N-glycosylation of colorectal cancer tissues: a liquid chromatography and mass spectrometry-based investigation.

Science.gov (United States)

Balog, Crina I A; Stavenhagen, Kathrin; Fung, Wesley L J; Koeleman, Carolien A; McDonnell, Liam A; Verhoeven, Aswin; Mesker, Wilma E; Tollenaar, Rob A E M; Deelder, André M; Wuhrer, Manfred

2012-09-01

Colorectal cancer is the third most common cancer worldwide with an annual incidence of ~1 million cases and an annual mortality rate of ~655,000 individuals. There is an urgent need for identifying novel targets to develop more sensitive, reliable, and specific tests for early stage detection of colon cancer. Post-translational modifications are known to play an important role in cancer progression and immune surveillance of tumors. In the present study, we compared the N-glycan profiles from 13 colorectal cancer tumor tissues and corresponding control colon tissues. The N-glycans were enzymatically released, purified, and labeled with 2-aminobenzoic acid. Aliquots were profiled by hydrophilic interaction liquid chromatography (HILIC-HPLC) with fluorescence detection and by negative mode MALDI-TOF-MS. Using partial least squares discriminant analysis to investigate the N-glycosylation changes in colorectal cancer, an excellent separation and prediction ability were observed for both HILIC-HPLC and MALDI-TOF-MS data. For structure elucidation, information from positive mode ESI-ion trap-MS/MS and negative mode MALDI-TOF/TOF-MS was combined. Among the features with a high separation power, structures containing a bisecting GlcNAc were found to be decreased in the tumor, whereas sulfated glycans, paucimannosidic glycans, and glycans containing a sialylated Lewis type epitope were shown to be increased in tumor tissues. In addition, core-fucosylated high mannose N-glycans were detected in tumor samples. In conclusion, the combination of HILIC and MALDI-TOF-MS profiling of N-glycans with multivariate statistical analysis demonstrated its potential for identifying N-glycosylation changes in colorectal cancer tissues and provided new leads that might be used as candidate biomarkers.

Characterization of kallikrein-related peptidase 4 glycosylations.

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Yamakoshi, Yasuo; Yamakoshi, Fumiko; Hu, Jan C-C; Simmer, James P

2011-12-01

Kallikrein-related peptidase 4 (KLK4) is a glycosylated serine protease that functions in the maturation (hardening) of dental enamel. Pig and mouse KLK4 contain three potential N-glycosylation sites. We isolated KLK4 from developing pig and mouse molars and characterized their N-glycosylations. N-glycans were enzymatically released by digestion with N-glycosidase F and fluorescently labeled with 2-aminobenzoic acid. Normal-phase high-performance liquid chromatography (NP-HPLC) revealed N-glycans with no, or with one, two, or three sialic acid attachments in pig KLK4 and with no, or with one or two sialic acid attachments in mouse KLK4. The labeled N-glycans were digested with sialidase to generate the asialo N-glycan cores that were fractionated by reverse-phase HPLC, and their retention times were compared with similarly labeled glycan standards. The purified cores were characterized by mass spectrometric and monosaccharide composition analyses. We determined that pig and mouse KLK4 have NA2 and NA2F biantennary N-glycan cores. The pig triantennary core is NA3. The mouse triantennary core is NA3 with a fucose connected by an α1-6 linkage, indicating that it is attached to the first N-acetyglucosamine (NA3F). We conclude that pig KLK4 has NA2, NA2F, and NA3 N-glycan cores with no, or with one, two, or three sialic acids. Mouse KLK4 has NA2, NA2F, and NA3F N-glycan cores with no, or with one or two sialic acids. © 2011 Eur J Oral Sci.

Integrated proteomic and N-glycoproteomic analyses of doxorubicin sensitive and resistant ovarian cancer cells reveal glycoprotein alteration in protein abundance and glycosylation

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Hou, Junjie; Zhang, Chengqian; Xue, Peng; Wang, Jifeng; Chen, Xiulan; Guo, Xiaojing; Yang, Fuquan

2017-01-01

Ovarian cancer is one of the most common cancer among women in the world, and chemotherapy remains the principal treatment for patients. However, drug resistance is a major obstacle to the effective treatment of ovarian cancers and the underlying mechanism is not clear. An increased understanding of the mechanisms that underline the pathogenesis of drug resistance is therefore needed to develop novel therapeutics and diagnostic. Herein, we report the comparative analysis of the doxorubicin sensitive OVCAR8 cells and its doxorubicin-resistant variant NCI/ADR-RES cells using integrated global proteomics and N-glycoproteomics. A total of 1525 unique N-glycosite-containing peptides from 740 N-glycoproteins were identified and quantified, of which 253 N-glycosite-containing peptides showed significant change in the NCI/ADR-RES cells. Meanwhile, stable isotope labeling by amino acids in cell culture (SILAC) based comparative proteomic analysis of the two ovarian cancer cells led to the quantification of 5509 proteins. As about 50% of the identified N-glycoproteins are low-abundance membrane proteins, only 44% of quantified unique N-glycosite-containing peptides had corresponding protein expression ratios. The comparison and calibration of the N-glycoproteome versus the proteome classified 14 change patterns of N-glycosite-containing peptides, including 8 up-regulated N-glycosite-containing peptides with the increased glycosylation sites occupancy, 35 up-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy, 2 down-regulated N-glycosite-containing peptides with the decreased glycosylation sites occupancy, 46 down-regulated N-glycosite-containing peptides with the unchanged glycosylation sites occupancy. Integrated proteomic and N-glycoproteomic analyses provide new insights, which can help to unravel the relationship of N-glycosylation and multidrug resistance (MDR), understand the mechanism of MDR, and discover the new diagnostic and

Expression of cancer-associated simple mucin-type O-glycosylated antigens in parasites.

Science.gov (United States)

Osinaga, Eduardo

2007-01-01

Simple mucin-type O-glycan structures, such as Tn, TF, sialyl-Tn and Tk antigens, are among of the most specific human cancer-associated structures. These antigens are involved in several types of receptor-ligand interactions, and they are potential targets for immunotherapy. In the last few years several simple mucin-type O-glycan antigens were identified in different species belonging to the main two helminth parasite phyla, and sialyl-Tn bearing glycoproteins were detected in Trypanosoma cruzi. These results are of interest to understand new aspects in parasite glycoimmunology and may help identify new biological characteristics of parasites as well of the host-parasite relationship. Considering that different groups reported a negative correlation between certain parasite infections and cancer development, we could hypothesize that simple mucin-type O-glycosylated antigens obtained from parasites could be good potential targets for cancer immunotherapy.

Immunolocalisation of members of the polypeptide N-acetylgalactosaminyl transferase (ppGalNAc-T) family is consistent with biologically relevant altered cell surface glycosylation in breast cancer

DEFF Research Database (Denmark)

Brooks, Susan A; Carter, Tracey M; Bennett, Eric P

2007-01-01

understood, may mediate the synthesis of varied glycoforms of cellular proteins with different biological activities. Disruptions in glycosylation are a common feature of cancer and may have functional significance. Immunocytochemistry with confocal scanning laser microscopy was employed to detect members...... of the ppGalNAc-T family, ppGalNAc-T1, -T2, -T3, -T4 and -T6 in a range of breast cell lines. The cells were chosen to represent a range of phenotypes from 'normal'/benign (HMT 3,522), primary, non-metastatic breast cancer (BT 474), to aggressive, metastatic breast cancer (ZR75-1, T47D, MCF-7, DU 4...... tightly restricted ppGalNAc-T's may result in initiation of O-linked glycosylation at normally unoccupied potential glycosylation sites leading to altered glycoforms of proteins with changed biological activity which may contribute to the pathogenesis of cancer....

Autoantibodies to aberrantly glycosylated MUC1 in early stage breast cancer are associated with a better prognosis

DEFF Research Database (Denmark)

Blixt, Ola; Bueti, Deanna; Burford, Brian

2011-01-01

associated glycoforms of MUC1 in a proportion of early breast cancer patients (54/198). Five positive sera were selected for detailed definition of the reactive epitopes using on chip glycosylation technology and a panel of glycopeptides based on a single MUC1 tandem repeat carrying specific glycans...

DMBT1 expression and glycosylation during the adenoma-carcinoma sequence in colorectal cancer

DEFF Research Database (Denmark)

Robbe, C; Paraskeva, C; Mollenhauer, J

2005-01-01

, location and its mode of secretion during malignant transformation in colorectal cancer. Using human colorectal PC/AA cell lines and tissue sections from individual patients, we have examined the expression of DMBT1 and its glycosylation in the adenoma-carcinoma sequence leading to the adenocarcinoma......The gene DMBT1 (deleted in malignant brain tumour-1) has been proposed to play a role in brain and epithelial cancer, but shows unusual features for a classical tumour-suppressor gene. On the one hand, DMBT1 has been linked to mucosal protection, whereas, on the other, it potentially plays a role...... in epithelial differentiation. Thus its function in a particular tissue is of mechanistic importance for its role in cancer. Because the former function requires secretion to the lumen and the latter function may depend on its presence in the extracellular matrix, we decided to investigate DMBT1 expression...

Glycosylation Engineering

DEFF Research Database (Denmark)

Clausen, Henrik; Wandall, Hans H.; Steentoft, Catharina

2017-01-01

Knowledge of the cellular pathways of glycosylation across phylogeny provides opportunities for designing glycans via genetic engineering in a wide variety of cell types including bacteria, fungi, plant cells, and mammalian cells. The commercial demand for glycosylation engineering is broad......, including production of biological therapeutics with defined glycosylation (Chapter 57). This chapter describes how knowledge of glycan structures and their metabolism (Parts I–III of this book) has led to the current state of glycosylation engineering in different cell types. Perspectives for rapid...

Diagnostic accuracy of urinary prostate protein glycosylation profiling in prostatitis diagnosis.

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Vermassen, Tijl; Van Praet, Charles; Poelaert, Filip; Lumen, Nicolaas; Decaestecker, Karel; Hoebeke, Piet; Van Belle, Simon; Rottey, Sylvie; Delanghe, Joris

2015-01-01

Although prostatitis is a common male urinary tract infection, clinical diagnosis of prostatitis is difficult. The developmental mechanism of prostatitis is not yet unraveled which led to the elaboration of various biomarkers. As changes in asparagine-linked-(N-)-glycosylation were observed between healthy volunteers (HV), patients with benign prostate hyperplasia and prostate cancer patients, a difference could exist in biochemical parameters and urinary N-glycosylation between HV and prostatitis patients. We therefore investigated if prostatic protein glycosylation could improve the diagnosis of prostatitis. Differences in serum and urine biochemical markers and in total urine N-glycosylation profile of prostatic proteins were determined between HV (N=66) and prostatitis patients (N=36). Additionally, diagnostic accuracy of significant biochemical markers and changes in N-glycosylation was assessed. Urinary white blood cell (WBC) count enabled discrimination of HV from prostatitis patients (Pprostatitis patients from HV (Pprostatitis patients compared to HV (Pprostatitis. Further research is required to unravel the developmental course of prostatic inflammation.

Glycosylation as a Main Regulator of Growth and Death Factor Receptors Signaling

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Inês Gomes Ferreira

2018-02-01

Full Text Available Glycosylation is a very frequent and functionally important post-translational protein modification that undergoes profound changes in cancer. Growth and death factor receptors and plasma membrane glycoproteins, which upon activation by extracellular ligands trigger a signal transduction cascade, are targets of several molecular anti-cancer drugs. In this review, we provide a thorough picture of the mechanisms bywhich glycosylation affects the activity of growth and death factor receptors in normal and pathological conditions. Glycosylation affects receptor activity through three non-mutually exclusive basic mechanisms: (1 by directly regulating intracellular transport, ligand binding, oligomerization and signaling of receptors; (2 through the binding of receptor carbohydrate structures to galectins, forming a lattice thatregulates receptor turnover on the plasma membrane; and (3 by receptor interaction with gangliosides inside membrane microdomains. Some carbohydrate chains, for example core fucose and β1,6-branching, exert a stimulatory effect on all receptors, while other structures exert opposite effects on different receptors or in different cellular contexts. In light of the crucial role played by glycosylation in the regulation of receptor activity, the development of next-generation drugs targeting glyco-epitopes of growth factor receptors should be considered a therapeutically interesting goal.

Biochemical Importance of Glycosylation of Plasminogen Activator Inhibitor-1

DEFF Research Database (Denmark)

Gils, Ann; Pedersen, Katrine Egelund; Skottrup, Peter Durand

2003-01-01

The serpin plasminogen activator inhibitor-1 (PAI-1) is a potential target for anti-thrombotic and anti-cancer therapy. PAI-1 has 3 potential sites for N-linked glycosylation. We demonstrate here that PAI-1 expressed recombinantly or naturally by human cell lines display a heterogeneous glycosyla...

N-glycosylation in sugarcane

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Maia Ivan G.

2001-01-01

Full Text Available The N-linked glycosylation of secretory and membrane proteins is the most complex posttranslational modification known to occur in eukaryotic cells. It has been shown to play critical roles in modulating protein function. Although this important biological process has been extensively studied in mammals, much less is known about this biosynthetic pathway in plants. The enzymes involved in plant N-glycan biosynthesis and processing are still not well defined and the mechanism of their genetic regulation is almost completely unknown. In this paper we describe our first attempt to understand the N-linked glycosylation mechanism in a plant species by using the data generated by the Sugarcane Expressed Sequence Tag (SUCEST project. The SUCEST database was mined for sugarcane gene products potentially involved in the N-glycosylation pathway. This approach has led to the identification and functional assignment of 90 expressed sequence tag (EST clusters sharing significant sequence similarity with the enzymes involved in N-glycan biosynthesis and processing. The ESTs identified were also analyzed to establish their relative abundance.

Functional importance of PAI-1 glycosylation

DEFF Research Database (Denmark)

Christensen, Anni; Naessens, Dominik; Skottrup, Peter

2001-01-01

Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-glycosylated PAI-1 could be conferred upon PAI-1 expressed in HEK293 cells by mutational inactivation of one or the other glycosylation site. These findings reveal a novel functional role for glycosylation of a serpin. The glycosylation sites are localised between a-helix H and b-strand 2C and b-strand 3C and a...

The relative contribution of mannose salvage pathways to glycosylation in PMI-deficient mouse embryonic fibroblast cells.

Science.gov (United States)

Fujita, Naonobu; Tamura, Ayako; Higashidani, Aya; Tonozuka, Takashi; Freeze, Hudson H; Nishikawa, Atsushi

2008-02-01

Mannose for mammalian glycan biosynthesis can be imported directly from the medium, derived from glucose or salvaged from endogenous or external glycans. All pathways must generate mannose 6-phosphate, the activated form of mannose. Imported or salvaged mannose is directly phosphorylated by hexokinase, whereas fructose 6-phosphate from glucose is converted to mannose 6-phosphate by phosphomannose isomerase (PMI). Normally, PMI provides the majority of mannose for glycan synthesis. To assess the contribution of PMI-independent pathways, we used PMI-null fibroblasts to study N-glycosylation of DNase I, a highly sensitive indicator protein. In PMI-null cells, imported mannose and salvaged mannose make a significant contribution to N-glycosylation. When these cells were grown in mannose-free medium along with the mannosidase inhibitor, swainsonine, to block the salvage pathways, N-glycosylation of DNase I was almost completely eliminated. Adding approximately 13 microm mannose to the medium completely restored normal glycosylation. Treatment with bafilomycin A(1), an inhibitor of lysosomal acidification, also markedly reduced N-glycosylation of DNase I, but in this case only 8 microm mannose was required to restore full glycosylation, indicating that a nonlysosomal source of mannose made a significant contribution. Glycosylation levels were greatly also reduced in glycoconjugate-free medium, when endosomal membrane trafficking was blocked by expression of a mutant SKD1. From these data, we conclude that PMI-null cells can salvage mannose from both endogenous and external glycoconjugates via lysosomal and nonlysosomal degradation pathways.

Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

Science.gov (United States)

de Jongh, Harmen H. J.; Robles, Carlos López; Nordlee, Julie A.; Lee, Poi-Wah; Baumert, Joseph L.; Hamilton, Robert G.; Taylor, Steve L.; Koppelman, Stef J.

2013-01-01

Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa), the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein's digestibility is not affected by such processing. PMID:23878817

Digestibility and IgE-Binding of Glycosylated Codfish Parvalbumin

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Harmen H. J. de Jongh

2013-01-01

Full Text Available Food-processing conditions may alter the allergenicity of food proteins by different means. In this study, the effect of the glycosylation as a result of thermal treatment on the digestibility and IgE-binding of codfish parvalbumin is investigated. Native and glycosylated parvalbumins were digested with pepsin at various conditions relevant for the gastrointestinal tract. Intact proteins and peptides were analysed for apparent molecular weight and IgE-binding. Glycosylation did not substantially affect the digestion. Although the peptides resulting from digestion were relatively large (3 and 4 kDa, the IgE-binding was strongly diminished. However, the glycosylated parvalbumin had a strong propensity to form dimers and tetramers, and these multimers bound IgE intensely, suggesting stronger IgE-binding than monomeric parvalbumin. We conclude that glycosylation of codfish parvalbumin does not affect the digestibility of parvalbumin and that the peptides resulting from this digestion show low IgE-binding, regardless of glycosylation. Glycosylation of parvalbumin leads to the formation of higher order structures that are more potent IgE binders than native, monomeric parvalbumin. Therefore, food-processing conditions applied to fish allergen can potentially lead to increased allergenicity, even while the protein’s digestibility is not affected by such processing.

ECM Proteins Glycosylation and Relation to Diabetes

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Pernodet, Nadine; Bloomberg, Ayla; Sood, Vandana; Slutsky, Lenny; Ge, Shouren; Clark, Richard; Rafailovich, Miriam

2004-03-01

The chemical modification and crosslinking of proteins by sugar glycosylation contribute to the aging of tissue proteins, and acceleration of this reaction during hyperglycemia is implicated in the pathogenesis of diabetic complications, such as disorder of the wound healing. Advanced glycation endproducts (AGEs) formation and protein crosslinking are irreversible processes that alter the structural and functional properties of proteins, lipid components and nucleic acids. And the mechanism, by which it happens, is not clear. Fibrinogen and fibronectin are plasma proteins, which play a major role in human wound healing. Fibrinogen converts to an insoluble fibrin "gel" following a cut, which eventually forms a clot to prevent blood loss, to direct cell adhesion and migration for forming scars. Fibronectin is a critical protein for cell adhesion and migration in wound healing. The effects of glucose on the binding of these plasma proteins from the extra cellular matrix (ECM) were followed at different concentrations by atomic force microscopy and lateral force modulation to measure the mechanical response of the samples. Glucose solutions (1, 2, and 3mg/mL) were incubated with the protein (100 mg/ml) and silicon (Si) substrates spun with sulfonated polystyrene (SPS) 28% for five days. Data showed that not only the organization of the protein on the surface was affected but also its mechanical properties. At 3 mg/mL glucose, Fn fibers were observed to be harder than those of the control, in good agreement with our hypothesis that glycosylation hardens tissues by crosslinking of proteins in the ECM and might cause fibers to break more easily.

Efficient synthesis of glycosylated phenazine natural products and analogs with DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors

DEFF Research Database (Denmark)

Laursen, Jane B.; Petersen, Lars; Jensen, K.J.

2003-01-01

Inspired by the occurrence and function of phenazines in natural products, new glycosylated analogs were designed and synthesized. DISAL (methyl 3,5-dinitrosalicylate) glycosyl donors were used in an efficient and easily-handled glycosylation protocol compatible with combinatorial chemistry....... Benzoylated D-glucose, D-galactose and L-quinovose DISAL glycosyl donors were synthesized in high yields and used under mild conditions to glycosylate methyl saphenate and 2-hydroxyphenazine. The glycosides were screened for biological activity and one compound showed inhibitory activity towards topoisomerase...

Diversity and functions of protein glycosylation in insects.

Science.gov (United States)

Walski, Tomasz; De Schutter, Kristof; Van Damme, Els J M; Smagghe, Guy

2017-04-01

The majority of proteins is modified with carbohydrate structures. This modification, called glycosylation, was shown to be crucial for protein folding, stability and subcellular location, as well as protein-protein interactions, recognition and signaling. Protein glycosylation is involved in multiple physiological processes, including embryonic development, growth, circadian rhythms, cell attachment as well as maintenance of organ structure, immunity and fertility. Although the general principles of glycosylation are similar among eukaryotic organisms, insects synthesize a distinct repertoire of glycan structures compared to plants and vertebrates. Consequently, a number of unique insect glycans mediate functions specific to this class of invertebrates. For instance, the core α1,3-fucosylation of N-glycans is absent in vertebrates, while in insects this modification is crucial for the development of wings and the nervous system. At present, most of the data on insect glycobiology comes from research in Drosophila. Yet, progressively more information on the glycan structures and the importance of glycosylation in other insects like beetles, caterpillars, aphids and bees is becoming available. This review gives a summary of the current knowledge and recent progress related to glycan diversity and function(s) of protein glycosylation in insects. We focus on N- and O-glycosylation, their synthesis, physiological role(s), as well as the molecular and biochemical basis of these processes. Copyright © 2017 Elsevier Ltd. All rights reserved.

Control of mucin-type O-glycosylation

DEFF Research Database (Denmark)

Bennett, Eric P; Mandel, Ulla; Clausen, Henrik

2012-01-01

residues, is one of the most abundant forms of protein glycosylation in animals. Although most protein glycosylation is controlled by one or two genes encoding the enzymes responsible for the initiation of glycosylation, i.e. the step where the first glycan is attached to the relevant amino acid residue...... in the protein, mucin-type O-glycosylation is controlled by a large family of up to 20 homologous genes encoding UDP-GalNAc:polypeptide GalNAc-transferases (GalNAc-Ts) (EC 2.4.1.41). Therefore, mucin-type O-glycosylation has the greatest potential for differential regulation in cells and tissues. The Gal...

N- and O-glycosylation Analysis of Human C1-inhibitor Reveals Extensive Mucin-type O-Glycosylation.

Science.gov (United States)

Stavenhagen, Kathrin; Kayili, H Mehmet; Holst, Stephanie; Koeleman, Carolien A M; Engel, Ruchira; Wouters, Diana; Zeerleder, Sacha; Salih, Bekir; Wuhrer, Manfred

2018-06-01

Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N - and O -glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O -glycosylated N-terminal region. Five novel and five known O -glycosylation sites were identified, carrying mainly core1-type O -glycans. In addition, we detected a heavily O -glycosylated portion spanning from Thr 82 -Ser 121 with up to 16 O -glycans attached. Likewise, all known six N -glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N -glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Prediction of glycosylation sites using random forests

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Hirst Jonathan D

2008-11-01

Full Text Available Abstract Background Post translational modifications (PTMs occur in the vast majority of proteins and are essential for function. Prediction of the sequence location of PTMs enhances the functional characterisation of proteins. Glycosylation is one type of PTM, and is implicated in protein folding, transport and function. Results We use the random forest algorithm and pairwise patterns to predict glycosylation sites. We identify pairwise patterns surrounding glycosylation sites and use an odds ratio to weight their propensity of association with modified residues. Our prediction program, GPP (glycosylation prediction program, predicts glycosylation sites with an accuracy of 90.8% for Ser sites, 92.0% for Thr sites and 92.8% for Asn sites. This is significantly better than current glycosylation predictors. We use the trepan algorithm to extract a set of comprehensible rules from GPP, which provide biological insight into all three major glycosylation types. Conclusion We have created an accurate predictor of glycosylation sites and used this to extract comprehensible rules about the glycosylation process. GPP is available online at http://comp.chem.nottingham.ac.uk/glyco/.

Multidimensional fractionation is a requirement for quantitation of Golgi-resident glycosylation enzymes from cultured human cells.

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Lin, Chi-Hung; Chik, Jenny H L; Packer, Nicolle H; Molloy, Mark P

2015-02-06

Glycosylation results from the concerted action of glycosylation enzymes in the secretory pathway. In general, gene expression serves as the primary control mechanism, but post-translational fine-tuning of glycosylation enzyme functions is often necessary for efficient synthesis of specific glycan epitopes. While the field of glycomics has rapidly advanced, there lacks routine proteomic methods to measure expression of specific glycosylation enzymes needed to fill the gap between mRNA expression and the glycomic profile in a "reverse genomics" workflow. Toward developing this workflow we enriched Golgi membranes from two human colon cancer cell lines by sucrose density centrifugation and further mass-based fractionation by SDS-PAGE. We then applied mass spectrometry to demonstrate a doubling in the number of Golgi resident proteins identified, compared to the unenriched, low speed centrifuged supernatant of lysed cells. A total of 35 Golgi-resident glycosylation enzymes, of which 23 were glycosyltransferases, were identified making this the largest protein database so far of Golgi resident glycosylation enzymes experimentally identified in cultured human cells. We developed targeted mass spectrometry assays for specific quantitation of many of these glycosylation enzymes. Our results show that alterations in abundance of glycosylation enzymes at the protein level were generally consistent with the resultant glycomic profiles, but not necessarily with the corresponding glycosyltransferase mRNA expression as exemplified by the case of O-glycan core 1 T synthase.

DISAL glycosyl donors for the synthesis of a linear hexasaccharide under mild conditions

DEFF Research Database (Denmark)

Petersen, Lars; Laursen, Jane B.; Larsen, K.

2003-01-01

The new class of glycosyl donors with a methyl 3,5-dinitrosalicylate (DISAL) anomeric leaving group has proved efficient for glycosylation under strictly neutral, mildly basic, or mildly acidic conditions. Here, we report the synthesis of novel DISAL disaccharide glycosyl donors prepared by easy...... nucleophilic aromatic substitution. These DISAL donors proved efficient in the synthesis of a starch-related hexasaccharide under very mild conditions. Glycosylations proceeded with alpha-selectivity and were compatible with Trt protecting groups....

N-Glycosylation of Lipocalin 2 Is Not Required for Secretion or Exosome Targeting

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Erawan Borkham-Kamphorst

2018-04-01

Full Text Available Lipocalin 2 (LCN2 is a highly conserved secreted adipokine acting as a serum transport protein for small hydrophobic molecules such as fatty acids and steroids. In addition, LCN2 limits bacterial growth by sequestering iron-containing siderophores and further protects against intestinal inflammation and tumorigenesis associated with alterations in the microbiota. Human LCN2 contains one N-glycosylation site conserved in other species. It was postulated that this post-translational modification could facilitate protein folding, protects from proteolysis, is required for proper trafficking from the Golgi apparatus to the cell surface, and might be relevant for effective secretion. We here show that the homologous nucleoside antibiotic tunicamycin blocks N-linked glycosylation but not secretion of LCN2 in primary murine hepatocytes, derivatives thereof, human lung carcinoma cell line A549, and human prostate cancer cell line PC-3. Moreover, both the glycosylated and the non-glycosylated LCN2 variants are equally targeted to exosomes, demonstrating that this post-translational modification is not necessary for proper trafficking of LCN2 into these membranous extracellular vesicles. Furthermore, a hydrophobic cluster analysis revealed that the N-glycosylation site is embedded in a highly hydrophobic evolutionarily conserved surrounding. In sum, our data indicate that the N-glycosylation of LCN2 is not required for proper secretion and exosome cargo recruitment in different cell types, but might be relevant to increase overall solubility.

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

International Nuclear Information System (INIS)

Kalra, Rajkumar S.; Wadhwa, Renu

2015-01-01

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein

A bioinformatics prediction approach towards analyzing the glycosylation, co-expression and interaction patterns of epithelial membrane antigen (EMA/MUC1)

Energy Technology Data Exchange (ETDEWEB)

Kalra, Rajkumar S., E-mail: renu-wadhwa@aist.go.jp; Wadhwa, Renu, E-mail: renu-wadhwa@aist.go.jp [Cell Proliferation Research Group and DBT-AIST International Laboratory for Advanced Biomedicine, National Institute of Advanced Industrial Science and Technology (AIST Central 4), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562 (Japan)

2015-02-27

Epithelial membrane antigen (EMA or MUC1) is a heavily glycosylated, type I transmembrane glycoprotein commonly expressed by epithelial cells of duct organs. It has been shown to be aberrantly glycosylated in several diseases including cancer. Protein sequence based annotation and analysis of glycosylation profile of glycoproteins by robust computational and comprehensive algorithms provides possible insights to the mechanism(s) of anomalous glycosylation. In present report, by using a number of bioinformatics applications we studied EMA/MUC1 and explored its trans-membrane structural domain sequence that is widely subjected to glycosylation. Exploration of different extracellular motifs led to prediction of N and O-linked glycosylation target sites. Based on the putative O-linked target sites, glycosylated moieties and pathways were envisaged. Furthermore, Protein network analysis demonstrated physical interaction of EMA with a number of proteins and confirmed its functional involvement in cell growth and proliferation pathways. Gene Ontology analysis suggested an involvement of EMA in a number of functions including signal transduction, protein binding, processing and transport along with glycosylation. Thus, present study explored potential of bioinformatics prediction approach in analyzing glycosylation, co-expression and interaction patterns of EMA/MUC1 glycoprotein.

A compound heterozygous mutation in DPAGT1 results in a congenital disorder of glycosylation with a relatively mild phenotype

NARCIS (Netherlands)

Iqbal, Z.; Shahzad, M.; Vissers, L.E.L.M.; Scherpenzeel, M. van; Gilissen, C.; Razzaq, A.; Zahoor, M.Y.; Khan, S.N.; Kleefstra, T.; Veltman, J.A.; Brouwer, A.P.M. de; Lefeber, D.J.; Bokhoven, H. van; Riazuddin, S.

2013-01-01

Congenital disorders of glycosylation (CDG) are a large group of recessive multisystem disorders caused by impaired protein or lipid glycosylation. The CDG-I subgroup is characterized by protein N-glycosylation defects originating in the endoplasmic reticulum. The genetic defect is known for 17

Distribution of N-glycosylation sequons in proteins: how apart are they?

DEFF Research Database (Denmark)

Rao, Shyama Prasad; Buus, Ole Thomsen; Wollenweber, Bernd

2011-01-01

of experimentally confirmed eukaryotic N-glycoproteins we analyzed the relative position and distribution of sequons. N-Glycosylation probability was found to be lower in the termini of protein sequences compared to the mid region. N-glycosylated sequons were found much farther from C terminus compared to the N......N-glycosylation is a common protein modification process, which affects a number of properties of proteins. Little is known about the distribution of N-glycosylation sequons, for example, the distance between glycosylated sites and their position in the protein primary sequence. Using a large set......-terminus of the protein sequence and this effect was more pronounced for NXS sequons. The distribution of sequons, modeled based on balls-in-boxes classical occupancy, showed a near-maximum probability. Considerable proportion of sequons was found within a distance of ten amino acids, indicating that the steric hindrance...

Functional importance of PAI-1 glycosylation

DEFF Research Database (Denmark)

Christensen, Anni; Naessens, Dominik; Skottrup, Peter

susceptible PAI-1 variant was not necessarily the one used when raising the antibody. This and other observations indicated that the carbohydrate moieties or the glycosylation sites are unlikely to be part of the epitopes for these antibodies. The antibody susceptibility characteristic for non......Structure-function studies of plasminogen activator inhibitor-1 (PAI-1) have previously been performed mostly with non-glycosylated material expressed in E. coli. We have now studied the importance of PAI-1 glycosylation for its functional properties. PAI-1 has 3 potential sites for N......-linked glycosylation. Biochemical analysis of PAI-1 variants with substitutions of the Asn residues in each of these sites and expression in human embryonic kidney 293 (HEK293) cells showed that only Asn211 and Asn 267, but not Asn331 are glycosylated, and revealed a differential composition of the carbohydrate...

