicated to assimilation of host-derived glycans, especially human milk oligosaccharides (HMOs). Among gut microbes, the presence of enzymes required for degrading HMOs with type-1 chains is essentially limited to infant-gut-associated bifidobacteria, suggesting HMOs serve as selected nutrients for the bacteria. In this study, I shortly discuss the research on bifidobacteria and HMOs from a historical perspective and summarize the roles of bifidobacterial enzymes in the assimilation of HMOs with type-1 chains. Based on this overview, I suggest the co-evolution between bifidobacteria and human beings mediated by HMOs. PMID:26838671

  8. Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis

    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

  9. Quantitative analysis of N-glycans from human alfa-acid-glycoprotein using stable isotope labeling and zwitterionic hydrophilic interaction capillary liquid chromatography electrospray mass spectrometry as tool for pancreatic disease diagnosis

    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 [12C]- and [13C]-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 α1-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 candidate

  10. Glycans affect DNA extraction and induce substantial differences in gut metagenomic studies

    Angelakis, E; Bachar, D.; Henrissat, B; Armougom, Fabrice; Audoly, G.; Lagier, J. C.; Robert, C.; Raoult, Didier

    2016-01-01

    Exopolysaccharides produced by bacterial species and present in feces are extremely inhibitory to DNA restriction and can cause discrepancies in metagenomic studies. We determined the effects of different DNA extraction methods on the apparent composition of the gut microbiota using Illumina MiSeq deep sequencing technology. DNA was extracted from the stool from an obese female using 10 different methods and the choice of DNA extraction method affected the proportional abundance at the phylum...

  11. Glycan analysis of Fonsecaea monophora from clinical and environmental origins reveals different structural profile and human antigenic response

    Juliana Reis Burjack

    2014-10-01

    Full Text Available Dematiaceous fungi constitute a large and heterogeneous group, characterized by having a dark pigment, the dihydroxynaftalen melanin - DHN, inside their cell walls. In nature they are found mainly as soil microbiota or decomposing organic matter, and are spread in tropical and subtropical regions. The fungus Fonsecaea monophora causes chromoblastomycosis in humans, and possesses essential mechanisms that may enhance pathogenicity, proliferation and dissemination inside the host. Glycoconjugates confer important properties to these pathogenic microorganisms. In this work, structural characterization of glycan structures present in two different strains of F. monophora MMHC82 and FE5p4, from clinical and environmental origins, respectively, was performed. Each one were grown on Minimal Medium (MM and Czapeck-Dox (CD medium, and the water soluble cell wall glycoconjugates and exopolysaccharides (EPS were evaluated by NMR, methylation and principal component analysis (PCA. By combining the methylation and 2D NMR analyses, it was possible to visualize the glycosidic profiles of the complex carbohydrate mixtures. Significant differences were observed in β-D-Galf-(1→5 and (1→6 linkages, α- and β-D-Glcp-(1→3, (1→4 and (1→6 units, as well as in α-D-Manp. PCA from 1H-NMR data showed that MMHC82 from CD medium showed a higher variation in the cell wall carbohydrates, mainly related to O-2 substituted β-D-Galf (δ 106.0/5.23 and δ 105.3/5.23 units. In order to investigate the antigenic response of the glycoconjugates, these were screened against serum from chromoblastomycosis patients. The antigen which contained the cell wall of MMHC82 grown in MM had β-D-Manp units that promoted higher antigenic response. The distribution of these fungal species in nature and the knowledge of how cell wall polysaccharides and glycoconjugates structure vary, may contribute to the better understanding and the elucidation of the pathology caused by this

  12. Strategy integrating stepped fragmentation and glycan diagnostic ion-based spectrum refinement for the identification of core fucosylated glycoproteome using mass spectrometry.

    Cao, Qichen; Zhao, Xinyuan; Zhao, Qing; Lv, Xiaodong; Ma, Cheng; Li, Xianyu; Zhao, Yan; Peng, Bo; Ying, Wantao; Qian, Xiaohong

    2014-07-15

    Core fucosylation (CF) is a special glycosylation pattern of proteins that has a strong relationship with cancer. The Food and Drug Administration (FDA) has approved the core fucosylated α-fetoprotein as a biomarker for the early diagnosis of hepatocellular carcinoma (HCC). The technology for identifying core fucosylated proteins has significant practical value. The major method for core fucosylated glycoprotein/glycopeptide analysis is neutral loss-based MS(3) scanning under collision-induced dissociation (CID) by ion trap mass spectrometry. However, due to the limited speed and low resolution of the MS(3) scan mode, it is difficult to achieve high-throughput, with only dozens of core fucosylated proteins identified in a single run. In this work, we developed a novel strategy for the identification of CF glycopeptides at a large scale, integrating the stepped fragmentation function, one novel feature of quadrupole-orbitrap mass spectrometry, with "glycan diagnostic ion"-based spectrum optimization. By using stepped fragmentation, we were able to obtain both highly accurate glycan and peptide information of a simplified CF glycopeptide in one spectrum. Moreover, the spectrum could be recorded with the same high speed as the conventional MS(2) scan. By using the "glycan diagnostic ion"-based spectrum refinement method, the efficiency of the CF glycopeptide discovery was significantly improved. We demonstrated the feasibility and reproducibility of our method by analyzing CF glycoproteomes of mouse liver tissue and HeLa cell samples spiked with standard CF glycoprotein. In total, 1364 and 856 CF glycopeptides belonging to 702 and 449 CF glycoproteins were identified, respectively, within a 78-min gradient analysis, which was approximately a 7-fold increase in the identification efficiency of CF glycopeptides compared to the currently used method. In this work, we took core fucosylated glycopeptides as a practical example to demonstrate the great potential of our

  13. Core 2 mucin-type O-glycan inhibits EPEC or EHEC O157:H7 invasion into HT-29 epithelial cells

    Ye, Jun; Qiong PAN; Shang, Yangyang; Wei, Xiaolong; Peng, Zhihong; Chen, Wensheng; Chen, Lei; Wang, Rongquan

    2015-01-01

    Background How host cell glycosylation affects EPEC or EHEC O157:H7 invasion is unclear. This study investigated whether and how O-glycans were involved in EPEC or EHEC O157:H7 invasion into HT-29 cells. Results Lectin histochemical staining confirmed stronger staining with PNA, which labeled Galβ1, 3 GalNAc (core 1 structure) in HT-29-Gal-OBN and C2GnT2-sh2/HT-29 cells, compared with control cells. EPEC or EHEC O157:H7 invasion into HT-29 and its derived cells was based on the intracellular ...

  14. Characterization of a monoclonal antibody to a novel glycan-dependent epitope in the V1/V2 domain of the HIV-1 envelope protein, gp120.

    Doran, Rachel C; Morales, Javier F; To, Briana; Morin, Trevor J; Theolis, Richard; O'Rourke, Sara M; Yu, Bin; Mesa, Kathryn A; Berman, Phillip W

    2014-11-01

    Recent studies have described several broadly neutralizing monoclonal antibodies (bN-mAbs) that recognize glycan-dependent epitopes (GDEs) in the HIV-1 envelope protein, gp120. These were recovered from HIV-1 infected subjects, and several (e.g., PG9, PG16, CH01, CH03) target glycans in the first and second variable (V1/V2) domain of gp120. The V1/V2 domain is thought to play an important role in conformational masking, and antibodies to the V1/V2 domain were recently identified as the only immune response that correlated with protection in the RV144 HIV-1 vaccine trial. While the importance of antibodies to polymeric glycans is well established for vaccines targeting bacterial diseases, the importance of antibodies to glycans in vaccines targeting HIV has only recently been recognized. Antibodies to GDEs may be particularly significant in HIV vaccines based on gp120, where 50% of the molecular mass of the envelope protein is contributed by N-linked carbohydrate. However, few studies have reported antibodies to GDEs in humans or animals immunized with candidate HIV-1 vaccines. In this report, we describe the isolation of a mouse mAb, 4B6, after immunization with the extracellular domain of the HIV-1 envelope protein, gp140. Epitope mapping using glycopeptide fragments and in vitro mutagenesis showed that binding of this antibody depends on N-linked glycosylation at asparagine N130 (HXB2 numbering) in the gp120 V1/V2 domain. Our results demonstrate that, in addition to natural HIV-1 infection, immunization with recombinant proteins can elicit antibodies to the GDEs in the V1/V2 domain of gp120. Although little is known regarding conditions that favor antibody responses to GDEs, our studies demonstrate that these antibodies can arise from a short-term immunization regimen. Our results suggest that antibodies to GDEs are more common than previously suspected, and that further analysis of antibody responses to the HIV-1 envelope protein will lead to the discovery of

  15. Changes in the profile of simple mucin-type O-glycans and polypeptide GalNAc-transferases in human testis and testicular neoplasms are associated with germ cell maturation and tumour differentiation

    Rajpert-De Meyts, E; Poll, S N; Goukasian, I;

    2007-01-01

    Testicular germ cell tumours (TGCT) exhibit remarkable ability to differentiate into virtually all somatic tissue types. In this study, we investigated changes in mucin-type O-glycosylation, which have been associated with somatic cell differentiation and cancer. Expression profile of simple muci...... in testicular neoplasms recapitulated the developmental order: Pre-invasive carcinoma in situ (CIS) cells and seminoma expressed fetal type sialylated glycans in keeping with their gonocyte-like phenotype. Neither simple mucin-type O-glycans nor GalNAc-transferase isoforms were found in...

  16. A lectin recognizes differential arrangements of O-glycans on mucin repeats.

    Kato, Kentaro; Takeuchi, Hideyuki; Ohki, Takao; Waki, Michihiko; Usami, Katsuaki; Hassan, Helle; Clausen, Henrik; Irimura, Tatsuro

    2008-07-11

    Interaction of Vicia villosa agglutinin-B4 (VVA-B4) to glycopeptides with O-linked GalNAc residues was investigated by surface plasmon resonance. The affinity was shown to be influenced by the arrangement of O-glycosylation sites on a peptide, PTTTPITTTTK, representing the tandem repeat of MUC2. The association rate constant was relatively high with a particular category of GalNAc-peptides in which more than three amino acid residues were placed between GalNAc-Thr residues. PTT( *)T( *)PITT( *)T( *)TK (T( *) indicates GalNAc-Thr) had the highest association rate constant among the glycopeptides tested. The dissociation rate constant was low in the peptides containing consecutive GalNAc residues and PT( *)TTPIT( *)T( *)T( *)TK was the lowest of the glycopeptides tested. Dissociation constant (K(D)), calculated as k(d)/k(a) was the lowest with PTT( *)T( *)PITT( *)T( *)TK. Therefore, the arrangement but not the quantity of GalNAc residues apparently determines the affinity between VVA-B4 and peptides with attached GalNAc residues. PMID:18455506

  17. Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella -- their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxin.

    Nakanishi, Kazuko; Yaoi, Katsuro; Nagino, Yasushi; Hara, Hirotaka; Kitami, Madoka; Atsumi, Shogo; Miura, Nami; Sato, Ryoichi

    2002-05-22

    Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin-binding region of each APN isoform. The results suggest that APN isoforms have a common toxin-binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS-PAGE is determined by factors such as expression level in conjunction with differences in binding affinity. PMID:12023048

  18. Determination of fat tissue area in the abdomen and evaluation of degree of obesity. Pt. 1. A unique application of a densitometric technique of computed tomography for CT values of fat tissue area

    Computed tomography (CT) scanning images were taken from 26 normal subjects, 23 obesity patients and 11 with leanness to determine fat tissue values. Setting three regions of interest (ROIs) for fat tissues identified by a double-window display, a total of 52 images were employed. Histograms were constructed for each of the 3 ROIs, and the maximum, mean and minimum values were computed for each fat tissues. Areas of entire fat tissues were computed on each image with the above-cited CT software for thyroidal iodine contents by setting ROIs along the outline of body, the abdominal wall and the wall of colon, respectively. Areas of subcutaneous fat tissues were calculated by simply subtracting the values of visceral fat tissues from those of entire fat tissues. Means of maximum and minimum CT values of visceral fat tissues on 52 images were -34.7 HU and -162.1 HU, respectively. The double-window display indicated that the spectrum of CT values of fat tissue included not only visceral and subcutaneous fat tissues but fecal materials with air bubbles in the colon. Areas of fecal materials with the same CT values as that of the fat tissues occupied 2.5±3.0% of that of the visceral fat tissue. The areas of subcutaneous and visceral fat tissues were largest at the levels of -20 to 0 mm and 60 to 100 mm, respectively, on all images. At the level of 0 mm, the areas of visceral fat tissue did not show any differences among normal subjects, obesity patients and patients with leanness. It was concluded that the CT software is applicable to obtain satisfactory values for areas of visceral fat tissue, and that CT images at the levels of 0, 40, 60 and 100 mm are necessary to accurately determine areas of visceral fat tissues. (S.Y.)

  19. A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3).

    van Wijk, Xander M; Lawrence, Roger; Thijssen, Victor L; van den Broek, Sebastiaan A; Troost, Ran; van Scherpenzeel, Monique; Naidu, Natasha; Oosterhof, Arie; Griffioen, Arjan W; Lefeber, Dirk J; van Delft, Floris L; van Kuppevelt, Toin H

    2015-07-01

    Glycosaminoglycan (GAG) polysaccharides have been implicated in a variety of cellular processes, and alterations in their amount and structure have been associated with diseases such as cancer. In this study, we probed 11 sugar analogs for their capacity to interfere with GAG biosynthesis. One analog, with a modification not directly involved in the glycosidic bond formation, 6F-N-acetyl-d-galactosamine (GalNAc) (Ac3), was selected for further study on its metabolic and biologic effect. Treatment of human ovarian carcinoma cells with 50 μM 6F-GalNAc (Ac3) inhibited biosynthesis of GAGs (chondroitin/dermatan sulfate by ∼50-60%, heparan sulfate by ∼35%), N-acetyl-d-glucosamine (GlcNAc)/GalNAc containing glycans recognized by the lectins Datura stramonium and peanut agglutinin (by ∼74 and ∼43%, respectively), and O-GlcNAc protein modification. With respect to function, 6F-GalNAc (Ac3) treatment inhibited growth factor signaling and reduced in vivo angiogenesis by ∼33%. Although the analog was readily transformed in cells into the uridine 5'-diphosphate (UDP)-activated form, it was not incorporated into GAGs. Rather, it strongly reduced cellular UDP-GalNAc and UDP-GlcNAc pools. Together with data from the literature, these findings indicate that nucleotide sugar depletion without incorporation is a common mechanism of sugar analogs for inhibiting GAG/glycan biosynthesis. PMID:25868729

  20. N-Glycans on secretory component: mediators of the interaction between secretory IgA and gram-positive commensals sustaining intestinal homeostasis.

    Mathias, Amandine; Corthésy, Blaise

    2011-09-01

    Human beings live in symbiosis with billions of microorganisms colonizing mucosal surfaces. The understanding of the mechanisms underlying this fine-tuned intestinal balance has made significant processes during the last decades. We have recently demonstrated that the interaction of SIgA with Gram-positive bacteria is essentially based on Fab-independent, glycan-mediated recognition. Results obtained using mouse hybridoma- and colostrum-derived secretory IgA (SIgA) consistently show that N-glycans present on secretory component (SC) play a crucial role in the process. Natural coating may involve specific Gram-positive cell wall components, which may explain selective recognition at the molecular level. More widely, the existence of these complexes is involved in the modulation of intestinal epithelial cell (IEC) responses in vitro and the formation of intestinal biofilms. Thus, SIgA may act as one of the pillars in homeostatic maintenance of the microbiota in the gut, adding yet another facet to its multiple roles in the mucosal environment. PMID:22067937

  1. Different immunity elicited by recombinant H5N1 hemagglutinin proteins containing pauci-mannose, high-mannose, or complex type N-glycans.

    Shih-Chang Lin

    Full Text Available Highly pathogenic avian influenza H5N1 viruses can result in poultry and occasionally in human mortality. A safe and effective H5N1 vaccine is urgently needed to reduce the pandemic potential. Hemagglutinin (HA, a major envelope protein accounting for approximately 80% of spikes in influenza virus, is often used as a major antigen for subunit vaccine development. In this study, we conducted a systematic study of the immune response against influenza virus infection following immunization with recombinant HA proteins expressed in insect (Sf9 cells, insect cells that contain exogenous genes for elaborating N-linked glycans (Mimic and mammalian cells (CHO. While the antibody titers are higher with the insect cell derived HA proteins, the neutralization and HA inhibition titers are much higher with the mammalian cell produced HA proteins. Recombinant HA proteins containing tri- or tetra-antennary complex, terminally sialylated and asialyated-galactose type N-glycans induced better protective immunity in mice to lethal challenge. The results are highly relevant to issues that should be considered in the production of fragment vaccines.

  2. Analysis of O-glycan heterogeneity in IgA1 myeloma proteins by Fourier transform ion cyclotron resonance mass spectrometry: implications for IgA nephropathy

    Renfrow, MB; Mackay, CL; Chalmers, MJ;

    2007-01-01

    IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis. In IgAN, IgA1 molecules with incompletely galactosylated O-linked glycans in the hinge region (HR) are present in mesangial immunodeposits and in circulating immune complexes. It is not known whether the galactose...... deficiency in IgA1 proteins occurs randomly or preferentially at specific sites. We have previously demonstrated the first direct localization of multiple O-glycosylation sites on a single IgA1 myeloma protein by use of activated ion-electron capture dissociation (AI-ECD) Fourier transform ion cyclotron...... resonance (FT-ICR) tandem mass spectrometry. Here, we report the analysis of IgA1 O-glycan heterogeneity by use of FT-ICR MS and liquid chromatography FT-ICR MS to obtain unbiased accurate mass profiles of IgA1 HR glycopeptides from three different IgA1 myeloma proteins. Additionally, we report the first AI...

  3. Differences in the mannose oligomer specificities of the closely related lectins from Galanthus nivalis and Zea mays strongly determine their eventual anti-HIV activity

    Fouquaert Elke

    2011-02-01

    Full Text Available Abstract Background In a recent report, the carbohydrate-binding specificities of the plant lectins Galanthus nivalis (GNA and the closely related lectin from Zea mays (GNAmaize were determined by glycan array analysis and indicated that GNAmaize recognizes complex-type N-glycans whereas GNA has specificity towards high-mannose-type glycans. Both lectins are tetrameric proteins sharing 64% sequence similarity. Results GNAmaize appeared to be ~20- to 100-fold less inhibitory than GNA against HIV infection, syncytia formation between persistently HIV-1-infected HuT-78 cells and uninfected CD4+ T-lymphocyte SupT1 cells, HIV-1 capture by DC-SIGN and subsequent transmission of DC-SIGN-captured virions to uninfected CD4+ T-lymphocyte cells. In contrast to GNA, which preferentially selects for virus strains with deleted high-mannose-type glycans on gp120, prolonged exposure of HIV-1 to dose-escalating concentrations of GNAmaize selected for mutant virus strains in which one complex-type glycan of gp120 was deleted. Surface Plasmon Resonance (SPR analysis revealed that GNA and GNAmaize interact with HIV IIIB gp120 with affinity constants (KD of 0.33 nM and 34 nM, respectively. Whereas immobilized GNA specifically binds mannose oligomers, GNAmaize selectively binds complex-type GlcNAcβ1,2Man oligomers. Also, epitope mapping experiments revealed that GNA and the mannose-specific mAb 2G12 can independently bind from GNAmaize to gp120, whereas GNAmaize cannot efficiently bind to gp120 that contained prebound PHA-E (GlcNAcβ1,2man specific or SNA (NeuAcα2,6X specific. Conclusion The markedly reduced anti-HIV activity of GNAmaize compared to GNA can be explained by the profound shift in glycan recognition and the disappearance of carbohydrate-binding sites in GNAmaize that have high affinity for mannose oligomers. These findings underscore the need for mannose oligomer recognition of therapeutics to be endowed with anti-HIV activity and that mannose, but

  4. Negligible elongation of mucin glycans with Gal β1-3 units distinguishes the laminated layer of Echinococcus multilocularis from that of Echinococcus granulosus.

    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. PMID:26891615

  5. Neofunctionalization of the Sec1 α1,2fucosyltransferase paralogue in leporids contributes to glycan polymorphism and resistance to rabbit hemorrhagic disease virus.