Prion propagation in cells expressing PrP glycosylation mutants.

Science.gov (United States)

Salamat, Muhammad K; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-04-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrP(C)) to a disease-related isoform (PrP(Sc)). PrP(C) carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrP(C) glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrP(Sc) and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrP(Sc), while PrP(C) with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrP(C), were able to form infectious PrP(Sc). Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection.

Unusual glycosylation of proteins: Beyond the universal sequon and other amino acids.

Science.gov (United States)

Dutta, Devawati; Mandal, Chhabinath; Mandal, Chitra

2017-12-01

Glycosylation of proteins is the most common, multifaceted co- and post-translational modification responsible for many biological processes and cellular functions. Significant alterations and aberrations of these processes are related to various pathological conditions, and often turn out to be disease biomarkers. Conventional N-glycosylation occurs through the recognition of the consensus sequon, asparagine (Asn)-X-serine (Ser)/threonine (Thr), where X is any amino acid except for proline, with N-acetylglucosamine (GlcNAc) as the first glycosidic linkage. Usually, O-glycosylation adds a glycan to the hydroxyl group of Ser or Thr beginning with N-acetylgalactosamine (GalNAc). Protein glycosylation is further governed by additional diversifications in sequon and structure, which are yet to be fully explored. This review mainly focuses on the occurrence of N-glycosylation in non-consensus motifs, where Ser/Thr at the +2 position is substituted by other amino acids. Additionally, N-glycosylation is also observed in other amide/amine group-containing amino acids. Similarly, O-glycosylation occurs at hydroxyl group-containing amino acids other than serine/threonine. The neighbouring amino acids and local structural features around the potential glycosylation site also play a significant role in determining the extent of glycosylation. All of these phenomena that yield glycosylation at the atypical sites are reported in a variety of biological systems, including different pathological conditions. Therefore, the discovery of more novel sequence patterns for N- and O-glycosylation may help in understanding the functions of complex biological processes and cellular functions. Taken together, all these information provided in this review would be helpful for the biological readers. Copyright © 2017 Elsevier B.V. All rights reserved.

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

International Nuclear Information System (INIS)

Kilcoyne, Michelle; Sharma, Shashank; McDevitt, Niamh; O’Leary, Claire; Joshi, Lokesh; McMahon, Siobhán S.

2012-01-01

Highlights: ► Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. ► Neuronal glycosylation in injury and after ChABC treatment is unknown. ► In silico mining verified that glyco-related genes were differentially regulated after SCI. ► In vitro model system revealed abnormal sialylation in an injured environment. ► The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually α-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment with ChABC was successful in returning neuronal glycosylation to normal conditions at all timepoints for MAA, PNA and SNA-I staining

Neuronal glycosylation differentials in normal, injured and chondroitinase-treated environments

Energy Technology Data Exchange (ETDEWEB)

Kilcoyne, Michelle; Sharma, Shashank [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McDevitt, Niamh; O' Leary, Claire [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland); Joshi, Lokesh [Glycoscience Group, National Centre for Biomedical Engineering Science, National University of Ireland, Galway (Ireland); McMahon, Siobhan S., E-mail: siobhan.mcmahon@nuigalway.ie [Anatomy, School of Medicine, National University of Ireland, Galway (Ireland)

2012-04-13

Highlights: Black-Right-Pointing-Pointer Carbohydrates are important in the CNS and ChABC has been used for spinal cord injury (SCI) treatment. Black-Right-Pointing-Pointer Neuronal glycosylation in injury and after ChABC treatment is unknown. Black-Right-Pointing-Pointer In silico mining verified that glyco-related genes were differentially regulated after SCI. Black-Right-Pointing-Pointer In vitro model system revealed abnormal sialylation in an injured environment. Black-Right-Pointing-Pointer The model indicated a return to normal neuronal glycosylation after ChABC treatment. -- Abstract: Glycosylation is found ubiquitously throughout the central nervous system (CNS). Chondroitin sulphate proteoglycans (CSPGs) are a group of molecules heavily substituted with glycosaminoglycans (GAGs) and are found in the extracellular matrix (ECM) and cell surfaces. Upon CNS injury, a glial scar is formed, which is inhibitory for axon regeneration. Several CSPGs are up-regulated within the glial scar, including NG2, and these CSPGs are key inhibitory molecules of axonal regeneration. Treatment with chondroitinase ABC (ChABC) can neutralise the inhibitory nature of NG2. A gene expression dataset was mined in silico to verify differentially regulated glycosylation-related genes in neurons after spinal cord injury and identify potential targets for further investigation. To establish the glycosylation differential of neurons that grow in a healthy, inhibitory and ChABC-treated environment, we established an indirect co-culture system where PC12 neurons were grown with primary astrocytes, Neu7 astrocytes (which overexpress NG2) and Neu7 astrocytes treated with ChABC. After 1, 4 and 8 days culture, lectin cytochemistry of the neurons was performed using five fluorescently-labelled lectins (ECA MAA, PNA, SNA-I and WFA). Usually {alpha}-(2,6)-linked sialylation scarcely occurs in the CNS but this motif was observed on the neurons in the injured environment only at day 8. Treatment

Loci associated with N-glycosylation of human immunoglobulin G show pleiotropy with autoimmune diseases and haematological cancers

NARCIS (Netherlands)

Lauc, G.; Huffman, J.E.; Pucic, M.; Zgaga, L.; Adamczyk, B.; Muzinic, A.; Novokmet, M.; Polasek, O.; Gornik, O.; Kristic, J.; Keser, T.; Vitart, V.; Scheijen, B.; Uh, H.W.; Molokhia, M.; Patrick, A.L.; McKeigue, P.; Kolcic, I.; Lukic, I.K.; Swann, O.; Leeuwen, F.N. van; Ruhaak, L.R.; Houwing-Duistermaat, J.J.; Slagboom, P.E.; Beekman, M.; Craen, A.J. de; Deelder, A.M.; Zeng, Q.; Wang, W.; Hastie, N.D.; Gyllensten, U.; Wilson, J.F.; Wuhrer, M.; Wright, A.F.; Rudd, P.M.; Hayward, C.; Aulchenko, Y.; Campbell, H.; Rudan, I.

2013-01-01

Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative

GLYCOSYLATED YGHJ POLYPEPTIDES FROM ENTEROTOXIGENIC ESCHERICHIA COLI (ETEC)

DEFF Research Database (Denmark)

2017-01-01

The present invention relates to glycosylated YghJ polypeptides from or derived from enterotoxigenic Escherichia coli (ETEC) that are immunogenic. In particular, the present invention relates to compositions or vaccines comprising the polypeptides and their application in immunization, vaccination...

Engineering Mammalian Mucin-type O-Glycosylation in Plants

DEFF Research Database (Denmark)

Yang, Zhang; Drew, Damian P; Jørgensen, Bodil

2012-01-01

-glycans are attached to proteins, and which structures are formed, difficult. Because plants are devoid of GalNAc-type O-glycosylation, we have assessed requirements for establishing human GalNAc O-glycosylation de novo in plants with the aim of developing cell systems with custom-designed O-glycosylation capacity...... was glycosylated with up to three and five GalNAc residues when co-expressed with GalNAc-T2 and a combination of GalNAc-T2 and GalNAc-T4, respectively, as determined by mass spectrometry. O-Glycosylation was furthermore demonstrated on a tandem repeat of MUC16 and interferon a2b. In plants, prolines in certain...... classes of proteins are hydroxylated and further substituted with plant-specific O-glycosylation; unsubstituted hydroxyprolines were identified in our MUC1 construct. In summary, this study demonstrates that mammalian type O-glycosylation can be established in plants and that plants may serve as a host...

Alterations in expressed prostate secretion-urine PSA N-glycosylation discriminate prostate cancer from benign prostate hyperplasia.

Science.gov (United States)

Jia, Gaozhen; Dong, Zhenyang; Sun, Chenxia; Wen, Fuping; Wang, Haifeng; Guo, Huaizu; Gao, Xu; Xu, Chuanliang; Xu, Chuanliang; Yang, Chenghua; Sun, Yinghao

2017-09-29

The prostate specific antigen (PSA) test is widely used for early diagnosis of prostate cancer (PCa). However, its limited sensitivity has led to over-diagnosis and over-treatment of PCa. Glycosylation alteration is a common phenomenon in cancer development. Different PSA glycan subforms have been proposed as diagnostic markers to better differentiate PCa from benign prostate hyperplasia (BPH). In this study, we purified PSA from expressed prostate secretions (EPS)-urine samples from 32 BPH and 30 PCa patients and provided detailed PSA glycan profiles in Chinese population. We found that most of the PSA glycans from EPS-urine were complex type biantennary glycans. We observed two major patterns in PSA glycan profiles. Overall there was no distinct separation of PSA glycan profiles between BPH and PCa patients. However, we detected a significant increase of glycan FA2 and FM5A2G2S1 in PCa when compared with BPH patients. Furthermore, we observed that the composition of FA2 glycan increased significantly in advanced PCa with Gleason score ≥8, which potentially could be translated to clinic as a marker for aggressive PCa.

Trans-species Engineering of Glycosylated Therapeutic Proteins

DEFF Research Database (Denmark)

Yang, Zhang

important to address. Whenever glycosylation has been found to be an important PTM for function or bioactivity, human therapeutics have generally been produced in mammalian Chinese hamster ovary (CHO) cell line. Oglycosylation is one of the most complex regulated PTMs of proteins but also one of the least...... understood. Currently, mammalian cells are required for human O-glycosylation. Increasing efforts have been devoted to engineering non-mammalian cells for production of recombinant proteins with “human-like” glycosylation. Substantial success has been achieved with designed N-glycosylation in both lower......Recombinant expression of therapeutic proteins is one of the major tasks in modern biomedicine. One of the most important factors with respect to therapeutic use in human is posttranslational modifications (PTMs) of the recombinant proteins, of which protein glycosylation is by far the most...

Glycogenomics as a mass spectrometry-guided genome-mining method for microbial glycosylated molecules.

Science.gov (United States)

Kersten, Roland D; Ziemert, Nadine; Gonzalez, David J; Duggan, Brendan M; Nizet, Victor; Dorrestein, Pieter C; Moore, Bradley S

2013-11-19

Glycosyl groups are an essential mediator of molecular interactions in cells and on cellular surfaces. There are very few methods that directly relate sugar-containing molecules to their biosynthetic machineries. Here, we introduce glycogenomics as an experiment-guided genome-mining approach for fast characterization of glycosylated natural products (GNPs) and their biosynthetic pathways from genome-sequenced microbes by targeting glycosyl groups in microbial metabolomes. Microbial GNPs consist of aglycone and glycosyl structure groups in which the sugar unit(s) are often critical for the GNP's bioactivity, e.g., by promoting binding to a target biomolecule. GNPs are a structurally diverse class of molecules with important pharmaceutical and agrochemical applications. Herein, O- and N-glycosyl groups are characterized in their sugar monomers by tandem mass spectrometry (MS) and matched to corresponding glycosylation genes in secondary metabolic pathways by a MS-glycogenetic code. The associated aglycone biosynthetic genes of the GNP genotype then classify the natural product to further guide structure elucidation. We highlight the glycogenomic strategy by the characterization of several bioactive glycosylated molecules and their gene clusters, including the anticancer agent cinerubin B from Streptomyces sp. SPB74 and an antibiotic, arenimycin B, from Salinispora arenicola CNB-527.

Prion Propagation in Cells Expressing PrP Glycosylation Mutants ▿

Science.gov (United States)

Salamat, Muhammad K.; Dron, Michel; Chapuis, Jérôme; Langevin, Christelle; Laude, Hubert

2011-01-01

Infection by prions involves conversion of a host-encoded cell surface protein (PrPC) to a disease-related isoform (PrPSc). PrPC carries two glycosylation sites variably occupied by complex N-glycans, which have been suggested by previous studies to influence the susceptibility to these diseases and to determine characteristics of prion strains. We used the Rov cell system, which is susceptible to sheep prions, to generate a series of PrPC glycosylation mutants with mutations at one or both attachment sites. We examined their subcellular trafficking and ability to convert into PrPSc and to sustain stable prion propagation in the absence of wild-type PrP. The susceptibility to infection of mutants monoglycosylated at either site differed dramatically depending on the amino acid substitution. Aglycosylated double mutants showed overaccumulation in the Golgi compartment and failed to be infected. Introduction of an ectopic glycosylation site near the N terminus fully restored cell surface expression of PrP but not convertibility into PrPSc, while PrPC with three glycosylation sites conferred cell permissiveness to infection similarly to the wild type. In contrast, predominantly aglycosylated molecules with nonmutated N-glycosylation sequons, produced in cells expressing glycosylphosphatidylinositol-anchorless PrPC, were able to form infectious PrPSc. Together our findings suggest that glycosylation is important for efficient trafficking of anchored PrP to the cell surface and sustained prion propagation. However, properly trafficked glycosylation mutants were not necessarily prone to conversion, thus making it difficult in such studies to discern whether the amino acid changes or glycan chain removal most influences the permissiveness to prion infection. PMID:21248032

Effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1

International Nuclear Information System (INIS)

Watanabe, Ayahisa; Nishijima, Ken-ichi; Zhao, Songji; Tamaki, Nagara; Kuge, Yuji; Tanaka, Yoshikazu; Itoh, Takeshi; Takemoto, Hiroshi

2012-01-01

Glycosylation is generally applicable as a strategy for increasing the activity of bioactive proteins. In this study, we examined the effect of glycosylation on biodistribution of radiolabeled glucagon-like peptide 1 (GLP-1) as a bioactive peptide for type 2 diabetes. Noninvasive imaging studies were performed using a gamma camera after the intravenous administration of 123 I-GLP-1 or 123 I-α2, 6-sialyl N-acetyllactosamine (glycosylated) GLP-1 in rats. In ex vivo biodistribution studies using 125 I-GLP-1 or 125 I-glycosylated GLP-1, organ samples were measured for radioactivity. Plasma samples were added to 15% trichloroacetic acid (TCA) to obtain TCA-insoluble and TCA-soluble fractions. The radioactivity in the TCA-insoluble and TCA-soluble fractions was measured. In the noninvasive imaging studies, a relatively high accumulation level of 123 I-GLP-1 was found in the liver, which is the major organ to eliminate exogenous GLP-1. The area under the time-activity curve (AUC) of 123 I-glycosylated GLP-1 in the liver was significantly lower (89%) than that of 123 I-GLP-1. These results were consistent with those of ex vivo biodistribution studies using 125 I-labeled peptides. The AUC of 125 I-glycosylated GLP-1 in the TCA-insoluble fraction was significantly higher (1.7-fold) than that of GLP-1. This study demonstrated that glycosylation significantly decreased the distribution of radiolabeled GLP-1 into the liver and increased the concentration of radiolabeled GLP-1 in plasma. These results suggested that glycosylation is a useful strategy for decreasing the distribution into the liver of bioactive peptides as desirable pharmaceuticals. (author)

Location, location, location: new insights into O-GalNAc protein glycosylation

DEFF Research Database (Denmark)

Gill, David J; Clausen, Henrik; Bard, Frederic

2011-01-01

O-GalNAc glycosylation of proteins confers essential structural, protective and signaling roles in eumetazoans. Addition of O-glycans onto proteins is an extremely complex process that regulates both sites of attachment and the types of oligosaccharides added. Twenty distinct polypeptide GalNAc......-transferases (GalNAc-Ts) initiate O-glycosylation and fine-tuning their expression provides a mechanism for regulating this action. Recently, a new mode of regulation has emerged where activation of Src kinase selectively redistributes Golgi-localized GalNAc-Ts to the ER. This relocalization results in a strong...... increase in the density of O-glycan decoration. In this review, we discuss how different mechanisms can regulate the number and the types of O-glycans decorating proteins. In addition, we speculate how Src-dependent relocation of GalNAc-Ts could play an important role in cancerous cellular transformation....

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments.

Science.gov (United States)

Yamakoshi, Yasuo; Nagano, Takatoshi; Hu, Jan Cc; Yamakoshi, Fumiko; Simmer, James P

2011-02-03

Dentin sialophosphoprotein (Dspp) is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp), the N-terminal domain of dentin sialophosphoprotein (Dspp), is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB) and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG) attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were tentatively assigned at Thr200, Thr216 and Thr

Porcine dentin sialoprotein glycosylation and glycosaminoglycan attachments

Directory of Open Access Journals (Sweden)

Yamakoshi Fumiko

2011-02-01

Full Text Available Abstract Background Dentin sialophosphoprotein (Dspp is a multidomain, secreted protein that is critical for the formation of tooth dentin. Mutations in DSPP cause inherited dentin defects categorized as dentin dysplasia type II and dentinogenesis imperfecta type II and type III. Dentin sialoprotein (Dsp, the N-terminal domain of dentin sialophosphoprotein (Dspp, is a highly glycosylated proteoglycan, but little is known about the number, character, and attachment sites of its carbohydrate moieties. Results To identify its carbohydrate attachment sites we isolated Dsp from developing porcine molars and digested it with endoproteinase Glu-C or pronase, fractionated the digestion products, identified fractions containing glycosylated peptides using a phenol sulfuric acid assay, and characterized the glycopeptides by N-terminal sequencing, amino acid analyses, or LC/MSMS. To determine the average number of sialic acid attachments per N-glycosylation, we digested Dsp with glycopeptidase A, labeled the released N-glycosylations with 2-aminobenzoic acid, and quantified the moles of released glycosylations by comparison to labeled standards of known concentration. Sialic acid was released by sialidase digestion and quantified by measuring β-NADH reduction of pyruvic acid, which was generated stoichiometrically from sialic acid by aldolase. To determine its forms, sialic acid released by sialidase digestion was labeled with 1,2-diamino-4,5-methyleneoxybenzene (DMB and compared to a DMB-labeled sialic acid reference panel by RP-HPLC. To determine the composition of Dsp glycosaminoglycan (GAG attachments, we digested Dsp with chondroitinase ABC and compared the chromotagraphic profiles of the released disaccharides to commercial standards. N-glycosylations were identified at Asn37, Asn77, Asn136, Asn155, Asn161, and Asn176. Dsp averages one sialic acid per N-glycosylation, which is always in the form of N-acetylneuraminic acid. O-glycosylations were

The glycosylated IgII extracellular domain of EMMPRIN is implicated in the induction of MMP-2.

Science.gov (United States)

Papadimitropoulou, Adriana; Mamalaki, Avgi

2013-07-01

EMMPRIN is a widely expressed transmembrane glycoprotein that plays important roles in many physiological and pathological processes, such as tumor invasion and metastasis. It stimulates the production of matrix metalloproteinase (MMPs) by tumor-associated fibroblasts. In the present study, our aim was to (a) to investigate if the IgII loop domain of the extracellular domain (ECD) of EMMPRIN contributes to the MMP production by fibroblasts and (b) to evaluate the significance of glycosylation in this process. For this purpose, we expressed the ECD, IgI, or IgII domains of EMMPRIN, in their glycosylated and non-glycosylated forms, in the heterologous expression systems of P. pastoris and E. coli, respectively. Dermal fibroblasts were treated with purified recombinant domains and proteins from cell extracts and supernatants were analyzed by Western blot and zymography assays. Fibroblasts treated with ECD-, IgI-, and IgII-glycosylated domains of EMMPRIN significantly stimulated the gelatinolytic activity of MMP-2, compared to untreated fibroblasts, whereas no significant effect was observed after treatment with the non-glycosylated ECD, IgI, and IgII domains. Western blot analysis from cell extracts and supernatants revealed that only the glycosylated forms were able to stimulate MMP-2 production and secretion, respectively. Quantitative PCR revealed that this effect was not attributed to transcriptional alterations. This study showed that N-glycosylation was a prerequisite for efficient MMP-2 production, with the IgII loop domain contributing significantly to this process. Perturbation of the function of IgII-EMMPRIN loop could have potential therapeutic value in the inhibition of MMP-2-dependent cancer cell invasion and metastasis.

Competition between folding and glycosylation in the endoplasmic reticulum

DEFF Research Database (Denmark)

Holst, B; Bruun, A W; Kielland-Brandt, Morten

1996-01-01

Using carboxypeptidase Y in Saccharomyces cerevisiae as a model system, the in vivo relationship between protein folding and N-glycosylation was studied. Seven new sites for N-glycosylation were introduced at positions buried in the folded protein structure. The level of glycosylation of such new...... acceptor sites. In some cases, all the newly synthesized mutant protein was modified at the novel site while in others no modification took place. In the most interesting category of mutants, the level of glycosylation was dependent on the conditions for folding. This shows that folding and glycosylation...

Cancer cell spheroids are a better screen for the photodynamic efficiency of glycosylated photosensitizers.

Directory of Open Access Journals (Sweden)

Patrícia M R Pereira

Full Text Available Photodynamic Therapy (PDT relies on the use of non-toxic photosensitizers that are locally and selectively activated by light to induce cell death or apoptosis through reactive oxygen species generation. The conjugation of porphyrinoids with sugars that target cancer is increasingly viewed as an effective way to increase the selectivity of PDT. To date, in vitro PDT efficacy is mostly screened using two-dimensional monolayer cultures. Compared to monolayer cultures, three-dimensional spheroid cultures have unique spatial distributions of nutrients, metabolites, oxygen and signalling molecules; therefore better mimic in vivo conditions. We obtained 0.05 mm3 spheroids with four different human tumor cell lines (HCT-116, MCF-7, UM-UC-3 and HeLa with appropriate sizes for screening PDT agents. We observed that detachment from monolayer culture and growth as tumor spheroids was accompanied by changes in glucose metabolism, endogenous ROS levels, galectin-1 and glucose transporter GLUT1 protein levels. We compared the phototoxic responses of a porphyrin conjugated with four glucose molecules (PorGlu4 in monolayer and spheroid cultures. The uptake and phototoxicity of PorGlu4 is highly dependent on the monolayer versus spheroid model used and on the different levels of GLUT1 protein expressed by these in vitro platforms. This study demonstrates that HCT-116, MCF-7, UM-UC-3 and HeLa spheroids afford a more rational platform for the screening of new glycosylated-photosensitizers compared to monolayer cultures of these cancer cells.

Nonenzymatic glycosylation of bovine myelin basic protein

International Nuclear Information System (INIS)

Hitz, J.B.

1987-01-01

In the CNS myelin sheath the nonenzymatic glycosylation reaction (at the early stage of the Amadori product) occurs only with the myelin basic protein and not with the other myelin proteins. This was observed in isolated bovine myelin by in vitro incubation with [ 14 C]-galactose and [ 14 C]-glucose. The respective in-vitro incorporation rates for purified bovine myelin basic protein with D-galactose, D-glucose and D-mannose were 7.2, 2.4 and 2.4 mmoles/mole myelin basic protein per day at 37 0 C. A more rapid, HPLC method was devised and characterized to specifically analyze for the Amadori product. The HPLC method was correlated to the [ 14 C]-sugar incorporation method for myelin basic protein under a set of standard reaction conditions using [ 14 C]-glucose and [ 14 C]-mannose with HPLC values at 1/6 and 1/5 of the [ 14 C]-sugar incorporation method. A novel myelin basic protein purification step has been developed that yields a relativity proteolytic free preparation that is easy to work with, being totally soluble at a neutral pH. Nine new spots appear for a trypsinized glycosylated MBP in the paper peptide map of which eight correspond to positions of the [ 3 H]-labeled Amadori product in affinity isolated peptides. These studies provide a general characterization of and a structural basis for investigations on nonenzymatically glycosylated MBP as well as identifying MBP as the only nonenzymatically glycosylated protein in the CNS myelin sheath which may accumulate during aging, diabetes, and demyelinating diseases in general

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

International Nuclear Information System (INIS)

Jones, Meredith B.; Tomiya, Noboru; Betenbaugh, Michael J.; Krag, Sharon S.

2010-01-01

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man 5 GlcNAc 2 -P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man 9 GlcNAc 2 -P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc 3 Man 9 GlcNAc 2 -P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man 5 GlcNAc 2 -PP-Dol through Glc 1 Man 9 GlcNAc 2 -PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc 3 Man 9 GlcNAc 2 -P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

Analysis and metabolic engineering of lipid-linked oligosaccharides in glycosylation-deficient CHO cells

Energy Technology Data Exchange (ETDEWEB)

Jones, Meredith B., E-mail: mbauman7@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Tomiya, Noboru, E-mail: ntomiya1@jhu.edu [Department of Biology, Johns Hopkins University, 3400 North Charles Street, Mudd Hall 104A, Baltimore, MD 21218 (United States); Betenbaugh, Michael J., E-mail: beten@jhu.edu [Department of Chemical and Biomolecular Engineering, Johns Hopkins University, 3400 North Charles Street, Maryland Hall 221, Baltimore, MD 21218 (United States); Krag, Sharon S., E-mail: skrag@jhsph.edu [Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, MD 21205 (United States)

2010-04-23

Glycosylation-deficient Chinese Hamster Ovary (CHO) cell lines can be used to expand our understanding of N-glycosylation pathways and to study Congenital Disorders of Glycosylation, diseases caused by defects in the synthesis of N-glycans. The mammalian N-glycosylation pathway involves the step-wise assembly of sugars onto a dolichol phosphate (P-Dol) carrier, forming a lipid-linked oligosaccharide (LLO), followed by the transfer of the completed oligosaccharide onto the protein of interest. In order to better understand how deficiencies in this pathway affect the availability of the completed LLO donor for use in N-glycosylation, we used a non-radioactive, HPLC-based assay to examine the intermediates in the LLO synthesis pathway for CHO-K1 cells and for three different glycosylation-deficient CHO cell lines. B4-2-1 cells, which have a mutation in the dolichol phosphate-mannose synthase (DPM2) gene, accumulated LLO with the structure Man{sub 5}GlcNAc{sub 2}-P-P-Dol, while MI8-5 cells, which lack glucosyltransferase I (ALG6) activity, accumulated Man{sub 9}GlcNAc{sub 2}-P-P-Dol. CHO-K1 and MI5-4 cells both produced primarily the complete LLO, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol, though the relative quantity was lower in MI5-4. MI5-4 cells have reduced hexokinase activity which could affect the availability of many of the substrates required for LLO synthesis and, consequently, impair production of the final LLO donor. Increasing hexokinase activity by overexpressing hexokinase II in MI5-4 caused a decrease in the relative quantities of the incomplete LLO intermediates from Man{sub 5}GlcNAc{sub 2}-PP-Dol through Glc{sub 1}Man{sub 9}GlcNAc{sub 2}-PP-Dol, and an increase in the relative quantity of the final LLO donor, Glc{sub 3}Man{sub 9}GlcNAc{sub 2}-P-P-Dol. This study suggests that metabolic engineering may be a useful strategy for improving LLO availability for use in N-glycosylation.

Defectively N-glycosylated and non-O-glycosylated aminopeptidase N (CD13) is normally expressed at the cell surface and has full enzymatic activity

DEFF Research Database (Denmark)

Norén, K; Hansen, Gert Helge; Clausen, H

1997-01-01

In order to study the effects of the absence of O-glycosylation and modifications of N-glycosylation on a class II membrane protein, pig and human aminopeptidase N (CD13) were stably expressed in the ldl(D) cell line. This cell line carries a UDP-Gal/UDP-GalNAc-epimerase deficiency which blocks...... the conversion of glucose into galactose derivatives. Thus it is possible in the ldl(D) cell line to selectively block O-glycosylation by the omission of N-acetylgalactoseamine from the culture medium and to alter N-glycosylation by the omission of galactose. In this way selectively altered glycosylated forms...

Carbohydrates on Proteins: Site-Specific Glycosylation Analysis by Mass Spectrometry

Science.gov (United States)

Zhu, Zhikai; Desaire, Heather

2015-07-01

Glycosylation on proteins adds complexity and versatility to these biologically vital macromolecules. To unveil the structure-function relationship of glycoproteins, glycopeptide-centric analysis using mass spectrometry (MS) has become a method of choice because the glycan is preserved on the glycosylation site and site-specific glycosylation profiles of proteins can be readily determined. However, glycopeptide analysis is still challenging given that glycopeptides are usually low in abundance and relatively difficult to detect and the resulting data require expertise to analyze. Viewing the urgent need to address these challenges, emerging methods and techniques are being developed with the goal of analyzing glycopeptides in a sensitive, comprehensive, and high-throughput manner. In this review, we discuss recent advances in glycoprotein and glycopeptide analysis, with topics covering sample preparation, analytical separation, MS and tandem MS techniques, as well as data interpretation and automation.

Nutritional Therapies in Congenital Disorders of Glycosylation (CDG

Directory of Open Access Journals (Sweden)

Peter Witters

2017-11-01

Full Text Available Congenital disorders of glycosylation (CDG are a group of more than 130 inborn errors of metabolism affecting N-linked, O-linked protein and lipid-linked glycosylation. The phenotype in CDG patients includes frequent liver involvement, especially the disorders belonging to the N-linked protein glycosylation group. There are only a few treatable CDG. Mannose-Phosphate Isomerase (MPI-CDG was the first treatable CDG by high dose mannose supplements. Recently, with the successful use of d-galactose in Phosphoglucomutase 1 (PGM1-CDG, other CDG types have been trialed on galactose and with an increasing number of potential nutritional therapies. Current mini review focuses on therapies in glycosylation disorders affecting liver function and dietary intervention in general in N-linked glycosylation disorders. We also emphasize now the importance of early screening for CDG in patients with mild hepatopathy but also in cholestasis.

Halide-mediated regioselective 6-O-glycosylation of unprotected hexopyranosides with perbenzylated glycosyl bromide donors

DEFF Research Database (Denmark)

Niedbal, Dominika Alina; Madsen, Robert

2016-01-01

The regio- and stereoselective glycosylation at the 6-position in 2,3,4,6-unprotected hexopyranosides has been investigated with dibutyltin oxide as the directing agent. Perbenzylated hexopyranosyl bromides were employed as the donors and the glycosylations were promoted by tetrabutylammonium...... bromide. The couplings were completely selective for both glucose and galactose donors and acceptors as long as the stannylene acetal of the acceptor was soluble in dichloromethane. This gave rise to a number of 1,2-cis-linked disaccharides in reasonable yields. Mannose donors and acceptors, on the other...

Glycosylation of the self-recognizing Escherichia coli Ag43 autotransporter protein

DEFF Research Database (Denmark)

Sherlock, O.; Dobrindt, U.; Jensen, J.B.

2006-01-01

a novel member to this exclusive group, namely, antigen 43 (Ag43), a self-recognizing autotransporter protein. By mass spectrometry Ag43 was demonstrated to be glycosylated by addition of heptose residues at several positions in the passenger domain. Glycosylation of Ag43 by the action of the Aah and Tib......C glycosyltransferases was observed in laboratory strains. Importantly, Ag43 was also found to be glycosylated in a wild-type strain, suggesting that Ag43-glycosylation may be a widespread phenomenon. Glycosylation of Ag43 does not seem to interfere with its self-associating properties. However, the glycosylated form...

Functional Analysis of Glycosylation of Zika Virus Envelope Protein

Directory of Open Access Journals (Sweden)

Camila R. Fontes-Garfias

2017-10-01

Full Text Available Summary: Zika virus (ZIKV infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. : Zika virus (ZIKV causes devastating congenital abnormities and Guillain-Barré syndrome. Fontes-Garfias et al. showed that the glycosylation of ZIKV envelope protein plays an important role in infecting mosquito vectors and pathogenesis in mouse. Keywords: Zika virus, glycosylation, flavivirus entry, mosquito transmission, vaccine

Toward stable genetic engineering of human o-glycosylation in plants

DEFF Research Database (Denmark)

Yang, Zhang; Bennett, Eric Paul; Jørgensen, Bodil

2012-01-01

Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Mucin-type (N-acetylgalactosamine [GalNAc]-type) O-glycosylation is found in eumetazoan cells but absent in plants and yeast, making these cell types...... an obvious choice for de novo engineering of this O-glycosylation pathway. We previously showed that transient implementation of O-glycosylation capacity in plants requires introduction of the synthesis of the donor substrate UDP-GalNAc and one or more polypeptide GalNAc-transferases for incorporating Gal......NAc residues into proteins. Here, we have stably engineered O-glycosylation capacity in two plant cell systems, soil-grown Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture cells. Efficient GalNAc O-glycosylation of two stably coexpressed substrate O...