    Nyström, Kristina; Abrantes, Joana; Lopes, Ana Margarida; Le Moullac-Vaidye, Béatrice; Marchandeau, Stéphane; Rocher, Jézabel; Ruvoën-Clouet, Nathalie; Esteves, Pedro J; Le Pendu, Jacques

    2015-04-01

    RHDV (rabbit hemorrhagic disease virus), a virulent calicivirus, causes high mortalities in European rabbit populations (Oryctolagus cuniculus). It uses α1,2fucosylated glycans, histo-blood group antigens (HBGAs), as attachment factors, with their absence or low expression generating resistance to the disease. Synthesis of these glycans requires an α1,2fucosyltransferase. In mammals, there are three closely located α1,2fucosyltransferase genes rSec1, rFut2 and rFut1 that arose through two rounds of duplications. In most mammalian species, Sec1 has clearly become a pseudogene. Yet, in leporids, it does not suffer gross alterations, although we previously observed that rabbit Sec1 variants present either low or no activity. Still, a low activity rSec1 allele correlated with survival to an RHDV outbreak. We now confirm the association between the α1,2fucosyltransferase loci and survival. In addition, we show that rabbits express homogenous rFut1 and rFut2 levels in the small intestine. Comparison of rFut1 and rFut2 activity showed that type 2 A, B and H antigens recognized by RHDV strains were mainly synthesized by rFut1, and all rFut1 variants detected in wild animals were equally active. Interestingly, rSec1 RNA levels were highly variable between individuals and high expression was associated with low binding of RHDV strains to the mucosa. Co-transfection of rFut1 and rSec1 caused a decrease in rFut1-generated RHDV binding sites, indicating that in rabbits, the catalytically inactive rSec1 protein acts as a dominant-negative of rFut1. Consistent with neofunctionalization of Sec1 in leporids, gene conversion analysis showed extensive homogenization between Sec1 and Fut2 in leporids, at variance with its limited degree in other mammals. Gene conversion additionally involving Fut1 was also observed at the C-terminus. Thus, in leporids, unlike in most other mammals where it became extinct, Sec1 evolved a new function with a dominant-negative effect on rFut1

  6. Neofunctionalization of the Sec1 α1,2fucosyltransferase paralogue in leporids contributes to glycan polymorphism and resistance to rabbit hemorrhagic disease virus.

    Kristina Nyström

    2015-04-01

    Full Text Available RHDV (rabbit hemorrhagic disease virus, a virulent calicivirus, causes high mortalities in European rabbit populations (Oryctolagus cuniculus. It uses α1,2fucosylated glycans, histo-blood group antigens (HBGAs, as attachment factors, with their absence or low expression generating resistance to the disease. Synthesis of these glycans requires an α1,2fucosyltransferase. In mammals, there are three closely located α1,2fucosyltransferase genes rSec1, rFut2 and rFut1 that arose through two rounds of duplications. In most mammalian species, Sec1 has clearly become a pseudogene. Yet, in leporids, it does not suffer gross alterations, although we previously observed that rabbit Sec1 variants present either low or no activity. Still, a low activity rSec1 allele correlated with survival to an RHDV outbreak. We now confirm the association between the α1,2fucosyltransferase loci and survival. In addition, we show that rabbits express homogenous rFut1 and rFut2 levels in the small intestine. Comparison of rFut1 and rFut2 activity showed that type 2 A, B and H antigens recognized by RHDV strains were mainly synthesized by rFut1, and all rFut1 variants detected in wild animals were equally active. Interestingly, rSec1 RNA levels were highly variable between individuals and high expression was associated with low binding of RHDV strains to the mucosa. Co-transfection of rFut1 and rSec1 caused a decrease in rFut1-generated RHDV binding sites, indicating that in rabbits, the catalytically inactive rSec1 protein acts as a dominant-negative of rFut1. Consistent with neofunctionalization of Sec1 in leporids, gene conversion analysis showed extensive homogenization between Sec1 and Fut2 in leporids, at variance with its limited degree in other mammals. Gene conversion additionally involving Fut1 was also observed at the C-terminus. Thus, in leporids, unlike in most other mammals where it became extinct, Sec1 evolved a new function with a dominant-negative effect

  7. Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors

    Sathyanarayana Bangalore K

    2006-10-01

    Full Text Available Abstract Background The mucin MUC16 and the glycosylphosphatidylinositol anchored glycoprotein mesothelin likely facilitate the peritoneal metastasis of ovarian tumors. The biochemical basis and the kinetics of the binding between these two glycoproteins are not clearly understood. Here we have addressed this deficit and provide further evidence supporting the role of the MUC16-mesothelin interaction in facilitating cell-cell binding under conditions that mimic the peritoneal environment. Results In this study we utilize recombinant-Fc tagged human mesothelin to measure the binding kinetics of this glycoprotein to MUC16 expressed on the ovarian tumor cell line OVCAR-3. OVCAR-3 derived sublines that did not express MUC16 showed no affinity for mesothelin. In a flow cytometry-based assay mesothelin binds with very high affinity to the MUC16 on the OVCAR-3 cells with an apparent Kd of 5–10 nM. Maximum interaction occurs within 5 mins of incubation of the recombinant mesothelin with the OVCAR-3 cells and significant binding is observed even after 10 sec. A five-fold molar excess of soluble MUC16 was unable to completely inhibit the binding of mesothelin to the OVCAR-3 cells. Oxidation of the MUC16 glycans, removal of its N-linked oligosaccharides, and treatment of the mucin with wheat germ agglutinin and erythroagglutinating phytohemagglutinin abrogates its binding to mesothelin. These observations suggest that at least a subset of the MUC16-asscociated N-glycans is required for binding to mesothelin. We also demonstrate that MUC16 positive ovarian tumor cells exhibit increased adherence to A431 cells transfected with mesothelin (A431-Meso+. Only minimal adhesion is observed between MUC16 knockdown cells and A431-Meso+ cells. The binding between the MUC16 expressing ovarian tumor cells and the A431-Meso+ cells occurs even in the presence of ascites from patients with ovarian cancer. Conclusion The strong binding kinetics of the mesothelin-MUC16

  8. Deletion of the highly conserved N-glycan at Asn260 of HIV-1 gp120 affects folding and lysosomal degradation of gp120, and results in loss of viral infectivity.

    Leen Mathys

    Full Text Available N-linked glycans covering the surface of the HIV-1 glycoprotein gp120 are of major importance for the correct folding of this glycoprotein. Of the, on average, 24 N-linked glycans present on gp120, the glycan at Asn260 was reported to be essential for the correct expression of gp120 and gp41 in the virus particle and deletion of the N260 glycan in gp120 heavily compromised virus infectivity. We show here that gp160 containing the N260Q mutation reaches the Golgi apparatus during biosynthesis. Using pulse-chase experiments with [35S] methionine/cysteine, we show that oxidative folding was slightly delayed in case of mutant N260Q gp160 and that CD4 binding was markedly compromised compared to wild-type gp160. In the search of compensatory mutations, we found a mutation in the V1/V2 loop of gp120 (S128N that could partially restore the infectivity of mutant N260Q gp120 virus. However, the mutation S128N did not enhance any of the above-mentioned processes so its underlying compensatory mechanism must be a conformational effect that does not affect CD4 binding per se. Finally, we show that mutant N260Q gp160 was cleaved to gp120 and gp41 to a much lower extent than wild-type gp160, and that it was subject of lysosomal degradation to a higher extent than wild-type gp160 showing a prominent role of this process in the breakdown of N260-glycan-deleted gp160, which could not be counteracted by the S128N mutation. Moreover, at least part of the wild-type or mutant gp160 that is normally targeted for lysosomal degradation reached a conformation that enabled CD4 binding.

  9. Glycopeptide Site Heterogeneity and Structural Diversity Determined by Combined Lectin Affinity Chromatography/IMS/CID/MS Techniques

    Zhu, Feifei; Trinidad, Jonathan C.; Clemmer, David E.

    2015-07-01

    Glycopeptides from a tryptic digest of chicken ovomucoid were enriched using a simplified lectin affinity chromatography (LAC) platform, and characterized by high-resolution mass spectrometry (MS) as well as ion mobility spectrometry (IMS)-MS. The LAC platform effectively enriched the glycoproteome, from which a total of 117 glycopeptides containing 27 glycan forms were identified for this protein. IMS-MS analysis revealed a high degree of glycopeptide site heterogeneity. Comparison of the IMS distributions of the glycopeptides from different charge states reveals that higher charge states allow more structures to be resolved. Presumably the repulsive interactions between charged sites lead to more open configurations, which are more readily separated compared with the more compact, lower charge state forms of the same groups of species. Combining IMS with collision induced dissociation (CID) made it possible to determine the presence of isomeric glycans and to reconstruct their IMS profiles. This study illustrates a workflow involving hybrid techniques for determining glycopeptide site heterogeneity and evaluating structural diversity of glycans and glycopeptides.

  10. Synthesis of the tetrasaccharide repeating unit of the O-glycan from the polar flagellum flagellin of Azospirillum brasilense Sp7.

    Pal, Kumar Bhaskar; Mukhopadhyay, Balaram

    2014-12-01

    Chemical synthesis of the tetrasaccharide repeating unit of the O-glycan from the polar flagellum flagellin of Azospirillum brasilense Sp7 in the form of its p-methoxyphenyl glycoside is reported. The required glycosidic linkages have been accomplished by activation of thioglycosides with N-iodosuccinimide in the presence of H2SO4-silica. H2SO4-silica was found to be an effective alternative to the classical acid promoters like TfOH or TMSOTf and it can lead to the formation of both 1,2-cis and 1,2-trans glycosidic linkages depending on the protecting group manipulation and control of the reaction condition. PMID:25299938

  11. Heterogeneity in gamma-glutamyltransferase mRNA expression and glycan structures. Search for tumor-specific variants in human liver metastases and colon carcinoma cells.

    Pettersen, Ingvild; Andersen, Jeanette Hammer; Bjornland, Kristin; Mathisen, Øystein; Bremnes, Roy; Wellman, Maria; Visvikis, Athanase; Huseby, Nils-Erik

    2003-05-30

    The enzyme gamma-glutamyltransferase (GGT) is frequently overexpressed in cancer cells and tissues and has significant utility as a cancer marker. Significant heterogeneity of the enzyme has been described due to both transcriptional and post-translational variations. For possible use in diagnosis and follow-up of patients with colorectal cancer, a search was performed for specific mRNA subtypes and glycan structures of the enzyme in liver metastases. We found no differences in the distribution of three GGT mRNA subtypes (fetal liver, HepG2, placenta) in metastatic tissue and normal liver tissue. Furthermore, the three subtypes were present in leukocytes isolated from both normal individuals and cancer patients. Two colon carcinoma cell lines (Colo 205 and HCC 2998) also displayed the three forms and no consistent changes in mRNA composition were noted after butyrate-induced differentiation of the cells. Thus, neither of the GGT mRNA subforms appear to be tumor-specific, although some qualitative and quantitative variations were noted. Two distinct glycosylation features were detected for GGT in metastatic tissue in contrast to normal liver GGT; an extreme sialic acid heterogeneity and a significant increase in beta1,6GlcNAc branching. The GGT glycans from the two colon carcinoma cell lines also possessed these features. As butyrate treatment of the cells resulted in an increased sialic acid content and a reduced beta1,6GlcNAc branching, the described carbohydrate structures appear to be part of a tumor-related pattern. We were, however, unable to identify such GGT isoforms in serum from patients with advanced colorectal cancer. This indicates that their usefulness in diagnostic use is doubtful. PMID:12758164

  12. N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN.

    Phoenix, Inaia; Nishiyama, Shoko; Lokugamage, Nandadeva; Hill, Terence E; Huante, Matthew B; Slack, Olga A L; Carpio, Victor H; Freiberg, Alexander N; Ikegami, Tetsuro

    2016-01-01

    Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3) grabbing non-integrin (DC-SIGN) acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV) glycoproteins (Gn/Gc) encode five putative N-glycan sequons (asparagine (N)-any amino acid (X)-serine (S)/threonine (T)) at positions: N438 (Gn), and N794, N829, N1035, and N1077 (Gc). The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N) to glutamine (Q) mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: "Gc-large" and "Gc-small", and N1077 was responsible for "Gc-large" band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN. PMID:27223297

  13. N-Glycans on the Rift Valley Fever Virus Envelope Glycoproteins Gn and Gc Redundantly Support Viral Infection via DC-SIGN

    Inaia Phoenix

    2016-05-01

    Full Text Available Rift Valley fever is a mosquito-transmitted, zoonotic disease that infects humans and ruminants. Dendritic cell specific intercellular adhesion molecule 3 (ICAM-3 grabbing non-integrin (DC-SIGN acts as a receptor for members of the phlebovirus genus. The Rift Valley fever virus (RVFV glycoproteins (Gn/Gc encode five putative N-glycan sequons (asparagine (N–any amino acid (X–serine (S/threonine (T at positions: N438 (Gn, and N794, N829, N1035, and N1077 (Gc. The N-glycosylation profile and significance in viral infection via DC-SIGN have not been elucidated. Gc N-glycosylation was first evaluated by using Gc asparagine (N to glutamine (Q mutants. Subsequently, we generated a series of recombinant RVFV MP-12 strain mutants, which encode N-to-Q mutations, and the infectivity of each mutant in Jurkat cells stably expressing DC-SIGN was evaluated. Results showed that Gc N794, N1035, and N1077 were N-glycosylated but N829 was not. Gc N1077 was heterogeneously N-glycosylated. RVFV Gc made two distinct N-glycoforms: “Gc-large” and “Gc-small”, and N1077 was responsible for “Gc-large” band. RVFV showed increased infection of cells expressing DC-SIGN compared to cells lacking DC-SIGN. Infection via DC-SIGN was increased in the presence of either Gn N438 or Gc N1077. Our study showed that N-glycans on the Gc and Gn surface glycoproteins redundantly support RVFV infection via DC-SIGN.

  14. Fasciola hepatica Surface Coat Glycoproteins Contain Mannosylated and Phosphorylated N-glycans and Exhibit Immune Modulatory Properties Independent of the Mannose Receptor.

    Ravidà, Alessandra; Aldridge, Allison M; Driessen, Nicole N; Heus, Ferry A H; Hokke, Cornelis H; O'Neill, Sandra M

    2016-04-01

    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. PMID:27104959

  15. Introduction of an N-glycan sequon into HEXA enhances human beta-hexosaminidase cellular uptake in a model of Sandhoff disease.

    Matsuoka, Kazuhiko; Tsuji, Daisuke; Aikawa, Sei-Ichi; Matsuzawa, Fumiko; Sakuraba, Hitoshi; Itoh, Kohji

    2010-08-01

    Human lysosomal beta-hexosaminidase A is a heterodimer composed of alpha- and beta-subunits encoded by HEXA and HEXB, respectively. We genetically introduced an additional N-glycosylation sequon into HEXA, which caused amino acid substitutions (S51 to N and A53 to T) at homologous positions to N84 and T86 in the beta-subunit. The mutant HexA (NgHexA) obtained from a Chinese hamster ovary (CHO) cell line co-expressing the mutated HEXA and wild-type HEXB complementary DNAs was demonstrated to contain an additional mannose-6-phosphate (M6P)-type-N-glycan. NgHexA was more efficiently taken up than the wild-type HexA and delivered to lysosomes, where it degraded accumulated substrates including GM2 ganglioside (GM2) when administered to cultured fibroblasts derived from a Sandhoff disease (SD) patient. On intracerebroventricular (i.c.v.) administration of NgHexA to SD model mice, NgHexA more efficiently restored the HexA activity and reduced the GM2 and GA2 (asialoGM2) accumulated in neural cells of the brain parenchyma than the wild-type HexA. These findings indicate that i.c.v. administration of the modified human HexA with an additional M6P-type N-glycan is applicable for enzyme replacement therapy (ERT) involving an M6P-receptor as a molecular target for HexA deficiencies including Tay-Sachs disease and SD. PMID:20571546

  16. Galectin-1-mediated cell adhesion, invasion and cell death in human anaplastic large cell lymphoma: regulatory roles of cell surface glycans.

    Suzuki, Osamu; Abe, Masafumi

    2014-05-01

    Galectin-1 is known to be one of the extracellular matrix proteins. To elucidate the biological roles of galectin-1 in cell adhesion and invasion of human anaplastic large cell lymphoma, we performed cell adhesion and invasion assays using the anaplastic large cell lymphoma cell line H-ALCL, which was previously established in our laboratory. From the cell surface lectin array, treatment with neuraminidase from Arthrobacter ureafaciens which cleaves all linkage types of cell surface sialic acid enhanced Arachis hypogaea (PNA), Helix pomatia (HPA) and Phaseolus vulgaris-L (L-PHA) lectin binding reactivity to cell surface of lymphoma cells suggesting that neuraminidase removes cell surface sialic acid. In cell adhesion and invasion assays treatment with neuraminidase markedly enhanced cell adhesion to galectin-1 and decreased cell invasive capacity through galectin-1. α2,6-linked sialic acid may be involved in masking the effect of the interaction between galectin-1 and cell surface glycans. H-ALCL cells expressed the β-galactoside-α2,6-sialyltransferase ST6Gal1. On resialylation assay by recombinant ST6Gal1 with CMP-Neu5Ac, α2,6-resialylation of L-PHA reactive oligosaccharide by ST6Gal1 resulted in inhibition of H-ALCL cell adhesion to galectin-1 compared to the desialylated H-ALCL cells. On knockdown experiments, knockdown of ST6Gal1 dramatically enhanced cell adhesion to galectin-1. N-glycosylation inhibitor swainsonine treatment resulted in enhancement of cell adhesion to galectin-1. In glycomic analysis using the lectin blocking assay treatment with PNA, Artocarpus integrifolia (Jacalin), Glycine max (SBA), Helix pomatia (HPA), Vicia villosa (VVA), Ulex europaeus (UEA-1), Triticum vulgaris (WGA), Canavalia ensiformis (ConA), Phaseolus vulgaris-L (L-PHA), Phaseolus vulgaris-E4 (E-PHA), Datura stramonium (DSA) lectins resulted in modulation of lymphoma cell to galectin-1 suggesting that several types of glycans may regulate cell adhesion to galectin-1 by

  17. P53 and Cancer-Associated Sialylated Glycans Are Surrogate Markers of Cancerization of the Bladder Associated with Schistosoma haematobium Infection

    Lima, Luís; Tavares, Ana; Peixoto, Andreia; Parreira, Beatriz; Correia da Costa, José Manuel; Brindley, Paul J.; Lopes, Carlos

    2014-01-01

    Background Bladder cancer is a significant health problem in rural areas of Africa and the Middle East where Schistosoma haematobium is prevalent, supporting an association between malignant transformation and infection by this blood fluke. Nevertheless, the molecular mechanisms linking these events are poorly understood. Bladder cancers in infected populations are generally diagnosed at a late stage since there is a lack of non-invasive diagnostic tools, hence enforcing the need for early carcinogenesis markers. Methodology/Principal Findings Forty-three formalin-fixed paraffin-embedded bladder biopsies of S. haematobium-infected patients, consisting of bladder tumours, tumour adjacent mucosa and pre-malignant/malignant urothelial lesions, were screened for bladder cancer biomarkers. These included the oncoprotein p53, the tumour proliferation rate (Ki-67>17%), cell-surface cancer-associated glycan sialyl-Tn (sTn) and sialyl-Lewisa/x (sLea/sLex), involved in immune escape and metastasis. Bladder tumours of non-S. haematobium etiology and normal urothelium were used as controls. S. haematobium-associated benign/pre-malignant lesions present alterations in p53 and sLex that were also found in bladder tumors. Similar results were observed in non-S. haematobium associated tumours, irrespectively of their histological nature, denoting some common molecular pathways. In addition, most benign/pre-malignant lesions also expressed sLea. However, proliferative phenotypes were more prevalent in lesions adjacent to bladder tumors while sLea was characteristic of sole benign/pre-malignant lesions, suggesting it may be a biomarker of early carcionogenesis associated with the parasite. A correlation was observed between the frequency of the biomarkers in the tumor and adjacent mucosa, with the exception of Ki-67. Most S. haematobium eggs embedded in the urothelium were also positive for sLea and sLex. Reinforcing the pathologic nature of the studied biomarkers, none was observed

  18. Monetarism Beyond M1A

    Pablo García; Rodrigo O. Valdés

    2004-01-01

    This work analyzes, from an empirical standpoint, the information contained in the M1A aggregate in Chile and compares it with other aggregates. The findings reveal that, at least for now, the M1A aggregate does not seem to have particularly valuable information that would make it a better indicator of inflationary pressures within the monetary policy decision horizon. This is consistent with the practice that central banks followed in the past, when they used quantitative targets for money a...

  19. Main: PYRIMIDINEBOXOSRAMY1A [PLACE

    Full Text Available PYRIMIDINEBOXOSRAMY1A S000259 19-August-2004 (last modified) kehi Pyrimidine box found in rice ( ... GARE and pyrimidine box are partially involved in sugar ... repression; Found in the promoter of barley alpha- ... ifically to this site; See S000265; alpha-amylase; sugar ... repression; GARE; pyrimidine box; feed-back metabo ...