Thermophilic and thermoacidophilic glycosylation genes and enzymes from Alicyclobacillus acidocaldarius and related organisms, methods

Science.gov (United States)

Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Reed, David W.; Lacey, Jeffrey A.

2016-01-12

Isolated and/or purified polypeptides and nucleic acid sequences encoding polypeptides from Alicyclobacillus acidocaldarius are provided. Further provided are methods for glycosylating and/or post-translationally modifying proteins using isolated and/or purified polypeptides and nucleic acid sequences from Alicyclobacillus acidocaldarius.

Trans-species Engineering of Glycosylated Therapeutic Proteins

DEFF Research Database (Denmark)

Yang, Zhang

eukaryotes and even prokaryotes. Insect and yeast cells produce O-glycosylation incompatible with use in humans, however recently the yeast Pichia was engineered to perform the first step of human-like O-glycosylation. This review provides an overview of past and current engineering efforts of N...

cancer metastasis and anti-cancer vaccines

Indian Academy of Sciences (India)

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 ...

Similarities and Differences in the Glycosylation Mechanisms in Prokaryotes and Eukaryotes

Directory of Open Access Journals (Sweden)

Anne Dell

2010-01-01

Full Text Available Recent years have witnessed a rapid growth in the number and diversity of prokaryotic proteins shown to carry N- and/or O-glycans, with protein glycosylation now considered as fundamental to the biology of these organisms as it is in eukaryotic systems. This article overviews the major glycosylation pathways that are known to exist in eukarya, bacteria and archaea. These are (i oligosaccharyltransferase (OST-mediated N-glycosylation which is abundant in eukarya and archaea, but is restricted to a limited range of bacteria; (ii stepwise cytoplasmic N-glycosylation that has so far only been confirmed in the bacterial domain; (iii OST-mediated O-glycosylation which appears to be characteristic of bacteria; and (iv stepwise O-glycosylation which is common in eukarya and bacteria. A key aim of the review is to integrate information from the three domains of life in order to highlight commonalities in glycosylation processes. We show how the OST-mediated N- and O-glycosylation pathways share cytoplasmic assembly of lipid-linked oligosaccharides, flipping across the ER/periplasmic/cytoplasmic membranes, and transferring “en bloc” to the protein acceptor. Moreover these hallmarks are mirrored in lipopolysaccharide biosynthesis. Like in eukaryotes, stepwise O-glycosylation occurs on diverse bacterial proteins including flagellins, adhesins, autotransporters and lipoproteins, with O-glycosylation chain extension often coupled with secretory mechanisms.

Glycosylation of the N-terminal potential N-glycosylation sites in the human α1,3-fucosyltransferase V and -VI (hFucTV and -VI)

DEFF Research Database (Denmark)

Christensen, Lise Lotte; Bross, Peter Gerd; Ørntoft, Torben Falck

2000-01-01

Human alpha1,3-fucosyltransferase V and -VI (hFucTV and -VI) each contain four potential N-glycosylation sites (hFucTV: Asn60, Asn105, Asn167 and Asn198 and hFucTVI: Asn46, Asn91, Asn153 and Asn184). Glycosylation of the two N-terminal potential N-glycosylation sites (hFucTV: Asn60, Asn105 and h......FucTVI: Asn46 and Asn91) have never been studied in detail. In the present study, we have analysed the glycosylation of these potential N-glycosylation sites. Initially, we compared the molecular mass of hFucTV and -VI expressed in COS-7 cells treated with tunicamycin with the mass of the proteins...... in untreated cells. The difference in molecular mass between the proteins in treated and untreated cells corresponded to the presence of at least three N-linked glycans. We then made a series of mutants, in which the asparagine residues in the N-terminal potential N-glycosylation sites were replaced...

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions.

Science.gov (United States)

Wang, Shengjun; Mao, Yang; Narimatsu, Yoshiki; Ye, Zilu; Tian, Weihua; Goth, Christoffer K; Lira-Navarrete, Erandi; Pedersen, Nis B; Benito-Vicente, Asier; Martin, Cesar; Uribe, Kepa B; Hurtado-Guerrero, Ramon; Christoffersen, Christina; Seidah, Nabil G; Nielsen, Rikke; Christensen, Erik I; Hansen, Lars; Bennett, Eric P; Vakhrushev, Sergey Y; Schjoldager, Katrine T; Clausen, Henrik

2018-05-11

The low-density lipoprotein receptor (LDLR) and related receptors are important for the transport of diverse biomolecules across cell membranes and barriers. Their functions are especially relevant for cholesterol homeostasis and diseases, including neurodegenerative and kidney disorders. Members of the LDLR-related protein family share LDLR class A (LA) repeats providing binding properties for lipoproteins and other biomolecules. We previously demonstrated that short linker regions between these LA repeats contain conserved O -glycan sites. Moreover, we found that O -glycan modifications at these sites are selectively controlled by the GalNAc-transferase isoform, GalNAc-T11. However, the effects of GalNAc-T11-mediated O -glycosylation on LDLR and related receptor localization and function are unknown. Here, we characterized O -glycosylation of LDLR-related proteins and identified conserved O -glycosylation sites in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O -glycosylation is not required for transport and cell-surface expression and stability of these receptors but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O -glycosylation increased affinity for LDL by ∼5-fold. The molecular basis for this observation is currently unknown, but these findings open up new avenues for exploring the roles of LDLR-related proteins in disease. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Comparative Glycoproteome Analysis: Dynamics of Protein Glycosylation during Metamorphic Transition from Pelagic to Benthic Life Stages in Three Invertebrates

KAUST Repository

Chandramouli, Kondethimmanahalli

2012-02-03

The life cycle of most benthic marine invertebrates has two distinct stages: the pelagic larval stage and the sessile juvenile stage. The transition between the larval stage and the juvenile stage is often abrupt and may be triggered by post-translational modification of proteins. Glycosylation, a very important post-translational modification, influences the biological activity of proteins. We used two-dimensional gel electrophoresis (2-DE) followed by glycoprotein-specific fluorescence staining and mass spectrometry with the goal of identifying glycosylation pattern changes during larval settlement and metamorphosis in barnacles, bryozoans, and polychaetes. Our results revealed substantial changes in the protein glycosylation patterns from larval to juvenile stages. Before metamorphosis, the degree of protein glycosylation was high in the barnacle Balanus (=Amphibalanus) amphitrite and the spionid polychaete Pseudopolydora vexillosa, whereas it increased after metamorphosis in the bryozoan Bugula neritina. We identified 19 abundant and differentially glycosylated proteins in these three species. Among the proteins, cellular stress- and metabolism-related proteins exhibited distinct glycosylation in B. amphitrite and B. neritina, whereas fatty acid metabolism-related proteins were abundantly glycosylated in P. vexillosa. Furthermore, the protein and gene expression analysis of some selected glycoproteins revealed that the degree of protein glycosylation did not always complement with transcriptional and translational changes associated with the larval-juvenile transition. The current study provides preliminary information on protein glycosylation in marine invertebrates that will serve as a solid basis for future comprehensive analysis of glycobiology during larval settlement and metamorphosis. © 2011 American Chemical Society.

NetOglyc: prediction of mucin type O-glycosylation sites based on sequence context and surface accessibility

DEFF Research Database (Denmark)

Hansen, Jan Erik; Lund, Ole; Tolstrup, Niels

1998-01-01

-glycosylated serine and threonine residues in independent test sets, thus proving more accurate than matrix statistics and vector projection methods. Predicition of O-glycosylation sites in the envelope glycoprotein gp120 from the primate lentiviruses HIV-1, HIV-2 and SIV are presented. The most conserved O...... structure and surface accessibility. The sequence context of glycosylated threonines was found to differ from that of serine, and the sites were found to cluster. Non-clustered sites had a sequence context different from that of clustered sites. charged residues were disfavoured at postition -1 and +3......-glycosylation signals in these evolutionary-related glycoproteins were found in their first hypervariable loop, V1. However, the strain variation for HIV-1 gp120 was significant. A computer server, available through WWW or E-mail, has been developed for prediction of mucin type O-glycosylation sites in proteins based...

Glycosylation in HIV-1 envelope glycoprotein and its biological implications

KAUST Repository

Ho, Yung Shwen

2013-08-01

Glycosylation of HIV-1 envelope proteins (Env gp120/gp41) plays a vital role in viral evasion from the host immune response, which occurs through the masking of key neutralization epitopes and the presentation of the Env glycosylation as \\'self\\' to the host immune system. Env glycosylation is generally conserved, yet its continual evolution plays an important role in modulating viral infectivity and Env immunogenicity. Thus, it is believed that Env glycosylation, which is a vital part of the HIV-1 architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may assist in effectively designing Env-based immunogens and in clearly understanding HIV vaccines. This review provides an in-depth perspective of various aspects of Env glycosylation in the context of HIV-1 pathogenesis. © 2013 Future Medicine Ltd.

[Non-enzymatic glycosylation of dietary protein in vitro].

Science.gov (United States)

Bednykh, B S; Evdokimov, I A; Sokolov, A I

2015-01-01

Non-enzymatic glycosylation of proteins, based on discovered by Mayarn reaction of carbohydrate aldehyde group with a free amino group of a protein molecule, is well known to experts in biochemistry of food industry. Generated brown solid in some cases give the product marketable qualities--crackling bread--in others conversely, worsen the product. The biological effects of far-advanced products of non-enzymatic protein glycosylation reaction have not been studied enough, although it was reported previously that they are not split by digestive enzymes and couldn't be absorbed by animals. The objective of this work was to compare the depth of glycosylation of different food proteins of animal and vegetable origin. The objects of the study were proteins of animal (casein, lactoglobulin, albumin) and vegetable (soy isolate, proteins of rice flour, buckwheat, oatmeal) origin, glucose and fructose were selected as glycosylation agents, exposure 15 days at 37 degrees C. Lactoglobulin was glycosylated to a lesser extent among the proteins of animal origin while protein of oatmeal was glycosylated in the least degree among vegetable proteins. Conversely, such proteins as casein and soya isolate protein bound rather large amounts of carbohydrates. Fructose binding with protein was generally higher than the binding of glucose. The only exception was a protein of oatmeal. When of glucose and fructose simultaneously presented in the incubation medium, glucose binding usually increased while binding of fructose, in contrast, reduced. According to the total amount of carbohydrate (mcg), which is able to attach a protein (mg) the studied food proteins located in the following order: albumin (38) > soy protein isolate (23) > casein (15,) > whey protein rice flour protein (6) > protein from buckwheat flour (3) > globulin (2) > protein of oatmeal (0.3). The results obtained are to be used to select the optimal combination of proteins and carbohydrates, in which the glycosylation

Functional Analysis of Glycosylation of Zika Virus Envelope Protein.

Science.gov (United States)

Fontes-Garfias, Camila R; Shan, Chao; Luo, Huanle; Muruato, Antonio E; Medeiros, Daniele B A; Mays, Elizabeth; Xie, Xuping; Zou, Jing; Roundy, Christopher M; Wakamiya, Maki; Rossi, Shannan L; Wang, Tian; Weaver, Scott C; Shi, Pei-Yong

2017-10-31

Zika virus (ZIKV) infection causes devastating congenital abnormities and Guillain-Barré syndrome. The ZIKV envelope (E) protein is responsible for viral entry and represents a major determinant for viral pathogenesis. Like other flaviviruses, the ZIKV E protein is glycosylated at amino acid N154. To study the function of E glycosylation, we generated a recombinant N154Q ZIKV that lacks the E glycosylation and analyzed the mutant virus in mammalian and mosquito hosts. In mouse models, the mutant was attenuated, as evidenced by lower viremia, decreased weight loss, and no mortality; however, knockout of E glycosylation did not significantly affect neurovirulence. Mice immunized with the mutant virus developed a robust neutralizing antibody response and were completely protected from wild-type ZIKV challenge. In mosquitoes, the mutant virus exhibited diminished oral infectivity for the Aedes aegypti vector. Collectively, the results demonstrate that E glycosylation is critical for ZIKV infection of mammalian and mosquito hosts. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Diversity in protein glycosylation among insect species.

Directory of Open Access Journals (Sweden)

Gianni Vandenborre

Full Text Available BACKGROUND: A very common protein modification in multicellular organisms is protein glycosylation or the addition of carbohydrate structures to the peptide backbone. Although the Class of the Insecta is the largest animal taxon on Earth, almost all information concerning glycosylation in insects is derived from studies with only one species, namely the fruit fly Drosophila melanogaster. METHODOLOGY/PRINCIPAL FINDINGS: In this report, the differences in glycoproteomes between insects belonging to several economically important insect orders were studied. Using GNA (Galanthus nivalis agglutinin affinity chromatography, different sets of glycoproteins with mannosyl-containing glycan structures were purified from the flour beetle (Tribolium castaneum, the silkworm (Bombyx mori, the honeybee (Apis mellifera, the fruit fly (D. melanogaster and the pea aphid (Acyrthosiphon pisum. To identify and characterize the purified glycoproteins, LC-MS/MS analysis was performed. For all insect species, it was demonstrated that glycoproteins were related to a broad range of biological processes and molecular functions. Moreover, the majority of glycoproteins retained on the GNA column were unique to one particular insect species and only a few glycoproteins were present in the five different glycoprotein sets. Furthermore, these data support the hypothesis that insect glycoproteins can be decorated with mannosylated O-glycans. CONCLUSIONS/SIGNIFICANCE: The results presented here demonstrate that oligomannose N-glycosylation events are highly specific depending on the insect species. In addition, we also demonstrated that protein O-mannosylation in insect species may occur more frequently than currently believed.

Mapping Sites of O-Glycosylation and Fringe Elongation on Drosophila Notch*

Science.gov (United States)

Harvey, Beth M.; Rana, Nadia A.; Moss, Hillary; Leonardi, Jessica; Jafar-Nejad, Hamed; Haltiwanger, Robert S.

2016-01-01

Glycosylation of the Notch receptor is essential for its activity and serves as an important modulator of signaling. Three major forms of O-glycosylation are predicted to occur at consensus sites within the epidermal growth factor-like repeats in the extracellular domain of the receptor: O-fucosylation, O-glucosylation, and O-GlcNAcylation. We have performed comprehensive mass spectral analyses of these three types of O-glycosylation on Drosophila Notch produced in S2 cells and identified peptides containing all 22 predicted O-fucose sites, all 18 predicted O-glucose sites, and all 18 putative O-GlcNAc sites. Using semiquantitative mass spectral methods, we have evaluated the occupancy and relative amounts of glycans at each site. The majority of the O-fucose sites were modified to high stoichiometries. Upon expression of the β3-N-acetylglucosaminyltransferase Fringe with Notch, we observed varying degrees of elongation beyond O-fucose monosaccharide, indicating that Fringe preferentially modifies certain sites more than others. Rumi modified O-glucose sites to high stoichiometries, although elongation of the O-glucose was site-specific. Although the current putative consensus sequence for O-GlcNAcylation predicts 18 O-GlcNAc sites on Notch, we only observed apparent O-GlcNAc modification at five sites. In addition, we performed mass spectral analysis on endogenous Notch purified from Drosophila embryos and found that the glycosylation states were similar to those found on Notch from S2 cells. These data provide foundational information for future studies investigating the mechanisms of how O-glycosylation regulates Notch activity. PMID:27268051

Optimization of a colorimetric assay for glycosylated human serum albumin

International Nuclear Information System (INIS)

Bohney, J.P.; Feldhoff, R.C.

1986-01-01

The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100 0 C. A NaBH 4 reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with [ 3 H]glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation

Links between CD147 Function, Glycosylation, and Caveolin-1

OpenAIRE

Tang, Wei; Chang, Sharon B.; Hemler, Martin E.

2004-01-01

Cell surface CD147 shows remarkable variations in size (31-65 kDa) because of heterogeneous N-glycosylation, with the most highly glycosylated forms functioning to induce matrix metalloproteinase (MMP) production. Here we show that all three CD147 N-glycosylation sites make similar contributions to both high and low glycoforms (HG- and LG-CD147). l-Phytohemagglutinin lectin binding and swainsonine inhibition experiments indicated that HG-CD147 contains N-acetylglucosaminyltransferase V-cataly...

Fasting serum glucose and glycosylated hemoglobin level in obesity.

Science.gov (United States)

Das, R K; Nessa, A; Hossain, M A; Siddiqui, N I; Hussain, M A

2014-04-01

Obesity is a condition in which the body fat stores are increased to an extent which impairs health and leads to serious health consequences. The amount of body fat is difficult to measure directly, and is usually determined from an indirect measure - the body mass index (BMI). Increased BMI in obese persons is directly associated with an increase in metabolic disease, such as type 2 diabetes mellitus. This Analytical cross sectional study was undertaken to assess the relation between obesity and glycemic control of body by measuring fasting serum glucose and glycosylated hemoglobin. This study was carried out in the Department of Physiology, Mymensingh Medical College, Mymensingh from 1st July 2011 to 30th June 2012 on 120 equally divided male and female persons within the age range of 25 to 55 years. Age more than 55 years and less than 25 years and diagnosed case of Hypothyroidism, Cushing's syndrome, polycystic ovary, Antipsychotic drug user and regular steroid users were excluded. Non probability purposive type of sampling technique was used for selecting the study subjects. Measurement of body mass index was done as per procedure. Fasting serum glucose was estimated by glucose oxidase method and Glycosylated hemoglobin by Boronate Affinity method. Statistical analysis was done by SPSS (version 17.0). Data were expressed as Mean±SE and statistical significance of difference among the groups were calculated by unpaired student's 't' test and Pearson's correlation coefficient tests were done as applicable. The Mean±SE of fasting serum glucose was significant at 1% level (P value obese group of BMI. There was no significant difference of glycosylated hemoglobin level between control and study groups. But there was positive correlation within each group. Fasting serum glucose also showed a bit stronger positive correlation with BMI. Both obese male and female persons showed higher levels of fasting serum glucose and glycosylated hemoglobin. The observed positive

N-linked glycosylation of the immunoglobulin variable region

NARCIS (Netherlands)

van de Bovenkamp, Fleur S.; Derksen, Ninotska I. L.; Ooijevaar-de Heer, Pleuni; van Schie, Karin A.; Kruithof, Simone; Berkowska, Magdalena A.; van der Schoot, C. Ellen; Ijspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E. M.; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S. Marieke; Vidarsson, Gestur; Rispens, Theo

2018-01-01

N-glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we

N-glycosylation of asparagine 8 regulates surface expression of major histocompatibility complex class I chain-related protein A (MICA) alleles dependent on threonine 24

DEFF Research Database (Denmark)

Pedersen, Maiken Mellergaard; Skovbakke, Sarah Line; Schneider, Christine L.

2014-01-01

for cell-surface expression and sought to identify the essential residues. We found that a single N-glycosylation site (N8) was important for MICA018 surface expression. The frequently expressed MICA allele 008, with an altered transmembrane and intracellular domain, was not affected by mutation of this N......-glycosylation site. Mutational analysis revealed that a single amino acid (T24) in the extracellular domain of MICA018 was essential for the N-glycosylation dependency, while the intracellular domain was not involved. The HHV7 immunoevasin, U21, was found to inhibit MICA018 surface expression by affecting N......-glycosylation and the retention was rescued by T24A substitution. Our study reveals N-glycosylation as an allele-specific regulatory mechanism important for regulation of surface expression of MICA018 and we pinpoint the residues essential for this N-glycosylation dependency. In addition we show that this regulatory mechanism...

[The role of protein glycosylation in immune system].

Science.gov (United States)

Ząbczyńska, Marta; Pocheć, Ewa

2015-01-01

Glycosylation is one of the most frequent post-translational modifications of proteins. The majority of cell surface and secreted proteins involved in immune response is glycosylated. The structural diversity of glycans depends on monosaccharide composition, type of glycosidic linkage and branching. These structural modifications determine a great variability of glycoproteins. The oligosaccharide components of proteins are regulated mostly by expression of glycosyltransferases and glycosidases and many environmental factors. Glycosylation influences the function of all immune cells. Glycans play a crucial role in intercellular contacts and leukocytes migration. These interactions are important in activation and proliferation of leukocytes and during immune response. The key immune proteins, such as TCR, MHC, TLR and antibodies are glycosylated. Sugars on the surface of pathogens and self-surface glycoproteins are recognized by special carbohydrate binding proteins called lectins. Changes of glycan structure are common in many pathological processes occurring in immune system, therefore they are used as molecular markers of different diseases.

Hydrophobic Man-1-P derivatives correct abnormal glycosylation in Type I congenital disorder of glycosylation fibroblasts.

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Eklund, Erik A; Merbouh, Nabyl; Ichikawa, Mie; Nishikawa, Atsushi; Clima, Jessica M; Dorman, James A; Norberg, Thomas; Freeze, Hudson H

2005-11-01

Patients with Type I congenital disorders of glycosylation (CDG-I) make incomplete lipid-linked oligosaccharides (LLO). These glycans are poorly transferred to proteins resulting in unoccupied glycosylation sequons. Mutations in phosphomannomutase (PMM2) cause CDG-Ia by reducing the activity of PMM, which converts mannose (Man)-6-P to Man-1-P before formation of GDP-Man. These patients have reduced Man-1-P and GDP-Man. To replenish intracellular Man-1-P pools in CDG-Ia cells, we synthesized two hydrophobic, membrane permeable acylated versions of Man-1-P and determined their ability to normalize LLO size and N-glycosylation in CDG-Ia fibroblasts. Both compounds, compound I (diacetoxymethyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate) (C-I) and compound II (diacetoxymethyl 2,3,4,6-tetra-O-ethyloxycarbonyl-alpha-D-mannopyranosyl phosphate) (C-II), contain two acetoxymethyl (CH2OAc) groups O-linked to phosphorous. C-I contains acetyl esters and C-II contains ethylcarbonate (CO2Et) esters on the Man residue. Both C-I and C-II normalized truncated LLO, but C-II was about 2-fold more efficient than C-I. C-II replenished the GDP-Man pool in CDG-Ia cells and was more efficiently incorporated into glycoproteins than exogenous Man at low concentrations (25-75 mM). In a glycosylation assay of DNaseI in CDG-Ia cells, C-II restored glycosylation to control cell levels. C-II also corrected impaired LLO biosynthesis in cells from a Dolichol (Dol)-P-Man deficient patient (CDG-Ie) and partially corrected LLO in cells from an ALG12 mannosyltransferase-deficient patient (CDG-Ig), whereas cells from an ALG3-deficient patient (CDG-Id) and from an MPDU1-deficient patient (CDG-If) were not corrected. These results validate the general concept of using pro-Man-1-P substrates as potential therapeutics for CDG-I patients.

N-glycosylation by N-acetylglucosaminyltransferase V enhances the interaction of CD147/basigin with integrin β1 and promotes HCC metastasis.

Science.gov (United States)

Cui, Jian; Huang, Wan; Wu, Bo; Jin, Jin; Jing, Lin; Shi, Wen-Pu; Liu, Zhen-Yu; Yuan, Lin; Luo, Dan; Li, Ling; Chen, Zhi-Nan; Jiang, Jian-Li

2018-05-01

While the importance of protein N-glycosylation in cancer cell migration is well appreciated, the precise mechanisms by which N-acetylglucosaminyltransferase V (GnT-V) regulates cancer processes remain largely unknown. In the current study, we report that GnT-V-mediated N-glycosylation of CD147/basigin, a tumor-associated glycoprotein that carries β1,6-N-acetylglucosamine (β1,6-GlcNAc) glycans, is upregulated during TGF-β1-induced epithelial-to-mesenchymal transition (EMT), which correlates with tumor metastasis in patients with hepatocellular carcinoma (HCC). Interruption of β1,6-GlcNAc glycan modification of CD147/basigin decreased matrix metalloproteinase (MMP) expression in HCC cell lines and affected the interaction of CD147/basigin with integrin β1. These results reveal that β1,6-branched glycans modulate the biological function of CD147/basigin in HCC metastasis. Moreover, we showed that the PI3K/Akt pathway regulates GnT-V expression and that inhibition of GnT-V-mediated N-glycosylation suppressed PI3K signaling. In summary, β1,6-branched N-glycosylation affects the biological function of CD147/basigin and these findings provide a novel approach for the development of therapeutic strategies targeting metastasis. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Polymorphism in the GALNT1 gene and epithelial ovarian cancer in non-Hispanic white women: the Ovarian Cancer Association Consortium

DEFF Research Database (Denmark)

Phelan, Catherine M; Tsai, Ya-Yu; Goode, Ellen L

2010-01-01

Aberrant glycosylation is a well-described hallmark of cancer. In a previous ovarian cancer case control study that examined polymorphisms in 26 glycosylation-associated genes, we found strong statistical evidence (P = 0.00017) that women who inherited two copies of a single-nucleotide polymorphism...... sites but significant differences compared with the original study population (P = 0.03). This study underscores the need for replication of putative findings in genetic association studies....

Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry.

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Zhang, Yong; Xie, Xinfang; Zhao, Xinyuan; Tian, Fang; Lv, Jicheng; Ying, Wantao; Qian, Xiaohong

2018-01-06

Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O-glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macroheterogeneity of O-glycosylation, site-specific O-glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O-glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O-glycopeptides, 173 O-glycosylation sites and 6 types of O-glycans originating from 49 O-glycoprotein groups were identified in human serum, including 121 novel O-glycosylation sites. Currently, this is the largest data set of site-specific native O-glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O-glycosylation in human health and diseases. The altered O-glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O-glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O-glycosylation. In this work, we developed a systematic strategy for intact O-glycopeptide characterization. This study took

Optimization of a colorimetric assay for glycosylated human serum albumin

Energy Technology Data Exchange (ETDEWEB)

Bohney, J.P.; Feldhoff, R.C.

1986-05-01

The thiobarbituric acid (TBA) assay has been used for several years to quantitate the amount of glucose which has been non-enzymatically linked to hemoglobin and other proteins. The ketoamine-protein adduct is converted to 5-hydroxymethylfurfural (HMF) by mild hydrolysis with oxalic acid. Reaction of HMF with TBA yields a colored product which has an absorbance maximum at 443 nm. Several modifications of the original procedure has been published, but none permit the unambiguous quantitation of glycosylated human serum albumin (glc-HSA). Problems relate to reagent preparation and stability, the time and temperature of hydrolysis, the choice of standards, and background color corrections. The authors have found that maximum color yield occurs after hydrolysis in an autoclave for 2 h. This increases the sensitivity 3-fold and cuts the assay time in half relative to hydrolysis for 4.5 h at 100/sup 0/C. A NaBH/sub 4/ reduction of a parallel protein sample must be performed to correct for variable background color associated with different sample sources and amounts. HMF can be used as a standard, however, corrections must be made for HMF degradation. Fructose is a better standard, but HMF formation from fructose is faster than formation from glc-HSA. This may result in an underestimate of percent glycosylation. The best standard appears to be glc-HSA prepared with (/sup 3/H)glucose. It appears that with proper controls and standards the TBA assay can be used to determine actual rather than relative percent glycosylation.

Glycosylation-related gene expression in HT29-MTX-E12 cells upon infection by Helicobacter pylori.

Science.gov (United States)

Cairns, Michael T; Gupta, Ananya; Naughton, Julie A; Kane, Marian; Clyne, Marguerite; Joshi, Lokesh

2017-10-07

To identify glycosylation-related genes in the HT29 derivative cell line, HT29-MTX-E12, showing differential expression on infection with Helicobacter pylori ( H. pylori ). Polarised HT29-MTX-E12 cells were infected for 24 h with H. pylori strain 26695. After infection RNA was isolated from both infected and non-infected host cells. Sufficient infections were carried out to provide triplicate samples for microarray analysis and for qRT-PCR analysis. RNA was isolated and hybridised to Affymetrix arrays. Analysis of microarray data identified genes significantly differentially expressed upon infection. Genes were grouped into gene ontology functional categories. Selected genes associated with host glycan structure (glycosyltransferases, hydrolases, lectins, mucins) were validated by real-time qRT-PCR analysis. Infection of host cells was confirmed by the isolation of live bacteria after 24 h incubation and by PCR amplification of bacteria-specific genes from the host cell RNA. H. pylori do not survive incubation under the adopted culture conditions unless they associate with the adherent mucus layer of the host cell. Microarray analysis identified a total of 276 genes that were significantly differentially expressed ( P < 0.05) upon H. pylori infection and where the fold change in expression was greater than 2. Six of these genes are involved in glycosylation-related processes. Real-time qRT-PCR demonstrated significant downregulation (1.8-fold, P < 0.05) of the mucin MUC20. REG4 was heavily expressed and significantly downregulated (3.1-fold, P < 0.05) upon infection. Gene ontology analysis was consistent with previous studies on H. pylori infection. Gene expression data suggest that infection with H. pylori causes a decrease in glycan synthesis, resulting in shorter and simpler glycan structures.

Glycosylation Helps Cellulase Enzymes Bind to Plant Cell Walls (Fact Sheet)

Energy Technology Data Exchange (ETDEWEB)

2012-06-01

Computer simulations suggest a new strategy to design enhanced enzymes for biofuels production. Large-scale computer simulations predict that the addition of glycosylation on carbohydrate-binding modules can dramatically improve the binding affinity of these protein domains over amino acid mutations alone. These simulations suggest that glycosylation can be used as a protein engineering tool to enhance the activity of cellulase enzymes, which are a key component in the conversion of cellulose to soluble sugars in the production of biofuels. Glycosylation is the covalent attachment of carbohydrate molecules to protein side chains, and is present in many proteins across all kingdoms of life. Moreover, glycosylation is known to serve a wide variety of functions in biological recognition, cell signaling, and metabolism. Cellulase enzymes, which are responsible for deconstructing cellulose found in plant cell walls to glucose, contain glycosylation that when modified can affect enzymatic activity-often in an unpredictable manner. To gain insight into the role of glycosylation on cellulase activity, scientists at the National Renewable Energy Laboratory (NREL) used computer simulation to predict that adding glycosylation on the carbohydrate-binding module of a cellulase enzyme dramatically boosts the binding affinity to cellulose-more than standard protein engineering approaches in which amino acids are mutated. Because it is known that higher binding affinity in cellulases leads to higher activity, this work suggests a new route to designing enhanced enzymes for biofuels production. More generally, this work suggests that tuning glycosylation in cellulase enzymes is a key factor to consider when engineering biochemical conversion processes, and that more work is needed to understand how glycosylation affects cellulase activity at the molecular level.

Surface glycosylation profiles of urine extracellular vesicles.

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Jared Q Gerlach

Full Text Available Urinary extracellular vesicles (uEVs are released by cells throughout the nephron and contain biomolecules from their cells of origin. Although uEV-associated proteins and RNA have been studied in detail, little information exists regarding uEV glycosylation characteristics. Surface glycosylation profiling by flow cytometry and lectin microarray was applied to uEVs enriched from urine of healthy adults by ultracentrifugation and centrifugal filtration. The carbohydrate specificity of lectin microarray profiles was confirmed by competitive sugar inhibition and carbohydrate-specific enzyme hydrolysis. Glycosylation profiles of uEVs and purified Tamm Horsfall protein were compared. In both flow cytometry and lectin microarray assays, uEVs demonstrated surface binding, at low to moderate intensities, of a broad range of lectins whether prepared by ultracentrifugation or centrifugal filtration. In general, ultracentrifugation-prepared uEVs demonstrated higher lectin binding intensities than centrifugal filtration-prepared uEVs consistent with lesser amounts of co-purified non-vesicular proteins. The surface glycosylation profiles of uEVs showed little inter-individual variation and were distinct from those of Tamm Horsfall protein, which bound a limited number of lectins. In a pilot study, lectin microarray was used to compare uEVs from individuals with autosomal dominant polycystic kidney disease to those of age-matched controls. The lectin microarray profiles of polycystic kidney disease and healthy uEVs showed differences in binding intensity of 6/43 lectins. Our results reveal a complex surface glycosylation profile of uEVs that is accessible to lectin-based analysis following multiple uEV enrichment techniques, is distinct from co-purified Tamm Horsfall protein and may demonstrate disease-specific modifications.

Aberrant Glycosylation in the Left Ventricle and Plasma of Rats with Cardiac Hypertrophy and Heart Failure.

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Chiaki Nagai-Okatani

Full Text Available Targeted proteomics focusing on post-translational modifications, including glycosylation, is a useful strategy for discovering novel biomarkers. To apply this strategy effectively to cardiac hypertrophy and resultant heart failure, we aimed to characterize glycosylation profiles in the left ventricle and plasma of rats with cardiac hypertrophy. Dahl salt-sensitive hypertensive rats, a model of hypertension-induced cardiac hypertrophy, were fed a high-salt (8% NaCl diet starting at 6 weeks. As a result, they exhibited cardiac hypertrophy at 12 weeks and partially impaired cardiac function at 16 weeks compared with control rats fed a low-salt (0.3% NaCl diet. Gene expression analysis revealed significant changes in the expression of genes encoding glycosyltransferases and glycosidases. Glycoproteome profiling using lectin microarrays indicated upregulation of mucin-type O-glycosylation, especially disialyl-T, and downregulation of core fucosylation on N-glycans, detected by specific interactions with Amaranthus caudatus and Aspergillus oryzae lectins, respectively. Upregulation of plasma α-l-fucosidase activity was identified as a biomarker candidate for cardiac hypertrophy, which is expected to support the existing marker, atrial natriuretic peptide and its related peptides. Proteomic analysis identified cysteine and glycine-rich protein 3, a master regulator of cardiac muscle function, as an O-glycosylated protein with altered glycosylation in the rats with cardiac hypertrophy, suggesting that alternations in O-glycosylation affect its oligomerization and function. In conclusion, our data provide evidence of significant changes in glycosylation pattern, specifically mucin-type O-glycosylation and core defucosylation, in the pathogenesis of cardiac hypertrophy and heart failure, suggesting that they are potential biomarkers for these diseases.

Chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines formed by cross-link of bone and synovium collagen.

Science.gov (United States)

Anastasia, Luigi; Rota, Paola; Anastasia, Mario; Allevi, Pietro

2013-09-21

This review focuses on the chemical structure, biosynthesis and synthesis of free and glycosylated pyridinolines (Pyds), fluorescent collagen cross-links, with a pyridinium salt structure. Pyds derive from the degradation of bone collagen and have attracted attention for their use as biochemical markers of bone resorption and to assess fracture risk prediction in persons suffering from osteoporosis, bone cancer and other bone or collagen diseases. We consider and critically discuss all reported syntheses of free and glycosylated Pyds evidencing an unrevised chemistry, original and of general utility, analysis of which allows us to also support a previously suggested non-enzymatic formation of Pyds in collagen better rationalizing and justifying the chemical events.

Role of structure and glycosylation of adsorbed protein films in biolubrication.

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Deepak H Veeregowda

Full Text Available Water forms the basis of lubrication in the human body, but is unable to provide sufficient lubrication without additives. The importance of biolubrication becomes evident upon aging and disease, particularly under conditions that affect secretion or composition of body fluids. Insufficient biolubrication, may impede proper speech, mastication and swallowing, underlie excessive friction and wear of articulating cartilage surfaces in hips and knees, cause vaginal dryness, and result in dry, irritated eyes. Currently, our understanding of biolubrication is insufficient to design effective therapeutics to restore biolubrication. Aim of this study was to establish the role of structure and glycosylation of adsorbed protein films in biolubrication, taking the oral cavity as a model and making use of its dynamics with daily perturbations due to different glandular secretions, speech, drinking and eating, and tooth brushing. Using different surface analytical techniques (a quartz crystal microbalance with dissipation monitoring, colloidal probe atomic force microscopy, contact angle measurements and X-ray photo-electron spectroscopy, we demonstrated that adsorbed salivary conditioning films in vitro are more lubricious when their hydrophilicity and degree of glycosylation increase, meanwhile decreasing their structural softness. High-molecular-weight, glycosylated proteins adsorbing in loops and trains, are described as necessary scaffolds impeding removal of water during loading of articulating surfaces. Comparing in vitro and in vivo water contact angles measured intra-orally, these findings were extrapolated to the in vivo situation. Accordingly, lubricating properties of teeth, as perceived in 20 volunteers comprising of equal numbers of male and female subjects, could be related with structural softness and glycosylation of adsorbed protein films on tooth surfaces. Summarizing, biolubrication is due to a combination of structure and glycosylation

Quantifying risk of penile prosthesis infection with elevated glycosylated hemoglobin.

Science.gov (United States)

Wilson, S K; Carson, C C; Cleves, M A; Delk, J R

1998-05-01

Elevation of glycosylated hemoglobin above levels of 11.5 mg.% has been considered a contraindication to penile prosthesis implantation in diabetic patients. We determine the predictive value of glycosylated hemoglobin A1C in penile prosthesis infections in diabetic and nondiabetic patients to confirm or deny this prevalent opinion. We conducted a 2-year prospective study of 389 patients, including 114 diabetics, who underwent 3-piece penile prosthesis implantation. All patients had similar preoperative preparation without regard to diabetic status, control or glycosylated hemoglobin A1C level. Risk of infection was statistically analyzed for diabetics versus nondiabetics, glycosylated hemoglobin A1C values above and below 11.5 mg.%, insulin dependent versus oral medication diabetics, and fasting blood sugars above and below 180 mg.%. Prosthesis infections developed in 10 diabetics (8.7%) and 11 nondiabetics (4.0%). No increased infection rate was observed in diabetics with high fasting sugars or diabetics on insulin. There was no statistically significant increased infection risk with increased levels of glycosylated hemoglobin A1C among all patients or among only the diabetics. In fact, there was no meaningful difference in the median or mean level of glycosylated hemoglobin A1C in the infected and noninfected patients regardless of diabetes. Use of glycosylated hemoglobin A1C values to identify and exclude surgical candidates with increased risk of infections is not proved by this study. Elevation of fasting sugar or insulin dependence also does not increase risk of infection in diabetics undergoing prosthesis implantation.

Method Development in the Regioselective Glycosylation of Unprotected Carbohydrates

DEFF Research Database (Denmark)

Niedbal, Dominika Alina

and the glycosylations were promoted by tetrabutylammonium bromide. The couplings were completely selective and gave rise to a number of 1,6-linked disaccharides with 1,2- cis-linked orientation. Project 2: Boron-mediated glycosylation of unprotected carbohydrates Boron-mediated regioselective Koenigs...

Marked increase in rat red blood cell membrane protein glycosylation by one-month treatment with a cafeteria diet

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Laia Oliva

2015-07-01

RBC were lower than those of plasma, even when expressed in molal units, and were practically nil in cafeteria-diet fed rats compared with controls; there was no effect of sex.Conclusions. RBC membrane glycosylation is a sensitive indicator of developing metabolic syndrome-related hyperglycemia, more sensitive than the general measurement of plasma or RBC protein glycosylation. The extensive glycosylation of blood proteins does not seem to be markedly affected by sex; and could be hardly justified from an assumedly sustained plasma hyperglycemia. The low levels of glucose found within RBC, especially in rats under the cafeteria diet, could hardly justify the extensive glycosylation of hemoglobin and the lack of differences with controls, which contained sizeable levels of intracellular glucose. Additional studies are needed to study the dynamics of glucose in vivo in the RBC to understand how such extensive protein glycosylation could take place.

Molecular partners of hNOT/ALG3, the human counterpart of the Drosophila NOT and yeast ALG3 gene, suggest its involvement in distinct cellular processes relevant to congenital disorders of glycosylation, cancer, neurodegeneration and a variety of further pathologies.

Science.gov (United States)

Hacker, Benedikt; Schultheiß, Christoph; Döring, Michael; Kurzik-Dumke, Ursula

2018-06-01

This study provides first insights into the involvement of hNOT/ALG3, the human counterpart of the Drosophila Neighbour of TID and yeast ALG3 gene, in various putative molecular networks. HNOT/ALG3 encodes two translated transcripts encoding precursor proteins differing in their N-terminus and showing 33% identity with the yeast asparagine-linked glycosylation 3 (ALG3) protein. Experimental evidence for the functional homology of the proteins of fly and man in the N-glycosylation has still to be provided. In this study, using the yeast two-hybrid technique we identify 17 molecular partners of hNOT-1/ALG3-1. We disclose the building of hNOT/ALG3 homodimers and provide experimental evidence for its in vivo interaction with the functionally linked proteins OSBP, OSBPL9 and LRP1, the SYPL1 protein and the transcription factor CREB3. Regarding the latter, we show that the 55 kDa N-glycosylated hNOT-1/ALG3-1 molecule binds the N-glycosylated CREB3 precursor but does not interact with CREB3's proteolytic products specific to the endoplasmic reticulum and to the nucleus. The interaction between the two partners is a prerequisite for the proteolytic activation of CREB3. In case of the further binding partners, our data suggest that hNOT-1/ALG3-1 interacts with both OSBPs and with their direct targets LRP1 and VAMP/VAP-A. Moreover, our results show that various partners of hNOT-1/ALG3-1 interact with its diverse post translationally processed products destined to distinct cellular compartments. Generally, our data suggest the involvement of hNOT-1/ALG3-1 in various molecular contexts determining essential processes associated with distinct cellular machineries and related to various pathologies, such as cancer, viral infections, neuronal and immunological disorders and CDG.

N-Glycosylation of cholera toxin B subunit: serendipity for novel plant-made vaccines?

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Nobuyuki eMatoba

2015-12-01

Full Text Available The non-toxic B subunit of cholera toxin (CTB has attracted considerable interests from vaccinologists due to strong mucosal immunomodulatory effects and potential utility as a vaccine scaffold for heterologous antigens. Along with other conventional protein expression systems, various plant species have been used as recombinant production hosts for CTB and its fusion proteins. However, it has recently become clear that the protein is N-glycosylated within the endoplasmic reticulum of plant cells – a eukaryotic post-translational modification that is not present in native CTB. While functionally active aglycosylated variants have been successfully engineered to circumvent potential safety and regulatory issues related to glycosylation, this modification may actually provide advantageous characteristics to the protein as a vaccine platform. Based on data from our recent studies, I discuss the unique features of N-glycosylated CTB produced in plants for the development of novel vaccines.

General N-and O-Linked Glycosylation of Lipoproteins in Mycoplasmas and Role of Exogenous Oligosaccharide.

Science.gov (United States)

Daubenspeck, James M; Jordan, David S; Simmons, Warren; Renfrow, Matthew B; Dybvig, Kevin

2015-01-01

The lack of a cell wall, flagella, fimbria, and other extracellular appendages and the possession of only a single membrane render the mycoplasmas structurally simplistic and ideal model organisms for the study of glycoconjugates. Most species have genomes of about 800 kb and code for few proteins predicted to have a role in glycobiology. The murine pathogens Mycoplasma arthritidis and Mycoplasma pulmonis have only a single gene annotated as coding for a glycosyltransferase but synthesize glycolipid, polysaccharide and glycoproteins. Previously, it was shown that M. arthritidis glycosylated surface lipoproteins through O-linkage. In the current study, O-linked glycoproteins were similarly found in M. pulmonis and both species of mycoplasma were found to also possess N-linked glycans at residues of asparagine and glutamine. Protein glycosylation occurred at numerous sites on surface-exposed lipoproteins with no apparent amino acid sequence specificity. The lipoproteins of Mycoplasma pneumoniae also are glycosylated. Glycosylation was dependent on the glycosidic linkages from host oligosaccharides. As far as we are aware, N-linked glycoproteins have not been previously described in Gram-positive bacteria, the organisms to which the mycoplasmas are phylogenetically related. The findings indicate that the mycoplasma cell surface is heavily glycosylated with implications for the modulation of mycoplasma-host interactions.

Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment.

Science.gov (United States)

Song, Ehwang; Mechref, Yehia

2015-01-01

Protein glycosylation is an important and common post-translational modification. More than 50% of human proteins are believed to be glycosylated to modulate the functionality of proteins. Aberrant glycosylation has been correlated to several diseases, such as inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer's and prion diseases, and cancer. Many approved cancer biomarkers are glycoproteins which are not highly abundant proteins. Therefore, effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. This chapter summarizes glycoprotein enrichment methods, including lectin affinity, immunoaffinity, hydrazide chemistry, hydrophilic interaction liquid chromatography, and click chemistry. The use of these enrichment approaches in assessing the qualitative and quantitative changes of glycoproteins in different types of cancers are presented and discussed. This chapter highlights the importance of glycoprotein enrichment techniques for the identification and characterization of new reliable cancer biomarkers.

Crystallization and preliminary X-ray diffraction of human interleukin-7 bound to unglycosylated and glycosylated forms of its α-receptor

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Wickham, Joseph Jr; Walsh, Scott T. R., E-mail: walsh.220@osu.edu [Department of Molecular and Cellular Biochemistry, Comprehensive Cancer Center, Ohio State University, 467 Hamilton Hall, 1645 Neil Avenue, Columbus, OH 43210 (United States)

2007-10-01

Bacterial and insect cell expression systems have been developed to produce unglycosylated and glycosylated forms of human interleukin-7 (IL-7) and the extracellular domain of its α receptor, IL-7Rα. We report the crystallization and X-ray diffraction of IL-7 complexes to both unglycosylated and glycosylated forms of the IL-7Rα to 2.7 and 3.0 Å, respectively. The interleukin-7 (IL-7) signaling pathway plays an essential role in the development, proliferation and homeostasis of T and B cells in cell-mediated immunity. Understimulation and overstimulation of the IL-7 signaling pathway leads to severe combined immunodeficiency, autoimmune reactions, heart disease and cancers. Stimulation of the IL-7 pathway begins with IL-7 binding to its α-receptor, IL-7Rα. Protein crystals of unglycosylated and glycosylated complexes of human IL-7–IL-7Rα extracellular domain (ECD) obtained using a surface entropy-reduction approach diffract to 2.7 and 3.0 Å, respectively. Anomalous dispersion methods will be used to solve the unglycosylated IL-7–IL-7Rα ECD complex structure and this unglycosylated structure will then serve as a model in molecular-replacement attempts to solve the structure of the glycosylated IL-7–α-receptor complex.

Crystallization and preliminary X-ray diffraction of human interleukin-7 bound to unglycosylated and glycosylated forms of its α-receptor

International Nuclear Information System (INIS)

Wickham, Joseph Jr; Walsh, Scott T. R.

2007-01-01

Bacterial and insect cell expression systems have been developed to produce unglycosylated and glycosylated forms of human interleukin-7 (IL-7) and the extracellular domain of its α receptor, IL-7Rα. We report the crystallization and X-ray diffraction of IL-7 complexes to both unglycosylated and glycosylated forms of the IL-7Rα to 2.7 and 3.0 Å, respectively. The interleukin-7 (IL-7) signaling pathway plays an essential role in the development, proliferation and homeostasis of T and B cells in cell-mediated immunity. Understimulation and overstimulation of the IL-7 signaling pathway leads to severe combined immunodeficiency, autoimmune reactions, heart disease and cancers. Stimulation of the IL-7 pathway begins with IL-7 binding to its α-receptor, IL-7Rα. Protein crystals of unglycosylated and glycosylated complexes of human IL-7–IL-7Rα extracellular domain (ECD) obtained using a surface entropy-reduction approach diffract to 2.7 and 3.0 Å, respectively. Anomalous dispersion methods will be used to solve the unglycosylated IL-7–IL-7Rα ECD complex structure and this unglycosylated structure will then serve as a model in molecular-replacement attempts to solve the structure of the glycosylated IL-7–α-receptor complex

The Emerging Importance of IgG Fab Glycosylation in Immunity.

Science.gov (United States)

van de Bovenkamp, Fleur S; Hafkenscheid, Lise; Rispens, Theo; Rombouts, Yoann

2016-02-15

Human IgG is the most abundant glycoprotein in serum and is crucial for protective immunity. In addition to conserved IgG Fc glycans, ∼15-25% of serum IgG contains glycans within the variable domains. These so-called "Fab glycans" are primarily highly processed complex-type biantennary N-glycans linked to N-glycosylation sites that emerge during somatic hypermutation. Specific patterns of Fab glycosylation are concurrent with physiological and pathological conditions, such as pregnancy and rheumatoid arthritis. With respect to function, Fab glycosylation can significantly affect stability, half-life, and binding characteristics of Abs and BCRs. Moreover, Fab glycans are associated with the anti-inflammatory activity of IVIgs. Consequently, IgG Fab glycosylation appears to be an important, yet poorly understood, process that modulates immunity. Copyright © 2016 by The American Association of Immunologists, Inc.

The S-Layer Glycoprotein of the Crenarchaeote Sulfolobus acidocaldarius Is Glycosylated at Multiple Sites with Chitobiose-Linked N-Glycans

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Elham Peyfoon

2010-01-01

Full Text Available Glycosylation of the S-layer of the crenarchaea Sulfolobus acidocaldarius has been investigated using glycoproteomic methodologies. The mature protein is predicted to contain 31 N-glycosylation consensus sites with approximately one third being found in the C-terminal domain spanning residues L1004-Q1395. Since this domain is rich in Lys and Arg and therefore relatively tractable to glycoproteomic analysis, this study has focused on mapping its N-glycosylation. Our analysis identified nine of the 11 consensus sequence sites, and all were found to be glycosylated. This constitutes a remarkably high glycosylation density in the C-terminal domain averaging one site for each stretch of 30–40 residues. Each of the glycosylation sites observed was shown to be modified with a heterogeneous family of glycans, with the largest having a composition Glc1Man2GlcNAc2 plus 6-sulfoquinovose (QuiS, consistent with the tribranched hexasaccharide previously reported in the cytochrome b558/566 of S. acidocaldarius. S. acidocaldarius is the only archaeal species whose N-glycans are known to be linked via the chitobiose core disaccharide that characterises the N-linked glycans of Eukarya.

Deciphering a pathway of Halobacterium salinarum N-glycosylation

Science.gov (United States)

Kandiba, Lina; Eichler, Jerry

2015-01-01

Genomic analysis points to N-glycosylation as being a common posttranslational modification in Archaea. To date, however, pathways of archaeal N-glycosylation have only been described for few species. With this in mind, the similarities of N-linked glycans decorating glycoproteins in the haloarchaea Haloferax volcanii and Halobacterium salinarum directed a series of bioinformatics, genetic, and biochemical experiments designed to describe that Hbt. salinarum pathway responsible for biogenesis of one of the two N-linked oligosaccharides described in this species. As in Hfx. volcanii, where agl (archaeal glycosylation) genes that encode proteins responsible for the assembly and attachment of a pentasaccharide to target protein Asn residues are clustered in the genome, Hbt. salinarum also contains a group of clustered homologous genes (VNG1048G-VNG1068G). Introduction of these Hbt. salinarum genes into Hfx. volcanii mutant strains deleted of the homologous sequence restored the lost activity. Moreover, transcription of the Hbt. salinarum genes in the native host, as well as in vitro biochemical confirmation of the predicted functions of several of the products of these genes provided further support for assignments made following bioinformatics and genetic experiments. Based on the results obtained in this study, the first description of an N-glycosylation pathway in Hbt. salinarum is offered. PMID:25461760

Flagellar glycosylation in Clostridium botulinum.

Science.gov (United States)

Twine, Susan M; Paul, Catherine J; Vinogradov, Evgeny; McNally, David J; Brisson, Jean-Robert; Mullen, James A; McMullin, David R; Jarrell, Harold C; Austin, John W; Kelly, John F; Logan, Susan M

2008-09-01

Flagellins from Clostridium botulinum were shown to be post-translationally modified with novel glycan moieties by top-down MS analysis of purified flagellin protein from strains of various toxin serotypes. Detailed analyses of flagellin from two strains of C. botulinum demonstrated that the protein is modified by a novel glycan moiety of mass 417 Da in O-linkage. Bioinformatic analysis of available C. botulinum genomes identified a flagellar glycosylation island containing homologs of genes recently identified in Campylobacter coli that have been shown to be responsible for the biosynthesis of legionaminic acid derivatives. Structural characterization of the carbohydrate moiety was completed utilizing both MS and NMR spectroscopy, and it was shown to be a novel legionaminic acid derivative, 7-acetamido-5-(N-methyl-glutam-4-yl)-amino-3,5,7,9-tetradeoxy-D-glycero-alpha-D-galacto-nonulosonic acid, (alphaLeg5GluNMe7Ac). Electron transfer dissociation MS with and without collision-activated dissociation was utilized to map seven sites of O-linked glycosylation, eliminating the need for chemical derivatization of tryptic peptides prior to analysis. Marker ions for novel glycans, as well as a unique C-terminal flagellin peptide marker ion, were identified in a top-down analysis of the intact protein. These ions have the potential for use in for rapid detection and discrimination of C. botulinum cells, indicating botulinum neurotoxin contamination. This is the first report of glycosylation of Gram-positive flagellar proteins by the 'sialic acid-like' nonulosonate sugar, legionaminic acid.

Glycosylation profiles of therapeutic antibody pharmaceuticals.

Science.gov (United States)

Wacker, Christoph; Berger, Christoph N; Girard, Philippe; Meier, Roger

2011-11-01

Recombinant antibodies specific for human targets are often used as therapeutics and represent a major class of drug products. Their therapeutic efficacy depends on the formation of antibody complexes resulting in the elimination of a target molecule or the modulation of specific signalling pathways. The physiological effects of antibody therapeutics are known to depend on the structural characteristics of the antibody molecule, specifically on the glycosylation which is the result of posttranslational modifications. Hence, production of therapeutic antibodies with a defined and consistent glycoform profile is needed which still remains a considerable challenge to the biopharmaceutical industry. To provide an insight into the industries capability to control their manufacturing process and to provide antibodies of highest quality, we conducted a market surveillance study and compared major oligosaccharide profiles of a number of monoclonal antibody pharmaceuticals sampled on the Swiss market. Product lot-to-lot variability was found to be generally low, suggesting that a majority of manufacturers have implemented high quality standards in their production processes. However, proportions of G0, G1 and G2 core-fucosylated chains derived from different products varied considerably and showed a bias towards the immature agalactosidated G0 form. Interestingly, differences in glycosylation caused by the production cell type seem to be of less importance compared with process related parameters such as cell growth. Copyright © 2011 Elsevier B.V. All rights reserved.

Altered protein glycosylation predicts Alzheimer's disease and modulates its pathology in disease model Drosophila.

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Frenkel-Pinter, Moran; Stempler, Shiri; Tal-Mazaki, Sharon; Losev, Yelena; Singh-Anand, Avnika; Escobar-Álvarez, Daniela; Lezmy, Jonathan; Gazit, Ehud; Ruppin, Eytan; Segal, Daniel

2017-08-01

The pathological hallmarks of Alzheimer's disease (AD) are pathogenic oligomers and fibrils of misfolded amyloidogenic proteins (e.g., β-amyloid and hyper-phosphorylated tau in AD), which cause progressive loss of neurons in the brain and nervous system. Although deviations from normal protein glycosylation have been documented in AD, their role in disease pathology has been barely explored. Here our analysis of available expression data sets indicates that many glycosylation-related genes are differentially expressed in brains of AD patients compared with healthy controls. The robust differences found enabled us to predict the occurrence of AD with remarkable accuracy in a test cohort and identify a set of key genes whose expression determines this classification. We then studied in vivo the effect of reducing expression of homologs of 6 of these genes in transgenic Drosophila overexpressing human tau, a well-established invertebrate AD model. These experiments have led to the identification of glycosylation genes that may augment or ameliorate tauopathy phenotypes. Our results indicate that OstDelta, l(2)not and beta4GalT7 are tauopathy suppressors, whereas pgnat5 and CG33303 are enhancers, of tauopathy. These results suggest that specific alterations in protein glycosylation may play a causal role in AD etiology. Copyright © 2017 Elsevier Inc. All rights reserved.

The effect of glycosylation on cytotoxicity of Ibaraki virus nonstructural protein NS3

Science.gov (United States)

URATA, Maho; WATANABE, Rie; IWATA, Hiroyuki

2015-01-01

The cytotoxicity of Ibaraki virus nonstructural protein NS3 was confirmed, and the contribution of glycosylation to this activity was examined by using glycosylation mutants of NS3 generated by site-directed mutagenesis. The expression of NS3 resulted in leakage of lactate dehydrogenase to the culture supernatant, suggesting the cytotoxicity of this protein. The lack of glycosylation impaired the transport of NS3 to the plasma membrane and resulted in reduced cytotoxicity. Combined with the previous observation that NS3 glycosylation was specifically observed in mammalian cells (Urata et al., Virus Research 2014), it was suggested that the alteration of NS3 cytotoxicity through modulating glycosylation is one of the strategies to achieve host specific pathogenisity of Ibaraki virus between mammals and vector arthropods. PMID:26178820

N-Glycosylation of Carnosinase Influences Protein Secretion and Enzyme Activity Implications for Hyperglycemia

NARCIS (Netherlands)

Riedl, Eva; Koeppel, Hannes; Pfister, Frederick; Peters, Verena; Sauerhoefer, Sibylle; Sternik, Paula; Brinkkoetter, Paul; Zentgraf, Hanswalter; Navis, Gerjan; Henning, Robert H.; Van Den Born, Jacob; Bakker, Stephan J. L.; Janssen, Bart; van der Woude, Fokko J.; Yard, Benito A.

OBJECTIVE-The (CTG)(n) polymorphism in the serum carnosinase (CN-1) gene affects CN-1 secretion Since CN-1 is heavily glycosylated and glycosylation might influence protein secretion as well, we tested the role of N-glycosylation for CN-1 secretion and enzyme activity. We also tested whether CN-1

Rapid phenolic O-glycosylation of small molecules and complex unprotected peptides in aqueous solvent

Science.gov (United States)

Wadzinski, Tyler J.; Steinauer, Angela; Hie, Liana; Pelletier, Guillaume; Schepartz, Alanna; Miller, Scott J.

2018-06-01

Glycosylated natural products and synthetic glycopeptides represent a significant and growing source of biochemical probes and therapeutic agents. However, methods that enable the aqueous glycosylation of endogenous amino acid functionality in peptides without the use of protecting groups are scarce. Here, we report a transformation that facilitates the efficient aqueous O-glycosylation of phenolic functionality in a wide range of small molecules, unprotected tyrosine, and tyrosine residues embedded within a range of complex, fully unprotected peptides. The transformation, which uses glycosyl fluoride donors and is promoted by Ca(OH)2, proceeds rapidly at room temperature in water, with good yields and selective formation of unique anomeric products depending on the stereochemistry of the glycosyl donor. High functional group tolerance is observed, and the phenol glycosylation occurs selectively in the presence of virtually all side chains of the proteinogenic amino acids with the singular exception of Cys. This method offers a highly selective, efficient, and operationally simple approach for the protecting-group-free synthesis of O-aryl glycosides and Tyr-O-glycosylated peptides in water.

[Conformation analysis of the N-glycosylation site Asn-X-Thr/Ser in glycoproteins].

Science.gov (United States)

Avanov, A Ia; Lipkind, G M

1990-03-01

Theoretical conformational analysis of oligopeptides CH3CO-Asn-X-Thr-NHCH3 (X = Gly, Ala, Pro), modelling N-glycosylation site, and their glycosylated derivatives CH3CO-(GlcNAc beta 1-4GlcNAc beta 1) Asn-X-Thr-NHCH3 has been carried out. Active conformations of the site are found, corresponding to structural prerequisities of N-glycosylation: Asn residue's position in beta-turn and hydrogen bond formation between side chains of Asn and Thr/Ser residues. In this case the L conformation of the central residue X is most probable. Since Pro residue does not possess this conformation, sequences with X = Pro are not glycosylated. It is shown that glycosylation of the above-mentioned sites is accompanied by reorientation of the Asn residue's side chains.

A computational framework for the automated construction of glycosylation reaction networks.

Science.gov (United States)

Liu, Gang; Neelamegham, Sriram

2014-01-01

Glycosylation is among the most common and complex post-translational modifications identified to date. It proceeds through the catalytic action of multiple enzyme families that include the glycosyltransferases that add monosaccharides to growing glycans, and glycosidases which remove sugar residues to trim glycans. The expression level and specificity of these enzymes, in part, regulate the glycan distribution or glycome of specific cell/tissue systems. Currently, there is no systematic method to describe the enzymes and cellular reaction networks that catalyze glycosylation. To address this limitation, we present a streamlined machine-readable definition for the glycosylating enzymes and additional methodologies to construct and analyze glycosylation reaction networks. In this computational framework, the enzyme class is systematically designed to store detailed specificity data such as enzymatic functional group, linkage and substrate specificity. The new classes and their associated functions enable both single-reaction inference and automated full network reconstruction, when given a list of reactants and/or products along with the enzymes present in the system. In addition, graph theory is used to support functions that map the connectivity between two or more species in a network, and that generate subset models to identify rate-limiting steps regulating glycan biosynthesis. Finally, this framework allows the synthesis of biochemical reaction networks using mass spectrometry (MS) data. The features described above are illustrated using three case studies that examine: i) O-linked glycan biosynthesis during the construction of functional selectin-ligands; ii) automated N-linked glycosylation pathway construction; and iii) the handling and analysis of glycomics based MS data. Overall, the new computational framework enables automated glycosylation network model construction and analysis by integrating knowledge of glycan structure and enzyme biochemistry. All

A computational framework for the automated construction of glycosylation reaction networks.

Directory of Open Access Journals (Sweden)

Gang Liu

Full Text Available Glycosylation is among the most common and complex post-translational modifications identified to date. It proceeds through the catalytic action of multiple enzyme families that include the glycosyltransferases that add monosaccharides to growing glycans, and glycosidases which remove sugar residues to trim glycans. The expression level and specificity of these enzymes, in part, regulate the glycan distribution or glycome of specific cell/tissue systems. Currently, there is no systematic method to describe the enzymes and cellular reaction networks that catalyze glycosylation. To address this limitation, we present a streamlined machine-readable definition for the glycosylating enzymes and additional methodologies to construct and analyze glycosylation reaction networks. In this computational framework, the enzyme class is systematically designed to store detailed specificity data such as enzymatic functional group, linkage and substrate specificity. The new classes and their associated functions enable both single-reaction inference and automated full network reconstruction, when given a list of reactants and/or products along with the enzymes present in the system. In addition, graph theory is used to support functions that map the connectivity between two or more species in a network, and that generate subset models to identify rate-limiting steps regulating glycan biosynthesis. Finally, this framework allows the synthesis of biochemical reaction networks using mass spectrometry (MS data. The features described above are illustrated using three case studies that examine: i O-linked glycan biosynthesis during the construction of functional selectin-ligands; ii automated N-linked glycosylation pathway construction; and iii the handling and analysis of glycomics based MS data. Overall, the new computational framework enables automated glycosylation network model construction and analysis by integrating knowledge of glycan structure and enzyme

Cell Surface Glycosylation Is Required for Efficient Mating of Haloferax volcanii

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Yarden Shalev

2017-07-01

Full Text Available Halophilic archaea use a fusion-based mating system for lateral gene transfer across cells, yet the molecular mechanisms involved remain unknown. Previous work implied that cell fusion involves cell–cell recognition since fusion occurs more efficiently between cells from the same species. Long believed to be restricted only to Eukarya, it is now known that cells of all three domains of life perform N-glycosylation, the covalent attachment of glycans to select target asparagine residues in proteins, and that this post-translational modification is common for archaeal cell surface proteins. Here, we show that differences in glycosylation of the Haloferax volcanii surface-layer glycoprotein, brought about either by changing medium salinity or by knocking out key glycosylation genes, reduced mating success. Thus, different glycosylation patterns are likely to underlie mating preference in halophilic archaea, contributing to speciation processes.