  20. Comparative Profiling of N-Glycans Isolated from Serum Samples of Ovarian Cancer Patients and Analyzed by Microchip Electrophoresis

    Mitra, Indranil; Alley, William R.; Goetz, John A.; Vasseur, Jacqueline A.; Novotny, Milos V.; Jacobson, Stephen C.

    2013-01-01

    Ovarian cancer is the fifth leading cause of cancer-related mortalities for women in the United States and the most lethal gynecological cancer. Aberrant glycosylation has been linked to several human diseases, including ovarian cancer, and accurate measurement of changes in glycosylation may provide relevant diagnostic and prognostic information. In this work, we used microchip electrophoresis coupled with laser-induced fluorescence detection to determine quantitative differences among the N...

  1. Overexpression of Galnt3 in Chondrocytes Resulted in Dwarfism Due to the Increase of Mucin-type O-Glycans and Reduction of Glycosaminoglycans*

    Yoshida, Carolina Andrea; Kawane, Tetsuya; Moriishi, Takeshi; Purushothaman, Anurag; Miyazaki, Toshihiro; Komori, Hisato; Mori, Masako; Qin, Xin; Hashimoto, Ayako; Sugahara, Kazuyuki; Yamana, Kei; Takada, Kenji; Komori, Toshihisa

    2014-01-01

    Galnt3, UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, transfers N-acetyl-d-galactosamine to serine and threonine residues, initiating mucin type O-glycosylation of proteins. We searched the target genes of Runx2, which is an essential transcription factor for chondrocyte maturation, in chondrocytes and found that Galnt3 expression was up-regulated by Runx2 and severely reduced in Runx2−/− cartilaginous skeletons. To investigate the function of Galnt3 in chondrocytes, we generated Galnt3−/− mice and chondrocyte-specific Galnt3 transgenic mice under the control of the Col2a1 promoter-enhancer. Galnt3−/− mice showed a delay in endochondral ossification and shortened limbs at embryonic day 16.5, suggesting that Galnt3 is involved in chondrocyte maturation. Galnt3 transgenic mice presented dwarfism, the chondrocyte maturation was retarded, the cell cycle in chondrocytes was accelerated, premature chondrocyte apoptosis occurred, and the growth plates were disorganized. The binding of Vicia villosa agglutinin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), was drastically increased in chondrocytes, and aggrecan (Acan) was highly enriched with Tn antigen. However, safranin O staining, which recognizes glycosaminoglycans (GAGs), and Acan were severely reduced. Chondroitin sulfate was reduced in amount, but the elongation of chondroitin sulfate chains had not been severely disturbed in the isolated GAGs. These findings indicate that overexpression of Galnt3 in chondrocytes caused dwarfism due to the increase of mucin-type O-glycans and the reduction of GAGs, probably through competition with xylosyltransferases, which initiate GAG chains by attaching O-linked xylose to serine residues, suggesting a negative effect of Galnt family proteins on Acan deposition in addition to the positive effect of Galnt3 on chondrocyte maturation. PMID:25107907

  2. Overexpression of Galnt3 in chondrocytes resulted in dwarfism due to the increase of mucin-type O-glycans and reduction of glycosaminoglycans.

    Yoshida, Carolina Andrea; Kawane, Tetsuya; Moriishi, Takeshi; Purushothaman, Anurag; Miyazaki, Toshihiro; Komori, Hisato; Mori, Masako; Qin, Xin; Hashimoto, Ayako; Sugahara, Kazuyuki; Yamana, Kei; Takada, Kenji; Komori, Toshihisa

    2014-09-19

    Galnt3, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, transfers N-acetyl-D-galactosamine to serine and threonine residues, initiating mucin type O-glycosylation of proteins. We searched the target genes of Runx2, which is an essential transcription factor for chondrocyte maturation, in chondrocytes and found that Galnt3 expression was up-regulated by Runx2 and severely reduced in Runx2(-/-) cartilaginous skeletons. To investigate the function of Galnt3 in chondrocytes, we generated Galnt3(-/-) mice and chondrocyte-specific Galnt3 transgenic mice under the control of the Col2a1 promoter-enhancer. Galnt3(-/-) mice showed a delay in endochondral ossification and shortened limbs at embryonic day 16.5, suggesting that Galnt3 is involved in chondrocyte maturation. Galnt3 transgenic mice presented dwarfism, the chondrocyte maturation was retarded, the cell cycle in chondrocytes was accelerated, premature chondrocyte apoptosis occurred, and the growth plates were disorganized. The binding of Vicia villosa agglutinin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), was drastically increased in chondrocytes, and aggrecan (Acan) was highly enriched with Tn antigen. However, safranin O staining, which recognizes glycosaminoglycans (GAGs), and Acan were severely reduced. Chondroitin sulfate was reduced in amount, but the elongation of chondroitin sulfate chains had not been severely disturbed in the isolated GAGs. These findings indicate that overexpression of Galnt3 in chondrocytes caused dwarfism due to the increase of mucin-type O-glycans and the reduction of GAGs, probably through competition with xylosyltransferases, which initiate GAG chains by attaching O-linked xylose to serine residues, suggesting a negative effect of Galnt family proteins on Acan deposition in addition to the positive effect of Galnt3 on chondrocyte maturation. PMID:25107907

  3. Flowability of JSC-1a

    Rame, Enrique; Wilkinson, Allen; Elliot, Alan; Young, Carolyn

    2009-01-01

    We have done a complete flowability characterization of the lunar soil simulant, JSC-1a, following closely the ASTM-6773 standard for the Schulze ring shear test. The measurements, which involve pre-shearing the material before each yield point, show JSC-1a to be cohesionless, with an angle of internal friction near 40 deg. We also measured yield loci after consolidating the material in a vibration table which show it to have significant cohesion (approximately equal to 1 kPa) and an angle of internal friction of about 60 deg. Hopper designs based on each type of flowability test differ significantly. These differences highlight the need to discern the condition of the lunar soil in the specific process where flowability is an issue. We close with a list not necessarily comprehensive of engineering rules of thumb that apply to powder flow in hoppers.

  4. Single-chain antibody-fragment M6P-1 possesses a mannose 6-phosphate monosaccharide-specific binding pocket that distinguishes N-glycan phosphorylation in a branch-specific manner†.

    Blackler, Ryan J; Evans, Dylan W; Smith, David F; Cummings, Richard D; Brooks, Cory L; Braulke, Thomas; Liu, Xinyu; Evans, Stephen V; Müller-Loennies, Sven

    2016-02-01

    The acquisition of mannose 6-phosphate (Man6P) on N-linked glycans of lysosomal enzymes is a structural requirement for their transport from the Golgi apparatus to lysosomes mediated by the mannose 6-phosphate receptors, 300 kDa cation-independent mannose 6-phosphate receptor (MPR300) and 46 kDa cation-dependent mannose 6-phosphate receptor (MPR46). Here we report that the single-chain variable domain (scFv) M6P-1 is a unique antibody fragment with specificity for Man6P monosaccharide that, through an array-screening approach against a number of phosphorylated N-glycans, is shown to bind mono- and diphosphorylated Man6 and Man7 glycans that contain terminal αMan6P(1 → 2)αMan(1 → 3)αMan. In contrast to MPR300, scFv M6P-1 does not bind phosphodiesters, monophosphorylated Man8 or mono- or diphosphorylated Man9 structures. Single crystal X-ray diffraction analysis to 2.7 Å resolution of Fv M6P-1 in complex with Man6P reveals that specificity and affinity is achieved via multiple hydrogen bonds to the mannose ring and two salt bridges to the phosphate moiety. In common with both MPRs, loss of binding was observed for scFv M6P-1 at pH values below the second pKa of Man6P (pKa = 6.1). The structures of Fv M6P-1 and the MPRs suggest that the change of the ionization state of Man6P is the main driving force for the loss of binding at acidic lysosomal pH (e.g. lysosome pH ∼ 4.6), which provides justification for the evolution of a lysosomal enzyme transport pathway based on Man6P recognition. PMID:26503547

  5. Brief report: a new profile of terminal N-acetyllactosamines glycans on pig red blood cells and different expression of alpha-galactose on Sika deer red blood cells and nucleated cells.

    Tan, Yingxia; Gong, Feng; Li, Subo; Ji, Shouping; Lu, Yanping; Gao, Hongwei; Xu, Hua; Zhang, Yangpei

    2010-05-01

    It has been reported that: (1) large variations were found in the number of sialic acid (SA) capped with N-acetyllactosamines (SA-Galbeta1-4GlcNAc-R) and alpha-Gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) or uncapped N-acetyllactosamines (Galbeta1-4GlcNAc-R) on different mammalian red blood cells, and on nucleated cells originating from a given tissue in various species; (2) goat, sheep, horse and mouse red blood cells lack alpha-Gal epitopes, despite the expression of this epitope on a variety of nucleated cells in these species, including lymphocytes differentiated from the same hematopoietic origin. In this study, flow cytometry and Western blot analyses of pig red blood cells showed that alpha-Gal epitopes on pig red cells developed concomitantly after treatment with neuraminidase, suggesting that the terminal N-acetyllactosaminide glycans were capped with SA-alpha-Gal epitopes. Whereas, the expression of the alpha-Gal epitopes on red blood cells from Sika deer (Cevus nippon hortulorum) were found to be absent even though the epitopes were present on their white blood cells. Thus, these results add new data not only for the terminal carbohydrate structures on cell surface glycans of various mammalian cells, but also for wide variety of epitope expression on the cells from different tissues, which might be useful for understanding their unique states resulting from differentiation and evolution. PMID:20422448

  6. The interaction between the first transmembrane domain and the thumb of ASIC1a is critical for its N-glycosylation and trafficking.

    Lan Jing

    Full Text Available Acid-sensing ion channel-1a (ASIC1a, the primary proton receptor in the brain, contributes to multiple diseases including stroke, epilepsy and multiple sclerosis. Thus, a better understanding of its biogenesis will provide important insights into the regulation of ASIC1a in diseases. Interestingly, ASIC1a contains a large, yet well organized ectodomain, which suggests the hypothesis that correct formation of domain-domain interactions at the extracellular side is a key regulatory step for ASIC1a maturation and trafficking. We tested this hypothesis here by focusing on the interaction between the first transmembrane domain (TM1 and the thumb of ASIC1a, an interaction known to be critical in channel gating. We mutated Tyr71 and Trp287, two key residues involved in the TM1-thumb interaction in mouse ASIC1a, and found that both Y71G and W287G decreased synaptic targeting and surface expression of ASIC1a. These defects were likely due to altered folding; both mutants showed increased resistance to tryptic cleavage, suggesting a change in conformation. Moreover, both mutants lacked the maturation of N-linked glycans through mid to late Golgi. These data suggest that disrupting the interaction between TM1 and thumb alters ASIC1a folding, impedes its glycosylation and reduces its trafficking. Moreover, reducing the culture temperature, an approach commonly used to facilitate protein folding, increased ASIC1a glycosylation, surface expression, current density and slowed the rate of desensitization. These results suggest that correct folding of extracellular ectodomain plays a critical role in ASIC1a biogenesis and function.

  7. Inhibitory effects of commonly used herbal extracts on UDP-glucuronosyltransferase 1A4, 1A6, and 1A9 enzyme activities.

    Mohamed, Mohamed-Eslam F; Frye, Reginald F

    2011-09-01

    The aim of this study was to investigate the effect of commonly used botanicals on UDP-glucuronosyltransferase (UGT) 1A4, UGT1A6, and UGT1A9 activities in human liver microsomes. The extracts screened were black cohosh, cranberry, echinacea, garlic, ginkgo, ginseng, milk thistle, saw palmetto, and valerian in addition to the green tea catechin epigallocatechin gallate (EGCG). Formation of trifluoperazine glucuronide, serotonin glucuronide, and mycophenolic acid phenolic glucuronide was used as an index reaction for UGT1A4, UGT1A6, and UGT1A9 activities, respectively, in human liver microsomes. Inhibition potency was expressed as the concentration of the inhibitor at 50% activity (IC(50)) and the volume in which the dose could be diluted to generate an IC(50)-equivalent concentration [volume/dose index (VDI)]. Potential inhibitors were EGCG for UGT1A4, milk thistle for both UGT1A6 and UGT1A9, saw palmetto for UGT1A6, and cranberry for UGT1A9. EGCG inhibited UGT1A4 with an IC(50) value of (mean ± S.E.) 33.8 ± 3.1 μg/ml. Milk thistle inhibited both UGT1A6 and UGT1A9 with IC(50) values of 59.5 ± 3.6 and 33.6 ± 3.1 μg/ml, respectively. Saw palmetto and cranberry weakly inhibited UGT1A6 and UGT1A9, respectively, with IC(50) values >100 μg/ml. For each inhibition, VDI was calculated to determine the potential of achieving IC(50)-equivalent concentrations in vivo. VDI values for inhibitors indicate a potential for inhibition of first-pass glucuronidation of UGT1A4, UGT1A6, and UGT1A9 substrates. These results highlight the possibility of herb-drug interactions through modulation of UGT enzyme activities. Further clinical studies are warranted to investigate the in vivo extent of the observed interactions. PMID:21632963

  8. Can voltammetry distinguish glycan isomers?

    Trefulka, Mojmír; Paleček, Emil

    2015-01-01

    Roč. 69, č. 1 (2015), s. 241-244. ISSN 0366-6352 R&D Projects: GA ČR(CZ) GAP301/11/2055 Institutional support: RVO:68081707 Keywords : oligosaccharide isomers * Os(VI) complexes * chemical modification Subject RIV: BO - Biophysics Impact factor: 1.468, year: 2014

  9. 肠膜明串珠菌BD1710在TSM中的产糖条件优化%Optimization of the Condition for Leuconostoc mesenteroides BD1710 to Biosynthesis Glycan in Tomato Juice-sucrose Medium

    韩瑨; 吴正钧; 游春苹; 徐晓芬

    2015-01-01

    The condition for Leuconostoc mesenteroides BD1710 to biosynthesis glycan in tomato juice-sucrose medium was optimizized, which was a combination of a medium composed of pure tomato juice supplemented with 15%sucrose, with the initial pH value of the medium adjusted to 7.0, and the fermentation undertaken at 28 ℃ with an inoculation ratio of 2.0 %. Under the optimized conditions , the glycan synthesized by L. mesenteroides BD1710 in the tomato juice-sucrose medium could reached 32.15 g/L , at the same level or a little higher than the yield of glycan by the same bacterial strain in a reported chemically defined medium suitable for Leuconostoc mesenteroides to express polysaccharides. The polymers obtained from the fermented tomato juice-sucrose medium by L.mesenteroides BD1710 via alchol participation was composed of 97.54 %carbohydrate and 0.72%protein, respectively. Therefore, tomato juice-sucrose medium could be an alternative of chemically defined medium for L. mesenteroides strains to biosynthesis glycan.%研究了肠膜明串珠菌BD1710(L. mesenteroides BD1710,CGMCC NO.6432)以TSM为基料时,接种量、初始pH、番茄汁浓度、蔗糖浓度、发酵温度对多糖产量的影响,同时,比较了肠膜明串珠菌BD1710在最优条件下发酵TSM和CDM的多糖产量,并对所获得的多糖组成进行了分析。结果表明:肠膜明串珠菌BD1710发酵TSM产多糖的最适条件分别为接种量2.0%(体积分数)、初始pH 7.0、番茄汁浓度100%(体积分数)、蔗糖浓度15%、发酵温度28℃。在优化条件下,肠膜明串珠菌BD1710的多糖产量最高可达32.15 g/L,与在CDM中多糖的产量相当。采用乙醇沉淀的方法从BD1710发酵TSM得到的多糖碳水化合物含量为97.54%,蛋白质含量为0.72%。因此,TSM可替代CDM来应用于明串珠菌合成多糖。

  10. 肠膜明串珠菌BD1710在TSM中的产糖条件优化%Optimization of the Condition for Leuconostoc mesenteroides BD1710 to Biosynthesis Glycan in Tomato Juice-sucrose Medium

    韩瑨; 吴正钧; 游春苹; 徐晓芬

    2015-01-01

    研究了肠膜明串珠菌BD1710(L. mesenteroides BD1710,CGMCC NO.6432)以TSM为基料时,接种量、初始pH、番茄汁浓度、蔗糖浓度、发酵温度对多糖产量的影响,同时,比较了肠膜明串珠菌BD1710在最优条件下发酵TSM和CDM的多糖产量,并对所获得的多糖组成进行了分析。结果表明:肠膜明串珠菌BD1710发酵TSM产多糖的最适条件分别为接种量2.0%(体积分数)、初始pH 7.0、番茄汁浓度100%(体积分数)、蔗糖浓度15%、发酵温度28℃。在优化条件下,肠膜明串珠菌BD1710的多糖产量最高可达32.15 g/L,与在CDM中多糖的产量相当。采用乙醇沉淀的方法从BD1710发酵TSM得到的多糖碳水化合物含量为97.54%,蛋白质含量为0.72%。因此,TSM可替代CDM来应用于明串珠菌合成多糖。%The condition for Leuconostoc mesenteroides BD1710 to biosynthesis glycan in tomato juice-sucrose medium was optimizized, which was a combination of a medium composed of pure tomato juice supplemented with 15%sucrose, with the initial pH value of the medium adjusted to 7.0, and the fermentation undertaken at 28 ℃ with an inoculation ratio of 2.0 %. Under the optimized conditions , the glycan synthesized by L. mesenteroides BD1710 in the tomato juice-sucrose medium could reached 32.15 g/L , at the same level or a little higher than the yield of glycan by the same bacterial strain in a reported chemically defined medium suitable for Leuconostoc mesenteroides to express polysaccharides. The polymers obtained from the fermented tomato juice-sucrose medium by L.mesenteroides BD1710 via alchol participation was composed of 97.54 %carbohydrate and 0.72%protein, respectively. Therefore, tomato juice-sucrose medium could be an alternative of chemically defined medium for L. mesenteroides strains to biosynthesis glycan.

  11. Adenovirus E1A gene autorepression: revertants of an E1A promoter mutation encode altered E1A proteins

    Larsen, P.L.; Tibbetts, C.

    1987-12-01

    Revertants have been isolated from Ad3hr15, a mutant of human adenovirus type 3 that carries a defective E1A promoter. Transcription of these revertant E1A genes is restored - from nil for Ad3hr15 mutant to levels exceeding that of the wild-type virus. The mutant Ad3hr15 virus and the revertants all have an aberrant E1A promoter that contains two short tandem duplications of viral DNA sequence. The E1A gene-coding region of the mutant is the same as that for wild-type adenovirus type 3, whereas the revertants are characterized by short in-frame deletions within the 5' exon region of their E1A genes. Location of these reverting, second-site deletions is discussed in relation to E1A gene autoregulation and the evolved diversity of E1A-related oncogenic potential among different human adenoviruses.

  12. Minimum Data Set Q1a Report

    U.S. Department of Health & Human Services — The MDS Q1a report summarizes, by state and county, percentages of residents that answered Yes to Q1a - Residents expresses or indicates preference to return to the...

  13. Interferon Beta-1a Intramuscular Injection

    Interferon beta-1a intramuscular injection is used to reduce the number of episodes of symptoms and slow ... and problems with vision, speech, and bladder control). Interferon beta-1a is in a class of medications ...

  14. Interferon Beta-1a Subcutaneous Injection

    Interferon beta-1a subcutaneous injection is used to reduce episodes of symptoms and slow the development of ... and problems with vision, speech, and bladder control). Interferon beta-1a is in a class of medications ...

  15. Interferon Beta-1a Intramuscular Injection

    Interferon beta-1a intramuscular injection is used to reduce the number of episodes of symptoms and slow the development of disability in patients ... Interferon beta-1a intramuscular injection comes as a powder in vials to be mixed into a solution for injection. Interferon beta-1a intramuscular ...

  16. Epigenetic regulation of REG1A and chemosensitivity of cutaneous melanoma

    Sato, Yusuke; Marzese, Diego M; OHTA, KATSUYA; Huang, Sharon K.; Sim, Myung Shin; Chong, Kelly; Hoon, Dave SB

    2013-01-01

    Regenerating gene 1A (REG1A) plays an important role in tissue regeneration and in cell proliferation in epithelium origin tumors; however, its role in melanoma has not been explored in details. The objective of this study was to identify whether REG1A is expressed in cutaneous melanoma and if REG1A expression status can predict prognosis in cutaneous melanoma patients with metastasis. We also determined whether epigenetic regulation of the promoter region regulates REG1A expression. AJCC sta...

  17. Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein

    Lee, Jeffrey E.; Fusco, Marnie L.; Abelson, Dafna M.; Hessell, Ann J.; Burton, Dennis R. [Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); Saphire, Erica Ollmann, E-mail: erica@scripps.edu [Department of Immunology and Microbial Science, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States); The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 (United States)

    2009-11-01

    Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing of the trimeric ebolavirus glycoprotein are described. The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008 ▶), Nature (London), 454, 177–182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the

  18. Radium-226 in certified uranium reference ores DL-1a, BL-4a, DH-1a and BL-5

    Radium-226 radioactivity in uranium reference ores BL-4a and BL-5 and uranium-thorium reference ores DL-1a and DH-1a was determined in an interlaboratory program. Twelve of thirteen participants used certified radium solutions from the United States National Bureau of Standards (NBS) for calibration purposes. Recommended values of sup(226)Ra activity and associated parameters were calculated by statistical treatment of the results. In all cases, the recommended values are within 2 percent of activities predicted assuming secular equilibrium in the sup(238)U decay series. The recommended values for radium activity are 1.40, 15.5, 31.5 and 857 Bq/ for DL-1a, BL-4a, DH-1a and BL-5, respectively

  19. Determining impurities

    A method of determining the content of impurities in organic-origin natural fibrous materials, in which a specimen of said material is compacted to a surface density of from 0.05 to 50 g/cm2, whereupon it is exposed to the effect of a soft gamma-radiation, the mass attenuation coefficient (μ) is determined and the percentage content (Csub(A)) of impurities is assessed. The method has applications in the textile industry. (author)

  20. 19 CFR 351.205 - Preliminary determination.

    2010-04-01

    ... determinations, postponement of preliminary determinations, notices of preliminary determinations, and the... its injury determination. (e) Postponement at the request of the petitioner. A petitioner must submit a request for postponement of the preliminary determination (see section 703(c)(1)(A) or section...

  1. μ-Theraphotoxin-An1a

    Rates, Breno; Prates, Maura V; Verano-Braga, Thiago;

    2013-01-01

    nM) induces membrane depolarization, increases the spontaneous firing frequency and reduces spike amplitude of cockroach dorsal unpaired median (DUM) neurons. In addition, under voltage-clamp conditions, μ-TRTX-An1a (100 nM) only partially blocks voltage-dependent sodium current amplitudes in DUM...

  2. 2000 Johnston Site 1A-P

    US Fish and Wildlife Service, Department of the Interior — Underwater Site 1A-P was established at Johnston Atoll by Dr. James Maragos, U.S. Fish & Wildlife Service, on June 29, 2000. With a start point (meter 0) at...

  3. 2006 Johnston Site 1A-P

    US Fish and Wildlife Service, Department of the Interior — Underwater Site 1A-P was established at Johnston Atoll by Dr. James Maragos, U.S. Fish & Wildlife Service, on June 29, 2000. With a start point (meter 0) at...

  4. Cyp1a reporter zebrafish reveals target tissues for dioxin

    Kim, Kun-Hee [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Park, Hye-Jeong [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Kim, Jin Hee [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Kim, Suhyun [Graduate School of Medicine, Korea University, Ansan (Korea, Republic of); Williams, Darren R. [New Drug Targets Laboratory, School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju (Korea, Republic of); Kim, Myeong-Kyu [Department of Neurology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Jung, Young Do [Department of Biochemistry, Chonnam National University Medical School, Gwangju (Korea, Republic of); Teraoka, Hiroki [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Park, Hae-Chul [Graduate School of Medicine, Korea University, Ansan (Korea, Republic of); Choy, Hyon E., E-mail: hyonchoy@chonnam.ac.kr [Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Shin, Boo Ahn, E-mail: bashin@chonnam.ac.kr [Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Choi, Seok-Yong, E-mail: zebrafish@chonnam.ac.kr [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); School of Biological Sciences and Technology, Chonnam National University, Gwangju (Korea, Republic of)

    2013-06-15

    Highlights: •2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic anthropogenic substance ever identified. •Transgenic cyp1a reporter zebrafish reveals target tissues for TCDD. •The retinal bipolar cells, otic vesicle, lateral line, pancreas, cloaca and pectoral fin bud are novel targets in zebrafish for TCDD. •Our findings will further understanding of human health risks by TCDD. -- Abstract: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the unintentional byproduct of various industrial processes, is classified as human carcinogen and could disrupt reproductive, developmental and endocrine systems. Induction of cyp1a1 is used as an indicator of TCDD exposure. We sought to determine tissues that are vulnerable to TCDD toxicity using a transgenic zebrafish (Danio rerio) model. We inserted a nuclear enhanced green fluorescent protein gene (EGFP) into the start codon of a zebrafish cyp1a gene in a fosmid clone using DNA recombineering. The resulting recombineered fosmid was then used to generate cyp1a reporter zebrafish, embryos of which were exposed to TCDD. Expression pattern of EGFP in the reporter zebrafish mirrored that of endogenous cyp1a mRNA. In addition, exposure of the embryos to TCDD at as low as 10 pM for 72 h, which does not elicit morphological abnormalities of embryos, markedly increased GFP expression. Furthermore, the reporter embryos responded to other AhR ligands as well. Exposure of the embryos to TCDD revealed previously reported (the cardiovascular system, liver, pancreas, kidney, swim bladder and skin) and unreported target tissues (retinal bipolar cells, otic vesicle, lateral line, cloaca and pectoral fin bud) for TCDD. Transgenic cyp1a reporter zebrafish we have developed can further understanding of ecotoxicological relevance and human health risks by TCDD. In addition, they could be used to identify agonists of AhR and antidotes to TCDD toxicity.

  5. Yilmaz Theory of SNe 1a Redshift

    Robertson, Stanley L.

    2015-01-01

    A redshift-luminosity distance relation in excellent agreement with observations is calculated here for SNe 1a using the Yilmaz gravitational theory. In contrast to the current conventional explanation based on general relativity, the Yilmaz theory does not require a cosmological constant term that implies the existence of "dark energy". The Yilmaz theory requires only one parameter; a mean mass-energy density of the cosmos. The required value is essentially the same as the critical density f...

  6. Structure of a HIV-1 Protease-Inhibitor Complex determined at 1.1A resolution

    Brynda, Jiří; Řezáčová, Pavlína; Štouračová, Renata; Fábry, Milan; Hradilek, Martin; Souček, Milan; Konvalinka, Jan; Sedláček, Juraj

    Jena : Jena, 2002, s. -. [International Conference on the crystal lization of Biological Macromolecules /9./. Jena, Německo (DE), 23.03.2002-28.03.2002] Institutional research plan: CEZ:AV0Z4055905; CEZ:AV0Z5052915 Keywords : Structure of a HIV-1 * protease * Inhibitor complex Subject RIV: EB - Genetics ; Molecular Biology

  7. Mammalian α-1,6-Fucosyltransferase (FUT8) Is the Sole Enzyme Responsible for the N-Acetylglucosaminyltransferase I-independent Core Fucosylation of High-mannose N-Glycans.

    Yang, Qiang; Wang, Lai-Xi

    2016-05-20

    Understanding the biosynthetic pathway of protein glycosylation in various expression cell lines is important for controlling and modulating the glycosylation profiles of recombinant glycoproteins. We found that expression of erythropoietin (EPO) in a HEK293S N-acetylglucosaminyltransferase I (GnT I)(-/-) cell line resulted in production of the Man5GlcNAc2 glycoforms, in which more than 50% were core-fucosylated, implicating a clear GnT I-independent core fucosylation pathway. Expression of GM-CSF and the ectodomain of FcγIIIA receptor led to ∼30% and 3% core fucosylation, suggesting that the level of core fucosylation also depends on the nature of the recombinant proteins. To elucidate the GnT I-independent core fucosylation pathway, we generated a stable HEK293S GnT I(-/-) cell line with either knockdown or overexpression of FUT8 by a highly efficient lentivirus-mediated gene transfer approach. We found that the EPO produced from the FUT8 knockdown cell line was the pure Man5GlcNAc2 glycoform, whereas that produced from the FUT8-overexpressing cell line was found to be fully core-fucosylated oligomannose glycan (Man5GlcNAc2Fuc). These results provide direct evidence that FUT8, the mammalian α1,6-fucosyltransferase, is the sole enzyme responsible for the GnT I-independent core fucosylation pathway. The production of the homogeneous core-fucosylated Man5GlcNAc2 glycoform of EPO in the FUT8-overexpressed HEK293S GnT I(-/-) cell line represents the first example of production of fully core-fucosylated high-mannose glycoforms. PMID:27008861

  8. Jack in the Box, Unit 1A

    Gammelgaard Nielsen, Anders

    around a communi- cative situation between two peo- ple. The assignment is carried out in groups of four to five students. It introduces students to a synthe- sised working method, in which a number of form-generating pa- rameters determine the final result. This technique of working is well- known in...

  9. Visualisation of serotonin-1A (5-HT1A) receptors in the central nervous system

    Passchier, Jan; van Waarde, A

    2001-01-01

    The 5-HT1A subtype of receptors for the neurotransmitter serotonin is predominantly located in the limbic forebrain and is involved in the modulation of emotion and the function of the hypothalamus. Since 5-HT1A receptors are implicated in the pathogenesis of anxiety, depression, hallucinogenic beha

  10. Earth and Sky, Unit 1A

    Gammelgaard Nielsen, Anders

    2011-01-01

    The assignment known as ‘Earth and sky’ is the final first year course at Unit 1a. The aim of the assignment is to strengthen the student’s abilities to manage a project process individu- ally. The process involves develop- ing the ability to make independent decisions.The point of departure for...... the ‘Earth and sky’ assignment is ex- perience students acquired during their group study tour to Austra- lia. Building in particular on the re- search conducted on the Sydney Opera House and the architectur- al principles of spatial creation that this building represents....

  11. Climate determinism or Geomagnetic determinism?

    Gallet, Y.; Genevey, A.; Le Goff, M.; Fluteau, F.; Courtillot, V.

    2006-12-01

    A number of episodes of sharp geomagnetic field variations (in both intensity and direction), lasting on the order of a century, have been identified in archeomagnetic records from Western Eurasia and have been called "archeomagnetic jerks". These seem to correlate well with multi-decadal cooling episodes detected in the North Atlantic Ocean and Western Europe, suggesting a causal link between both phenomena. A possible mechanism could be a geomagnetic modulation of the cosmic ray flux that would control the nucleation rate of clouds. We wish to underline the remarkable coincidence between archeomagnetic jerks, cooling events in Western Europe and drought periods in tropical and sub-tropical regions of the northern hemisphere. The latter two can be interpreted in terms of global teleconnections among regional climates. It has been suggested that these climatic variations had caused major changes in the history of ancient civilizations, such as in Mesopotamia, which were critically dependent on water supply and particularly vulnerable to lower rainfall amounts. This is one of the foundations of "climate determinism". Our studies, which suggest a geomagnetic origin for at least some of the inferred climatic events, lead us to propose the idea of a "geomagnetic determinism" in the history of humanity.

  12. Carnitine palmitoyltransferase 1A (CPT1A) P479L prevalence in live newborns in Yukon, Northwest Territories, and Nunavut.

    Collins, Sorcha A; Sinclair, Graham; McIntosh, Sarah; Bamforth, Fiona; Thompson, Robert; Sobol, Isaac; Osborne, Geraldine; Corriveau, Andre; Santos, Maria; Hanley, Brendan; Greenberg, Cheryl R; Vallance, Hilary; Arbour, Laura

    2010-01-01

    Carnitine palmitoyltransferase 1A (CPT1A), encoded by the gene CPT1A, is the hepatic isoform of CPT1 and is a major regulatory point in long-chain fatty acid oxidation. CPT1A deficiency confers risk for hypoketotic hypoglycaemia, hepatic encephalopathy, seizures, and sudden unexpected death in infancy (SUDI). It remains controversial whether the CPT1A gene variant, c.1436C>T (p.P479L), identified in Inuit, First Nations, and Alaska Native infants, causes susceptibility to decompensation, in particular during times of fever and intercurrent illness. Although newborn screening for the P479L variant occurs in some jurisdictions, background knowledge about the presence of the variant in Canadian Aboriginal populations is lacking. In an effort to understand the population implications of the variant in northern Canada, overall frequencies of the variant were assessed. Further studies are underway to determine associated risk. Ethics approval was obtained from university REBs, local research institutes, and with consultation with territorial Aboriginal groups. Newborn screening blood spots from all infants born in 2006 in the three territories were genotyped for the p.P479L variant. p.P479L (c.1436C>T) allele frequencies in the three territories were 0.02, 0.08, and 0.77 in Yukon (n=325), Northwest Territories (n=564), and Nunavut (n=695), respectively. Homozygosity rates were 0%, 3%, and 64%. Aboriginal status was available only in NWT, with allele frequencies of 0.04, 0.44, 0.00, and 0.01 for First Nations, Inuvialuit/Inuit, Métis, and non-Aboriginal populations. Although individual blood spots were not identified for Aboriginal ethnicity in Nunavut infants, ~90% of infants in Nunavut are born to Inuit women. The allele frequency and rate of homozygosity for the CPT1A P479L variant were high in Inuit and Inuvialuit who reside in northern coastal regions. The variant is present at a low frequency in First Nations populations, who reside in areas less coastal than the

  13. Modulator of Apoptosis 1: A Highly Regulated RASSF1A-Interacting BH3-Like Protein

    Jennifer Law

    2012-01-01

    Full Text Available Modulator of apoptosis 1 (MOAP-1 is a BH3-like protein that plays key roles in both the intrinsic and extrinsic modes of cell death or apoptosis. MOAP-1 is part of the Ras association domain family 1A (RASSF1A/MOAP-1 pro-apoptotic extrinsic signaling pathway that regulates apoptosis by utilizing death receptors such as tumor necrosis factor α (TNFα or TNF-related apoptosis-inducing ligand (TRAIL to inhibit abnormal growth. RASSF1A is a bona fide tumor suppressor gene that is epigenetically silenced by promoter-specific methylation in numerous human cancers. MOAP-1 is a downstream effector of RASSF1A that promotes Bax activation and cell death and is highly regulated during apoptosis. We speculate that MOAP-1 and RASSF1A are important elements of an “apoptotic checkpoint” that directly influences the outcome of cell death. The failure to regulate this pro-apoptotic pathway may result in the appearance of cancer and possibly other disorders. Although loss of RASSF1A expression is frequently observed in human cancers, it is currently unknown if MOAP-1 expression may also be affected during carcinogenesis to result in uncontrolled malignant growth. In this article, we will summarize what is known about the biological role(s of MOAP-1 and how it functions as a downstream effector to RASSF1A.

  14. Induction of diphenytriazol on cytochrome CYP1A

    Yun-zhen HU; Tong-wei YAO

    2004-01-01

    AIM: To study the effects of diphenytriazol on cytochrome P-450 (CYP) enzymes. METHODS: SD rats were pretreated with diphenytriazol. The catalytic activities of rat liver microsomes were determined by assaying ethoxyresorufin-O-deethylase (EROD) and pentoxyresorufin-O-dealkylase. Phenacetin and aminopyrine were selected as the substrate of CYP1A and CYP2B, respectively. The concentration of remaining substrate in microsomal incubates was determined by reversed-phase high-performance liquid chromatography (RP-HPLC). The inhibition of fluvoxamine or α-naphthoflavone on phenacetin metabolism was measured. RESULTS: Phenacetin was significantly metabolized in the diphenytriazol-treated microsomes and the metabolic degree increased according to the diphenytriazol-treatment days. There existed a significant correlation between the metabolic degree of phenacetin and EROD in the microsomes pretreated with diphenytriazol. Both fluvoxamine and α-naphthofiavone inhibited the metabolism of phenacetin significantly, and the inhibition constants (Ki) were (5.4± 1.0) μmol/L and (10.4±0.5)μmol/L, respectively. The activity of microsomes pretreated with diphenytriazol for 4 d was similar to that in β-naphthoflavone group, but was significantly different from those in control group and phenobarbital group.CONCLUSION: These results reveal that diphenytriazol is a novel inducer of CYP1A.

  15. Motor axon loss is associated with hand dysfunction in Charcot-Marie-Tooth disease 1a.

    Videler, A.J.; Dijk, J.P. van; Beelen, A.; Visser, M. de; Nollet, F.; Schaik, I.N. van

    2008-01-01

    BACKGROUND: Charcot Marie Tooth type 1a (CMT1a) is a primarily demyelinating neuropathy, characterized by slowly progressive muscle weakness, atrophy, and sensory loss, and is most pronounced in both feet and hands. There is increasing evidence that muscle weakness is determined by motor axonal dysf

  16. TRPA1: A Gatekeeper for Inflammation

    Bautista, Diana M.; Pellegrino, Maurizio; Tsunozaki, Makoto

    2014-01-01

    Tissue damage evokes an inflammatory response that promotes the removal of harmful stimuli, tissue repair, and protective behaviors to prevent further damage and encourage healing. However, inflammation may outlive its usefulness and become chronic. Chronic inflammation can lead to a host of diseases, including asthma, itch, rheumatoid arthritis, and colitis. Primary afferent sensory neurons that innervate target organs release inflammatory neuropeptides in the local area of tissue damage to promote vascular leakage, the recruitment of immune cells, and hypersensitivity to mechanical and thermal stimuli. TRPA1 channels are required for neuronal excitation, the release of inflammatory neuropeptides, and subsequent pain hypersensitivity. TRPA1 is also activated by the release of inflammatory agents from nonneuronal cells in the area of tissue injury or disease. This dual function of TRPA1 as a detector and instigator of inflammatory agents makes TRPA1 a gatekeeper of chronic inflammatory disorders of the skin, airways, and gastrointestinal tract. PMID:23020579

  17. The Glycan Role in the Glycopeptide Immunogenicity Revealed by Atomistic Simulations and Spectroscopic Experiments on the Multiple Sclerosis Biomarker CSF114(Glc)

    Bruno, Agostino; Scrima, Mario; Novellino, Ettore; D'Errico, Gerardino; D'Ursi, Anna Maria; Limongelli, Vittorio

    2015-03-01

    Glycoproteins are often recognized as not-self molecules by antibodies triggering the onset of severe autoimmune diseases such as Multiple Sclerosis (MS). Thus, the development of antigen-mimicking biomarkers represents an attractive strategy for an early diagnosis of the disease. An example is the synthetic glycopeptide CSF114(Glc), which was designed and tested as MS biomarker and whose clinical application was limited by its reduced ability to detect autoantibodies in MS patients. In the attempt to improve the efficacy of CSF114(Glc), we have characterized all the events leading to the final binding of the biomarker to the autoantibody using atomistic simulations, ESR and NMR experiments. The glycosydic moiety plays a primary role in the whole process. In particular, in an environment mimicking that used in the clinical tests the glycopeptide assumes a α-helix structure that is functional for the interaction with the antibody. In this conformation CSF114(Glc) binds the monoclonal antibody mAb8-18C5 similarly to the myelin oligodendrocyte glycoprotein MOG, which is a known MS auto-antigen, thus explaining its diagnostic activity. Our study offers new molecular bases to design more effective biomarkers and provides a most valid protocol to investigate other systems where the environment effect is determinant for the biological activity.

  18. X-1A in flight over lakebed

    1953-01-01

    The Bell Aircraft Corporation X-1A (48-1384) returning from an Air Force test flight over Edwards Air Force Base, California in late 1953. A North American F-86A Sabre as chase plane will follow the X-1A to touchdown. The Rogers Dry Lake is the whitish area under the planes with the airfield at the edge of the dry lake. Bell test pilot Jean 'Skip' Ziegler made six flights between 14 February and 25 April 1953. Air Force test pilots Maj. Charles 'Chuck' Yeager and Maj. Arthur 'Kit' Murray made 18 test flights between 21 November 1953 and 26 August 1954. NACA test pilot Joseph Walker made one successful flight on 20 July 1955. During a second flight attempt, on 8 August 1955, an explosion damaged the aircraft shortly before launch. Walker, unhurt, climbed up into the JTB-29A mothership, and the X-1A was jettisoned over the Edwards AFB bombing range. There were five versions of the Bell X-1 rocket-powered research aircraft that flew at the NACA High-Speed Flight Research Station, Edwards, California. The bullet-shaped X-1 aircraft were built by Bell Aircraft Corporation, Buffalo, N.Y. for the U.S. Army Air Forces (after 1947, U.S. Air Force) and the National Advisory Committee for Aeronautics (NACA). The X-1 Program was originally designated the XS-1 for EXperimental Sonic. The X-1's mission was to investigate the transonic speed range (speeds from just below to just above the speed of sound) and, if possible, to break the 'sound barrier.' Three different X-1s were built and designated: X-1-1, X-1-2 (later modified to become the X-1E), and X-1-3. The basic X-1 aircraft were flown by a large number of different pilots from 1946 to 1951. The X-1 Program not only proved that humans could go beyond the speed of sound, it reinforced the understanding that technological barriers could be overcome. The X-1s pioneered many structural and aerodynamic advances including extremely thin, yet extremely strong wing sections; supersonic fuselage configurations; control system

  19. Identification and molecular cloning of a novel glycoside hydrolase family of core 1 type O-glycan-specific endo-alpha-N-acetylgalactosaminidase from Bifidobacterium longum.