A Novel Strategy for Characterization of Glycosylated Proteins Separated by Gel Electrophoresis

DEFF Research Database (Denmark)

Larsen, Martin; Skottrup, Peter; Enghild, Jan Johannes

Protein glycosylation can be vital for changing the function or physiochemical properties of a protein. Abnormal glycosylation can lead to protein malfunction, resulting in severe diseases. Therefore, it is important to develop techniques for characterization of such modifications in proteins...... graphite powder micro-columns in combination with mass spectrometry. The method is faster and more sensitive than previous approaches and would be ideal for proteomics studies and verification of correct glycosylation of recombinant glycoproteins....

HEK293T cell lines defective for O-linked glycosylation.

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James M Termini

Full Text Available Here we describe derivatives of the HEK293T cell line that are defective in their ability to generate mucin-type O-linked glycosylation. Using CRISPR/Cas9 and a single-cell GFP-sorting procedure, the UDP-galactose-4-epimerase (GALE, galactokinase 1 (GALK1, and galactokinase 2 (GALK2 genes were knocked out individually and in combinations with greater than 90% of recovered clones having the desired mutations. Although HEK293T cells are tetraploid, we found this approach to be an efficient method to target and disrupt all 4 copies of the target gene. Deficient glycosylation in the GALE knockout cell line could be rescued by the addition of galactose and N-acetylgalactosamine (GalNAc to the cell culture media. However, when key enzymes of the galactose/GalNAc salvage pathways were disrupted in tandem (GALE+GALK1 or GALE+GALK2, O-glycosylation was eliminated and could not be rescued by the addition of either galactose plus GalNAc or UDP-galactose plus UDP-GalNAc. GALK1 and GALK2 are key enzymes of the galactose/GalNAc salvage pathways. Mass spectrometry was performed on whole cell lysate of the knockout cell lines to verify the glycosylation phenotype. As expected, the GALE knockout was almost completely devoid of all O-glycosylation, with minimal glycosylation as a result of functional salvage pathways. However, the GALE+GALK1 and GALE+GALK2 knockout lines were devoid of all O-glycans. Mass spectrometry analysis revealed that the disruption of GALE, GALK1, and GALE+GALK2 had little effect on the N-glycome. But when GALE was knocked out in tandem with GALK1, N-glycans were exclusively of the high mannose type. Due to the well-characterized nature of these five knockout cell lines, they will likely prove useful for a wide variety of applications.

Diagnosis and activity assessment of immunoglobulin A nephropathy: current perspectives on noninvasive testing with aberrantly glycosylated immunoglobulin A-related biomarkers

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Suzuki Y

2014-10-01

Full Text Available Yusuke Suzuki,1 Hitoshi Suzuki,1 Yuko Makita,1 Akiko Takahata,1 Keiko Takahashi,1 Masahiro Muto,1 Yohei Sasaki,1 Atikemu Kelimu,1 Keiichi Matsuzaki,2 Hiroyuki Yanagawa,1 Keiko Okazaki,1 Yasuhiko Tomino1 1Division of Nephrology, Department of Internal Medicine, Juntendo University Faculty of Medicine, Tokyo, 2Kyoto University Health Service, Kyoto, Japan Abstract: Immunoglobulin (Ig A nephropathy (IgAN is the most common form of glomerular disease worldwide and is associated with a poor prognosis. Thus, development of a curative treatment and strategies for early diagnosis and treatment are urgently needed. Pathological analysis of renal biopsy is the gold standard for the diagnosis and assessment of disease activity; however, immediate and frequent assessment based on biopsy specimens is difficult. Therefore, a simple and safe alternative is desirable. On the other hand, it is now widely accepted that multi-hit steps, including production of aberrantly glycosylated serum IgA1 (first hit, and IgG or IgA autoantibodies that recognize glycan containing epitopes on glycosylated serum IgA1 (second hit and their subsequent immune complex formation (third hit and glomerular deposition (fourth hit, are required for continued progression of IgAN. Although the prognostic and predictive values of several markers have been discussed elsewhere, we recently developed a highly sensitive and specific diagnostic method by measuring serum levels of glycosylated serum IgA1 and related IgA immune complex. In addition, we confirmed a significant correlation between serum levels of these essential effector molecules and disease activity after treatment, suggesting that each can be considered as a practical surrogate marker of therapeutic effects in this slowly progressive disease. Such a noninvasive diagnostic and activity assessment method using these disease-oriented specific biomarkers may be useful in the early diagnosis of and intervention in IgAN, with

Blood pressure reduction due to hemoglobin glycosylation in type 2 diabetic patients

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Pedro Cabrales

2008-08-01

Full Text Available Pedro Cabrales1, Miguel A Salazar Vázquez2,3, Beatriz Y Salazar Vázquez3,4, Martha Rodríguez-Morán5, Marcos Intaglietta4, Fernando Guerrero-Romero51La Jolla Bioengineering Institute, La Jolla, California, USA; 2Hospital Regional No. 1, of the Mexican Social Security Institute, Victoria de Durango, Dgo. Mexico; 3Faculty of Medicine and Dept. of Physical Chemistry, Universidad Juárez del Estado de Durango, Victoria de Durango, Dgo. Mexico; 4Department of Bioengineering, University of California, San Diego, La Jolla, California, USA; 5Biomedical Research Unit, of the Mexican Social Security Institute, Victoria de Durango, Dgo. MexicoObjective: To test the hypothesis that glycosylation of hemoglobin constitutes a risk factor for hypertension.Methods: A total of 129 relative uniform diabetic subjects (86 women and 42 men were enrolled in a cross-sectional study. Exclusion criteria included alcohol consumption, smoking, ischemic heart disease, stroke, neoplasia, renal, hepatic, and chronic inflammatory disease. Systolic and diastolic pressures were recorded in subsequent days and mean arterial blood pressure (MAP was determined. Hemoglobin glycosylation was measured by determining the percentage glycosylated hemoglobin (HbA1c by means of the automated microparticle enzyme immunoassay test.Results: MAP was found to be independent of the concentration of HbA1c; however, correcting MAP for the variability in hematocrit, to evidence the level of vasoconstriction (or vasodilatation showed that MAP is negatively correlated with the concentration of HbA1c (p for trend <0.05, when patients treated for hypertension are excluded from the analysis. Patients treated for hypertension showed the opposite trend with increasing MAP as HbA1c increased (p for the difference in trends <0.05.Conclusions: Glycosylation per se appears to lead to blood pressure reduction in type 2 diabetic patients untreated for hypertension. Treatment for hypertension may be

Glycosylation patterns of kidney proteins differ in rat diabetic nephropathy.

Science.gov (United States)

Ravidà, Alessandra; Musante, Luca; Kreivi, Marjut; Miinalainen, Ilkka; Byrne, Barry; Saraswat, Mayank; Henry, Michael; Meleady, Paula; Clynes, Martin; Holthofer, Harry

2015-05-01

Diabetic nephropathy often progresses to end-stage kidney disease and, ultimately, to renal replacement therapy. Hyperglycemia per se is expected to have a direct impact on the biosynthesis of N- and O-linked glycoproteins. This study aims to establish the link between protein glycosylation and progression of experimental diabetic kidney disease using orthogonal methods. Kidneys of streptozotocin-diabetic and control rats were harvested at three different time points post streptozotocin injection. A panel of 12 plant lectins was used in the screening of lectin blots. The lectins UEAI, PHA-E, GSI, PNA, and RCA identified remarkable disease-associated differences in glycoprotein expression. Lectin affinity chromatography followed by mass spectrometric analyses led to the identification of several glycoproteins involved in salt-handling, angiogenesis, and extracellular matrix degradation. Our data confirm a substantial link between glycosylation signature and diabetes progression. Furthermore, as suggested by our findings on dipeptidyl peptidase-IV, altered protein glycosylation may reflect changes in biochemical properties such as enzymatic activity. Thus, our study demonstrates the unexplored potential of protein glycosylation analysis in the discovery of molecules linked to diabetic kidney disease.

Model-based analysis of N-glycosylation in Chinese hamster ovary cells

DEFF Research Database (Denmark)

Krambeck, Frederick J.; Bennun, Sandra V; Andersen, Mikael Rørdam

2017-01-01

The Chinese hamster ovary (CHO) cell is the gold standard for manufacturing of glycosylated recombinant proteins for production of biotherapeutics. The similarity of its glycosylation patterns to the human versions enable the products of this cell line favorable pharmacokinetic properties and lower...

Oxytocin analogues with O-glycosylated serine and threonine in position 4

Czech Academy of Sciences Publication Activity Database

Marcinkowska, A.; Borovičková, Lenka; Slaninová, Jiřina; Grzonka, Z.

2007-01-01

Roč. 81, č. 7 (2007), s. 1335-1344 ISSN 0137- 5083 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z90210515 Keywords : oxytocin * glycosylated serin * glycosylated threonin * position 4 Subject RIV: CE - Biochemistry Impact factor: 0.483, year: 2007

Differential dependence on N-glycosylation of anthrax toxin receptors CMG2 and TEM8.

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Sarah Friebe

Full Text Available ANTXR 1 and 2, also known as TEM8 and CMG2, are two type I membrane proteins, which have been extensively studied for their role as anthrax toxin receptors, but with a still elusive physiological function. Here we have analyzed the importance of N-glycosylation on folding, trafficking and ligand binding of these closely related proteins. We find that TEM8 has a stringent dependence on N-glycosylation. The presence of at least one glycan on each of its two extracellular domains, the vWA and Ig-like domains, is indeed necessary for efficient trafficking to the cell surface. In the absence of any N-linked glycans, TEM8 fails to fold correctly and is recognized by the ER quality control machinery. Expression of N-glycosylation mutants reveals that CMG2 is less vulnerable to sugar loss. The absence of N-linked glycans in one of the extracellular domains indeed has little impact on folding, trafficking or receptor function of the wild type protein expressed in tissue culture cells. N-glycans do, however, seem required in primary fibroblasts from human patients. Here, the presence of N-linked sugars increases the tolerance to mutations in cmg2 causing the rare genetic disease Hyaline Fibromatosis Syndrome. It thus appears that CMG2 glycosylation provides a buffer towards genetic variation by promoting folding of the protein in the ER lumen.

Micropinocytic ingestion of glycosylated albumin by isolated microvessels: possible role in pathogenesis of diabetic microangiopathy.

OpenAIRE

Williams, S K; Devenny, J J; Bitensky, M W

1981-01-01

Microvessels isolated from rat epididymal fat exhibit differential vesicular ingestion rates for unmodified and non-enzymatically glycosylated rat albumin. While unmodified rat albumin is excluded from ingestion by endothelial micropinocytic vesicles, glycosylated albumin is avidly taken up by endocytosis. Interaction of albumin and glycosylated albumin with endothelium was studied with a double-label fluorescence assay of micropinocytosis. When glycosylated albumin was present at a concentra...

N-glycosylated catalytic unit meets O-glycosylated propeptide: complex protein architecture in a fungal hexosaminidase

Czech Academy of Sciences Publication Activity Database

Plíhal, Ondřej; Sklenář, Jan; Kmoníčková, J.; Man, Petr; Pompach, Petr; Havlíček, Vladimír; Křen, Vladimír; Bezouška, Karel

2004-01-01

Roč. 32, č. 5 (2004), s. 764-765 ISSN 0300-5127 R&D Projects: GA ČR GA203/04/1045 Institutional research plan: CEZ:AV0Z5020903 Keywords : asperillus oryzoe * glycosyl hydrolase * preproprotein Subject RIV: EE - Microbiology, Virology Impact factor: 2.267, year: 2004

In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins.

Science.gov (United States)

Fang, Pan; Wang, Xin-Jian; Xue, Yu; Liu, Ming-Qi; Zeng, Wen-Feng; Zhang, Yang; Zhang, Lei; Gao, Xing; Yan, Guo-Quan; Yao, Jun; Shen, Hua-Li; Yang, Peng-Yuan

2016-06-21

N-glycosylation is one of the most prominent and abundant posttranslational modifications of proteins. It is estimated that over 50% of mammalian proteins undergo glycosylation. However, the analysis of N-glycoproteins has been limited by the available analytical technology. In this study, we comprehensively mapped the N-glycosylation sites in the mouse brain proteome by combining complementary methods, which included seven protease treatments, four enrichment techniques and two fractionation strategies. Altogether, 13492 N-glycopeptides containing 8386 N-glycosylation sites on 3982 proteins were identified. After evaluating the performance of the above methods, we proposed a simple and efficient workflow for large-scale N-glycosylation site mapping. The optimized workflow yielded 80% of the initially identified N-glycosylation sites with considerably less effort. Analysis of the identified N-glycoproteins revealed that many of the mouse brain proteins are N-glycosylated, including those proteins in critical pathways for nervous system development and neurological disease. Additionally, several important biomarkers of various diseases were found to be N-glycosylated. These data confirm that N-glycosylation is important in both physiological and pathological processes in the brain, and provide useful details about numerous N-glycosylation sites in brain proteins.

Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy.

Science.gov (United States)

Bondt, Albert; Wuhrer, Manfred; Kuijper, T Martijn; Hazes, Johanna M W; Dolhain, Radboud J E M

2016-11-25

Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. IgGs were captured from RA and control sera obtained before (RA only), during and after pregnancy, followed by Fc and Fab separation, glycan release, and mass spectrometric detection. In parallel, glycans from intact IgG were analysed. The data was used to calculate glycosylation traits, and to estimate the level of Fab glycosylation. The overall level of Fab glycosylation was increased in RA patients compared to controls, while no differences in Fab glycosylation patterns were found. For the Fc and intact IgG (Total) previously observed differences in galactosylation and bisection were confirmed. Furthermore, increased galactosylation of Fc and Total were associated with lower disease activity and autoantibody positivity. In addition, the change in Fc galactosylation associated with the change in disease activity during pregnancy and after delivery, while this was not the case for Fab. In contrast to changes in Fc glycosylation, changes in Fab glycosylation are not associated with improvement of RA during pregnancy and arthritis flare after delivery.

Analysis of expression and glycosylation of avian metapneumovirus attachment glycoprotein from recombinant baculoviruses.

Science.gov (United States)

Luo, Lizhong; Nishi, Krista; MacLeod, Erin; Sabara, Marta I; Li, Yan

2010-11-01

Recently, we reported the expression and glycosylation of avian metapneumovirus attachment glycoprotein (AMPV/C G protein) in eukaryotic cell lines by a transient-expression method. In the present study, we investigated the biosynthesis and O-linked glycosylation of the AMPV/C G protein in a baculovirus expression system. The results showed that the insect cell-produced G protein migrated more rapidly in SDS-PAGE as compared to LLC-MK2 cell-derived G proteins owing to glycosylation differences. The fully processed, mature form of G protein migrated between 78 and 86 kDa, which is smaller than the 110 kDa mature form of G expressed in LLC-MK2 cells. In addition, several immature G gene products migrating at 40-48 and 60-70 kDa were also detected by SDS-PAGE and represented glycosylated intermediates. The addition of the antibiotic tunicamycin, which blocks early steps of glycosylation, to insect cell culture resulted in the disappearance of two glycosylated forms of the G protein and identified a 38 kDa unglycosylated precursor. The maturation of the G protein was completely blocked by monensin, suggesting that the O-linked glycosylation of G initiated in the trans-Golgi compartment. The presence of O-linked sugars on the mature protein was further confirmed by lectin Arachis hypogaea binding assay. Furthermore, antigenic features of the G protein expressed in insect cells were evaluated by ELISA. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

N-Glycosylation optimization of recombinant antibodies in CHO cell through process and metabolic engineering

DEFF Research Database (Denmark)

Fan, Yuzhou

, analysis, control and optimization of N-glycosylation were thoroughly reviewed. In particular, how to control and optimize N-glycosylation in CHO cells was exclusively studied. The main focus of this PhD project is to find effective approaches of modulating N-glycosylation of CHO-derived recombinant...... galactose as feed additives, changing process parameters such as seeding density and cultivation duration are all demonstrated to be effective. The causal explanation of their impact on glycosylation can be various, including product, metabolism, proteome and physiology-associated mechanism. In the middle...... part of the thesis, both literature reviews and experimental applications were provided to demonstrate how to use omics data and implement systems biology to understand biological activities, especially N-glycosylation in CHO cells. In the last part of the thesis, the second strategy that apply genetic...

Cancer Risks for Relatives of Children with Cancer

International Nuclear Information System (INIS)

Heath, J. A.; Smibert, E.; Algar, E. M.; Heath, J. A.; Dite, G. S.; Hopper, J. L.; Algar, E. M.

2014-01-01

We determined the extent and distribution of cancers in relatives of 379 children newly diagnosed with cancer. Family history was collected from 1,337 first-degree and 3,399 second-degree relatives and incidence compared with national age- and gender-specific rates. Overall, 14 children (3.7%) had a relative with a history of childhood cancer and 26 children (6.9%) had a first-degree relative with a history of cancer, with only one of these having an identifiable familial cancer syndrome. There was a higher than expected incidence of childhood cancer among first-degree relatives (parents and siblings) (standardized incidence ratio (SIR) 1.43; 95% CI 0.54-5.08). There was also a higher than expected incidence of adult cancers among first-degree relatives (SIR 1.45; 95% CI 0.93-2.21), particularly in females (SIR 1.82; 95% CI 1.26-3.39). The increased family cancer history in first-degree females was largely attributable to an effect in mothers (SIR 1.78; 95% CI 1.27-3.33). The gender-specific association was reflected in higher than expected incidence rates of breast cancer in both mothers (SIR 1.92; 95% CI 0.72-6.83) and aunts (SIR 1.64; 95% CI 0.98-2.94). These findings support the hypothesis that previously undetected familial cancer syndromes contribute to childhood cancer.

Enzymatic glycosylation of multivalent scaffolds

Czech Academy of Sciences Publication Activity Database

Bojarová, Pavla; Rosencrantz, R. R.; Elling, L.; Křen, Vladimír

2013-01-01

Roč. 42, č. 11 (2013), s. 4774-4797 ISSN 0306-0012 R&D Projects: GA MŠk(CZ) LD13042; GA ČR GAP207/10/0321 Institutional support: RVO:61388971 Keywords : N-ACETYLGLUCOSAMINYLTRANSFERASE-III * MUCIN TANDEM REPEAT * NEIGHBORING RESIDUE GLYCOSYLATION Subject RIV: CC - Organic Chemistry Impact factor: 30.425, year: 2013

Polymorphism in the GALNT1 gene and epithelial ovarian cancer in non-Hispanic white women: the Ovarian Cancer Association Consortium

DEFF Research Database (Denmark)

Phelan, Catherine M; Tsai, Ya-Yu; Goode, Ellen L

2010-01-01

Aberrant glycosylation is a well-described hallmark of cancer. In a previous ovarian cancer case control study that examined polymorphisms in 26 glycosylation-associated genes, we found strong statistical evidence (P = 0.00017) that women who inherited two copies of a single-nucleotide polymorphism...... in the UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferase, GALNT1, had decreased ovarian cancer risk. The current study attempted to replicate this observation. The GALNT1 single-nucleotide polymorphism rs17647532 was genotyped in 6,965 cases and 8,377 controls from 14 studies forming...... the Ovarian Cancer Association Consortium. The fixed effects estimate per rs17647532 allele was null (odds ratio, 0.99; 95% confidence interval, 0.92-1.07). When a recessive model was fit, the results were unchanged. Test for heterogeneity of the odds ratios revealed consistency across the 14 replication...

COMPARISON OF FRUCTOSAMINE AND GLYCOSYLATED HEMOGLOBIN IN A NON-INSULIN DEPENDENT DIABETIC POPULATION

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M. Amini

1999-08-01

Full Text Available In an attempt to determine the clinical value of frnctosamine assay for monitoring type II diabetic patients, correlation of frnctosamine with glycosylated hemoglobin was studied. 100 patients with type II diabetes mcllitus were compared with 100 normal subjects. Fasting blood glucose, glycosylated hemoglobin, albumin and frnctosamine were measured in alt subjects. In the diabetic patients, a significant correlation was observed between fasting blood glucose and glycosylated hemoglobin (r = 0.64, p < 0.01 and scrum frnctosamine (r = 0.7, P < 0.01. Tlicrc was also a significant correlation between glycosylated hemoglobin and scrum frtictosmine (r = .94, I'<0.01. Frnctosamine, assay can be used as an index of diabetes control.

Importance of glycosylation on function of a potassium channel in neuroblastoma cells.

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M K Hall

Full Text Available The Kv3.1 glycoprotein, a voltage-gated potassium channel, is expressed throughout the central nervous system. The role of N-glycans attached to the Kv3.1 glycoprotein on conducting and non-conducting functions of the Kv3.1 channel are quite limiting. Glycosylated (wild type, partially glycosylated (N220Q and N229Q, and unglycosylated (N220Q/N229Q Kv3.1 proteins were expressed and characterized in a cultured neuronal-derived cell model, B35 neuroblastoma cells. Western blots, whole cell current recordings, and wound healing assays were employed to provide evidence that the conducting and non-conducting properties of the Kv3.1 channel were modified by N-glycans of the Kv3.1 glycoprotein. Electrophoretic migration of the various Kv3.1 proteins treated with PNGase F and neuraminidase verified that the glycosylation sites were occupied and that the N-glycans could be sialylated, respectively. The unglycosylated channel favored a different whole cell current pattern than the glycoform. Further the outward ionic currents of the unglycosylated channel had slower activation and deactivation rates than those of the glycosylated Kv3.1 channel. These kinetic parameters of the partially glycosylated Kv3.1 channels were also slowed. B35 cells expressing glycosylated Kv3.1 protein migrated faster than those expressing partially glycosylated and much faster than those expressing the unglycosylated Kv3.1 protein. These results have demonstrated that N-glycans of the Kv3.1 glycoprotein enhance outward ionic current kinetics, and neuronal migration. It is speculated that physiological changes which lead to a reduction in N-glycan attachment to proteins will alter the functions of the Kv3.1 channel.

Cancer biomarkers defined by autoantibody signatures to aberrant O-glycopeptide epitopes

DEFF Research Database (Denmark)

Wandall, Hans H; Blixt, Ola; Tarp, Mads A

2010-01-01

Autoantibodies to cancer antigens hold promise as biomarkers for early detection of cancer. Proteins that are aberrantly processed in cancer cells are likely to present autoantibody targets. The extracellular mucin MUC1 is overexpressed and aberrantly glycosylated in many cancers; thus, we evalua...

Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase.

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Jason M Shapiro

Full Text Available We previously isolated AF20, a murine monoclonal antibody that recognizes a cell surface glycoprotein of approximately 90-110 kDa. The AF20 antigen is specifically expressed in human hepatoma and colon cancer cell lines, and thus could serve as a cancer biomarker. To uncover the molecular identity of the AF20 antigen, a combination of ion-exchange chromatography, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis was employed to purify the AF20 antigen followed by trypsin digestion and mass spectrometry. Surprisingly, three host proteins were thus purified from human hepatoma and colon cancer cell lines: transferrin receptor 1 (TFR1, heat shock protein 90 (HSP90, and Na+/K+ ATPase or Mg++ ATPase. Co-immunoprecipitation followed by Western blot analysis confirmed interaction among the three proteins. However, only the cDNA encoding TFR1 conferred strong cell surface staining by the AF20 antibody following its transient transfection into a cell line lacking endogenous AF20. In support of the molecular identity of AF20 as TFR1, diferric but not iron-free transferrin could prevent AF20 antigen-antibody interaction during immunoprecipitation. Moreover, very similar patterns of AF20 and TFR1 overexpression was documented in colon cancer tissues. In conclusion, AF20 is glycosylated TFR1. This finding could explain the molecular structure of AF20, its cell surface localization, as well as overexpression in cancer cells. Glycosylated TFR1 should serve as a usefulness target for anti-cancer therapy, or a vehicle for delivery of anti-tumor drugs with high affinity and specificity. The biological significance of the complex formation between TFR1, HSP90, and/or transporting ATPase warrants further investigation.

Biological role of site-specific O-glycosylation in cell adhesion activity and phosphorylation of osteopontin.

Science.gov (United States)

Oyama, Midori; Kariya, Yoshinobu; Kariya, Yukiko; Matsumoto, Kana; Kanno, Mayumi; Yamaguchi, Yoshiki; Hashimoto, Yasuhiro

2018-05-09

Osteopontin (OPN) is an extracellular glycosylated phosphoprotein that promotes cell adhesion by interacting with several integrin receptors. We previously reported that an OPN mutant lacking five O-glycosylation sites (Thr 134 /Thr 138 /Thr 143 /Thr 147 /Thr 152 ) in the threonine/proline-rich region increased cell adhesion activity and phosphorylation compared with the wild type. However, the role of O-glycosylation in cell adhesion activity and phosphorylation of OPN remains to be clarified. Here, we show that site-specific O-glycosylation in the threonine/proline-rich region of OPN affects its cell adhesion activity and phosphorylation independently and/or synergistically. Using site-directed mutagenesis, we found that OPN mutants with substitution sets of Thr 134 /Thr 138 or Thr 143 /Thr 147 /Thr 152 had decreased and increased cell adhesion activity, respectively. In contrast, the introduction of a single mutation into the O-glycosylation sites had no effect on OPN cell adhesion activity. An adhesion assay using function-blocking antibodies against αvβ3 and β1 integrins, as well as αvβ3 integrin-overexpressing A549 cells, revealed that site-specific O-glycosylation affected the association of OPN with the two integrins. Phosphorylation analyses using phos-tag and LC-MS/MS indicated that phosphorylation levels and sites were influenced by the O-glycosylation status, although the number of O-glycosylation sites was not correlated with the phosphorylation level in OPN. Furthermore, a correlation analysis between phosphorylation level and cell adhesion activity in OPN mutants with the site-specific O-glycosylation showed that they were not always correlated. These results provide conclusive evidence of a novel regulatory mechanism of cell adhesion activity and phosphorylation of OPN by site-specific O-glycosylation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Heterogeneity of rabbit endogenous pyrogens is not attributable to glycosylated variants of a single polypeptide chain.

Science.gov (United States)

Murphy, P A; Cebula, T A; Windle, B E

1981-10-01

Rabbit endogenous pyrogens were of about the same molecular size, but showed considerable heterogeneity of their isoelectric points. We attempted to show that this heterogeneity was attributable to variable glycosylation of a single polypeptide chain. When peritoneal exudate cells were stimulated to make pyrogens in the presence of 2-deoxy-D-glucose, there was a relatively trivial suppression of pyrogen release, and analysis by isoelectric focusing showed parallel inhibition of secretion of all the forms of endogenous pyrogen. When cells were stimulated in the presence of 3H-labeled amino acids and 14C-labeled glucosamine or glucose, the purified pyrogens were labeled with 3H but not with 14C. Macrophage membrane preparations were made which contained glycosyl transferases and could transfer sugar residues from sugar nucleotides to deglycosylated fetuin. These macrophage membrane preparations did not transfer sugars to the pI 7.3 endogenous pyrogen. Treatment of endogenous pyrogens with neuraminidase or with periodate produced no evidence suggesting that the pyrogens were glycosylated. Last, endogenous pyrogens did not bind to any of four lectins with different carbohydrate specificities. This evidence suggests that the heterogeneity of rabbit endogenous pyrogens is not attributable to glycosylation and must have some other cause.

Glycosyltransferase Gene Expression Profiles Classify Cancer Types and Propose Prognostic Subtypes

Science.gov (United States)

Ashkani, Jahanshah; Naidoo, Kevin J.

2016-05-01

Aberrant glycosylation in tumours stem from altered glycosyltransferase (GT) gene expression but can the expression profiles of these signature genes be used to classify cancer types and lead to cancer subtype discovery? The differential structural changes to cellular glycan structures are predominantly regulated by the expression patterns of GT genes and are a hallmark of neoplastic cell metamorphoses. We found that the expression of 210 GT genes taken from 1893 cancer patient samples in The Cancer Genome Atlas (TCGA) microarray data are able to classify six cancers; breast, ovarian, glioblastoma, kidney, colon and lung. The GT gene expression profiles are used to develop cancer classifiers and propose subtypes. The subclassification of breast cancer solid tumour samples illustrates the discovery of subgroups from GT genes that match well against basal-like and HER2-enriched subtypes and correlates to clinical, mutation and survival data. This cancer type glycosyltransferase gene signature finding provides foundational evidence for the centrality of glycosylation in cancer.

Macrocyclic bis-thioureas catalyze stereospecific glycosylation reactions.

Science.gov (United States)

Park, Yongho; Harper, Kaid C; Kuhl, Nadine; Kwan, Eugene E; Liu, Richard Y; Jacobsen, Eric N

2017-01-13

Carbohydrates are involved in nearly all aspects of biochemistry, but their complex chemical structures present long-standing practical challenges to their synthesis. In particular, stereochemical outcomes in glycosylation reactions are highly dependent on the steric and electronic properties of coupling partners; thus, carbohydrate synthesis is not easily predictable. Here we report the discovery of a macrocyclic bis-thiourea derivative that catalyzes stereospecific invertive substitution pathways of glycosyl chlorides. The utility of the catalyst is demonstrated in the synthesis of trans-1,2-, cis-1,2-, and 2-deoxy-β-glycosides. Mechanistic studies are consistent with a cooperative mechanism in which an electrophile and a nucleophile are simultaneously activated to effect a stereospecific substitution reaction. Copyright © 2017, American Association for the Advancement of Science.

O-GLYCOBASE version 4.0: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Gupta, Ramneek; Birch, Hanne; Rapacki, Krzysztof

1999-01-01

O-GLYCBASE is a database of glycoproteins with O-linked glycosylation sites. Entries with at least one experimentally verified O-glycosylation site have been complied from protein sequence databases and literature. Each entry contains information about the glycan involved, the species, sequence, ...

SEM visualization of glycosylated surface molecules using lectin-coated microspheres

Science.gov (United States)

Duke, J.; Janer, L.; Campbell, M.

1985-01-01

There are several techniques currently used to localize glycosylated surface molecules by scanning electron microscopy (Grinnell, 1980; Molday, 1976; Linthicum and Sell, 1975; Nicolson, 1974; Lo Buglio, et al, 1972). A simple and rapid method, using a modification of Grinnell's technique is reported here. Essentially, microspheres coated with Concavalin A are used to bind to glycosylated regions of the palatal shelf epithelium and are visualized in the scanning electron microscope (SEM).