    Fujita, Kiyotaka; Oura, Fusako; Nagamine, Noriko; Katayama, Takane; Hiratake, Jun; Sakata, Kanzo; Kumagai, Hidehiko; Yamamoto, Kenji

    2005-11-11

    We found endo-alpha-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl beta1,3-N-acetyl-D-galactosamine (Galbeta1,3GalNAc) alpha-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590-1381 amino acid residues) exhibited 31-53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Galbeta1,3GalNAc disaccharide from Galbeta1,3GalNAcalpha1pNP and asialofetuin, but did not release GalNAc, Galbeta1,3(GlcNAcbeta1,6)GalNAc, GlcNAcbeta1,3GalNAc, and Galbeta1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by (1)H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-alpha-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101). PMID:16141207

  20. UGT1A1 and UGT1A9 functional variants, meat intake, and colon cancer, among Caucasians and African Americans

    Girard, Hugo; Butler, Lesley M.; Villeneuve, Lyne; Millikan, Robert C.; Sinha, Rashmi; Sandler, Robert S.; Guillemette, Chantal

    2008-01-01

    Glucuronidation by the UDP-glucuronosyltransferase enzymes (UGTs) is one of the primary detoxification pathways of dietary heterocyclic amines (HCAs) and polycyclic aromatic hydrocarbons (PAHs). In a population-based case-control study of 537 cases and 866 controls, we investigated whether colon cancer was associated with genetic variations in UGT1A1 and UGT1A9 genes and we determined if those variations modify the association between colon cancer and dietary HCA and PAH exposure. We measured...

  1. Structure and Active Stie Residues of Pg1D, an N-Acetyltransferase from the Bacillosamine Synthetic Pathway Required for N-Glycan Synthesis in Campylobacter jejuni

    Rangarajan,E.; Ruane, K.; Sulea, T.; Watson, D.; Proteau, A.; Leclerc, S.; Cygler, M.; Matte, A.; Young, N.

    2008-01-01

    Campylobacter jejuni is highly unusual among bacteria in forming N-linked glycoproteins. The heptasaccharide produced by its pgl system is attached to protein Asn through its terminal 2, 4-diacetamido-2, 4,6-trideoxy-d-Glc (QuiNAc4NAc or N, N'-diacetylbacillosamine) moiety. The crucial, last part of this sugar's synthesis is the acetylation of UDP-2-acetamido-4-amino-2, 4,6-trideoxy-d-Glc by the enzyme PglD, with acetyl-CoA as a cosubstrate. We have determined the crystal structures of PglD in CoA-bound and unbound forms, refined to 1.8 and 1.75 Angstroms resolution, respectively. PglD is a trimer of subunits each comprised of two domains, an N-terminal {alpha}/{beta}-domain and a C-terminal left-handed {beta}-helix. Few structural differences accompany CoA binding, except in the C-terminal region following the {beta}-helix (residues 189-195), which adopts an extended structure in the unbound form and folds to extend the {beta}-helix upon binding CoA. Computational molecular docking suggests a different mode of nucleotide-sugar binding with respect to the acetyl-CoA donor, with the molecules arranged in an 'L-shape', compared with the 'in-line' orientation in related enzymes. Modeling indicates that the oxyanion intermediate would be stabilized by the NH group of Gly143', with His125' the most likely residue to function as a general base, removing H+ from the amino group prior to nucleophilic attack at the carbonyl carbon of acetyl-CoA. Site-specific mutations of active site residues confirmed the importance of His125', Glu124', and Asn118. We conclude that Asn118 exerts its function by stabilizing the intricate hydrogen bonding network within the active site and that Glu124' may function to increase the pKa of the putative general base, His125'.

  2. Functionality of promoter microsatellites of arginine vasopressin receptor 1A (AVPR1A: implications for autism

    Tansey Katherine E

    2011-03-01

    Full Text Available Abstract Background Arginine vasopressin (AVP has been hypothesized to play a role in aetiology of autism based on a demonstrated involvement in the regulation of social behaviours. The arginine vasopressin receptor 1A gene (AVPR1A is widely expressed in the brain and is considered to be a key receptor for regulation of social behaviour. Moreover, genetic variation at AVPR1A has been reported to be associated with autism. Evidence from non-human mammals implicates variation in the 5'-flanking region of AVPR1A in variable gene expression and social behaviour. Methods We examined four tagging single nucleotide polymorphisms (SNPs (rs3803107, rs1042615, rs3741865, rs11174815 and three microsatellites (RS3, RS1 and AVR at the AVPR1A gene for association in an autism cohort from Ireland. Two 5'-flanking region polymorphisms in the human AVPR1A, RS3 and RS1, were also tested for their effect on relative promoter activity. Results The short alleles of RS1 and the SNP rs11174815 show weak association with autism in the Irish population (P = 0.036 and P = 0.008, respectively. Both RS1 and RS3 showed differences in relative promoter activity by length. Shorter repeat alleles of RS1 and RS3 decreased relative promoter activity in the human neuroblastoma cell line SH-SY5Y. Conclusions These aligning results can be interpreted as a functional route for this association, namely that shorter alleles of RS1 lead to decreased AVPR1A transcription, which may proffer increased susceptibility to the autism phenotype.

  3. Functionality of promoter microsatellites of arginine vasopressin receptor 1A (AVPR1A): implications for autism

    Tansey, Katherine E

    2011-03-31

    Abstract Background Arginine vasopressin (AVP) has been hypothesized to play a role in aetiology of autism based on a demonstrated involvement in the regulation of social behaviours. The arginine vasopressin receptor 1A gene (AVPR1A) is widely expressed in the brain and is considered to be a key receptor for regulation of social behaviour. Moreover, genetic variation at AVPR1A has been reported to be associated with autism. Evidence from non-human mammals implicates variation in the 5\\'-flanking region of AVPR1A in variable gene expression and social behaviour. Methods We examined four tagging single nucleotide polymorphisms (SNPs) (rs3803107, rs1042615, rs3741865, rs11174815) and three microsatellites (RS3, RS1 and AVR) at the AVPR1A gene for association in an autism cohort from Ireland. Two 5\\'-flanking region polymorphisms in the human AVPR1A, RS3 and RS1, were also tested for their effect on relative promoter activity. Results The short alleles of RS1 and the SNP rs11174815 show weak association with autism in the Irish population (P = 0.036 and P = 0.008, respectively). Both RS1 and RS3 showed differences in relative promoter activity by length. Shorter repeat alleles of RS1 and RS3 decreased relative promoter activity in the human neuroblastoma cell line SH-SY5Y. Conclusions These aligning results can be interpreted as a functional route for this association, namely that shorter alleles of RS1 lead to decreased AVPR1A transcription, which may proffer increased susceptibility to the autism phenotype.

  4. Basal HIF-1a expression levels are not predictive for radiosensitivity of human cancer cell lines

    Schilling, D.; Multhoff, G. [Klinikum rechts der Isar der Technischen Univ. Muenchen (Germany). Dept. of Radiation Oncology; Helmholtz Center Munich, CCG - Innate Immunity in Tumor Biology, Munich (Germany). German Research Center for Environmental Health - Inst. of Pathology; Bayer, C.; Emmerich, K.; Molls, M.; Vaupel, P. [Klinikum rechts der Isar der Technischen Univ. Muenchen (Germany). Dept. of Radiation Oncology; Huber, R.M. [Klinikum der Univ. Muenchen (Germany). Dept. of Pneumology

    2012-04-15

    High levels of hypoxia inducible factor (HIF)-1a in tumors are reported to be associated with tumor progression and resistance to therapy. To examine the impact of HIF-1a on radioresistance under normoxia, the sensitivity towards irradiation was measured in human tumor cell lines that differ significantly in their basal HIF-1a levels. HIF-1a levels were quantified in lysates of H1339, EPLC-272H, A549, SAS, XF354, FaDu, BHY, and CX- tumor cell lines by ELISA. Protein levels of HIF-1a, HIF-2a, carbonic anhydrase IX (CA IX), and GAPDH were assessed by Western blot analysis. Knock-down experiments were performed using HIF-1a siRNA. Clonogenic survival after irradiation was determined by the colony forming assay. According to their basal HIF-1a status, the tumor cell lines were divided into low (SAS, XF354, FaDu, A549, CX-), intermediate (EPLC-272H, BHY), and high (H1339) HIF-1a expressors. The functionality of the high basal HIF-1a expression in H1339 cells was proven by reduced CA IX expression after knocking-down HIF-1a. Linear regression analysis revealed no correlation between basal HIF-1a levels and the survival fraction at either 2 or 4 Gy in all tumor cell lines investigated. Our data suggest that basal HIF-1a levels in human tumor cell lines do not predict their radiosensitivity under normoxia. (orig.)

  5. The single N-glycan deletion mutant of soluble ErbB3 protein attenuates heregulin β1-induced tumor progression by blocking of the HIF-1 and Nrf2 pathway

    Takamiya, Rina, E-mail: rinataka0429@gmail.com; Takahashi, Motoko; Uehara, Yasuaki; Ariki, Shigeru; Hashimoto, Jiro; Hasegawa, Yoshihiro; Kuroki, Yoshio

    2014-11-21

    Highlights: • The sErbB3 N418Q mutant blocks heregulin β1 induced nuclear accumulation of HIF-1α. • The sErbB3 N418Q mutant attenuates cancer cell migration induced by heregulin β1. • The sErbB3 N418Q mutant blocks heregulin β1 induced nuclear accumulation of Nrf2. • The sErbB3 N418Q mutant may be a potential therapeutic application for tumor. - Abstract: It has been well documented that activation of the ErbB3–PI3K–Akt pathway is implicated in tumor survival and progression. We previously demonstrated that the single N-glycan deletion mutant of soluble ErbB3 protein (sErbB3 N418Q) attenuates heregulin β1-induced ErbB3 signaling. The active PI3K–Akt pathway augments the nuclear accumulation of hypoxia inducible factor (HIF)-1α, which activates the transcription of many target genes and drives cancer progression. In this study, we focused on the effects of sErbB3 N418Q mutant on nuclear accumulation of HIF-1α. Pretreatment with the sErbB3 N418Q mutant suppressed heregulin β1-induced HIF-1α activation in MCF7 cells. Similar results were also obtained in other breast cancer cell lines, T47D and BT474. Interestingly, these suppressive effects were not observed with the sErbB3 wild type. In addition, pretreatment with the sErbB3 N418Q mutant suppressed the cell migration of MCF7 cells induced by heregulin β1. Furthermore, incubation with heregulin β1 also induced the nuclear accumulation of Nrf2, and this effect was also reduced by the sErbB3 N418Q mutant, but not the sErbB3 wild type. These findings indicated that the sErbB3 N418Q mutant suppressed malignant formation of cancer cells by blocking of the HIF-1α and Nrf2 pathways.

  6. Coordinate deletion of N-glycans from the heptad repeats of the fusion F protein of Newcastle disease virus yields a hyperfusogenic virus with increased replication, virulence, and immunogenicity.

    Samal, Sweety; Khattar, Sunil K; Kumar, Sachin; Collins, Peter L; Samal, Siba K

    2012-03-01

    The role of N-linked glycosylation of the Newcastle disease virus (NDV) fusion (F) protein in viral replication and pathogenesis was examined by eliminating potential acceptor sites using a reverse genetics system for the moderately pathogenic strain Beaudette C (BC). The NDV-BC F protein contains six potential acceptor sites for N-linked glycosylation at residues 85, 191, 366, 447, 471, and 541 (sites Ng1 to Ng6, respectively). The sites at Ng2 and Ng5 are present in heptad repeat (HR) domains HR1 and HR2, respectively, and thus might affect fusion. Each N-glycosylation site was eliminated individually by replacing asparagine (N) with glutamine (Q), and a double mutant (Ng2 + 5) involving the two HR domains was also made. Each mutant was successfully recovered by reverse genetics except for the one involving Ng6, which is present in the cytoplasmic domain. All of the F proteins expressed by the recovered mutant viruses were efficiently cleaved and transported to the infected-cell surface. None of the individual mutations affected viral fusogenicity, but the double mutation at Ng2 and Ng5 in HR1 and HR2 increased fusogenicity >12-fold. The single mutations at sites Ng1, Ng2, and Ng5 resulted in modestly reduced multicycle growth in vitro. These three single mutations were also the most attenuating in eggs and 1-day-old chicks and were associated with decreased replication and spread in 2-week-old chickens. In contrast, the combination of the mutations at Ng2 and Ng5 yielded a virus that, compared to the BC parent, replicated >100-fold more efficiently in vitro, was more virulent in eggs and chicks, replicated more efficiently in chickens with enhanced tropism for the brain and gut, and elicited stronger humoral cell responses. These results illustrate the effects of N-glycosylation of the F protein on NDV pathobiology and suggest that the N-glycans in HR1 and HR2 coordinately downregulate viral fusion and virulence. PMID:22205748

  7. Neuroticism and serotonin 5-HT1A receptors in healthy subjects

    Hirvonen, Jussi; Tuominen, Lauri; Någren, Kjell; Hietala, Jarmo

    2015-01-01

    Neuroticism is a personality trait associated with vulnerability for mood and anxiety disorders. Serotonergic mechanisms likely contribute to neuroticism. Serotonin 5-HT1A receptors are altered in mood and anxiety disorders, but whether 5-HT1A receptors are associated with neuroticism in healthy...... subjects is unclear. We measured brain serotonin 5-HT1A receptor in 34 healthy subjects in vivo using positron emission tomography (PET) and [carbonyl-(11)C]WAY-100635. Binding potential (BPP) was determined using the golden standard of kinetic compartmental modeling using arterial blood samples and...... radiometabolite determination. Personality traits were assessed using the Karolinska Scales of Personality. We found a strong negative association between serotonin 5-HT1A receptor BPP and neuroticism. That is, individuals with high neuroticism tended to have lower 5-HT1A receptor binding than individuals with...

  8. Systematic Screening of the Serotonin Receptor 1A (5-HT1A) Gene in Chronic Tinnitus

    Kleinjung T; Langguth B; Fischer B; Hajak G; Eichhammer P; Sand PG

    2006-01-01

    Objective Chronic tinnitus is a highly prevalent condition and has been hypothesized to result from an innate disturbance in central nervous serotonergic transmission. Given the frequent comorbidity with major depression and anxiety, we argue that candidate genes for these disorders are likely to overlap. The present study addresses the gene encoding for the 5-HT1A receptor as a putative risk factor for tinnitus. Methods In 88 subjects with a diagnosis of chronic subjective tinnitus who underwent a detailed neurootological examination, the entire 5-HT1A gene was amplified using overlapping PCR products. Amplicons were custom sequenced bidirectionally and were screened for variants in multiple alignments against the human genome reference. Results We identified a synonymous C > T exchange at residue 184 (Pro) in 7/88 subjects, but detected no missense variants in the population under study. Specifically, the following residues were fully conserved: 16 (Pro), 22 (Gly), 28 (Ile), 98 (Val), 220(Arg), 267 (Val), 273 (Gly), and 418 (Asn). Discussion The present data count against the causation of chronic tinnitus by a change in the 5-HT1A receptor's amino acid sequence. However, the allele frequency for the 184Pro minor allele (0.04) reached twice the frequency reported in control cohorts from the same ethnicity.Additional investigations are invited to clarify the role of the 5-HT1A polymorphism in larger samples, and to control for comorbid affective disorders.

  9. Adsorption of Water on JSC-1A Lunar Simulant Samples

    Goering, John; Sah, Shweta; Burghaus, Uwe; Street, Kenneth W.

    2008-01-01

    Remote sensing probes sent to the moon in the 1990s indicated that water may exist in areas such as the bottoms of deep, permanently shadowed craters at the lunar poles, buried under regolith. Water is of paramount importance for any lunar exploration and colonization project which would require self-sustainable systems. Therefore, investigating the interaction of water with lunar regolith is pertinent to future exploration. The lunar environment can be approximated in ultra-high vacuum systems such as those used in thermal desorption spectroscopy (TDS). Questions about water dissociation, surface wetting, degree of crystallization, details of water-ice transitions, and cluster formation kinetics can be addressed by TDS. Lunar regolith specimens collected during the Apollo missions are still available though precious, so testing with simulant is required before applying to use lunar regolith samples. Hence, we used for these studies JSC-1a, mostly an aluminosilicate glass and basaltic material containing substantial amounts of plagioclase, some olivine and traces of other minerals. Objectives of this project include: 1) Manufacturing samples using as little raw material as possible, allowing the use of surface chemistry and kinetics tools to determine the feasibility of parallel studies on regolith, and 2) Characterizing the adsorption kinetics of water on the regolith simulant. This has implications for the probability of finding water on the moon and, if present, for recovery techniques. For condensed water films, complex TDS data were obtained containing multiple features, which are related to subtle rearrangements of the water adlayer. Results from JSC-1a TDS studies indicate: 1) Water dissociation on JSC-1a at low exposures, with features detected at temperatures as high as 450 K and 2) The formation of 3D water clusters and a rather porous condensed water film. It appears plausible that the sub- m sized particles act as nucleation centers.

  10. Lack of association between UGT1A7, UGT1A9, ARP, SPINK1 and CFTR gene polymorphisms and pancreatic cancer in Italian patients

    Ada Piepoli; Annamaria Gentile; Maria Rosa Valvano; Daniela Barana; Cristina Oliani; Rosa Cotugno; Michele Quitadamo; Angelo Andriulli; Francesco Perri

    2006-01-01

    AIM: To investigate simultaneously UGT1A7, UGT1A9,ARP, SPINK and CFTR genes to verify whether genetic polymorphisms predispose to the development of pancreatic cancer (PC).METHODS: Genomic DNA of 61 pancreatic cancer patients and 105 healthy controls (HC) were analyzed.UGT1,47 genotyping was determined by PCR-RFLP analysis. Specific PCR and sequencing were used to analyze genetic variants of UGT1A9, ARP, SPINK1 and CFTR genes.RESULTS: Four different alleles (*1: WT;*2: N129Kand R131K;*3: N129K, R131K, and W208R;and *4:W208R) in UGT1A7 and three different alleles (*1: WT;*4: Y242X;and *5: D256N) in UGT1A9 were detected.All UGT1A polymorphisms were observed at similar frequency in PC patients and HC. Seven different alleles in ARP were found in PC patients and HC at similar frequency. The SPINK1 mutations N34S and P55Soccurred in five PC patients with a prevalence (4.1%) not significantly different from that observed (2.0%) in HC.The only CFTR ΔF508 mutation was recognized in three PC patients with a prevalence (4.9%) similar to HC.CONCLUSION: UGT1A7, UGT1A9, ARP, SPINK1 and CFTR gene polymorphisms are not associated with PC in Italian patients.

  11. Methylation – an uncommon modification of glycans*

    Staudacher, Erika

    2012-01-01

    A methyl group on a sugar residue is a rarely reported event. Until now this kind of modification has been found in the kingdom of animals only in worms and molluscs, whereas it is more frequently present in some species of bacteria, fungi, algae and plants, but not in mammals. The monosaccharides involved as well as the positions of the methyl groups on the sugar vary with the species. Methylation seems to play a role in some recognition events but details are still unknown. This review summ...

  12. Targeted identification of Schistosoma mansoni egg glycans

    Robijn, Marjolein Louise Maria

    2008-01-01

    Schistosomiasis, also known as bilharzia, is a disease that still occurs in many parts of Africa, the Middle East and Southern America, mainly Brasil and the Caribbean. Schistosomiasis is, after malaria, the second most common parasitic infection. Currently 200 million people are infected with the w

  13. Raloxifene glucuronidation in liver and intestinal microsomes of humans and monkeys: contribution of UGT1A1, UGT1A8 and UGT1A9.

    Kishi, Naoki; Takasuka, Akane; Kokawa, Yuki; Isobe, Takashi; Taguchi, Maho; Shigeyama, Masato; Murata, Mikio; Suno, Manabu; Hanioka, Nobumitsu

    2016-04-01

    1. Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4'-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9). 2. Although the Km and CLint values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in Vmax values (liver microsomes, humans > monkeys; intestinal microsomes, humans  UGT1A8 >UGT1A9 for humans, and UGT1A8 > UGT1A1 > UGT1A9 for monkeys. The activities of 4'-glucuronidation were UGT1A8 > UGT1A1 > UGT1A9 in humans and monkeys. 4. These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys. PMID:26247833

  14. Visualisation of serotonin-1A (5-HT1A) receptors in the central nervous system

    The 5-HT1A subtype of receptors for the neurotransmitter serotonin is predominantly located in the limbic forebrain and is involved in the modulation of emotion and the function of the hypothalamus. Since 5-HT1A receptors are implicated in the pathogenesis of anxiety, depression, hallucinogenic behaviour, motion sickness and eating disorders, they are an important target for drug therapy. Here, we review the radioligands which are available for visualisation and quantification of this important neuroreceptor in the human brain, using positron emission tomography (PET) or single-photon emission tomography (SPET). More than 20 compounds have been labelled with carbon-11 (half-life 20 min), fluorine-18 (half-life 109.8 min) or iodine-123 (half-life 13.2 h): structural analogues of the agonist, 8-OH-DPAT, structural analogues of the antagonist, WAY 100635, and apomorphines. The most successful radioligands thus far are [carbonyl-11C] WAY-100635 (WAY), [carbonyl-11C]desmethyl-WAY-100635 (DWAY), p-[18F]MPPF and [11C]robalzotan (NAD-299). The high-affinity ligands WAY and DWAY produce excellent images of 5-HT1A receptor distribution in the brain (even the raphe nuclei are visualised), but they cannot be distributed to remote facilities and they probably cannot be used to measure changes in endogenous serotonin. Binding of the moderate-affinity ligands MPPF and NAD-299 may be more sensitive to serotonin competition and MPPF can be distributed to PET centres within a flying distance of a few hours. Future research should be directed towards: (a) improvement of the metabolic stability in primates; (b) development of a fluorinated radioligand which can be produced in large quantities and (c) production of a radioiodinated or technetium-labelled ligand for SPET. (orig.)

  15. Polyclonal T-cells express CD1a in Langerhans cell histiocytosis (LCH lesions.

    Jennifer A West

    Full Text Available Langerhans cell histiocytosis (LCH is a complex and poorly understood disorder that has characteristics of both inflammatory and neoplastic disease. By using eight-colour flow cytometry, we have identified a previously unreported population of CD1a(+/CD3(+ T-cells in LCH lesions. The expression of CD1a is regarded as a hallmark of this disease; however, it has always been presumed that it was only expressed by pathogenic Langerhans cells (LCs. We have now detected CD1a expression by a range of T-cell subsets within all of the LCH lesions that were examined, establishing that CD1a expression in these lesions is no longer restricted to pathogenic LCs. The presence of CD1a(+ T-cells in all of the LCH lesions that we have studied to date warrants further investigation into their biological function to determine whether these cells are important in the pathogenesis of LCH.

  16. The biofilm matrix of Campylobacter jejuni determined by fluorescence lectin-binding analysis.

    Turonova, Hana; Neu, Thomas R; Ulbrich, Pavel; Pazlarova, Jarmila; Tresse, Odile

    2016-05-01

    Campylobacter jejuni is responsible for the most common bacterial foodborne gastroenteritis. Despite its fastidious growth, it can survive harsh conditions through biofilm formation. In this work, fluorescence lectin-binding analysis was used to determine the glycoconjugates present in the biofilm matrix of two well-described strains. Screening of 72 lectins revealed strain-specific patterns with six lectins interacting with the biofilm matrix of both strains. The most common sugar moiety contained galactose and N-acetylgalactosamine. Several lectins interacted with N-acetylglucosamine and sialic acid, probably originated from the capsular polysaccharides, lipooligosaccharides and N-glycans of C. jejuni. In addition, glycoconjugates containing mannose and fucose were detected within the biofilm, which have not previously been found in the C. jejuni envelope. Detection of thioflavin T and curcumin highlighted the presence of amyloids in the cell envelope without association with specific cell appendages. The lectins ECA, GS-I, HMA and LEA constitute a reliable cocktail to detect the biofilm matrix of C. jejuni. PMID:27097059

  17. Epigenetic Modulation of Collagen 1A1: Therapeutic Implications in Fibrosis and Endometriosis.

    Zheng, Ye; Khan, Zaraq; Zanfagnin, Valentina; Correa, Luiz F; Delaney, Abigail A; Daftary, Gaurang S

    2016-04-01

    Progressive fibrosis is recalcitrant to conventional therapy and commonly complicates chronic diseases and surgical healing. We evaluate here a novel mechanism that regulates scar-tissue collagen (COL1A1/Col1a1) expression and characterizes its translational relevance as a targeted therapy for fibrosis in an endometriosis disease model. Endometriosis is caused by displacement and implantation of uterine endometrium onto abdominal organs and spreads with progressive scarring. Transcription factor KLF11 is specifically diminished in endometriosis lesions. Loss of KLF11-mediated repression of COL1A1/Col1a1 expression resulted in increased fibrosis. To determine the biological significance of COL1A1/Col1a1 expression on fibrosis, we modulated its expression. In human endometrial-stromal fibroblasts, KLF11 recruited SIN3A/HDAC (histone deacetylase), resulting in COL1A1-promoter deacetylation and repression. This role of KLF11 was pharmacologically replicated by a histone acetyl transferase inhibitor (garcinol). In contrast, opposite effects were obtained with a HDAC inhibitor (suberoyl anilide hydroxamic acid), confirming regulatory specificity for these reciprocally active epigenetic mechanisms. Fibrosis was concordantly reversed in Klf11(-/-)animals by histone acetyl transferase inhibitor and in wild-type animals by HDAC inhibitor treatments. Aberrant lesional COL1A1 regulation is significant because fibrosis depended on lesion rather than host genotype. This is the first report demonstrating feasibility for targeted pharmacological reversal of fibrosis, an intractable phenotype of diverse chronic diseases. PMID:26935598

  18. 26 CFR 1.148-1A - Definitions and elections.

    2010-04-01

    ... 26 Internal Revenue 2 2010-04-01 2010-04-01 false Definitions and elections. 1.148-1A Section 1.148-1A Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF THE TREASURY (CONTINUED) INCOME TAX....148-1A Definitions and elections. (a) . For guidance see § 1.148-1. (b) Certain...

  19. Cytochrome P450 1A2 Metabolizes 17β-Estradiol to Suppress Hepatocellular Carcinoma

    Ren, Jianwai; Chen, George G.; Liu, Yi; Su, Xianwei; Hu, Baoguang; Leung, Billy C. S.; Wang, Y.; Ho, Rocky L. K.; Yang, Shengli; Lu, Gang; Lee, C. G.; Lai, Paul B. S.

    2016-01-01

    Hepatocellular carcinoma (HCC) occurs more frequently in men than in women. It is commonly agreed that estrogen plays important roles in suppressing HCC development, however, the underlying mechanism remains largely unknown. Since estrogen is mainly metabolized in liver and its metabolites affect cell proliferation, we sought to investigate if the liver-specific cytochrome P450 1A2 (CYP1A2) mediated the inhibitory effect of estrogen on HCC. In this study, the expression of estrogen-metabolizing enzyme CYP1A2 was determined in HCC tissues and cell lines. Cell proliferation and apoptosis were assessed in cells with or without CYP1A2 overexpression. The levels of 17β-estradiol (E2) and its metabolite 2-methoxyestradiol (2-ME) were determined. A xenograft tumor model in mice was established to confirm the findings. It was found that CYP1A2 expression was greatly repressed in HCC. E2 suppressed HCC cell proliferation and xenograft tumor development by inducing apoptosis. The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME. E2 in combination with sorafenib showed an additive effect on HCC. The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib. In addition, HDAC inhibitors greatly induced CYP1A2 promoter activities in cancer cells, especially liver cancer cells, but not in non-tumorigenic cells. Collectively, CYP1A2 metabolizes E2 to generate the potent anti-tumor agent 2-ME in HCC. The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy. PMID:27093553

  20. The clinical application of UGT1A1 pharmacogenetic testing: Gene-environment interactions

    Marques Sara

    2010-04-01

    Full Text Available Abstract Over the past decade, the number of pharmacogenetic tests has increased considerably, allowing for the development of our knowledge of their clinical application. The uridine diphosphate glucuronosyltransferase 1A1 gene (UGT1A1 assay is an example of a pharmacogenetic test. Numerous variants have been found in UGT1A1, the main conjugating enzyme of bilirubin and drugs such as the anticancer drug irinotecan. Recently, the US Food and Drug Administration (FDA recommended testing for the presence of UGT1A1*28, an allele correlated with decreased transcriptional activity, to predict patients at risk of irinotecan toxicity. The administration of other drugs -- such as inhibitors of the UGT1A1 enzyme -- can clinically mimic the *28 phenotype, whereas inducers of UGT1A1 can increase the glucuronidation rate of the enzyme. The *28 polymorphism is not present in all ethnicities at a similar frequency, which suggests that it is important to study different populations to determine the clinical relevance of testing for UGT1A1*28 and to identify other clinically relevant UGT1A1 variants. Environmental factors such as lifestyle can also affect UGT1A1 activity. This review is a critical analysis of studies on drugs that can be affected by the presence of UGT1A1*28, the distribution of this polymorphism around the globe, distinct variants that may be clinically significant in African and Asian populations and how lifestyle can affect treatment outcomes that depend on UGT1A1 activity.

  1. Complete misloading of a mark IV MCO with mark 1A fuel

    The purpose of this analysis is to determine the worst case scenario for a total misload of a Mark IV MCO with Mark 1A fuel and scrap. This study is not intended to classify any of the components of the baskets

  2. Alpha-defensin DEFA1A3 gene copy number elevation in Danish Crohn's disease patients

    Jespersgaard, Cathrine; Fode, Peder; Dybdahl, Marianne;

    2011-01-01

    BACKGROUND AND PURPOSE OF STUDY: Extensive copy number variation is observed for the DEFA1A3 gene encoding alpha-defensins 1-3. The objective of this study was to determine the involvement of alpha-defensins in colonic tissue from Crohn's disease (CD) patients and the possible genetic association...

  3. Lectin histochemistry of intestinal carbohydrate determinants in representatives of different classes of vertebrates

    Antonyuk R.V.

    2015-12-01

    Full Text Available Background. Glycoproteins (including mucin of vertebrate’s intestine play an important role in its protection against chemical and mechanical damage and bacterial attacks. Their diversity was described by many authors, but understanding of their chemical structure remains far from complete. These data can be extended by methods of lectin histochemistry. Objective. To investigate the rearrangement of intestinal carbohydrate determinants in the context of vertebrate evolution. Methods. Distal and proximal segments of small and large intestines of humans (Homo sapiens, laboratory (Wistar rat (Rattus norvegicus f. Domesticus, rock pigeon (Columba livia, smooth snake (Coronella austriaca, common frog (Rana temporaria, common carp (Cyprinus carpio that belong to different classes of vertebrates were taken for the experiment. Nine lectins with different carbohydrate specificities: wheat germ (WGA, potato (STA, elderberry bark (SNA, golden rain bark (LABA, locust bark (RPBA, roe carp (CCRA, Phaseolus vulgaris erytroagglutinin (PHA-E, peanut (PNA and jack fruit (AIA – were included into the panel. Results. Differences in lectin staining between small and large intestine were more pronounced in higher (human, rat than in lower (frog, carp vertebrates. Lectin receptors were more diverse in frog intestine in comparison with carp. Lectin interaction with mucin secretory granules of smooth snake revealed lack of N-acetyl-D-glucosamine residues and abundance of N-acetyl-D-galactosamine determinants. Conclusion. Intestines of all studied vertebrate species demonstrate high content of secretory mucins that exposed terminal acidic carbohydrates including sialic acid. The diversity and differences in the structure of glycans of the digestive tract of vertebrates is apparently determined by several factors – diet, environmental and living conditions, intestinal microbiota interactions etc. Citation: Antonyuk RV, Lutsyk AD. [Lectin histochemistry of intestinal

  4. Analysis list: ARID1A [Chip-atlas[Archive

    Full Text Available ARID1A Liver + hg19 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/ARID1A.1.tsv http://dbar...chive.biosciencedbc.jp/kyushu-u/hg19/target/ARID1A.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/AR...ID1A.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/ARID1A.Liver.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Liver.gml ...

  5. Analysis list: DCP1A [Chip-atlas[Archive

    Full Text Available DCP1A Uterus + hg19 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/DCP1A.1....tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/DCP1A.5.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/DCP...1A.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/DCP1A.Uterus.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Uterus.gml ...

  6. The growth and tumor suppressors NORE1A and RASSF1A are targets for calpain-mediated proteolysis.

    Sergey Kuznetsov

    Full Text Available BACKGROUND: NORE1A and RASSF1A are growth and tumour suppressors inactivated in a variety of cancers. Methylation of NORE1A and RASSF1A promoters is the predominant mechanism for downregulation of these proteins; however, other mechanisms are likely to exist. METHODOLOGY/PRINCIPAL FINDINGS: Here we describe a proteolysis of NORE1A and RASSF1A by calpains as alternative mechanism of their downregulation. Extracts of H358 cell line, a human bronchoalveolar carcinoma, and H460, a large cell carcinoma, were capable of proteolysis of NORE1A protein in the calpain-dependent manner. Likewise, RASSF1A tumor suppressor was proteolyzed by the H358 cell extract. Addition of calpain inhibitor to H358 and H460 cells growing in tissue culture resulted in re-expression of endogenous NORE1A. A survey of 10 human lung tumours revealed that three of them contain an activity capable of inducing NORE1A degradation. CONCLUSIONS/SIGNIFICANCE: Thus, degradation by calpains is a novel mechanism for downregulation of NORE1A and RASSF1A proteins and might be the mechanism allowing cancer cells to escape growth suppression.

  7. Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

    The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[a]anthracene, β-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression

  8. PPARGC1A DNA methylation in subcutaneous adipose tissue in low birth weight subjects

    Gillberg, Linn; Jacobsen, Stine; Rönn, Tina;

    2014-01-01

    insulin-stimulated SAT from LBW and matched normal birth weight (NBW) subjects during control and high-fat overfeeding. MATERIALS/METHODS: Nineteen young healthy men with LBW and 26 NBW controls were studied after both a 5-day high-fat overfeeding and a control diet in a randomized crossover setting. DNA......OBJECTIVE: Increased DNA methylation of the metabolic regulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A) has been reported in skeletal muscle from type 2 diabetes (T2D) subjects and from low birth weight (LBW) subjects with an increased risk of T2D. High......-fat overfeeding increases PPARGC1A DNA methylation in muscle in a birth weight dependent manner. However, PPARGC1A DNA methylation in subcutaneous adipose tissue (SAT) in LBW subjects has not previously been investigated. Our objective was to determine PPARGC1A DNA methylation and mRNA expression in basal and...

  9. Age-related changes in motor unit number estimates in adult patients with Charcot-Marie-Tooth type 1A

    J.P. van Dijk; C. Verhamme; I.N. van Schaik; H.J. Schelhaas; E. Mans; L.J. Bour; D.F. Stegeman; M.J. Zwarts

    2010-01-01

    Background: Charcot-Marie-Tooth disease type 1A (CMT1A) is known as a demyelinating hereditary neuropathy. Secondary axonal dysfunction is the most important determinant of disease severity. In adult patients, clinical progression may be because of further axonal deterioration as was shown with comp

  10. Age-related changes in motor unit number estimates in adult patients with Charcot-Marie-Tooth type 1A.

    Dijk, J.P. van; Verhamme, C.; Schaik, I.N. van; Schelhaas, H.J.; Mans, E.; Bour, L.J.; Stegeman, D.F.; Zwarts, M.J.

    2010-01-01

    BACKGROUND: Charcot-Marie-Tooth disease type 1A (CMT1A) is known as a demyelinating hereditary neuropathy. Secondary axonal dysfunction is the most important determinant of disease severity. In adult patients, clinical progression may be because of further axonal deterioration as was shown with comp

  11. The antagonistic effect of antipsychotic drugs on a HEK293 cell line stably expressing human alpha(1A1)-adrenoceptors

    Nourian, Zahra; Mulvany, Michael J; Nielsen, Karsten Bork;

    2008-01-01

    Antipsychotic drugs often cause orthostatic hypotension, probably through antagonist action on resistance vessel alpha(1A)-adrenoceptors. Here we have tested this possibility directly using cells transfected with a relevant human alpha(1A)-adrenoceptor splice variant. To determine a splice varian...

  12. NFKB1: a suppressor of inflammation, ageing and cancer.

    Cartwright, Tyrell; Perkins, Neil D; L Wilson, Caroline

    2016-05-01

    The pleiotropic consequences of nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) pathway activation result from the combinatorial effects of the five subunits that form the homo- and heterodimeric NF-κB complexes. Although biochemical and gene knockout studies have demonstrated overlapping and distinct functions for these proteins, much is still not known about the mechanisms determining context-dependent functions, the formation of different dimer complexes and transcriptional control in response to diverse stimuli. Here we discuss recent results that reveal that the nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (NFKB1) (p105/p50) subunit is an important regulator of NF-κB activity in vivo. These effects are not restricted to being a dimer partner for other NF-κB subunits. Rather p50 homodimers have a critical role as suppressors of the NF-κB response, while the p105 precursor has a variety of NF-κB-independent functions. The importance of Nfkb1 function can be seen in mouse models, where Nfkb1(-/-) mice display increased inflammation and susceptibility to certain forms of DNA damage, leading to cancer, and a rapid ageing phenotype. In humans, low expression of Kip1 ubiquitination-promoting complex 1 (KPC1), a ubiquitin ligase required for p105 to p50 processing, was shown to correlate with a reduction in p50 and glioblastoma incidence. Therefore, while the majority of research in this field has focused on the upstream signalling pathways leading to NF-κB activation or the function of other NF-κB subunits, such as RelA (p65), these data demonstrate a critical role for NFKB1, potentially revealing new strategies for targeting this pathway in inflammatory diseases and cancer. PMID:26663363

  13. Abiotic and biotic stress tolerance in Arabidopsis overexpressing the multiprotein bridging factor 1a (MBF1a) transcriptional coactivator gene.

    Kim, Min-Jung; Lim, Gah-Hyun; Kim, Eun-Seon; Ko, Chang-Beom; Yang, Kwang-Yeol; Jeong, Jin-An; Lee, Myung-Chul; Kim, Cheol Soo

    2007-03-01

    We conducted a genetic yeast screen to identify salt tolerance (SAT) genes in a maize kernel cDNA library. During the screening, we identified a maize clone (SAT41) that seemed to confer elevated salt tolerance in comparison to control cells. SAT41 cDNA encodes a 16-kDa protein which is 82.4% identical to the Arabidopsis Multiprotein bridging factor 1a (MBF1a) transcriptional coactivator gene. To further examine salinity tolerance in Arabidopsis, we functionally characterized the MBF1a gene and found that dehydration as well as heightened glucose (Glc) induced MBF1a expression. Constitutive expression of MBF1a in Arabidopsis led to elevated salt tolerance in transgenic lines. Interestingly, plants overexpressing MBF1a exhibited insensitivity to Glc and resistance to fungal disease. Our results suggest that MBF1a is involved in stress tolerance as well as in ethylene and Glc signaling in Arabidopsis. PMID:17234157

  14. CD1a Reactivity in Non-neoplastic Adenohypophysis.

    Pisapia, David J; Rosenblum, Marc K; Lavi, Ehud

    2016-06-01

    Within the differential diagnosis of patients presenting with sellar or suprasellar lesions is Langerhans cell histiocytosis (LCH). CD1a staining is frequently used to identify the presence of an abnormal proliferation of Langerhans cells on histologic sections and contributes to the diagnosis of LCH. Here, we report that the MTB-1 monoclonal antibody against the CD1a antigen reacts to native adenohypophyseal epithelial cells. We show that immunohistochemistry for CD1a exhibits strong positivity in all autopsy and surgically resected non-neoplastic adenohypophysis tested. Thus, CD1a positivity by itself should be interpreted with caution, and we recommend the routine use of a panel of stains including CD1a, Langerin, and synaptophysin in conjunction with morphologic analysis before a diagnosis of LCH is rendered. In addition, we find that pituitary adenomas fail to stain for CD1a prompting consideration of the utility of this stain as a marker for non-neoplastic gland. PMID:26999501

  15. Serotonin type-1A receptor imaging in depression

    Regional 5-hydroxytryptamine1A (5-HT1A) receptor binding potential (BP) of depressed subjects with primary, recurrent, familial mood disorders was compared to that of healthy controls by using positron emission tomography and [carbonyl-11C]WAY-100635 {[11C]N-(2-(4-(2-methoxyphenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide}. The mean 5-HT1A receptor BP was reduced 42% in the midbrain raphe and 25-33% in limbic and neocortical areas in the mesiotemporal, occipital, and parietal cortex. The magnitude of these abnormalities was most prominent in bipolar depressives and unipolar depressives who had bipolar relatives. These abnormal reductions in 5-HT1A receptor BP are consistent with in vivo evidence that 5-HT1A receptor sensitivity is reduced in major depressive disorder and postmortem data showing a widespread deficit of 5-HT1A receptor expression in primary mood disorders

  16. Analysis list: DYRK1A [Chip-atlas[Archive

    Full Text Available DYRK1A Neural,Uterus + hg19 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/...DYRK1A.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/DYRK1A.5.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/target/DYRK1A.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/DYRK1A.Neural....tsv,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/DYRK1A.Uterus.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/Neural.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Uterus.gml ...