Glycosylation of KSHV Encoded vGPCR Functions in Its Signaling and Tumorigenicity

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Hui Wu

2015-03-01

Full Text Available Kaposi’s sarcoma-associated herpesvirus (KSHV is a tumor virus and the etiologic agent of Kaposi’s Sarcoma (KS. KSHV G protein-coupled receptor (vGPCR is an oncogene that is implicated in malignancies associated with KHSV infection. In this study, we show that vGPCR undergoes extensive N-linked glycosylation within the extracellular domains, specifically asparagines 18, 22, 31 and 202. An immunofluorescence assay demonstrates that N-linked glycosylation are necessary for vGPCR trafficking to the cellular membrane. Employing vGPCR mutants whose glycosylation sites were ablated, we show that these vGPCR mutants failed to activate downstream signaling in cultured cells and were severely impaired to induce tumor formation in the xenograph nude mouse model. These findings support the conclusion that glycosylation is critical for vGPCR tumorigenesis and imply that chemokine regulation at the plasma membrane is crucial for vGPCR mediated signaling.

O-GLYCBASE: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Hansen, Jan; Lund, Ole; Nielsen, Jens O.

1996-01-01

O-GLYCBASE is a comprehensive database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the SWISS-PROT and PIR databases as well as directly from recently published reports. Nineteen percent of the entries extracted from the databases n...... of mucin type O-glycosylation sites in mammalian glycoproteins exclusively from the primary sequence is made available by E-mail or WWW. The O-GLYCBASE database is also available electronically through our WWW server or by anonymous FTP....

Structural and Functional Consequences of Increased Tubulin Glycosylation in Diabetes Mellitus

Science.gov (United States)

Williams, Stuart K.; Howarth, Nancy L.; Devenny, James J.; Bitensky, Mark W.

1982-11-01

The extent of in vitro nonenzymatic glycosylation of purified rat brain tubulin was dependent on time and glucose concentration. Tubulin glycosylation profoundly inhibited GTP-dependent tubulin polymerization. Electron microscopy and NaDodSO4/polyacrylamide gel electrophoresis showed that glycosylated tubulin forms high molecular weight amorphous aggregates that are not disrupted by detergents or reducing agents. The amount of covalently bound NaB3H4-reducible sugars in tubulin recovered from brain of streptozotocin-induced diabetic rats was dramatically increased as compared with tubulin recovered from normal rat brain. Moreover, tubulin recovered from diabetic rat brain exhibited less GTP-induced polymerization than tubulin from nondiabetic controls. The possible implications of these data for diabetic neuropathy are discussed.

Understanding Alzheimer's disease by global quantification of protein phosphorylation and sialylated N-linked glycosylation profiles

DEFF Research Database (Denmark)

Lassen, Pernille S.; Thygesen, Camilla; Larsen, Martin R.

2017-01-01

elucidated them in neurodegenerative diseases such as Alzheimer's disease. Here, we comprehensively review Alzheimer's pathology in relation to protein phosphorylation and glycosylation on synaptic plasticity from neuroproteomics data. Moreover, we highlight several mass spectrometry-based sample processing...

O-GLYCBASE version 3.0: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Hansen, Jan; Lund, Ole; Nilsson, Jette

1998-01-01

O-GLYCBASE is a revised database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the literature, and from the sequence databases. Entries include informations about species, sequence, glycosylation sites and glycan type and is fully cr...

Patterns of glycemic control using glycosylated hemoglobin in diabetics.

Science.gov (United States)

Kahlon, Arunpreet Singh; Pathak, Rambha

2011-07-01

Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS) is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3%) had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came out to be .311. This correlation was found to be statistically

Diagnostic serum glycosylation profile in patients with intellectual disability as a result of MAN1B1 deficiency

DEFF Research Database (Denmark)

Van Scherpenzeel, Monique; Timal, Sharita; Rymen, Daisy

2014-01-01

Congenital disorders of glycosylation comprise a group of genetic defects with a high frequency of intellectual disability, caused by deficient glycosylation of proteins and lipids. The molecular basis of the majority of the congenital disorders of glycosylation type I subtypes, localized...... in the cytosol and endoplasmic reticulum, has been solved. However, elucidation of causative genes for defective Golgi glycosylation (congenital disorders of glycosylation type II) remains challenging because of a lack of sufficiently specific diagnostic serum methods. In a single patient with intellectual...... disability, whole-exome sequencing revealed MAN1B1 as congenital disorder of glycosylation type II candidate gene. A novel mass spectrometry method was applied for high-resolution glycoprofiling of intact plasma transferrin. A highly characteristic glycosylation signature was observed with hybrid type N...

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

Energy Technology Data Exchange (ETDEWEB)

Palmieri, D. [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy); Valli, M.; Viglio, S. [Department of Biochemistry, University of Pavia (Italy); Ferrari, N. [Istituto Nazionale per la ricerca sul Cancro, Genova (Italy); Ledda, B.; Volta, C. [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy); Manduca, P., E-mail: man-via@unige.it [Genetics, DIBIO, University of Genova, Corso Europa 26, 16132 Genova (Italy)

2010-03-10

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

Osteoblasts extracellular matrix induces vessel like structures through glycosylated collagen I

International Nuclear Information System (INIS)

Palmieri, D.; Valli, M.; Viglio, S.; Ferrari, N.; Ledda, B.; Volta, C.; Manduca, P.

2010-01-01

Extracellular matrix (ECM) plays a fundamental role in angiogenesis affecting endothelial cells proliferation, migration and differentiation. Vessels-like network formation in vitro is a reliable test to study the inductive effects of ECM on angiogenesis. Here we utilized matrix deposed by osteoblasts as substrate where the molecular and structural complexity of the endogenous ECM is preserved, to test if it induces vessel-like network formation by endothelial cells in vitro. ECM is more similar to the physiological substrate in vivo than other substrates previously utilized for these studies in vitro. Osteogenic ECM, prepared in vitro from mature osteoblasts at the phase of maximal deposition and glycosylation of collagen I, induces EAhy926, HUVEC, and HDMEC endothelial cells to form vessels-like structures and promotes the activation of metalloproteinase-2 (MMP-2); the functionality of the p-38/MAPK signaling pathway is required. Osteogenic ECM also induces a transient increase of CXCL12 and a decrease of the receptor CXCR4. The induction of vessel-like networks is dependent from proper glycosylation of collagens and does not occur on osteogenic ECMs if deglycosylated by -galactosidase or on less glycosylated ECMs derived from preosteoblasts and normal fibroblasts, while is sustained on ECM from osteogenesis imperfecta fibroblasts only when their mutation is associated with over-glycosylation of collagen type I. These data support that post-translational glycosylation has a role in the induction in endothelial cells in vitro of molecules conductive to self-organization in vessels-like structures.

Doxorubicin attached to HPMA copolymer via amide bond modifies the glycosylation pattern of EL4 cells.

Science.gov (United States)

Kovar, Lubomir; Etrych, Tomas; Kabesova, Martina; Subr, Vladimir; Vetvicka, David; Hovorka, Ondrej; Strohalm, Jiri; Sklenar, Jan; Chytil, Petr; Ulbrich, Karel; Rihova, Blanka

2010-08-01

To avoid the side effects of the anti-cancer drug doxorubicin (Dox), we conjugated this drug to a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone. Dox was conjugated via an amide bond (Dox-HPMA(AM), PK1) or a hydrazone pH-sensitive bond (Dox-HPMA(HYD)). In contrast to Dox and Dox-HPMA(HYD), Dox-HPMA(AM) accumulates within the cell's intracellular membranes, including those of the Golgi complex and endoplasmic reticulum, both involved in protein glycosylation. Flow cytometry was used to determine lectin binding and cell death, immunoblot to characterize the presence of CD7, CD43, CD44, and CD45, and high-performance anion exchange chromatography with pulsed amperometric detector analysis for characterization of plasma membrane saccharide composition. Incubation of EL4 cells with Dox-HPMA(AM) conjugate, in contrast to Dox or Dox-HPMA(HYD), increased the amounts of membrane surface-associated glycoproteins, as well as saccharide moieties recognized by peanut agglutinin, Erythrina cristagalli, or galectin-1 lectins. Only Dox-HPMA(AM) increased expression of the highly glycosylated membrane glycoprotein CD43, while expression of others (CD7, CD44, and CD45) was unaffected. The binding sites for galectin-1 are present on CD43 molecule. Furthermore, we present that EL4 treated with Dox-HPMA(AM) possesses increased sensitivity to galectin-1-induced apoptosis. In this study, we demonstrate that Dox-HPMA(AM) treatment changes glycosylation of the EL4 T cell lymphoma surface and sensitizes the cells to galectin-1-induced apoptosis.

A Knowledge-Based System for Display and Prediction of O-Glycosylation Network Behaviour in Response to Enzyme Knockouts.

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Andrew G McDonald

2016-04-01

Full Text Available O-linked glycosylation is an important post-translational modification of mucin-type protein, changes to which are important biomarkers of cancer. For this study of the enzymes of O-glycosylation, we developed a shorthand notation for representing GalNAc-linked oligosaccharides, a method for their graphical interpretation, and a pattern-matching algorithm that generates networks of enzyme-catalysed reactions. Software for generating glycans from the enzyme activities is presented, and is also available online. The degree distributions of the resulting enzyme-reaction networks were found to be Poisson in nature. Simple graph-theoretic measures were used to characterise the resulting reaction networks. From a study of in-silico single-enzyme knockouts of each of 25 enzymes known to be involved in mucin O-glycan biosynthesis, six of them, β-1,4-galactosyltransferase (β4Gal-T4, four glycosyltransferases and one sulfotransferase, play the dominant role in determining O-glycan heterogeneity. In the absence of β4Gal-T4, all Lewis X, sialyl-Lewis X, Lewis Y and Sda/Cad glycoforms were eliminated, in contrast to knockouts of the N-acetylglucosaminyltransferases, which did not affect the relative abundances of O-glycans expressing these epitopes. A set of 244 experimentally determined mucin-type O-glycans obtained from the literature was used to validate the method, which was able to predict up to 98% of the most common structures obtained from human and engineered CHO cell glycoforms.

Other cancers in lung cancer families are overwhelmingly smoking-related cancers

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Hongyao Yu

2017-06-01

Full Text Available Familial risks of lung cancer are well-established, but whether lung cancer clusters with other discordant cancers is less certain, particularly beyond smoking-related sites, which may provide evidence on genetic contributions to lung cancer aetiology. We used a novel approach to search for familial associations in the Swedish Family-Cancer Database. This involved assessment of familial relative risk for cancer X in families with increasing numbers of lung cancer patients and, conversely, relative risks for lung cancer in families with increasing numbers of patients with cancers X. However, we lacked information on smoking. The total number of lung cancers in the database was 125 563. We applied stringent statistical criteria and found that seven discordant cancers were associated with lung cancer among family members, and six of these were known to be connected with smoking: oesophageal, upper aerodigestive tract, liver, cervical, kidney and urinary bladder cancers. A further novel finding was that cancer of unknown primary also associated with lung cancer. We also factored in histological evidence and found that anal and connective tissue cancers could be associated with lung cancer for reasons other than smoking. For endometrial and prostate cancers, suggestive negative associations with lung cancer were found. Although we lacked information on smoking it is prudent to conclude that practically all observed discordant associations of lung cancer were with cancers for which smoking is a risk factor.

Glucosamine derived DISAL donors for stereoselective glycosylations under neutral conditions

DEFF Research Database (Denmark)

Grathe, S.; Thygesen, M.B.; Larsen, K.

2005-01-01

DISAL (methyl 3,5-dinitrosa/icylate) D-glcosyl, D-galactosyl, D-mannosyl, and L-quinovosyl donors have previously provided the efficient glycosylation of a range of substrates under either strictly neutral, mildly basic, or very mildly Lewis acidic (LiClO4) conditions. Herein we report the synthe......DISAL (methyl 3,5-dinitrosa/icylate) D-glcosyl, D-galactosyl, D-mannosyl, and L-quinovosyl donors have previously provided the efficient glycosylation of a range of substrates under either strictly neutral, mildly basic, or very mildly Lewis acidic (LiClO4) conditions. Herein we report...... the synthesis of new glucosamine DISAL donors, carrying N-TCP, -Troc, or -TFAc protecting groups, and their use in beta-(1,2-trans) selective glycosylations, primarily in NMP in the absence of any added Lewis acids, or in CH3NO2 with LiClO4. Finally, precise microwave heating proved effective in promoting...

Cancer-related anemia

International Nuclear Information System (INIS)

Abdel-Rzaeq, Hikmat N.

2004-01-01

Anemia is the most common hematological abnormality in cancer patients is often under-recognized and undertreated. The pathogenesis of cancer anemia is complex and most of time multifactorial; involving factors related to the tumor itself or its therapy. While anemia can be present in a wide range of symptoms, involing almost every organ, it is beleived that it contributes much to cancer-related-fatigue, one of the most common symptoms in cancer patients. In addition there is increasing evidence to suggest that anemia is an independent factor adversely affecting tumor reponse and patient survival. While blood transfusion was the only option to treat cancer related anemia, the use of recombinant human erythropoietin (rHuEPO) is becomig the new standard of care, more so with the recent studies demonstrating the feasibility of a sigle weekly injection .Things are even getting better with the recent approval of a new form of rHuEPO; Darbepoetin an analogue with a 3-fold longer half-life. In addition to its effects in raising homoglobin, several well controlled studies demonstrated decrease in transfusion requirementsand better qualify of life assessed objectively using standard assesments scales. (author)

Patterns of glycemic control using glycosylated hemoglobin in diabetics

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Arunpreet Singh Kahlon

2011-01-01

Full Text Available Aim : Till now estimation of blood glucose is the highly effective method for diagnosing diabetes mellitus but it provides a short-term picture of control. More evidence is required to prove that plasma glucose and glycosylated hemoglobin levels together gives a better estimate of glycemic control and compliance with treatment. Indian diabetes risk score (IDRS is a simplified screening tool for identifying undiagnosed diabetic subjects, requires minimum time, and effort and can help to considerably reduce the costs of screening. Objective : To study patterns of glycemic control using glycosylated hemoglobin in diabetic patients. To find out correlation between levels of plasma glucose and glycosylated hemoglobin in diabetics and to calculate IDRS of the study population. Materials and Methods : A cross sectional study was conducted among 300 known diabetic patients attending outpatient department of a rural medical college in Haryana, India. Following standard procedures and protocols FPG and glycosylated hemoglobin were measured to find out a pattern of glycemic control in them after taking their written and informed consent. A correlation between the levels of glycosylated hemoglobin and fasting blood glucose was also calculated. These patients were made to fill a performa and their demographic and clinical risk factors were noted and based on this, their IDRS was calculated. This was done to validate the IDRS in Indian rural population. Results : Fifty-two percent of the population had fasting plasma glucose level between 125-150 mg/dl, 21% had this level between 151-175 mg/dl. Thirteen percent of the study subjects had HbA1C between 6.5-7.5, more than half (57.3% had this value between 7.5-8.5, 12% and 18% had values between 8.5-9.5 and 9.5-10.5, respectively. Twelve percent of the participants had HbA1C level higher than 10.5. Correlation of fasting plasma glucose level and HbA1C was also studied and found that correlation coefficient came

Human Cell Line-Derived Monoclonal IgA Antibodies for Cancer Immunotherapy

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Felix Hart

2017-05-01

Full Text Available IgA antibodies have great potential to improve the functional diversity of current IgG antibody-based cancer immunotherapy options. However, IgA production and purification is not well established, which can at least in part be attributed to the more complex glycosylation as compared to IgG antibodies. IgA antibodies possess up to five N-glycosylation sites within their constant region of the heavy chain as compared to one site for IgG antibodies. The human GlycoExpress expression system was developed to produce biotherapeutics with optimized glycosylation and used here to generate a panel of IgA isotype antibodies directed against targets for solid (TA-mucin 1, Her2, EGFR, Thomsen–Friedenreich and hematological (CD20 cancer indications. The feasibility of good manufacturing practice was shown by the production of 11 g IgA within 35 days in a one liter perfusion bioreactor, and IgA antibodies in high purity were obtained after purification. The monoclonal IgA antibodies possessed a high sialylation degree, and no non-human glycan structures were detected. Kinetic analysis revealed increased avidity antigen binding for IgA dimers as compared to monomeric antibodies. The IgA antibodies exhibited potent Fab- and Fc-mediated functionalities against cancer cell lines, whereby especially granulocytes are recruited. Therefore, for patients who do not sufficiently benefit from therapeutic IgG antibodies, IgA antibodies may complement current regiment options and represent a promising strategy for cancer immunotherapy. In conclusion, a panel of novel biofunctional IgA antibodies with human glycosylation was successfully generated.

Endoplasmic reticulum stress and N-glycosylation modulate expression of WFS1 protein

International Nuclear Information System (INIS)

Yamaguchi, Suguru; Ishihara, Hisamitsu; Tamura, Akira; Yamada, Takahiro; Takahashi, Rui; Takei, Daisuke; Katagiri, Hideki; Oka, Yoshitomo

2004-01-01

Mutations of the WFS1 gene are responsible for two hereditary diseases, Wolfram syndrome and low frequency sensorineural hearing loss. The WFS1 protein is a glycoprotein located in the endoplasmic reticulum (ER) membrane but its function is poorly understood. Herein we show WFS1 mRNA and protein levels in pancreatic islets to be increased with ER-stress inducers, thapsigargin and dithiothreitol. Another ER-stress inducer, the N-glycosylation inhibitor tunicamycin, also raised WFS1 mRNA but not protein levels. Site-directed mutagenesis showed both Asn-663 and Asn-748 to be N-glycosylated in mouse WFS1 protein. The glycosylation-defective WFS1 protein, in which Asn-663 and Asn-748 had been substituted with aspartate, exhibited an increased protein turnover rate. Consistent with this, the WFS1 protein was more rapidly degraded in the presence of tunicamycin. These data indicate that ER-stress and N-glycosylation play important roles in WFS1 expression and stability, and also suggest regulatory roles for this protein in ER-stress induced cell death

EDEM2 and OS-9 are required for ER-associated degradation of non-glycosylated sonic hedgehog.

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Hsiang-Yun Tang

Full Text Available Misfolded proteins of the endoplasmic reticulum (ER are eliminated by the ER-associated degradation (ERAD in eukaryotes. In S. cerevisiae, ER-resident lectins mediate substrate recognition through bipartite signals consisting of an unfolded local structure and the adjacent glycan. Trimming of the glycan is essential for the directional delivery of the substrates. Whether a similar recognition and delivery mechanism exists in mammalian cells is unknown. In this study, we systematically study the function and substrate specificity of known mammalian ER lectins, including EDEM1/2/3, OS-9 and XTP-3B using the recently identified ERAD substrate sonic hedgehog (SHH, a soluble protein carrying a single N-glycan, as well as its nonglycosylated mutant N278A. Efficient ERAD of N278A requires the core processing complex of HRD1, SEL1L and p97, similar to the glycosylated SHH. While EDEM2 was required for ERAD of both glycosylated and non-glycosylated SHHs, EDEM3 was only necessary for glycosylated SHH and EDEM1 was dispensable for both. Degradation of SHH and N278A also required OS-9, but not the related lectin XTP3-B. Robust interaction of both EDEM2 and OS-9 with a non-glycosylated SHH variant indicates that the misfolded polypeptide backbone, rather than a glycan signature, functions as the predominant signal for recognition for ERAD. Notably, SHH-N278A is the first nonglycosylated substrate to require EDEM2 for recognition and targeting for ERAD. EDEM2 also interacts with calnexin and SEL1L, suggesting a potential avenue by which misfolded glycoproteins may be shunted towards SEL1L and ERAD rather than being released into the secretory pathway. Thus, ER lectins participate in the recognition and delivery of misfolded ER substrates differently in mammals, with an underlying mechanism distinct from that of S. cerevisiae.

IN VITRO STUDY ON INHIBITION OF GLYCOSYLATION OF ...

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complications of diabetes mellitus (Makita et al., 1991). Apart from protein ... enzymes; inhibition of regulatory molecule binding; crosslinking of glycosylated .... further investigation specific bio active compound responsible for such activities.

Evolutionary Pattern of N-Glycosylation Sequon Numbers  in Eukaryotic ABC Protein Superfamilies

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R. Shyama Prasad Rao

2010-02-01

Full Text Available Many proteins contain a large number of NXS/T sequences (where X is any amino acid except proline which are the potential sites of asparagine (N linked glycosylation. However, the patterns of occurrence of these N-glycosylation sequons in related proteins or groups of proteins and their underlying causes have largely been unexplored. We computed the actual and probabilistic occurrence of NXS/T sequons in ABC protein superfamilies from eight diverse eukaryotic organisms. The ABC proteins contained significantly higher NXS/T sequon numbers compared to respective genome-wide average, but the sequon density was significantly lower owing to the increase in protein size and decrease in sequon specific amino acids. However, mammalian ABC proteins have significantly higher sequon density, and both serine and threonine containing sequons (NXS and NXT have been positively selected—against the recent findings of only threonine specific Darwinian selection of sequons in proteins. The occurrence of sequons was positively correlated with the frequency of sequon specific amino acids and negatively correlated with proline and the NPS/T sequences. Further, the NPS/T sequences were significantly higher than expected in plant ABC proteins which have the lowest number of NXS/T sequons. Accord- ingly, compared to overall proteins, N-glycosylation sequons in ABC protein superfamilies have a distinct pattern of occurrence, and the results are discussed in an evolutionary perspective.

Measurement of Glycosylated Alpha-Fetoprotein Improves Diagnostic Power over the Native Form in Hepatocellular Carcinoma

Science.gov (United States)

Jin, Jonghwa; Park, Jiyoung; Yu, Su Jong; Yoon, Jung-Hwan; Kim, Youngsoo

2014-01-01

Serum alpha-fetoprotein (AFP) has long been used as a diagnostic marker for hepatocellular carcinoma (HCC), albeit controversially. Although it remains widely used in clinics, the value of AFP in HCC diagnosis has recently been challenged due to its significant rates of false positive and false negative findings. To improve the efficacy of AFP as HCC diagnostic marker, we developed a method of measuring total and glycosylated AFP by multiple reaction monitoring (MRM)-MS. In this study, we verified the total amount of AFP (nonglycopeptide levels) and the degree of glycosylated AFP (deglycopeptide levels) in 60 normal (41 men and 19 women; mean age 53 years; range 32–74 years), 35 LC (23 men and 12 women; mean age 56 years; range 43–78 years; HBV-related), and 60 HCC subjects (42 men and 18 women; mean age 58 years; range 38–76 years; HBV-related; 30 stage I, 15 stage II, and 10 stage III). By MRM-MS analysis, the nonglycopeptide had 56.7% sensitivity, 68.3% specificity, and an AUC of 0.687 [cutoff value: ≥0.02 (light/heavy ratio)], comparing the normal and HCC group, whereas the deglycopeptide had 93.3% sensitivity, 68.3% specificity, and an AUC of 0.859 [cutoff value: ≥0.02 (light/heavy ratio)]. In comparing the stage I HCC subgroup with the LC group, the nonglycopeptide had a sensitivity of 66.7%, specificity of 80.0%, and an AUC of 0.712 [cutoff value: ≥0.02 (light/heavy ratio)], whereas the deglycopeptide had a sensitivity of 96.7%, specificity of 80.0%, and an AUC of 0.918 [cutoff value: ≥0.02 (light/heavy ratio)]. These data demonstrate that the discriminatory power of the deglycopeptide is greater than that of the nonglycopeptide. We conclude that deglycopeptide can distinguish cancer status between normal subjects and HCC patients better than nonglycopeptide. PMID:25310463

Genome-scale metabolic model of Pichia pastoris with native and humanized glycosylation of recombinant proteins.

Science.gov (United States)

Irani, Zahra Azimzadeh; Kerkhoven, Eduard J; Shojaosadati, Seyed Abbas; Nielsen, Jens

2016-05-01

Pichia pastoris is used for commercial production of human therapeutic proteins, and genome-scale models of P. pastoris metabolism have been generated in the past to study the metabolism and associated protein production by this yeast. A major challenge with clinical usage of recombinant proteins produced by P. pastoris is the difference in N-glycosylation of proteins produced by humans and this yeast. However, through metabolic engineering, a P. pastoris strain capable of producing humanized N-glycosylated proteins was constructed. The current genome-scale models of P. pastoris do not address native nor humanized N-glycosylation, and we therefore developed ihGlycopastoris, an extension to the iLC915 model with both native and humanized N-glycosylation for recombinant protein production, but also an estimation of N-glycosylation of P. pastoris native proteins. This new model gives a better prediction of protein yield, demonstrates the effect of the different types of N-glycosylation of protein yield, and can be used to predict potential targets for strain improvement. The model represents a step towards a more complete description of protein production in P. pastoris, which is required for using these models to understand and optimize protein production processes. © 2015 Wiley Periodicals, Inc.

Congenital disorders of glycosylation: The Saudi experience.

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Alsubhi, Sarah; Alhashem, Amal; Faqeih, Eissa; Alfadhel, Majid; Alfaifi, Abdullah; Altuwaijri, Waleed; Alsahli, Saud; Aldhalaan, Hesham; Alkuraya, Fowzan S; Hundallah, Khalid; Mahmoud, Adel; Alasmari, Ali; Mutairi, Fuad Al; Abduraouf, Hanem; AlRasheed, Layan; Alshahwan, Saad; Tabarki, Brahim

2017-10-01

We retrospectively reviewed Saudi patients who had a congenital disorder of glycosylation (CDG). Twenty-seven Saudi patients (14 males, 13 females) from 13 unrelated families were identified. Based on molecular studies, the 27 CDG patients were classified into different subtypes: ALG9-CDG (8 patients, 29.5%), ALG3-CDG (7 patients, 26%), COG6-CDG (7 patients, 26%), MGAT2-CDG (3 patients, 11%), SLC35A2-CDG (1 patient), and PMM2-CDG (1 patient). All the patients had homozygous gene mutations. The combined carrier frequency of CDG for the encountered founder mutations in the Saudi population is 11.5 per 10,000, which translates to a minimum disease burden of 14 patients per 1,000,000. Our study provides comprehensive epidemiologic information and prevalence figures for each of these CDG in a large cohort of congenital disorder of glycosylation patients. © 2017 Wiley Periodicals, Inc.

Optimal Synthetic Glycosylation of a Therapeutic Antibody.

Science.gov (United States)

Parsons, Thomas B; Struwe, Weston B; Gault, Joseph; Yamamoto, Keisuke; Taylor, Thomas A; Raj, Ritu; Wals, Kim; Mohammed, Shabaz; Robinson, Carol V; Benesch, Justin L P; Davis, Benjamin G

2016-02-12

Glycosylation patterns in antibodies critically determine biological and physical properties but their precise control is a significant challenge in biology and biotechnology. We describe herein the optimization of an endoglycosidase-catalyzed glycosylation of the best-selling biotherapeutic Herceptin, an anti-HER2 antibody. Precise MS analysis of the intact four-chain Ab heteromultimer reveals nonspecific, non-enzymatic reactions (glycation), which are not detected under standard denaturing conditions. This competing reaction, which has hitherto been underestimated as a source of side products, can now be minimized. Optimization allowed access to the purest natural form of Herceptin to date (≥90 %). Moreover, through the use of a small library of sugars containing non-natural functional groups, Ab variants containing defined numbers of selectively addressable chemical tags (reaction handles at Sia C1) in specific positions (for attachment of cargo molecules or "glycorandomization") were readily generated.

Dengue Virus Glycosylation: What Do We Know?

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Sally S. L. Yap

2017-07-01

Full Text Available In many infectious diseases caused by either viruses or bacteria, pathogen glycoproteins play important roles during the infection cycle, ranging from entry to successful intracellular replication and host immune evasion. Dengue is no exception. Dengue virus glycoproteins, envelope protein (E and non-structural protein 1 (NS1 are two popular sub-unit vaccine candidates. E protein on the virion surface is the major target of neutralizing antibodies. NS1 which is secreted during DENV infection has been shown to induce a variety of host responses through its binding to several host factors. However, despite their critical role in disease and protection, the glycosylated variants of these two proteins and their biological importance have remained understudied. In this review, we seek to provide a comprehensive summary of the current knowledge on protein glycosylation in DENV, and its role in virus biogenesis, host cell receptor interaction and disease pathogenesis.

Conformationally superarmed S-ethyl glycosyl donors as effective building blocks for chemoselective oligosaccharide synthesis in one pot

DEFF Research Database (Denmark)

Bandara, Mithila D.; Yasomanee, Jagodige P.; Rath, Nigam P.

2017-01-01

A new series of superarmed glycosyl donors has been investigated. It was demonstrated that the S-ethyl leaving group allows for high reactivity, which is much higher than that of equally equipped S-phenyl glycosyl donors that were previously investigated by our groups. The superarmed S......-ethyl glycosyl donors equipped with a 2-O-benzoyl group gave complete β-stereoselectivity. Utility of the new glycosyl donors has been demonstrated in a one-pot one-addition oligosaccharide synthesis with all of the reaction components present from the beginning...

Low Density Lipoprotein Receptor Class A Repeats Are O-Glycosylated in Linker Regions

DEFF Research Database (Denmark)

Pedersen, Nis Borbye; Wang, Shengjun; Narimatsu, Yoshiki

2014-01-01

, which in wild-type CHO cells is glycosylated with the typical sialylated core 1 structure. The glycosites in linker regions of LDLR class A repeats are conserved in LDLR from man to Xenopus and found in other homologous receptors. O-Glycosylation is controlled by a large family of polypeptide Gal...

Prediction, conservation analysis, and structural characterization of mammalian mucin-type O-glycosylation sites

DEFF Research Database (Denmark)

Julenius, Karin; Mølgaard, Anne; Gupta, Ramneek

2005-01-01

could be predicted from averaged properties together with the fact that glycosylation sites are not precisely conserved indicates that mucin-type glycosylation in most cases is a bulk property and not a very site-specific one. NetOGlyc 3.1 is made available at www.cbs.dtu.dk/services/netoglyc....

Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes.

Science.gov (United States)

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H J; Hensbergen, Paul J; Reiding, Karli R; Hazes, Johanna M W; Dolhain, Radboud J E M; Wuhrer, Manfred

2014-11-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼ 20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Immunoglobulin G (IgG) Fab Glycosylation Analysis Using a New Mass Spectrometric High-throughput Profiling Method Reveals Pregnancy-associated Changes*

Science.gov (United States)

Bondt, Albert; Rombouts, Yoann; Selman, Maurice H. J.; Hensbergen, Paul J.; Reiding, Karli R.; Hazes, Johanna M. W.; Dolhain, Radboud J. E. M.; Wuhrer, Manfred

2014-01-01

The N-linked glycosylation of the constant fragment (Fc) of immunoglobulin G has been shown to change during pathological and physiological events and to strongly influence antibody inflammatory properties. In contrast, little is known about Fab-linked N-glycosylation, carried by ∼20% of IgG. Here we present a high-throughput workflow to analyze Fab and Fc glycosylation of polyclonal IgG purified from 5 μl of serum. We were able to detect and quantify 37 different N-glycans by means of MALDI-TOF-MS analysis in reflectron positive mode using a novel linkage-specific derivatization of sialic acid. This method was applied to 174 samples of a pregnancy cohort to reveal Fab glycosylation features and their change with pregnancy. Data analysis revealed marked differences between Fab and Fc glycosylation, especially in the levels of galactosylation and sialylation, incidence of bisecting GlcNAc, and presence of high mannose structures, which were all higher in the Fab portion than the Fc, whereas Fc showed higher levels of fucosylation. Additionally, we observed several changes during pregnancy and after delivery. Fab N-glycan sialylation was increased and bisection was decreased relative to postpartum time points, and nearly complete galactosylation of Fab glycans was observed throughout. Fc glycosylation changes were similar to results described before, with increased galactosylation and sialylation and decreased bisection during pregnancy. We expect that the parallel analysis of IgG Fab and Fc, as set up in this paper, will be important for unraveling roles of these glycans in (auto)immunity, which may be mediated via recognition by human lectins or modulation of antigen binding. PMID:25004930

A novel cerebello-ocular syndrome with abnormal glycosylation due to abnormalities in dolichol metabolism.