  17. Analysis list: Hif1a [Chip-atlas[Archive

    Full Text Available Hif1a Blood,Embryo + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Hif1...a.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Hif1a.5.tsv http://dbarchive.biosciencedbc.jp/...kyushu-u/mm9/target/Hif1a.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Hif1a.Blood.tsv,http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/Hif1a.Embryo.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/mm9/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Embryo.gml ...

  18. Analysis list: HIF1A [Chip-atlas[Archive

    Full Text Available HIF1A Blood,Breast + hg19 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/HI...F1A.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/target/HIF1A.5.tsv http://dbarchive.biosciencedbc....jp/kyushu-u/hg19/target/HIF1A.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/HIF1A.Blood.tsv,http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/HIF1A.Breast.tsv http://dbarchive.bioscience...dbc.jp/kyushu-u/hg19/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/colo/Breast.gml ...

  19. Analysis list: Ptf1a [Chip-atlas[Archive

    Full Text Available Ptf1a Embryo,Pancreas + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Ptf1a.1.tsv http://dba...rchive.biosciencedbc.jp/kyushu-u/mm9/target/Ptf1a.5.tsv http://dbarchive.biosciencedbc....jp/kyushu-u/mm9/target/Ptf1a.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Ptf1a.Embryo.tsv,http://dba...rchive.biosciencedbc.jp/kyushu-u/mm9/colo/Ptf1a.Pancreas.tsv http://dbarch...ive.biosciencedbc.jp/kyushu-u/mm9/colo/Embryo.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Pancreas.gml ...

  20. Photodissociation of N2O: excitation of 1A" states.

    Schinke, Reinhard; Schmidt, Johan A

    2012-11-26

    We investigate the contributions of the lowest two (1)A" states in the UV photodissociation of N(2)O employing three-dimensional potential energy surfaces and transition dipole moment functions. Because the transition dipole moments are much smaller than for the 2 (1)A' state, we conclude that excitation of the (1)A" states has a marginal effect. The dense vibrational spectrum of the quasi-bound 2(1)A" state possibly explains some of the tiny, noise-like structures of the measured absorption spectrum. PMID:22536943

  1. Psychopharmacology of 5-HT{sub 1A} receptors

    Cowen, Philip J

    2000-07-01

    Serotonin{sub 1A} (5-HT{sub 1A}) receptors are located on both 5-HT cell bodies where they act as inhibitory autoreceptors and at postsynaptic sites where they mediate the effects of 5-HT released from nerve terminals. The sensitivity of 5-HT{sub 1A} receptors in humans can be measured using the technique of pharmacological challenge. For example, acute administration of a selective 5-HT{sub 1A} receptor agonist, such as ipsapirone, decreases body temperature and increases plasma cortisol through activation of pre- and postsynaptic 5-HT{sub 1A} receptors, respectively. Use of this technique has demonstrated that unmedicated patients with major depression have decreased sensitivity of both pre- and postsynaptic 5-HT{sub 1A} receptors. Treatment with selective serotonin reuptake inhibitors further down-regulates 5-HT{sub 1A} receptor activity. Due to the hypotheses linking decreased sensitivity of 5-HT{sub 1A} autoreceptors with the onset of antidepressant activity, there is current interest in the therapeutic efficacy of combined treatment with selective serotonin reuptake inhibitors and 5-HT{sub 1A} receptor antagonists.

  2. Stiffness of modified Type 1a linear external skeletal fixators.

    Reaugh, H F; Rochat, M C; Bruce, C W; Galloway, D S; Payton, M E

    2007-01-01

    Modifications of a Type 1a external skeletal fixator (ESF) frame were evaluated by alternately placing transfixation pins on opposite sides of the connecting rod (Type 1a-MOD) or by placing additional connecting rods on either of the two inside (Type 1a-INSIDE) or two outside (Type 1a-OUTSIDE) transfixation pins. The objective of this study was to evaluate the stiffness of these modifications in terms of axial compression (AC), cranial-caudal bending (CCB), and medial-lateral bending (MLB). We hypothesized that these designs would allow significant increase in unilateral frame stiffness, over Type 1a, without proportional increase in frame complexity or technical difficulty of application. All of the ESF frames were constructed using large IMEX SKtrade mark clamps, 3.2 mm threaded fixation pins, 9.5 mm carbon fibre connecting rods and Delrin rods as bone models. Nine, eight pin frames of each design were constructed, and subjected to repetitive non-destructive loading forces (AC, CCB, MLB) using a materials testing machine. Frame construct stiffness for each force (AC, CCB, MLB) was derived from load-deformation curve analysis and displayed in N/mm. Data revealed the 1a-MOD and 1a-OUTSIDE constructs had significantly increased stiffness in CCB and AC as compared to the Type 1a constructs while all of the modified constructs were significantly stiffer in MLB than the Type 1a constructs. PMID:18038001

  3. Pathway-targeted pharmacogenomics of CYP1A2 in human liver

    Kathrin eKlein

    2010-11-01

    Full Text Available The human drug metabolizing cytochrome P450 (CYP 1A2, is one of the major P450 isoforms contributing by about 5-20% to the hepatic P450 pool and catalyzing oxidative biotransformation of up to 10% of clinically relevant drugs including clozapine and caffeine. CYP1A2 activity is interindividually highly variable and although twin studies have suggested a high heritability, underlying genetic factors are still unknown. Here we adopted a pathway-oriented approach using a large human liver bank (n=150 to elucidate whether variants in candidate genes of constitutive, ligand-inducible, and pathophysiological inhibitory regulatory pathways may explain different hepatic CYP1A2 phenotypes. Samples were phenotyped for phenacetin O-deethylase activity, and the expression of CYP1A2 protein and mRNA was determined. CYP1A2 expression and function was increased in smokers and decreased in patients with inflammation and cholestasis. Of 169 SNPs in 17 candidate genes including the CYP1A locus, 136 non-redundant SNPs with minor allele frequency >5% were analyzed by univariate and multivariate methods. A total of 13 strong significant associations were identified, of which 10 SNPs in the ARNT, AhRR, HNF1α, IL1β, SRC-1, and VDR genes showed consistent changes for at least two phenotypes by univariate analysis. Multivariate linear modelling indicated that the polymorphisms and non-genetic factors together explained 42%, 38% and 33% of CYP1A2 variation at activity, protein and mRNA levels, respectively. In conclusion, we identified novel trans-associations between regulatory genes and hepatic CYP1A2 function and expression, but additional genetic factors must be assumed to explain the full extent of CYP1A2 heritability.

  4. Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging.

    Carnally, Stewart M; Dev, Harveer S; Stewart, Andrew P; Barrera, Nelson P; Van Bemmelen, Miguel X; Schild, Laurent; Henderson, Robert M; Edwardson, J Michael

    2008-08-01

    There has been confusion about the subunit stoichiometry of the degenerin family of ion channels. Recently, a crystal structure of acid-sensing ion channel (ASIC) 1a revealed that it assembles as a trimer. Here, we used atomic force microscopy (AFM) to image unprocessed ASIC1a bound to mica. We detected a mixture of subunit monomers, dimers and trimers. In some cases, triple-subunit clusters were clearly visible, confirming the trimeric structure of the channel, and indicating that the trimer sometimes disaggregated after adhesion to the mica surface. This AFM-based technique will now enable us to determine the subunit arrangement within heteromeric ASICs. PMID:18514062

  5. Direct visualization of the trimeric structure of the ASIC1a channel, using AFM imaging

    There has been confusion about the subunit stoichiometry of the degenerin family of ion channels. Recently, a crystal structure of acid-sensing ion channel (ASIC) 1a revealed that it assembles as a trimer. Here, we used atomic force microscopy (AFM) to image unprocessed ASIC1a bound to mica. We detected a mixture of subunit monomers, dimers and trimers. In some cases, triple-subunit clusters were clearly visible, confirming the trimeric structure of the channel, and indicating that the trimer sometimes disaggregated after adhesion to the mica surface. This AFM-based technique will now enable us to determine the subunit arrangement within heteromeric ASICs

  6. Carnitine palmitoyltransferase 1A (CPT1A): a transcriptional target of PAX3-FKHR and mediates PAX3-FKHR–dependent motility in alveolar rhabdomyosarcoma cells

    2012-01-01

    Background Alveolar rhabdomyosarcoma (ARMS) has a high propensity to metastasize, leading to its aggressiveness and a poor survival rate among those with the disease. More than 80% of aggressive ARMSs harbor a PAX3-FKHR fusion transcription factor, which regulates cell migration and promotes metastasis, most likely by regulating the fusion protein’s transcriptional targets. Therefore, identifying druggable transcription targets of PAX3-FKHR that are also downstream effectors of PAX3-FKHR–mediated cell migration and metastasis may lead to novel therapeutic approaches for treating ARMS. Methods To identify genes whose expression is directly affected by the level of PAX3-FKHR in an ARMS cellular-context, we first developed an ARMS cell line in which PAX3-FKHR is stably down-regulated, and showed that stably downregulating PAX3-FKHR in ARMS cells significantly decreased the cells’ motility. We used microarray analysis to identify genes whose expression level decreased when PAX3-FKHR was downregulated. We used mutational analysis, promoter reporter assays, and electrophoretic mobility shift assays to determine whether PAX3-FKHR binds to the promoter region of the target gene. We used siRNA and pharmacologic inhibitor to downregulate the target gene of PAX3-FKHR and investigated the effect of such downregulation on cell motility. Results We found that when PAX3-FKHR was downregulated, the expression of carnitine palmitoyltransferase 1A (CPT1A) decreased. We showed that PAX3-FKHR binds to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel transcriptional target of PAX3-FKHR. Furthermore, downregulating CPT1A decreased cell motility in ARMS cells, indicating that CPT1A is a downstream effector of PAX3-FKHR–mediated cell migration and metastasis. Conclusions Taken together, we have identified CPT1A as a novel transcriptional target of PAX3-FKHR and revealed the novel function of CPT1A in promoting cell motility. CPT1A may

  7. Carnitine palmitoyltransferase 1A (CPT1A: a transcriptional target of PAX3-FKHR and mediates PAX3-FKHR–dependent motility in alveolar rhabdomyosarcoma cells

    Liu Lingling

    2012-04-01

    Full Text Available Abstract Background Alveolar rhabdomyosarcoma (ARMS has a high propensity to metastasize, leading to its aggressiveness and a poor survival rate among those with the disease. More than 80% of aggressive ARMSs harbor a PAX3-FKHR fusion transcription factor, which regulates cell migration and promotes metastasis, most likely by regulating the fusion protein’s transcriptional targets. Therefore, identifying druggable transcription targets of PAX3-FKHR that are also downstream effectors of PAX3-FKHR–mediated cell migration and metastasis may lead to novel therapeutic approaches for treating ARMS. Methods To identify genes whose expression is directly affected by the level of PAX3-FKHR in an ARMS cellular-context, we first developed an ARMS cell line in which PAX3-FKHR is stably down-regulated, and showed that stably downregulating PAX3-FKHR in ARMS cells significantly decreased the cells’ motility. We used microarray analysis to identify genes whose expression level decreased when PAX3-FKHR was downregulated. We used mutational analysis, promoter reporter assays, and electrophoretic mobility shift assays to determine whether PAX3-FKHR binds to the promoter region of the target gene. We used siRNA and pharmacologic inhibitor to downregulate the target gene of PAX3-FKHR and investigated the effect of such downregulation on cell motility. Results We found that when PAX3-FKHR was downregulated, the expression of carnitine palmitoyltransferase 1A (CPT1A decreased. We showed that PAX3-FKHR binds to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel transcriptional target of PAX3-FKHR. Furthermore, downregulating CPT1A decreased cell motility in ARMS cells, indicating that CPT1A is a downstream effector of PAX3-FKHR–mediated cell migration and metastasis. Conclusions Taken together, we have identified CPT1A as a novel transcriptional target of PAX3-FKHR and revealed the novel function of CPT1A in promoting

  8. Carnitine palmitoyltransferase 1A (CPT1A): a transcriptional target of PAX3-FKHR and mediates PAX3-FKHR–dependent motility in alveolar rhabdomyosarcoma cells

    Alveolar rhabdomyosarcoma (ARMS) has a high propensity to metastasize, leading to its aggressiveness and a poor survival rate among those with the disease. More than 80% of aggressive ARMSs harbor a PAX3-FKHR fusion transcription factor, which regulates cell migration and promotes metastasis, most likely by regulating the fusion protein’s transcriptional targets. Therefore, identifying druggable transcription targets of PAX3-FKHR that are also downstream effectors of PAX3-FKHR–mediated cell migration and metastasis may lead to novel therapeutic approaches for treating ARMS. To identify genes whose expression is directly affected by the level of PAX3-FKHR in an ARMS cellular-context, we first developed an ARMS cell line in which PAX3-FKHR is stably down-regulated, and showed that stably downregulating PAX3-FKHR in ARMS cells significantly decreased the cells’ motility. We used microarray analysis to identify genes whose expression level decreased when PAX3-FKHR was downregulated. We used mutational analysis, promoter reporter assays, and electrophoretic mobility shift assays to determine whether PAX3-FKHR binds to the promoter region of the target gene. We used siRNA and pharmacologic inhibitor to downregulate the target gene of PAX3-FKHR and investigated the effect of such downregulation on cell motility. We found that when PAX3-FKHR was downregulated, the expression of carnitine palmitoyltransferase 1A (CPT1A) decreased. We showed that PAX3-FKHR binds to a paired-domain binding-site in the CPT1A promoter region, indicating that CPT1A is a novel transcriptional target of PAX3-FKHR. Furthermore, downregulating CPT1A decreased cell motility in ARMS cells, indicating that CPT1A is a downstream effector of PAX3-FKHR–mediated cell migration and metastasis. Taken together, we have identified CPT1A as a novel transcriptional target of PAX3-FKHR and revealed the novel function of CPT1A in promoting cell motility. CPT1A may represent a novel therapeutic target for

  9. Main: 1A0K [RPSD[Archive

    Full Text Available 1A0K シロイヌナズナ Arabidopsis Arabidopsis thaliana (L.) Heynh. Profilin 1 Name=Pro1; Synonyms= ... 580; G84580.|PDB; 1A0K; X-ray; @=1-131.|PDB; 1PLM; Model ; A=2-131.|PDB; 3NUL; X-ray; @=2-131.|InterPro; IPR ...

  10. 41 CFR 50-204.1a - Variances.

    2010-07-01

    ... 41 Public Contracts and Property Management 1 2010-07-01 2010-07-01 true Variances. 50-204.1a... and Application § 50-204.1a Variances. (a) Variances from standards in this part may be granted in the same circumstances in which variances may be granted under sections 6(b)(6)(A) or 6(d) of the...

  11. Paresev 1-A and Tow Plane with Crew and Pilot

    1962-01-01

    With the Paresev 1-A and the 450-hp Stearman Sport Biplane as a backdrop the pilot and crew pose for this picture in 1962. Starting at left: On the motorcycle is Walter Whiteside, in the Paresev 1-A is test pilot Milton Thompson, Frank Fedor, Richard Klein, Victor Horton, Tom Kelly, Jr., Fred Harris, owner of the Stearman, John Orahood, and Gary Layton.

  12. Mitochondrial Carnitine Palmitoyltransferase 1a (CPT1a) Is Part of an Outer Membrane Fatty Acid Transfer Complex*

    Lee, Kwangwon; Kerner, Janos; Hoppel, Charles L.

    2011-01-01

    CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immu...

  13. Direct determination of quasar redshifts

    De Bruijne, J H J; Perryman, M; Peacock, A; Favata, F; Rando, N; Martin, D; Verhoeve, P; Christlieb, N; Bruijne, Jos de; Reynolds, Alastair P; Perryman, Michael; Peacock, Anthony; Favata, Fabio; Rando, Nicola; Martin, Didier; Verhoeve, Peter; Christlieb, Norbert

    2001-01-01

    We present observations of 11 quasars, selected in the range z = 2.2-4.1, obtained with ESA's Superconducting Tunnel Junction (STJ) camera on the WHT. Using a single template quasar spectrum, we show that we can determine the redshifts of these objects to about 1%. A follow-up spectroscopic observation of one QSO for which our best-fit redshift (z = 2.976) differs significantly from the tentative literature value (z ~ 2.30) confirms that the latter was incorrect.

  14. Variation in Hsp70-1A Expression Contributes to Skin Color Diversity.

    Murase, Daiki; Hachiya, Akira; Fullenkamp, Rachel; Beck, Anita; Moriwaki, Shigeru; Hase, Tadashi; Takema, Yoshinori; Manga, Prashiela

    2016-08-01

    The wide range in human skin color results from varying levels of the pigment melanin. Genetic mechanisms underlying coloration differences have been explored, but identified genes do not account for all variation seen in the skin color spectrum. Post-transcriptional and post-translational regulation of factors that determine skin color, including melanin synthesis in epidermal melanocytes, melanosome transfer to keratinocytes, and melanosome degradation, is also critical for pigmentation. We therefore investigated proteins that are differentially expressed in melanocytes derived from either white or African American skin. Two-dimensional gel electrophoresis and mass spectrometry demonstrated that heat shock protein 70-1A (Hsp70-1A) protein levels were significantly higher in African American melanocytes compared with white melanocytes. Hsp70-1A expression significantly correlated with levels of tyrosinase, the rate-limiting melanogenic enzyme, consistent with a proposed role for Hsp70 family members in tyrosinase post-translational modification. In addition, pharmacologic inhibition and small interfering RNA-mediated downregulation of Hsp70-1A correlated with pigmentation changes in cultured melanocytes, modified human skin substitutes, and ex vivo skin. Furthermore, Hsp70-1A inhibition led to increased autophagy-mediated melanosome degradation in keratinocytes. Our data thus reveal that epidermal Hsp70-1A contributes to the diversity of skin color by regulating the amount of melanin synthesized in melanocytes and modulating autophagic melanosome degradation in keratinocytes. PMID:27094592

  15. Epigeneitc silencing of ribosomal RNA genes by Mybbp1a

    Tan Bertrand

    2012-06-01

    Full Text Available Abstract Background Transcription of the ribosomal RNA gene repeats by Pol I occurs in the nucleolus and is a fundamental step in ribosome biogenesis and protein translation. Due to tight coordination between ribosome biogenesis and cell proliferation, transcription of rRNA and stable maintenance of rDNA clusters are thought to be under intricate control by intercalated mechanisms, particularly at the epigenetic level. Methods and Results Here we identify the nucleolar protein Myb-binding protein 1a (Mybbp1a as a novel negative regulator of rRNA expression. Suppression of rDNA transcription by Mybbp1a was linked to promoter regulation as illustrated by its binding to the chromatin around the hypermethylated, inactive rDNA gene promoters. Our data further showed that downregulation of Mybbp1a abrogated the local DNA methylation levels and histone marks associated with gene silencing, and altered the promoter occupancy of various factors such UBF and HDACs, consequently leading to elevated rRNA expression. Mechanistically, we propose that Mybbp1a maintains rDNA repeats in a silenced state while in association with the negative epigenetic modifiers HDAC1/2. Conclusions Results from our present work reveal a previously unrecognized co-repressor role of Mybbp1a in rRNA expression. They are further consistent with the scenario that Mybbp1a is an integral constituent of the rDNA epigenetic regulation that underlies the balanced state of rDNA clusters.

  16. Mitochondrial Carnitine Palmitoyltransferase 1a (CPT1a) Is Part of an Outer Membrane Fatty Acid Transfer Complex*

    Lee, Kwangwon; Kerner, Janos; Hoppel, Charles L.