NARCIS (Netherlands)

Morava, E.; Wevers, R.A.; Cantagrel, V.; Hoefsloot, L.H.; Al-Gazali, L.; Schoots, J.; Rooij, A. van; Huijben, K.; Ravenswaaij-Arts, C.M.A. van; Jongmans, M.C.J.; Sykut-Cegielska, J.; Hoffmann, G.F.; Bluemel, P.; Adamowicz, M.; Reeuwijk, J. van; Ng, B.G.; Bergman, J.E.; Bokhoven, J.H.L.M. van; Korner, C.; Babovic-Vuksanovic, D.; Willemsen, M.A.A.P.; Gleeson, J.G.; Lehle, L.; Brouwer, A.P.M. de; Lefeber, D.J.

2010-01-01

Cerebellar hypoplasia and slowly progressive ophthalmological symptoms are common features in patients with congenital disorders of glycosylation type I. In a group of patients with congenital disorders of glycosylation type I with unknown aetiology, we have previously described a distinct phenotype

21 CFR 864.7470 - Glycosylated hemoglobin assay.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Glycosylated hemoglobin assay. 864.7470 Section 864.7470 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES HEMATOLOGY AND PATHOLOGY DEVICES Hematology Kits and Packages § 864.7470...

Development of on-chip fully automated immunoassay system "μTASWako i30" to measure the changes in glycosylation profiles of alpha-fetoprotein in patients with hepatocellular carcinoma.

Science.gov (United States)

Kurosawa, Tatsuo; Watanabe, Mitsuo

2016-12-01

Glycosylation profiles significantly change during oncogenesis. Aberrant glycosylation can be used as a cancer biomarker in clinical settings. Different glycoforms can be separately detected using lectin affinity electrophoresis and lectin array-based methods. However, most methodologies and procedures need experienced technique to perform the assays and expertise to interpret the results. To apply glycomarkers for clinical practice, a robust assay system with an easy-to-use workflow is required. Wako's μTASWako i30, a fully automated immunoanalyzer, was developed for in vitro diagnostics based on microfluidic technology. It utilizes the principles of liquid-phase binding assay, where immunoreactions are performed in a liquid phase, and electrokinetic analyte transport assay. Capillary electrophoresis on microfluidic chip has enabled the detection of different glycoform types of alpha-fetoprotein (AFP), a serum biomarker for hepatocellular carcinoma. AFP with altered glycosylation can be separated based on the reactivity to Lens culinaris agglutinin on electrophoresis. The glycoform AFP-L3 was reportedly more specific in hepatocellular carcinoma. This assay system can provide a high sensitivity and rapid results in 9 min. The test results for ratio of AFP-L3 to total AFP using μTASWako i30 are correlated with those of conventional methodology. The μTASWako assay system and the technology can be utilized for glycosylation analysis in the postgenomic era. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reduced apolipoprotein glycosylation in patients with the metabolic syndrome.

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Olga V Savinova

Full Text Available The purpose of this study was to compare the apolipoprotein composition of the three major lipoprotein classes in patients with metabolic syndrome to healthy controls.Very low density (VLDL, intermediate/low density (IDL/LDL, hereafter LDL, and high density lipoproteins (HDL fractions were isolated from plasma of 56 metabolic syndrome subjects and from 14 age-sex matched healthy volunteers. The apolipoprotein content of fractions was analyzed by one-dimensional (1D gel electrophoresis with confirmation by a combination of mass spectrometry and biochemical assays.Metabolic syndrome patients differed from healthy controls in the following ways: (1 total plasma--apoA1 was lower, whereas apoB, apoC2, apoC3, and apoE were higher; (2 VLDL--apoB, apoC3, and apoE were increased; (3 LDL--apoC3 was increased, (4 HDL--associated constitutive serum amyloid A protein (SAA4 was reduced (p<0.05 vs. controls for all. In patients with metabolic syndrome, the most extensively glycosylated (di-sialylated isoform of apoC3 was reduced in VLDL, LDL, and HDL fractions by 17%, 30%, and 25%, respectively (p<0.01 vs. controls for all. Similarly, the glycosylated isoform of apoE was reduced in VLDL, LDL, and HDL fractions by 15%, 26%, and 37% (p<0.01 vs. controls for all. Finally, glycosylated isoform of SAA4 in HDL fraction was 42% lower in patients with metabolic syndrome compared with controls (p<0.001.Patients with metabolic syndrome displayed several changes in plasma apolipoprotein composition consistent with hypertriglyceridemia and low HDL cholesterol levels. Reduced glycosylation of apoC3, apoE and SAA4 are novel findings, the pathophysiological consequences of which remain to be determined.

Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris.

Science.gov (United States)

Lei, Da; Xu, Yang; He, Qinghua; Pang, Yifeng; Chen, Bo; Xiong, Liang; Li, Yanping

2013-12-01

Neutral protease I from Aspergillus oryzae 3.042 was expressed in Pichia pastoris and its N-glycosylation properties were analyzed. After purification by nickel-affinity chromatography column, the recombinant neutral protease (rNPI) was confirmed to be N-glycosylated by periodicacid/Schiff's base staining and Endo H digestion. Moreover, the deglycosylated protein's molecular weight decreased to 43.3 kDa from 54.5 kDa analyzed by SDS-PAGE and MALDI-TOF-MS, and the hyperglycosylation extent was 21 %. The N-glycosylation site of rNPI was analyzed by nano LC-MS/MS after digesting by trypsin and Glu-C, and the unique potential site Asn41 of mature peptide was found to be glycosylated. Homology modeling of the 3D structure of rNPI indicated that the attached N-glycans hardly affected neutral protease's activity due to the great distance away from the active site of the enzyme.

Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN) on monocytes/macrophages.

Science.gov (United States)

Ge, Heng; Yuan, Wei; Liu, Jidong; He, Qing; Ding, Song; Pu, Jun; He, Ben

2015-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages. The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells) in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway. 1) It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN) that increased after being exposed to inflammatory signals (PMA and H2O2). 2) Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG) the simple type. 3) Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression). Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

Functional relevance of protein glycosylation to the pro-inflammatory effects of extracellular matrix metalloproteinase inducer (EMMPRIN on monocytes/macrophages.

Directory of Open Access Journals (Sweden)

Heng Ge

Full Text Available Extracellular matrix metalloproteinase inducer (EMMPRIN is an important pro-inflammatory protein involved in the cellular functions of monocytes/macrophages. We have hypothesized that high-level heterogeneousness of protein glycosylation of EMMPRIN may have functional relevance to its biological effects and affect the inflammatory activity of monocytes/macrophages.The glycosylation patterns of EMMPRIN expressed by monocytes/macrophages (THP-1 cells in response to different extracellular stimuli were observed, and the structures of different glycosylation forms were identified. After the purification of highly- and less-glycosylated proteins respectively, the impacts of different glycosylation forms on the pro-inflammatory effects of EMMPRIN were examined in various aspects, such as cell adhesion to endothelial cells, cell migrations, cytokine expression, and activation of inflammatory signalling pathway.1 It was mainly the highly-glycosylated form of EMMPRIN (HG-EMMPRIN that increased after being exposed to inflammatory signals (PMA and H2O2. 2 Glycosylation of EMMPRIN in monocytes/macrophages led to N-linked-glycans being added to the protein, with the HG form containing complex-type glycans and the less-glycosylated form (LG the simple type. 3 Only the HG-EMMPRIN but not the LG-EMMPRIN exhibited pro-inflammatory effects and stimulated inflammatory activities of the monocytes/macrophages (i.e., activation of ERK1/2 and NF-κB pathway, enhanced monocyte-endothelium adhesion, cell migration and matrix metalloproteinase -9 expression.Post-transcriptional glycosylation represents an important mechanism that determines the biological effects of EMMPRIN in monocytes/macrophages. Glycosylation of EMMPRIN may serve as a potential target for regulating the inflammatory activities of monocytes/macrophages.

The 'sweet' spot of cellular pluripotency: protein glycosylation in human pluripotent stem cells and its applications in regenerative medicine.

Science.gov (United States)

Wang, Yu-Chieh; Lin, Victor; Loring, Jeanne F; Peterson, Suzanne E

2015-05-01

Human pluripotent stem cells (hPSCs) promise for the future of regenerative medicine. The structural and biochemical diversity associated with glycans makes them a unique type of macromolecule modification that is involved in the regulation of a vast array of biochemical events and cellular activities including pluripotency in hPSCs. The primary focus of this review article is to highlight recent advances in stem cell research from a glycobiological perspective. We also discuss how our understanding of glycans and glycosylation may help overcome barriers hindering the clinical application of hPSC-derived cells. A literature survey using NCBI-PubMed and Google Scholar was performed in 2014. Regenerative medicine hopes to provide novel strategies to combat human disease and tissue injury that currently lack effective therapies. Although progress in this field is accelerating, many critical issues remain to be addressed in order for cell-based therapy to become a practical and safe treatment option. Emerging evidence suggests that protein glycosylation may significantly influence the regulation of cellular pluripotency, and that the exploitation of protein glycosylation in hPSCs and their differentiated derivatives may lead to transformative and translational discoveries for regenerative medicine. In addition, hPSCs represent a novel research platform for investigating glycosylation-related disease.

Is glycosylated haemoglobin a marker of fertility?

DEFF Research Database (Denmark)

Hjollund, N H; Jensen, Tina Kold; Bonde, Jens Peter

1999-01-01

We performed a follow-up study of time to pregnancy in a population of first-time pregnancy planners without previous reproductive experience. The objective of this paper is to report and discuss a finding of a strong relationship between glycosylated haemoglobin (HbA1C) and fertility. A total...

O-GLYCBASE version 2.0: a revised database of O-glycosylated proteins

DEFF Research Database (Denmark)

Hansen, Jan; Lund, Ole; Rapacki, Kristoffer

1997-01-01

O-GLYCBASE is an updated database of information on glycoproteins and their O-linked glycosylation sites. Entries are compiled and revised from the literature, and from the SWISS-PROT database. Entries include information about species, sequence, glycosylation sites and glycan type. O-GLYCBASE is...... patterns for the GalNAc, mannose and GlcNAc transferases are shown. The O-GLYCBASE database is available through WWW or by anonymous FTP....

In-Depth N-Glycosylation Reveals Species-Specific Modifications and Functions of the Royal Jelly Protein from Western (Apis mellifera) and Eastern Honeybees (Apis cerana).

Science.gov (United States)

Feng, Mao; Fang, Yu; Han, Bin; Xu, Xiang; Fan, Pei; Hao, Yue; Qi, Yuping; Hu, Han; Huo, Xinmei; Meng, Lifeng; Wu, Bin; Li, Jianke

2015-12-04

Royal jelly (RJ), secreted by honeybee workers, plays diverse roles as nutrients and defense agents for honeybee biology and human health. Despite being reported to be glycoproteins, the glycosylation characterization and functionality of RJ proteins in different honeybee species are largely unknown. An in-depth N-glycoproteome analysis and functional assay of RJ produced by Apis mellifera lingustica (Aml) and Apis cerana cerana (Acc) were conducted. RJ produced by Aml yielded 80 nonredundant N-glycoproteins carrying 190 glycosites, of which 23 novel proteins harboring 35 glycosites were identified. For Acc, all 43 proteins glycosylated at 138 glycosites were reported for the first time. Proteins with distinct N-glycoproteomic characteristics in terms of glycoprotein species, number of N-glycosylated sites, glycosylation motif, abundance level of glycoproteins, and N-glycosites were observed in this two RJ samples. The fact that the low inhibitory efficiency of N-glycosylated major royal jelly protein 2 (MRJP2) against Paenibacillus larvae (P. larvae) and the absence of antibacterial related glycosylated apidaecin, hymenoptaecin, and peritrophic matrix in the Aml RJ compared to Acc reveal the mechanism for why the Aml larvae are susceptible to P. larvae, the causative agent of a fatal brood disease (American foulbrood, AFB). The observed antihypertension activity of N-glycosylated MRJP1 in two RJ samples and a stronger activity found in Acc than in Aml reveal that specific RJ protein and modification are potentially useful for the treatment of hypertensive disease for humans. Our data gain novel understanding that the western and eastern bees have evolved species-specific strategies of glycosylation to fine-tune protein activity for optimizing molecular function as nutrients and immune agents for the good of honeybee and influence on the health promoting activity for human as well. This serves as a valuable resource for the targeted probing of the biological

Effects of N-glycosylation on protein conformation and dynamics: Protein Data Bank analysis and molecular dynamics simulation study.

Science.gov (United States)

Lee, Hui Sun; Qi, Yifei; Im, Wonpil

2015-03-09

N-linked glycosylation is one of the most important, chemically complex, and ubiquitous post-translational modifications in all eukaryotes. The N-glycans that are covalently linked to proteins are involved in numerous biological processes. There is considerable interest in developments of general approaches to predict the structural consequences of site-specific glycosylation and to understand how these effects can be exploited in protein design with advantageous properties. In this study, the impacts of N-glycans on protein structure and dynamics are systematically investigated using an integrated computational approach of the Protein Data Bank structure analysis and atomistic molecular dynamics simulations of glycosylated and deglycosylated proteins. Our study reveals that N-glycosylation does not induce significant changes in protein structure, but decreases protein dynamics, likely leading to an increase in protein stability. Overall, these results suggest not only a common role of glycosylation in proteins, but also a need for certain proteins to be properly glycosylated to gain their intrinsic dynamic properties.

Cancer-Related Fatigue in Cancer Survivorship.

Science.gov (United States)

Ebede, Chidinma C; Jang, Yongchang; Escalante, Carmen P

2017-11-01

Cancer-related fatigue (CRF) significantly interferes with usual functioning because of the distressing sense of physical, emotional, and cognitive exhaustion. Assessment of CRF is important and should be performed during the initial cancer diagnosis, throughout cancer treatment, and after treatment using a fatigue scoring scale (mild-severe). The general approach to CRF management applies to cancer survivors at all fatigue levels and includes education, counseling, and other strategies. Nonpharmacologic interventions include psychosocial interventions, exercise, yoga, physically based therapy, dietary management, and sleep therapy. Pharmacologic interventions include psychostimulants. Antidepressants may also benefit when CRF is accompanied by depression. Copyright © 2017 Elsevier Inc. All rights reserved.

Thermotolerance and protein glycosylation: Inhibition studies with sodium fluoride, azauridine and tunicamycin

International Nuclear Information System (INIS)

Bursey, D.L.; Henle, K.J.; Nagle, W.A.; Moss, A.J.

1987-01-01

The glycosylation hypothesis predicts increased incorporation of monosaccharides into 0-linked glycoproteins during thermotolerance development and inhibition of thermotolerance when this process is blocked. Specific inhibitors of 0-linked glycosylation are not available. The authors examined the effect of non-specific inhibition of glycosylation on thermotolerance development by: 1. restriction of both exogenous sugars and endogeneous sugar synthesis with NaF to block glycolysis while providing L-glutamine as a substrate for ATP synthesis in the TCA cycle; or 2. inhibition of UDP-sugar synthesis using azauridine and tunicamycin. Inhibitors were added to cell cultures after heat conditioning (10 min, 45 0 ) and removed after 6 hr prior to 45 0 -test heating. Sugar deprivation was achieved with 10mM NaF in glucose-free EBSS, supplemented with 2mM L-glutamine. Synthesis of UDP-sugars was inhibited with 1mM azauridine + 1μg/ml tunicamycin. Thermotolerance development was inhibited 87% by NaF/glutamine and 47% by azauridine/tunicamycin. For example, the D/sub o/ of the thermotolerant cells was 42.5 min (control D/sub o/ = 3 min), but only 5.5 min with inhibition by the NaF solution. These results support the absolute requirement of sugar precursors for thermotolerance development as predicted by the glycosylation hypothesis

A Systematic Study of Site-specific GalNAc-type O-Glycosylation Modulating Proprotein Convertase Processing

DEFF Research Database (Denmark)

Schjoldager, Katrine Ter-Borch Gram; Vester-Christensen, Malene B.; Goth, Christoffer K.

2011-01-01

Site-specific GalNAc-type O-glycosylation is emerging as an important co-regulator of proprotein convertase (PC) processing of proteins. PC processing is crucial in regulating many fundamental biological pathways and O-glycans in or immediately adjacent to processing sites may affect recognition...... and function of PCs. Thus, we previously demonstrated that deficiency in site-specific O-glycosylation in a PC site of the fibroblast growth factor, FGF23, resulted in marked reduction in secretion of active unprocessed FGF23, which cause familial tumoral calcinosis and hyperostosis hyperphosphatemia. GalNAc......-type O-glycosylation is found on serine and threonine amino acids and up to 20 distinct polypeptide GalNAc transferases catalyze the first addition of GalNAc to proteins making this step the most complex and differentially regulated steps in protein glycosylation. There is no reliable prediction model...

Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation.

Science.gov (United States)

Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian; Wagtberg Sen, Jette; Rasmussen, Søren Kofoed; Kontoravdi, Cleo; Weilguny, Dietmar; Andersen, Mikael Rørdam

2015-03-01

Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4(+) and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH4+ in the cell culture. Furthermore, galactosylation of the mAb Fc glycans

fasting blood glucose and glycosylated haemoglobin levels

African Journals Online (AJOL)

Prince Acheampong

(HbA1c) levels of diabetes mellitus patients as an index of glycaemic control. It was a prospective case- finding study using laboratory and general practice records. ... range of glycosylated haemoglobins, and the cut-off values for some clinical .... quality of glycaemic control by glycated haemoglobin in out-patient diabetic ...

Proteomics and pathway analysis of N-glycosylated mammary gland proteins in response to Escherichia coli mastitis in cattle.

Science.gov (United States)

Yang, Yongxin; Shen, Weijun; Zhao, Xiaowei; Zhao, Huiling; Huang, Dongwei; Cheng, Guanglong

2014-06-01

The aim of this study was to investigate the N-linked glycosylated protein profile of mammary tissue from healthy cows and cows with mastitis due to Escherichia coli, in order to understand the molecular mechanisms of the host response to mastitis. N-glycopeptides were enriched with a lectin mixture and identified through high-accuracy mass spectrometry. A total of 551 N-glycosylation sites, corresponding to 294 proteins, were identified in the mammary tissues of healthy cows; these glycoproteins were categorised into three functional groups and clustered into 11 specific pathways. A total of 511 N-glycosylation sites, corresponding to 283 glycosylated proteins, were detected in the mammary tissues of cows with E. coli mastitis. There were differences in N-glycosylation sites in 98 proteins in the mammary tissues of healthy cows and cows with mastitis due to E. coli. Most proteins with altered glycosylation were those involved in responses to stress, cell adhesion and the immune response, and were assigned to five specific pathways based on their gene ontology annotation. The results from this study show that the glycosylated protein profile in the mammary tissues of healthy and mastitic cows are different, and altered glycoproteins are associated with several pathways, including the lysosome and O-glycan biosynthesis pathways. Copyright © 2014. Published by Elsevier Ltd.

Structural differences between glycosylated, disulfide-linked heterodimeric Knob-into-Hole Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products.

Science.gov (United States)

Kuglstatter, A; Stihle, M; Neumann, C; Müller, C; Schaefer, W; Klein, C; Benz, J

2017-09-01

An increasing number of bispecific therapeutic antibodies are progressing through clinical development. The Knob-into-Hole (KiH) technology uses complementary mutations in the CH3 region of the antibody Fc fragment to achieve heavy chain heterodimerization. Here we describe the X-ray crystal structures of glycosylated and disulfide-engineered heterodimeric KiH Fc fragment and its homodimeric Knob-Knob and Hole-Hole side products. The heterodimer structure confirms the KiH design principle and supports the hypothesis that glycosylation stabilizes a closed Fc conformation. Both homodimer structures show parallel Fc fragment architectures, in contrast to recently reported crystal structures of the corresponding aglycosylated Fc fragments which in the absence of disulfide mutations show an unexpected antiparallel arrangement. The glycosylated Knob-Knob Fc fragment is destabilized as indicated by variability in the relative orientation of its CH3 domains. The glycosylated Hole-Hole Fc fragment shows an unexpected intermolecular disulfide bond via the introduced Y349C Hole mutation which results in a large CH3 domain shift and a new CH3-CH3 interface. The crystal structures of glycosylated, disulfide-linked KiH Fc fragment and its Knob-Knob and Hole-Hole side products reported here will facilitate further design of highly efficient antibody heterodimerization strategies. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Modeling the mechanism of glycosylation reactions between ethanol, 1,2-ethanediol and methoxymethanol.

Science.gov (United States)

Azofra, Luis Miguel; Alkorta, Ibon; Toro-Labbé, Alejandro; Elguero, José

2013-09-07

The mechanism of the S(N)2 model glycosylation reaction between ethanol, 1,2-ethanediol and methoxymethanol has been studied theoretically at the B3LYP/6-311+G(d,p) computational level. Three different types of reactions have been explored: (i) the exchange of hydroxyl groups between these model systems; (ii) the basic catalysis reactions by combination of the substrates as glycosyl donors (neutral species) and acceptors (enolate species); and (iii) the effect on the reaction profile of an explicit H2O molecule in the reactions considered in (ii). The reaction force, the electronic chemical potential and the reaction electronic flux have been characterized for the reaction path in each case. Energy calculations show that methoxymethanol is the worst glycosyl donor model among the ones studied here, while 1,2-ethanediol is the best, having the lowest activation barrier of 74.7 kJ mol(-1) for the reaction between this one and the ethanolate as the glycosyl acceptor model. In general, the presence of direct interactions between the atoms involved in the penta-coordinated TS increases the activation energies of the processes.

Glycation and transglutaminase mediated glycosylation of fish gelatin peptides with glucosamine enhance bioactivity.

Science.gov (United States)

Hong, Pui Khoon; Gottardi, Davide; Ndagijimana, Maurice; Betti, Mirko

2014-01-01

A mixture of novel glycopeptides from glycosylation between cold water fish skin gelatin hydrolysates and glucosamine (GlcN) via transglutaminase (TGase), as well as glycation between fish gelatin hydrolysate and GlcN were identified by their pattern of molecular distribution using MALDI-TOF-MS. Glycated/glycosylated hydrolysates showed superior bioactivity to their original hydrolysates. Alcalase-derived fish skin gelatin hydrolysate glycosylated with GlcN in the presence of TGase at 25°C (FAT25) possessed antioxidant activity when tested in a linoleic acid oxidation system, when measured according to its 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging activity and when tested at the cellular level with human hepatocarcinoma (HepG2) cells as target cells. In addition, Alcalase-derived glycosylated hydrolysates showed specificity toward the inhibition of Escherichia coli (E. coli). The Flavourzyme-derived glycopeptides prepared at 37°C (FFC37 and FFT37) showed better DPPH scavenging activity than their native hydrolysates. The glycated Flavourzyme-derived hydrolysates were found to act as potential antimicrobial agents when incubated with E. coli and Bacillus subtilis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fab glycosylation of immunoglobulin G does not associate with improvement of rheumatoid arthritis during pregnancy

NARCIS (Netherlands)

A. Bondt (Albert); M. Wuhrer (Manfred); T.M. Kuijper (Martijn); J.M.W. Hazes (Mieke); R.J.E.M. Dolhain (Radboud)

2016-01-01

textabstractBackground: Changes in immunoglobulin G (IgG) constant domain (Fc) glycosylation are associated with changes in rheumatoid arthritis (RA) disease activity in response to pregnancy. Here, we sought to determine whether the same holds true for variable domain (Fab) glycosylation. Methods:

SIKLODEKSTRIN GLIKOSIL TRANSFERASE DAN PEMANFAATANNYA DALAM INDUSTRI [Cyclodextrin Glycosyl Transferase and its application in industries

Directory of Open Access Journals (Sweden)

Budiasih Wahyuntari

2005-12-01

Full Text Available Cyclodextrin glycosyl transferase (CGT-ase is mainly produced by Bacilli. Systematical name of the enzyme is E.C. 2.4.1.19 a-1,4 glucan-4-glycosyl transferase. The enzyme catalyzes hydrolysis of starch intramolecular, and intermolecular transglycosylation of a-1,4, glucan chains. Cyclodextrins are a-1,4 linked cyclic oligosaccharides resulting from enzymatic degradation of starch by cyclodextrin glycosyl transferase through untramolecular transglycosylation. The major cyclodextrins are made up of 6, 7 and 8 glucopyranose units which are known as a-, b-, and y-cyclodextrin. All CGT-ase catalyze three kinds of cyclodextrins, the proportion of the cyclodextrins depends on the enzyme source and reaction conditions. The intermolecular transglycosylation ability of the enzyme has been applied in transfering glycosyl residues into suitable acceptor. Transglycosylation by the enzymes have been tested to improve solubility of some flavonoids and to favor precipitation ci some glycosides.

Impact of O-glycosylation on the molecular and cellular adhesion properties of the Escherichia coli autotransporter protein Ag43.

Science.gov (United States)

Reidl, Sebastian; Lehmann, Annika; Schiller, Roswitha; Salam Khan, A; Dobrindt, Ulrich

2009-08-01

Antigen 43 (Ag43) represents an entire family of closely related autotransporter proteins in Escherichia coli and has been described to confer aggregation and fluffing of cells, to promote biofilm formation, uptake and survival in macrophages as well as long-term persistence of uropathogenic E. coli in the murine urinary tract. Furthermore, it has been reported that glycosylation of the Ag43 passenger domain (alpha(43)) stabilizes its conformation and increases adhesion to Hep-2 cells. We characterized the role of Ag43 as an adhesin and the impact of O-glycosylation on the function of Ag43. To analyze whether structural variations in the alpha(43) domain correlate with different functional properties, we cloned 5 different agn43 alleles from different E. coli subtypes and tested them for autoaggregation, biofilm formation, adhesion to different eukaryotic cell lines as well as to purified components of the extracellular matrix. These experiments were performed with nonglycosylated and O-glycosylated Ag43 variants. We show for the first time that Ag43 mediates bacterial adhesion in a cell line-specific manner and that structural variations of the alpha(43) domain correlate with increased adhesive properties to proteins of the extracellular matrix such as collagen and laminin. Whereas O-glycosylation of many alpha(43) domains led to impaired autoaggregation and a significantly reduced adhesion to eukaryotic cell lines, their interaction with collagen was significantly increased. These data demonstrate that O-glycosylation is not a prerequisite for Ag43 function and that the different traits mediated by Ag43, i.e., biofilm formation, autoaggregation, adhesion to eukaryotic cells and extracellular matrix proteins, rely on distinct mechanisms.

Functional Divergence in the Role of N-Linked Glycosylation in Smoothened Signaling.

Directory of Open Access Journals (Sweden)

Suresh Marada

2015-08-01

Full Text Available The G protein-coupled receptor (GPCR Smoothened (Smo is the requisite signal transducer of the evolutionarily conserved Hedgehog (Hh pathway. Although aspects of Smo signaling are conserved from Drosophila to vertebrates, significant differences have evolved. These include changes in its active sub-cellular localization, and the ability of vertebrate Smo to induce distinct G protein-dependent and independent signals in response to ligand. Whereas the canonical Smo signal to Gli transcriptional effectors occurs in a G protein-independent manner, its non-canonical signal employs Gαi. Whether vertebrate Smo can selectively bias its signal between these routes is not yet known. N-linked glycosylation is a post-translational modification that can influence GPCR trafficking, ligand responsiveness and signal output. Smo proteins in Drosophila and vertebrate systems harbor N-linked glycans, but their role in Smo signaling has not been established. Herein, we present a comprehensive analysis of Drosophila and murine Smo glycosylation that supports a functional divergence in the contribution of N-linked glycans to signaling. Of the seven predicted glycan acceptor sites in Drosophila Smo, one is essential. Loss of N-glycosylation at this site disrupted Smo trafficking and attenuated its signaling capability. In stark contrast, we found that all four predicted N-glycosylation sites on murine Smo were dispensable for proper trafficking, agonist binding and canonical signal induction. However, the under-glycosylated protein was compromised in its ability to induce a non-canonical signal through Gαi, providing for the first time evidence that Smo can bias its signal and that a post-translational modification can impact this process. As such, we postulate a profound shift in N-glycan function from affecting Smo ER exit in flies to influencing its signal output in mice.

Por secretion system-dependent secretion and glycosylation of Porphyromonas gingivalis hemin-binding protein 35.

Directory of Open Access Journals (Sweden)

Mikio Shoji

Full Text Available The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS, which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs in their C-termini. Hemin-binding protein 35 (HBP35, which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study.

Adaptive antibody diversification through N-linked glycosylation of the immunoglobulin variable region.

Science.gov (United States)

van de Bovenkamp, Fleur S; Derksen, Ninotska I L; Ooijevaar-de Heer, Pleuni; van Schie, Karin A; Kruithof, Simone; Berkowska, Magdalena A; van der Schoot, C Ellen; IJspeert, Hanna; van der Burg, Mirjam; Gils, Ann; Hafkenscheid, Lise; Toes, René E M; Rombouts, Yoann; Plomp, Rosina; Wuhrer, Manfred; van Ham, S Marieke; Vidarsson, Gestur; Rispens, Theo

2018-02-20

A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N -linked glycans, a process conditional on the introduction of consensus amino acid motifs ( N -glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N -glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

Enhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry

NARCIS (Netherlands)

Wang, Guanbo; de Jong, Rob N; van den Bremer, Ewald T J; Parren, Paul W H I; Heck, Albert J R

2017-01-01

The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis

SnapShot: O-Glycosylation Pathways across Kingdoms

DEFF Research Database (Denmark)

Joshi, Hiren J.; Narimatsu, Yoshiki; Schjoldager, Katrine T.

2018-01-01

O-glycosylation is one of the most abundant and diverse types of post-translational modifications of proteins. O-glycans modulate the structure, stability, and function of proteins and serve generalized as well as highly specific roles in most biological processes. This ShapShot presents types of......-glycans found in different organisms and their principle biosynthetic pathways...

Role of protein glycosylation on the expression of muscarinic receptors of N4TG1 neuroblastoma cells

International Nuclear Information System (INIS)

Ahmad, A.; Chiang, P.K.

1986-01-01

Muscarinic acetylcholine receptors (mAChR) are glycoproteins. Experiments were conducted to determine whether active glycosylation of proteins in N4TG1 neuroblastoma cells could affect the expression of muscarinic receptors on the cell surface. The binding of radioactive N-methylscopolamine, a membrane impermeable ligand, to intact cells was used as a measure of mAChR. In the presence of the inhibitors of glycosylation, such as tunicamycin, monensin and amphomycin, N-linked glycosylation of proteins in the N4TG1 cells was inhibited, as measured by the incorporation of radioactive glucosamine or mannose in proteins. At the concentrations of tunicamycin and monensin used, the glycosylation of proteins after 3 hours were drastically reduced, but the number of mAChR in the cells was not altered. The apparent lack of effect within a short incubation period could be attributed to the presence of preformed oligosaccharide dolichol readily available for N-glycosylation. However, after 24 hours, tunicamycin (0.05 μg/ml) caused a decrease in the number of mAChR by 17% without having any effect on protein synthesis. Therefore, de novo glycosylation of proteins may be required for the expression of mAChR receptors in the N4TG1 neuroblastoma cell surface

An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins

DEFF Research Database (Denmark)

Hägglund, Per; Matthiesen, R.; Elortza, F.