    2011-01-01

    CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM. PMID:21622568

  17. Mitochondrial carnitine palmitoyltransferase 1a (CPT1a) is part of an outer membrane fatty acid transfer complex.

    Lee, Kwangwon; Kerner, Janos; Hoppel, Charles L

    2011-07-22

    CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM. PMID:21622568

  18. The Alpha-1A Adrenergic Receptor in the Rabbit Heart

    R Croft Thomas; Cowley, Patrick M.; Abhishek Singh; Bat-Erdene Myagmar; Swigart, Philip M.; Baker, Anthony J.; Simpson, Paul C.

    2016-01-01

    The alpha-1A-adrenergic receptor (AR) subtype is associated with cardioprotective signaling in the mouse and human heart. The rabbit is useful for cardiac disease modeling, but data on the alpha-1A in the rabbit heart are limited. Our objective was to test for expression and function of the alpha-1A in rabbit heart. By quantitative real-time reverse transcription PCR (qPCR) on mRNA from ventricular myocardium of adult male New Zealand White rabbits, the alpha-1B was 99% of total alpha-1-AR mR...

  19. Expression and localization of Aquaporin 1a in the sea-bass (Dicentrarchus labrax during ontogeny

    IvoneGiffard-Mena

    2011-07-01

    Full Text Available The successful establishment of a species in a given habitat depends on the ability of each of its developing stages to adapt to the environment. In order to understand this process we have studied the adaptation of a euryhaline fish, the sea-bass Dicentrarchus labrax, to various salinities during its ontogeny. The expression and localization of Aquaporin 1a (AQP1a mRNA and protein were determined in different osmoregulatory tissues. In larvae, the sites of AQP1a expression are variable and they shift according to age, implying functional changes. In juveniles after metamorphosis (D32-48 post hatch, 15 - 25 mm and in pre-adults, an increase in AQP1a transcript abundance was noted in the digestive tract, and the AQP1a location was observed in the intestine. In juveniles (D87-100 post hatch, 38 - 48 mm, the transcript levels of AQP1a in the digestive tract and in the kidney were higher in sea water than at lower salinity. These observations, in agreement with existing models, suggest that in sea water-acclimated fish, the imbibed water is absorbed via AQP1a through the digestive tract, particularly the intestine and the rectum. In addition, AQP1a may play a role in water reabsorption in the kidney. These mechanisms compensate dehydratation in sea water, and they contribute to the adaptation of juveniles to salinity changes during sea-lagoon migrations. These results contribute to the interpretation of the adaptation of populations to habitats where salinity varies.

  20. Production of 4He and tritium from Be in the COBRA-1A2 irradiation

    The production of 4He and tritium has been calculated for beryllium irradiated in the COBRA-1A2 experiment in the Experimental Breeder Reactor II. Reaction rates were based on adjusted neutron spectra determined from reactor dosimetry measurements at three different elevations in the region of the beryllium capsules. Equations are given so that gas production can be calculated for any specific capsule elevation

  1. Cytochrome P450 1A1 expression in cetacean skin biopsies from the Indian Ocean

    Jauniaux, Thierry; Farnir, Frédéric; Fontaine, Michael; Kiszka, Jeremy; Sarlet, Michaël; Coignoul, Freddy

    2011-01-01

    The study describes cytochrome P450 1A1 (CYPA1) expression in the skin of different cetacean species (Megaptera novaeangliae, n = 15; Stenella attenuata, n = 7 and Stenella longirostris, n = 24) from the Mozambique Channel island of Mayotte. Immunohistochemical examination was performed with a monoclonal antibody against scup cytochrome CYPA1. The sex was determined using a molecular approach consisting in the genotyping sex-specific genes. CYPA1 was detected at the junction between epidermis...

  2. Production of {sup 4}He and tritium from Be in the COBRA-1A2 irradiation

    Greenwood, L.R. [Pacific Northwest National Lab., Richland, WA (United States)

    1998-03-01

    The production of {sup 4}He and tritium has been calculated for beryllium irradiated in the COBRA-1A2 experiment in the Experimental Breeder Reactor II. Reaction rates were based on adjusted neutron spectra determined from reactor dosimetry measurements at three different elevations in the region of the beryllium capsules. Equations are given so that gas production can be calculated for any specific capsule elevation.

  3. Functional Analysis of In-frame Indel ARID1A Mutations Reveals New Regulatory Mechanisms of Its Tumor Suppressor Functions

    Bin Guan

    2012-10-01

    Full Text Available AT-rich interactive domain 1A (ARID1A has emerged as a new tumor suppressor in which frequent somatic mutations have been identified in several types of human cancers. Although most ARID1A somatic mutations are frame-shift or nonsense mutations that contribute to mRNA decay and loss of protein expression, 5% of ARID1A mutations are in-frame insertions or deletions (indels that involve only a small stretch of peptides. Naturally occurring in-frame indel mutations provide unique and useful models to explore the biology and regulatory role of ARID1A. In this study, we analyzed indel mutations identified in gynecological cancers to determine how these mutations affect the tumor suppressor function of ARID1A. Our results demonstrate that all in-frame mutants analyzed lost their ability to inhibit cellular proliferation or activate transcription of CDKN1A, which encodes p21, a downstream effector of ARID1A. We also showed that ARID1A is a nucleocytoplasmic protein whose stability depends on its subcellular localization. Nuclear ARID1A is less stable than cytoplasmic ARID1A because ARID1A is rapidly degraded by the ubiquitin-proteasome system in the nucleus. In-frame deletions affecting the consensus nuclear export signal reduce steady-state protein levels of ARID1A. This defect in nuclear exportation leads to nuclear retention and subsequent degradation. Our findings delineate a mechanism underlying the regulation of ARID1A subcellular distribution and protein stability and suggest that targeting the nuclear ubiquitin-proteasome system can increase the amount of the ARID1A protein in the nucleus and restore its tumor suppressor functions.

  4. MISR Level 1A Navigation Data V002

    National Aeronautics and Space Administration — This is the Reformatted Annotated Level 1A Product for the Navigation Data, which contains samples of the EOS-AM1 Platform position and attitude data. (Suggested...

  5. MISR Level 1A Calibration Data V002

    National Aeronautics and Space Administration — Level 1A Calibration data in DN.The data numbers have been commuted from 12-bit numbers to 16-bit,byte aligned half-words. (Suggested Usage: MISR SCF processing...

  6. Paresev 1-A on Lakebed with Tow Plane

    1962-01-01

    The Paresev 1-A (Paraglider Research Vehicle) and the tow airplane, 450-hp Stearman Sport Biplane sitting on Rogers dry lakebed, Edwards, California. The control system in the Paresev 1-A had a more conventional control stick position and was cable-operated. The main landing gear used shocks and bungees with the 150-square-foot wing membrane being made of 6-ounce unsealed Dacron.

  7. Musical Aptitude Is Associated with AVPR1A-Haplotypes

    Ukkola, Liisa T.; Onkamo, Päivi; Raijas, Pirre; Karma, Kai; Järvelä, Irma

    2009-01-01

    Artistic creativity forms the basis of music culture and music industry. Composing, improvising and arranging music are complex creative functions of the human brain, which biological value remains unknown. We hypothesized that practicing music is social communication that needs musical aptitude and even creativity in music. In order to understand the neurobiological basis of music in human evolution and communication we analyzed polymorphisms of the arginine vasopressin receptor 1A (AVPR1A),...

  8. Translation elongation factor-1A1 (eEF1A1 localizes to the spine by domain III

    Sun-Jung Cho

    2012-04-01

    Full Text Available In vertebrates, there are two variants of eukaryotic peptideelongation factor 1A (eEF1A; formerly eEF-1α, eEF1A1 andeEF1A2, which have three well-conserved domains (DI, DII,and DIII. In neurons, eEF1A1 is the embryonic type, which isexpressed during embryonic development as well as the firsttwo postnatal weeks. In the present study, EGFP-tagged eEF1A1truncates were expressed in cortical neurons isolated from ratembryo (E18-19. Live cell images of transfected neurons showedthat DIII-containing EGFP-fusion proteins (EGFP-DIII, -DII-III,-DI-III formed clusters that were confined within somatodendriticdomains, while DIII-missing ones (EGFP-DI, -DII, -DI-II andcontrol EGFP were homogeneously dispersed throughout theneuron including axons. In dendrites, EGFP-DIII was targeted tothe heads of spine- and filopodia-like protrusions, where it wascolocalized with SynGAPα, a postsynaptic marker. Our dataindicate that DIII of eEF1A1 mediates formation of clusters andlocalization to spines. [BMB reports 2012; 45(4: 227-232

  9. TAR-1 A programme for the determination of time behaviour of neutron density on a thermal reactor

    This programme, written for the UNIVAC-UCT of J.E.N., obtain the time behaviour of neutron density as a function of both positive and negative step change in reactivity. These results are obtained from solutions of the space-independent kinetic equations of a bare thermal reactor based on the Fermi continuous slowing down model and using six groups of delayed neutrons. (Author) 3 refs

  10. HPLC Determination of Caffeine and Paraxanthine in Urine: An Assay for Cytochrome P450 1A2 Activity

    Furge, Laura Lowe; Fletke, Kyle J.

    2007-01-01

    Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among…

  11. ZmHO-1, a maize haem oxygenase-1 gene, plays a role in determining lateral root development.

    Han, Bin; Xu, Sheng; Xie, Yan-Jie; Huang, Jing-Jing; Wang, Li-Juan; Yang, Zheng; Zhang, Chang-He; Sun, Ya; Shen, Wen-Biao; Xie, Gui-Shui

    2012-03-01

    Previous results revealed that haem oxygenase-1 (HO-1)/carbon monoxide (CO) system is involved in auxin-induced adventitious root formation. In this report, a cDNA for the gene ZmHO-1, encoding an HO-1 protein, was cloned from Zea mays seedlings. ZmHO-1 has a conserved HO signature sequence and shares highest homology with rice SE5 (OsHO-1) protein. We further discovered that N-1-naphthylacetic acid (NAA), haemin, and CO aqueous solution, led to the induction of ZmHO-1 expression as well as the thereafter promotion of lateral root development. These effects were specific for ZmHO-1 since the potent HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) differentially blocked the above actions. The addition of haemin and CO were able to reverse the auxin depletion-triggered inhibition of lateral root formation as well as the decreased ZmHO-1 transcripts. Molecular evidence showed that the haemin- or CO-mediated the modulation of target genes responsible for lateral root formation, including ZmCDK and ZmCKI2, could be blocked by ZnPPIX. Overexpression of ZmHO-1 in transgenic Arabidopsis plants resulted in promotion of lateral root development as well as the modulation of cell cycle regulatory gene expressions. Overall, our results suggested that a maize HO-1 gene is required for the lateral root formation. PMID:22284711

  12. Serotonin type-1A receptor imaging in depression

    Drevets, Wayne C. E-mail: drevets@pet.upmc.edu; Frank, Ellen; Price, Julie C.; Kupfer, David J.; Greer, Phil J.; Mathis, Chester

    2000-07-01

    Regional 5-hydroxytryptamine{sub 1A} (5-HT{sub 1A}) receptor binding potential (BP) of depressed subjects with primary, recurrent, familial mood disorders was compared to that of healthy controls by using positron emission tomography and [carbonyl-{sup 11}C]WAY-100635 {l_brace}[{sup 11}C]N-(2-(4-(2-methoxyphenyl)-1-piperazin-1-yl)ethyl)-N-(2-pyridyl) cyclohexanecarboxamide{r_brace}. The mean 5-HT{sub 1A} receptor BP was reduced 42% in the midbrain raphe and 25-33% in limbic and neocortical areas in the mesiotemporal, occipital, and parietal cortex. The magnitude of these abnormalities was most prominent in bipolar depressives and unipolar depressives who had bipolar relatives. These abnormal reductions in 5-HT{sub 1A} receptor BP are consistent with in vivo evidence that 5-HT{sub 1A} receptor sensitivity is reduced in major depressive disorder and postmortem data showing a widespread deficit of 5-HT{sub 1A} receptor expression in primary mood disorders.

  13. The First Chinese Ocean Color Satellite HY-1A

    PAN Delu; HE Xianqiang

    2004-01-01

    HY-1A is China's first ocean color satellite, and was launched on 15 May 2002as a piggyback satellite on the FY-1D satellite using the Long March rocket.There are two sensors on the satellite: China's Ocean Color and Temperature Scanner (COCTS), and the CCD Coastal Zone Imager (CZI). In the report,first, the properties and characteristics of HY-1A are briefly introduced; Second, the quality and availability of the data are evaluated by the mean of the Complex Signal Noise Ratio (CSNR) which is simulated theoretically; Third,the received HY-1A data are compared with SeaSTAR data to understand the accuracy of radiance measurement by the HY-1A; Finally, the remote sensing products of ocean color and temperature are mapped by HY-1A to study its application potentiality. The results show that the HY-1A has a latent capability for the application of marine environment detection, the management and protection of marine resources, and the furthering of national rights and interests.Meanwhile, some modifications are proposed for the next ocean satellite, suchas adding the case of tilt scanning, modification of the CCD element uniformity,and adding more ocean satellite receiving stations.

  14. Drosophila PIWI associates with chromatin and interacts directly with HP1a.

    Brower-Toland, Brent; Findley, Seth D; Jiang, Ling; Liu, Li; Yin, Hang; Dus, Monica; Zhou, Pei; Elgin, Sarah C R; Lin, Haifan

    2007-09-15

    The interface between cellular systems involving small noncoding RNAs and epigenetic change remains largely unexplored in metazoans. RNA-induced silencing systems have the potential to target particular regions of the genome for epigenetic change by locating specific sequences and recruiting chromatin modifiers. Noting that several genes encoding RNA silencing components have been implicated in epigenetic regulation in Drosophila, we sought a direct link between the RNA silencing system and heterochromatin components. Here we show that PIWI, an ARGONAUTE/PIWI protein family member that binds to Piwi-interacting RNAs (piRNAs), strongly and specifically interacts with heterochromatin protein 1a (HP1a), a central player in heterochromatic gene silencing. The HP1a dimer binds a PxVxL-type motif in the N-terminal domain of PIWI. This motif is required in fruit flies for normal silencing of transgenes embedded in heterochromatin. We also demonstrate that PIWI, like HP1a, is itself a chromatin-associated protein whose distribution in polytene chromosomes overlaps with HP1a and appears to be RNA dependent. These findings implicate a direct interaction between the PIWI-mediated small RNA mechanism and heterochromatin-forming pathways in determining the epigenetic state of the fly genome. PMID:17875665

  15. The molecular basis for the broad substrate specificity of human sulfotransferase 1A1.

    Ilana Berger

    Full Text Available Cytosolic sulfotransferases (SULTs are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.

  16. Porcine EEF1A1 and EEF1A2 genes: genomic structure, polymorphism, mapping and expression

    Svobodová, K.; Horák, Pavel; Stratil, Antonín; Bartenschlager, H.; Van Poucke, M.; Chalupová, P.; Dvořáková, Věra; Knorr, Ch.; Stupka, R.; Čítek, J.; Šprysl, M.; Palánová, Anna; Peelman, L. J.; Geldermann, H.; Knoll, A.

    2015-01-01

    Roč. 42, č. 8 (2015), s. 1257-1264. ISSN 0301-4851 R&D Projects: GA ČR(CZ) GA523/06/1302; GA ČR GA523/09/0844 Institutional support: RVO:67985904 Keywords : EEF1A1 * EEF1A2 * gene expression Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.024, year: 2014

  17. Disruption of thyroid hormone homeostasis in Ugt1a-deficient Gunn rats by microsomal enzyme inducers is not due to enhanced thyroxine glucuronidation☆

    Richardson, Terrilyn A.; Klaassen, Curtis D.

    2010-01-01

    Microsomal enzyme inducers (MEI) that increase UDP-glucuronosyltransferases (UGTs) are thought to increase glucuronidation of thyroxine (T4), thus reducing serum T4, and subsequently increasing thyroid stimulating hormone (TSH). Ugt1a1 and Ugt1a6 mediate T4 glucuronidation. Therefore, this experiment determined the involvement of Ugt1a enzymes in increased T4 glucuronidation, decreased serum T4, and increased TSH after MEI treatment. Male Wistar and Ugt1a-deficient Wistar (Gunn) rats were fed...

  18. The Alpha-1A Adrenergic Receptor in the Rabbit Heart.

    R Croft Thomas

    Full Text Available The alpha-1A-adrenergic receptor (AR subtype is associated with cardioprotective signaling in the mouse and human heart. The rabbit is useful for cardiac disease modeling, but data on the alpha-1A in the rabbit heart are limited. Our objective was to test for expression and function of the alpha-1A in rabbit heart. By quantitative real-time reverse transcription PCR (qPCR on mRNA from ventricular myocardium of adult male New Zealand White rabbits, the alpha-1B was 99% of total alpha-1-AR mRNA, with <1% alpha-1A and alpha-1D, whereas alpha-1A mRNA was over 50% of total in brain and liver. Saturation radioligand binding identified ~4 fmol total alpha-1-ARs per mg myocardial protein, with 17% alpha-1A by competition with the selective antagonist 5-methylurapidil. The alpha-1D was not detected by competition with BMY-7378, indicating that 83% of alpha-1-ARs were alpha-1B. In isolated left ventricle and right ventricle, the selective alpha-1A agonist A61603 stimulated a negative inotropic effect, versus a positive inotropic effect with the nonselective alpha-1-agonist phenylephrine and the beta-agonist isoproterenol. Blood pressure assay in conscious rabbits using an indwelling aortic telemeter showed that A61603 by bolus intravenous dosing increased mean arterial pressure by 20 mm Hg at 0.14 μg/kg, 10-fold lower than norepinephrine, and chronic A61603 infusion by iPRECIO programmable micro Infusion pump did not increase BP at 22 μg/kg/d. A myocardial slice model useful in human myocardium and an anthracycline cardiotoxicity model useful in mouse were both problematic in rabbit. We conclude that alpha-1A mRNA is very low in rabbit heart, but the receptor is present by binding and mediates a negative inotropic response. Expression and function of the alpha-1A in rabbit heart differ from mouse and human, but the vasopressor response is similar to mouse.

  19. The Alpha-1A Adrenergic Receptor in the Rabbit Heart

    Myagmar, Bat-Erdene; Swigart, Philip M.; Baker, Anthony J.; Simpson, Paul C.

    2016-01-01

    The alpha-1A-adrenergic receptor (AR) subtype is associated with cardioprotective signaling in the mouse and human heart. The rabbit is useful for cardiac disease modeling, but data on the alpha-1A in the rabbit heart are limited. Our objective was to test for expression and function of the alpha-1A in rabbit heart. By quantitative real-time reverse transcription PCR (qPCR) on mRNA from ventricular myocardium of adult male New Zealand White rabbits, the alpha-1B was 99% of total alpha-1-AR mRNA, with micro Infusion pump did not increase BP at 22 μg/kg/d. A myocardial slice model useful in human myocardium and an anthracycline cardiotoxicity model useful in mouse were both problematic in rabbit. We conclude that alpha-1A mRNA is very low in rabbit heart, but the receptor is present by binding and mediates a negative inotropic response. Expression and function of the alpha-1A in rabbit heart differ from mouse and human, but the vasopressor response is similar to mouse. PMID:27258143

  20. Expansion of B-1a cells with germline heavy chain sequence in lupus mice

    Nichol E Holodick

    2016-03-01

    Full Text Available B6.Sle1.Sle2.Sle3 (B6.TC lupus-prone mice carrying the NZB allele of Cdkn2c, encoding for the cyclin-dependent kinase inhibitor P18INK4, accumulate B-1a cells due to a higher rate of proliferative self-renewal. However, it is unclear whether this affects primarily early appearing B-1a cells of fetal origin or later appearing B-1a cells that emerge from bone marrow. B-1a cells are the major source of natural autoantibodies, and it has been shown that their protective nature is associated with a germline-like sequence, which is characterized by few N-nucleotide insertions and a repertoire skewed towards rearrangements predominated during fetal life, VH11 and VH12. To determine the nature of B-1a cells expanded in B6.TC mice, we amplified immunoglobulin genes by PCR from single cells in mice. Sequencing showed a significantly higher proportion of B-1a cell antibodies display fewer N-additions in B6.TC mice than in B6 control mice. Following this lower number of N-insertions within the CDR-H3 region, the B6.TC B-1a cells display shorter CDR-H3 length than B6 B-1a cells. The absence of N-additions is a surrogate for fetal origin, as TdT expression starts after birth in mice. Therefore, our results suggest that the B-1a cell population is not only expanded in autoimmune B6.TC mice but also qualitatively different with the majority of cells from fetal origin. Accordingly, our sequencing results also demonstrated overuse of VH11 and VH12 in autoimmune B6.TC mice as compared to B6 controls. These results suggest that the development of lupus autoantibodies in these mice is coupled with skewing of the B-1a cell repertoire and possible retention of protective natural antibodies.