2007-01-01

and N-acetyl-β-glucosaminidase) are also included. The two strategies were here applied to identify 103 N-glycosylation sites in the Cohn IV fraction of human plasma. In addition, Endo D/H digestion uniquely enabled identification of 23 fucosylated N-glycosylation sites. Several O-glycosylated peptides......, thereby reducing the complexity and facilitating glycosylation site determinations. Here, we have used two different enzymatic deglycosylation strategies for N-glycosylation site analysis. (1) Removal of entire N-glycan chains by peptide- N-glycosidase (PNGase) digestion, with concomitant deamidation...... of the released asparagine residue. The reaction is carried out in H218O to facilitate identification of the formerly glycosylated peptide by incorporatation of 18O into the formed aspartic acid residue. (2) Digestion with two endo-β- N-acetylglucosaminidases (Endo D and Endo H) that cleave the glycosidic bond...

Modulation and modeling of monoclonal antibody N-linked glycosylation in mammalian cell perfusion reactors.

Science.gov (United States)

Karst, Daniel J; Scibona, Ernesto; Serra, Elisa; Bielser, Jean-Marc; Souquet, Jonathan; Stettler, Matthieu; Broly, Hervé; Soos, Miroslav; Morbidelli, Massimo; Villiger, Thomas K

2017-09-01

Mammalian cell perfusion cultures are gaining renewed interest as an alternative to traditional fed-batch processes for the production of therapeutic proteins, such as monoclonal antibodies (mAb). The steady state operation at high viable cell density allows the continuous delivery of antibody product with increased space-time yield and reduced in-process variability of critical product quality attributes (CQA). In particular, the production of a confined mAb N-linked glycosylation pattern has the potential to increase therapeutic efficacy and bioactivity. In this study, we show that accurate control of flow rates, media composition and cell density of a Chinese hamster ovary (CHO) cell perfusion bioreactor allowed the production of a constant glycosylation profile for over 20 days. Steady state was reached after an initial transition phase of 6 days required for the stabilization of extra- and intracellular processes. The possibility to modulate the glycosylation profile was further investigated in a Design of Experiment (DoE), at different viable cell density and media supplement concentrations. This strategy was implemented in a sequential screening approach, where various steady states were achieved sequentially during one culture. It was found that, whereas high ammonia levels reached at high viable cell densities (VCD) values inhibited the processing to complex glycan structures, the supplementation of either galactose, or manganese as well as their synergy significantly increased the proportion of complex forms. The obtained experimental data set was used to compare the reliability of a statistical response surface model (RSM) to a mechanistic model of N-linked glycosylation. The latter outperformed the response surface predictions with respect to its capability and reliability in predicting the system behavior (i.e., glycosylation pattern) outside the experimental space covered by the DoE design used for the model parameter estimation. Therefore, we can

Enhanced SCAP glycosylation by inflammation induces macrophage foam cell formation.

Directory of Open Access Journals (Sweden)

Chao Zhou

Full Text Available Inflammatory stress promotes foam cell formation by disrupting LDL receptor feedback regulation in macrophages. Sterol Regulatory Element Binding Proteins (SREBPs Cleavage-Activating Protein (SCAP glycosylation plays crucial roles in regulating LDL receptor and 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCoAR feedback regulation. The present study was to investigate if inflammatory stress disrupts LDL receptor and HMGCoAR feedback regulation by affecting SCAP glycosylation in THP-1 macrophages. Intracellular cholesterol content was assessed by Oil Red O staining and quantitative assay. The expression of molecules controlling cholesterol homeostasis was examined using real-time quantitative RT-PCR and Western blotting. The translocation of SCAP from the endoplasmic reticulum (ER to the Golgi was detected by confocal microscopy. We demonstrated that exposure to inflammatory cytokines increased lipid accumulation in THP-1 macrophages, accompanying with an increased SCAP expression even in the presence of a high concentration of LDL. These inflammatory cytokines also prolonged the half-life of SCAP by enhancing glycosylation of SCAP due to the elevated expression of the Golgi mannosidase II. This may enhance translocation and recycling of SCAP between the ER and the Golgi, escorting more SREBP2 from the ER to the Golgi for activation by proteolytic cleavages as evidenced by an increased N-terminal of SREBP2 (active form. As a consequence, the LDL receptor and HMGCoAR expression were up-regulated. Interestingly, these effects could be blocked by inhibitors of Golgi mannosidases. Our results indicated that inflammation increased native LDL uptake and endogenous cholesterol de novo synthesis, thereby causing foam cell formation via increasing transcription and protein glycosylation of SCAP in macrophages. These data imply that inhibitors of Golgi processing enzymes might have a potential vascular-protective role in prevention of atherosclerotic foam

Stannylene‐Mediated Regioselective 6‐O‐Glycosylation of Unprotected Phenyl 1‐Thioglycopyranosides

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Maggi, Agnese; Madsen, Robert

2013-01-01

acetal, and then subjected to selective glycosylation at the 6‐position with the Koenigs–Knorr protocol. Peracylated glycosyl bromides of D‐glucose, D‐galactose, D‐mannose and D‐glucosamine were employed as the donors to give the corresponding (1→6)‐linked disaccharides in moderate to good yields......‐thio‐β‐D‐glucopyranoside gave rise to the corresponding (1→6)‐linked trisaccharides in moderate yields....

GtfA and GtfB Are Both Required for Protein O-Glycosylation in Lactobacillus plantarum

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Lee, I-Chiao; van Swam, Iris I.; Tomita, Satoru; Morsomme, Pierre; Rolain, Thomas; Hols, Pascal; Bron, Peter A.

2014-01-01

Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively. PMID:24532775

Transgenic rice seed expressing flavonoid biosynthetic genes accumulate glycosylated and/or acylated flavonoids in protein bodies

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Ogo, Yuko; Mori, Tetsuya; Nakabayashi, Ryo; Saito, Kazuki; Takaiwa, Fumio

2016-01-01

Plant-specialized (or secondary) metabolites represent an important source of high-value chemicals. In order to generate a new production platform for these metabolites, an attempt was made to produce flavonoids in rice seeds. Metabolome analysis of these transgenic rice seeds using liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometry was performed. A total of 4392 peaks were detected in both transgenic and non-transgenic rice, 20–40% of which were only detected in transgenic rice. Among these, 82 flavonoids, including 37 flavonols, 11 isoflavones, and 34 flavones, were chemically assigned. Most of the flavonols and isoflavones were O-glycosylated, while many flavones were O-glycosylated and/or C-glycosylated. Several flavonoids were acylated with malonyl, feruloyl, acetyl, and coumaroyl groups. These glycosylated/acylated flavonoids are thought to have been biosynthesized by endogenous rice enzymes using newly synthesized flavonoids whose biosynthesis was catalysed by exogenous enzymes. The subcellular localization of the flavonoids differed depending on the class of aglycone and the glycosylation/acylation pattern. Therefore, flavonoids with the intended aglycones were efficiently produced in rice seeds via the exogenous enzymes introduced, while the flavonoids were variously glycosylated/acylated by endogenous enzymes. The results suggest that rice seeds are useful not only as a production platform for plant-specialized metabolites such as flavonoids but also as a tool for expanding the diversity of flavonoid structures, providing novel, physiologically active substances. PMID:26438413

Discrimination between glycosylation patterns of therapeutic antibodies using a microfluidic platform, MALDI-MS and multivariate statistics.

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Thuy, Tran Thi; Tengstrand, Erik; Aberg, Magnus; Thorsén, Gunnar

2012-11-01

Optimal glycosylation with respect to the efficacy, serum half-life time, and immunogenic properties is essential in the generation of therapeutic antibodies. The glycosylation pattern can be affected by several different parameters during the manufacture of antibodies and may change significantly over cultivation time. Fast and robust methods for determination of the glycosylation patterns of therapeutic antibodies are therefore needed. We have recently presented an efficient method for the determination of glycans on therapeutic antibodies using a microfluidic CD platform for sample preparation prior to matrix-assisted laser-desorption mass spectrometry analysis. In the present work, this method is applied to analyse the glycosylation patterns of three commercially available therapeutic antibodies and one intended for therapeutic use. Two of the antibodies produced in mouse myeloma cell line (SP2/0) and one produced in Chinese hamster ovary (CHO) cells exhibited similar glycosylation patterns but could still be readily differentiated from each other using multivariate statistical methods. The two antibodies with most similar glycosylation patterns were also studied in an assessment of the method's applicability for quality control of therapeutic antibodies. The method presented in this paper is highly automated and rapid. It can therefore efficiently generate data that helps to keep a production process within the desired design space or assess that an identical product is being produced after changes to the process. Copyright © 2012 Elsevier B.V. All rights reserved.

Antibody glycosylation and its impact on the pharmacokinetics and pharmacodynamics of monoclonal antibodies and Fc-fusion proteins.

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Liu, Liming

2015-06-01

Understanding the impact of glycosylation and keeping a close control on glycosylation of product candidates are required for both novel and biosimilar monoclonal antibodies (mAbs) and Fc-fusion protein development to ensure proper safety and efficacy profiles. Most therapeutic mAbs are of IgG class and contain a glycosylation site in the Fc region at amino acid position 297 and, in some cases, in the Fab region. For Fc-fusion proteins, glycosylation also frequently occurs in the fusion partners. Depending on the expression host, glycosylation patterns in mAb or Fc-fusions can be significantly different, thus significantly impacting the pharmacokinetics (PK) and pharmacodynamics (PD) of mAbs. Glycans that have a major impact on PK and PD of mAb or Fc-fusion proteins include mannose, sialic acids, fucose (Fuc), and galactose (Gal). Mannosylated glycans can impact the PK of the molecule, leading to reduced exposure and potentially lower efficacy. The level of sialic acid, N-acetylneuraminic acid (NANA), can also have a significant impact on the PK of Fc-fusion molecules. Core Fuc in the glycan structure reduces IgG antibody binding to IgG Fc receptor IIIa relative to IgG lacking Fuc, resulting in decreased antibody-dependent cell-mediated cytotoxicity (ADCC) activities. Glycoengineered Chinese hamster ovary (CHO) expression systems can produce afucosylated mAbs that have increased ADCC activities. Terminal Gal in a mAb is important in the complement-dependent cytotoxicity (CDC) in that lower levels of Gal reduce CDC activity. Glycans can also have impacts on the safety of mAb. mAbs produced in murine myeloma cells such as NS0 and SP2/0 contain glycans such as Galα1-3Galβ1-4N-acetylglucosamine-R and N-glycolylneuraminic acid (NGNA) that are not naturally present in humans and can be immunogenic when used as therapeutics. © 2015 Wiley Periodicals, Inc. and the American Pharmacists Association.

Glycosylation of Hemagglutinin and Neuraminidase of Influenza A Virus as Signature for Ecological Spillover and Adaptation among Influenza Reservoirs

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Paul Kim

2018-04-01

Full Text Available Glycosylation of the hemagglutinin (HA and neuraminidase (NA of the influenza provides crucial means for immune evasion and viral fitness in a host population. However, the time-dependent dynamics of each glycosylation sites have not been addressed. We monitored the potential N-linked glycosylation (NLG sites of over 10,000 HA and NA of H1N1 subtype isolated from human, avian, and swine species over the past century. The results show a shift in glycosylation sites as a hallmark of 1918 and 2009 pandemics, and also for the 1976 “abortive pandemic”. Co-segregation of particular glycosylation sites was identified as a characteristic of zoonotic transmission from animal reservoirs, and interestingly, of “reverse zoonosis” of human viruses into swine populations as well. After the 2009 pandemic, recent isolates accrued glycosylation at canonical sites in HA, reflecting gradual seasonal adaptation, and a novel glycosylation in NA as an independent signature for adaptation among humans. Structural predictions indicated a remarkably pleiotropic influence of glycans on multiple HA epitopes for immune evasion, without sacrificing the receptor binding of HA or the activity of NA. The results provided the rationale for establishing the ecological niche of influenza viruses among the reservoir and could be implemented for influenza surveillance and improving pandemic preparedness.

Glycosylation of Hemagglutinin and Neuraminidase of Influenza A Virus as Signature for Ecological Spillover and Adaptation among Influenza Reservoirs

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Kim, Paul; Jang, Yo Han; Kwon, Soon Bin; Lee, Chung Min; Han, Gyoonhee; Seong, Baik Lin

2018-01-01

Glycosylation of the hemagglutinin (HA) and neuraminidase (NA) of the influenza provides crucial means for immune evasion and viral fitness in a host population. However, the time-dependent dynamics of each glycosylation sites have not been addressed. We monitored the potential N-linked glycosylation (NLG) sites of over 10,000 HA and NA of H1N1 subtype isolated from human, avian, and swine species over the past century. The results show a shift in glycosylation sites as a hallmark of 1918 and 2009 pandemics, and also for the 1976 “abortive pandemic”. Co-segregation of particular glycosylation sites was identified as a characteristic of zoonotic transmission from animal reservoirs, and interestingly, of “reverse zoonosis” of human viruses into swine populations as well. After the 2009 pandemic, recent isolates accrued glycosylation at canonical sites in HA, reflecting gradual seasonal adaptation, and a novel glycosylation in NA as an independent signature for adaptation among humans. Structural predictions indicated a remarkably pleiotropic influence of glycans on multiple HA epitopes for immune evasion, without sacrificing the receptor binding of HA or the activity of NA. The results provided the rationale for establishing the ecological niche of influenza viruses among the reservoir and could be implemented for influenza surveillance and improving pandemic preparedness. PMID:29642453

The human receptor for urokinase plasminogen activator. NH2-terminal amino acid sequence and glycosylation variants

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Behrendt, N; Rønne, E; Ploug, M

1990-01-01

-PA. The purified protein shows a single 55-60 kDa band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. It is a heavily glycosylated protein, the deglycosylated polypeptide chain comprising only 35 kDa. The glycosylated protein contains N-acetyl-D-glucosamine and sialic acid......, but no N-acetyl-D-galactosamine. Glycosylation is responsible for substantial heterogeneity in the receptor on phorbol ester-stimulated U937 cells, and also for molecular weight variations among various cell lines. The amino acid composition and the NH2-terminal amino acid sequence are reported...

Sensitive and comprehensive analysis of O-glycosylation in biotherapeutics: a case study of novel erythropoiesis stimulating protein.

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Kim, Unyong; Oh, Myung Jin; Seo, Youngsuk; Jeon, Yinae; Eom, Joon-Ho; An, Hyun Joo

2017-09-01

Glycosylation of recombinant human erythropoietins (rhEPOs) is significantly associated with drug's quality and potency. Thus, comprehensive characterization of glycosylation is vital to assess the biotherapeutic quality and establish the equivalency of biosimilar rhEPOs. However, current glycan analysis mainly focuses on the N-glycans due to the absence of analytical tools to liberate O-glycans with high sensitivity. We developed selective and sensitive method to profile native O-glycans on rhEPOs. O-glycosylation on rhEPO including O-acetylation on a sialic acid was comprehensively characterized. Details such as O-glycan structure and O-acetyl-modification site were obtained from tandem MS. This method may be applied to QC and batch analysis of not only rhEPOs but also other biotherapeutics bearing multiple O-glycosylations.

Mining the Virgin Land of Neurotoxicology: A Novel Paradigm of Neurotoxic Peptides Action on Glycosylated Voltage-Gated Sodium Channels

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Zhirui Liu

2012-01-01

Full Text Available Voltage-gated sodium channels (VGSCs are important membrane protein carrying on the molecular basis for action potentials (AP in neuronal firings. Even though the structure-function studies were the most pursued spots, the posttranslation modification processes, such as glycosylation, phosphorylation, and alternative splicing associating with channel functions captured less eyesights. The accumulative research suggested an interaction between the sialic acids chains and ion-permeable pores, giving rise to subtle but significant impacts on channel gating. Sodium channel-specific neurotoxic toxins, a family of long-chain polypeptides originated from venomous animals, are found to potentially share the binding sites adjacent to glycosylated region on VGSCs. Thus, an interaction between toxin and glycosylated VGSC might hopefully join the campaign to approach the role of glycosylation in modulating VGSCs-involved neuronal network activity. This paper will cover the state-of-the-art advances of researches on glycosylation-mediated VGSCs function and the possible underlying mechanisms of interactions between toxin and glycosylated VGSCs, which may therefore, fulfill the knowledge in identifying the pharmacological targets and therapeutic values of VGSCs.

Risk of ovarian cancer in women with first-degree relatives with cancer

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Soegaard, Marie; Frederiksen, Kirsten; Jensen, Allan

2009-01-01

OBJECTIVE: To assess the risk of ovarian cancer in women with first-degree relatives with cancer at one of the four most frequent hereditary sites based on validated cancer diagnoses and to examine the association according to age at diagnosis of ovarian cancer and histology. DESIGN: Case......-control study. SETTING AND POPULATION: First-degree relatives of 554 women with invasive epithelial ovarian cancer and 1,564 controls were included. METHODS: Analyses were performed using multiple logistic regression models. RESULTS: Ovarian cancer in a first-degree relative was significantly associated...... with increased risk of ovarian cancer (OR, 2.4; 95% CI, 1.4-4.1 (mother or sister)). Ovarian cancer in a first-degree relative appeared to be a stronger risk factor for early-onset (cancer than late-onset (OR, 5.3; 95% CI, 2.0-14.1 vs. OR, 1.8; 95% CI, 1.0-3.4). The positive association...

SLC39A8 Deficiency: A Disorder of Manganese Transport and Glycosylation.

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Park, Julien H; Hogrebe, Max; Grüneberg, Marianne; DuChesne, Ingrid; von der Heiden, Ava L; Reunert, Janine; Schlingmann, Karl P; Boycott, Kym M; Beaulieu, Chandree L; Mhanni, Aziz A; Innes, A Micheil; Hörtnagel, Konstanze; Biskup, Saskia; Gleixner, Eva M; Kurlemann, Gerhard; Fiedler, Barbara; Omran, Heymut; Rutsch, Frank; Wada, Yoshinao; Tsiakas, Konstantinos; Santer, René; Nebert, Daniel W; Rust, Stephan; Marquardt, Thorsten

2015-12-03

SLC39A8 is a membrane transporter responsible for manganese uptake into the cell. Via whole-exome sequencing, we studied a child that presented with cranial asymmetry, severe infantile spasms with hypsarrhythmia, and dysproportionate dwarfism. Analysis of transferrin glycosylation revealed severe dysglycosylation corresponding to a type II congenital disorder of glycosylation (CDG) and the blood manganese levels were below the detection limit. The variants c.112G>C (p.Gly38Arg) and c.1019T>A (p.Ile340Asn) were identified in SLC39A8. A second individual with the variants c.97G>A (p.Val33Met) and c.1004G>C (p.Ser335Thr) on the paternal allele and c.610G>T (p.Gly204Cys) on the maternal allele was identified among a group of unresolved case subjects with CDG. These data demonstrate that variants in SLC39A8 impair the function of manganese-dependent enzymes, most notably β-1,4-galactosyltransferase, a Golgi enzyme essential for biosynthesis of the carbohydrate part of glycoproteins. Impaired galactosylation leads to a severe disorder with deformed skull, severe seizures, short limbs, profound psychomotor retardation, and hearing loss. Oral galactose supplementation is a treatment option and results in complete normalization of glycosylation. SLC39A8 deficiency links a trace element deficiency with inherited glycosylation disorders. Copyright © 2015 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Synthesis of Curcumin Glycosides with Enhanced Anticancer Properties Using One-Pot Multienzyme Glycosylation Technique.

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Gurung, Rit Bahadur; Gong, So Youn; Dhakal, Dipesh; Le, Tuoi Thi; Jung, Na Rae; Jung, Hye Jin; Oh, Tae Jin; Sohng, Jae Kyung

2017-09-28

Curcumin is a natural polyphenolic compound, widely acclaimed for its antioxidant, antiinflammatory, antibacterial, and anticancerous properties. However, its use has been limited due to its low-aqueous solubility and poor bioavailability, rapid clearance, and low cellular uptake. In order to assess the effect of glycosylation on the pharmacological properties of curcumin, one-pot multienzyme (OPME) chemoenzymatic glycosylation reactions with UDP- α-D-glucose or UDP-α-D-2-deoxyglucose as donor substrate were employed. The result indicated significant conversion of curcumin to its glycosylated derivatives: curcumin 4'- O -β- glucoside, curcumin 4',4''-di- O -β-glucoside, curcumin 4'- O -β-2-deoxyglucoside, and curcumin 4',4''-di- O -β-2-deoxyglucoside. The products were characterized by ultra-fast performance liquid chromatography, high-resolution quadruple-time-of-flight electrospray ionization-mass spectrometry, and NMR analyses. All the products showed improved water solubility and comparable antibacterial activities. Additionally, the curcumin 4'- O -β-glucoside and curcumin 4'- O -β-2-deoxyglucoside showed enhanced anticancer activities compared with the parent aglycone and diglycoside derivatives. This result indicates that glycosylation can be an effective approach for enhancing the pharmaceutical properties of different natural products, such as curcumin.

Glycosyl-Nucleolipids as new bioinspired amphiphiles.

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Latxague, Laurent; Patwa, Amit; Amigues, Eric; Barthélémy, Philippe

2013-09-30

Four new Glycosyl-NucleoLipid (GNL) analogs featuring either a single fluorocarbon or double hydrocarbon chains were synthesized in good yields from azido thymidine as starting material. Physicochemical studies (surface tension measurements, differential scanning calorimetry) indicate that hydroxybutanamide-based GNLs feature endothermic phase transition temperatures like the previously reported double chain glycerol-based GNLs. The second generation of GNFs featuring a free nucleobase reported here presents a better surface activity (lower glim) compared to the first generation of GNFs.

Genes Involved in the Endoplasmic Reticulum N-Glycosylation Pathway of the Red Microalga Porphyridium sp.: A Bioinformatic Study

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Oshrat Levy-Ontman

2014-02-01

Full Text Available N-glycosylation is one of the most important post-translational modifications that influence protein polymorphism, including protein structures and their functions. Although this important biological process has been extensively studied in mammals, only limited knowledge exists regarding glycosylation in algae. The current research is focused on the red microalga Porphyridium sp., which is a potentially valuable source for various applications, such as skin therapy, food, and pharmaceuticals. The enzymes involved in the biosynthesis and processing of N-glycans remain undefined in this species, and the mechanism(s of their genetic regulation is completely unknown. In this study, we describe our pioneering attempt to understand the endoplasmic reticulum N-Glycosylation pathway in Porphyridium sp., using a bioinformatic approach. Homology searches, based on sequence similarities with genes encoding proteins involved in the ER N-glycosylation pathway (including their conserved parts were conducted using the TBLASTN function on the algae DNA scaffold contigs database. This approach led to the identification of 24 encoded-genes implicated with the ER N-glycosylation pathway in Porphyridium sp. Homologs were found for almost all known N-glycosylation protein sequences in the ER pathway of Porphyridium sp.; thus, suggesting that the ER-pathway is conserved; as it is in other organisms (animals, plants, yeasts, etc..

Cancer Communication on Social Media: Examining How Cancer Caregivers Use Facebook for Cancer-Related Communication.

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Gage-Bouchard, Elizabeth A; LaValley, Susan; Mollica, Michelle; Beaupin, Lynda Kwon

Americans are increasingly using social media (such as Facebook, YouTube, and Twitter) for health-related communication. Much of the previous research on social media and health communication has focused on Facebook groups related to a specific disease or Facebook pages related to an advocacy organization. Less is known about how people communicate about cancer on personal Facebook pages. In this study, we expand upon previous research by examining how cancer caregivers use personal Facebook pages for cancer-related communication. We examined themes in cancer-related exchanges through a content analysis of 12 months of data from 18 publically available Facebook pages hosted by parents of children with acute lymphoblastic leukemia (15 852 total posts). Six themes emerged: (1) documenting the cancer journey, (2) sharing emotional strain associated with caregiving, (3) promoting awareness and advocacy about pediatric cancer, (4) fundraising, (5) mobilizing support, and (6) expressing gratitude for support. Building upon previous research documenting the increasing use of social media for health-related communication and support, our findings show that personal Facebook pages offer a platform for cancer caregivers to share their cancer-related experiences, promote advocacy and awareness, and mobilize social support. Providers must recognize the importance of social media as a vehicle for support and communication for families of children with cancer. Nurses should educate parents on how to appraise information obtained through Facebook using evidence-based guidelines. Providers can encourage caregivers to use Facebook as a tool for communication, information, and support.

Prediction of mucin-type O-glycosylation sites in mammalian proteins using the composition of k-spaced amino acid pairs

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Sheng Zhi-Ya

2008-02-01

Full Text Available Abstract Background As one of the most common protein post-translational modifications, glycosylation is involved in a variety of important biological processes. Computational identification of glycosylation sites in protein sequences becomes increasingly important in the post-genomic era. A new encoding scheme was employed to improve the prediction of mucin-type O-glycosylation sites in mammalian proteins. Results A new protein bioinformatics tool, CKSAAP_OGlySite, was developed to predict mucin-type O-glycosylation serine/threonine (S/T sites in mammalian proteins. Using the composition of k-spaced amino acid pairs (CKSAAP based encoding scheme, the proposed method was trained and tested in a new and stringent O-glycosylation dataset with the assistance of Support Vector Machine (SVM. When the ratio of O-glycosylation to non-glycosylation sites in training datasets was set as 1:1, 10-fold cross-validation tests showed that the proposed method yielded a high accuracy of 83.1% and 81.4% in predicting O-glycosylated S and T sites, respectively. Based on the same datasets, CKSAAP_OGlySite resulted in a higher accuracy than the conventional binary encoding based method (about +5.0%. When trained and tested in 1:5 datasets, the CKSAAP encoding showed a more significant improvement than the binary encoding. We also merged the training datasets of S and T sites and integrated the prediction of S and T sites into one single predictor (i.e. S+T predictor. Either in 1:1 or 1:5 datasets, the performance of this S+T predictor was always slightly better than those predictors where S and T sites were independently predicted, suggesting that the molecular recognition of O-glycosylated S/T sites seems to be similar and the increase of the S+T predictor's accuracy may be a result of expanded training datasets. Moreover, CKSAAP_OGlySite was also shown to have better performance when benchmarked against two existing predictors. Conclusion Because of CKSAAP

Protein N-glycosylation in eukaryotic microalgae and its impact on the production of nuclear expressed biopharmaceuticals

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Elodie eMathieu-Rivet

2014-07-01

Full Text Available Microalgae are currently used for the production of food compounds. Recently, few microalgae species have been investigated as potential biofactories for the production of biopharmaceuticals. Indeed in this context, microalgae are cheap, classified as Generally Recognized As Safe (GRAS organisms and can be grown easily. However, problems remain to be solved before any industrial production of microalgae-made biopharmaceuticals. Among them, post-translational modifications of the proteins need to be considered. Especially, N-glycosylation acquired by the secreted recombinant proteins is of major concern since most of the biopharmaceuticals are N-glycosylated and it is well recognized that glycosylation represent one of their critical quality attribute. Therefore, the evaluation of microalgae as alternative cell factory for biopharmaceutical productions thus requires to investigate their N-glycosylation capability in order to determine to what extend it differs from their human counterpart and to determine appropriate strategies for remodelling the microalgae glycosylation into human-compatible oligosaccharides. Here, we review the secreted recombinant proteins which have been successfully produced in microalgae. We also report on recent bioinformatics and biochemical data concerning the structure of glycans N-linked to proteins from various microalgae phyla and comment the consequences on the glycan engineering strategies that may be necessary to render those microalgae-made biopharmaceuticals compatible with human therapy.

Statin use and reduced cancer-related mortality

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Nielsen, Sune F; Nordestgaard, Børge G; Bojesen, Stig E

2012-01-01

A reduction in the availability of cholesterol may limit the cellular proliferation required for cancer growth and metastasis. We tested the hypothesis that statin use begun before a cancer diagnosis is associated with reduced cancer-related mortality.......A reduction in the availability of cholesterol may limit the cellular proliferation required for cancer growth and metastasis. We tested the hypothesis that statin use begun before a cancer diagnosis is associated with reduced cancer-related mortality....

Financial Impact of Colorectal Cancer and Its Consequences: Associations Between Cancer-Related Financial Stress and Strain and Health-Related Quality of Life.

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Sharp, Linda; O'Leary, Eamonn; O'Ceilleachair, Alan; Skally, Mairead; Hanly, Paul

2018-01-01

The financial impact and consequences of cancer on the lives of survivors remain poorly understood. This is especially true for colorectal cancer. We investigated objective cancer-related financial stress, subjective cancer-related financial strain, and their association with health-related quality of life in colorectal cancer survivors. This was a cross-sectional postal survey. The study was conducted in Ireland, which has a mixed public-private healthcare system. Colorectal cancer survivors, diagnosed 6 to 37 months prior, were identified from the population-based National Cancer Registry. Cancer-related financial stress was assessed as impact of cancer on household ability to make ends meet and cancer-related financial strain by feelings about household financial situation since cancer diagnosis. Health-related quality of life was based on European Organisation for Research and Treatment of Cancer QLQ-C30 global health status. Logistic regression was used to identify associations between financial stress and strain and low health-related quality of life (lowest quartile, score ≤50). A total of 493 survivors participated. Overall, 41% reported cancer-related financial stress and 39% cancer-related financial strain; 32% reported both financial stress and financial strain. After adjustment for sociodemographic and clinical variables, the odds of low health-related quality of life were significantly higher in those who reported cancer-related financial stress postdiagnosis compared with those who reported no change in financial stress postcancer (OR = 2.54 (95% CI, 1.62-3.99)). The odds of low health-related quality of life were also significantly higher in those with worse financial strain postdiagnosis (OR =1.73 (95% CI, 1.09-2.72)). The OR for those with both cancer-related financial stress and financial strain was 2.59 (95% CI, 1.59-4.22). Survey responders were younger, on average, than nonresponders. Responders and nonresponders may have differed in cancer-related

High abundance of Serine/Threonine-rich regions predicted to be hyper-O-glycosylated in the secretory proteins coded by eight fungal genomes

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González Mario

2012-09-01

Full Text Available Abstract Background O-glycosylation of secretory proteins has been found to be an important factor in fungal biology and virulence. It consists in the addition of short glycosidic chains to Ser or Thr residues in the protein backbone via O-glycosidic bonds. Secretory proteins in fungi frequently display Ser/Thr rich regions that could be sites of extensive O-glycosylation. We have analyzed in silico the complete sets of putatively secretory proteins coded by eight fungal genomes (Botrytis cinerea, Magnaporthe grisea, Sclerotinia sclerotiorum, Ustilago maydis, Aspergillus nidulans, Neurospora crassa, Trichoderma reesei, and Saccharomyces cerevisiae in search of Ser/Thr-rich regions as well as regions predicted to be highly O-glycosylated by NetOGlyc (http://www.cbs.dtu.dk. Results By comparison with experimental data, NetOGlyc was found to overestimate the number of O-glycosylation sites in fungi by a factor of 1.5, but to be quite reliable in the prediction of highly O-glycosylated regions. About half of secretory proteins have at least one Ser/Thr-rich region, with a Ser/Thr content of at least 40% over an average length of 40 amino acids. Most secretory proteins in filamentous fungi were predicted to be O-glycosylated, sometimes in dozens or even hundreds of sites. Residues predicted to be O-glycosylated have a tendency to be grouped together forming hyper-O-glycosylated regions of varying length. Conclusions About one fourth of secretory fungal proteins were predicted to have at least one hyper-O-glycosylated region, which consists of 45 amino acids on average and displays at least one O-glycosylated Ser or Thr every four residues. These putative highly O-glycosylated regions can be found anywhere along the proteins but have a slight tendency to be at either one of the two ends.

Enzymatic Glycosylation of Small Molecules: Challenging Substrates Require Tailored Catalysts

Czech Academy of Sciences Publication Activity Database

Desmet, T.; Soetaert, W.; Bojarová, Pavla; Křen, Vladimír; Dijkhuizen, L.; Eastwick-Field, V.; Schiller, A.

2012-01-01

Roč. 18, č. 35 (2012), s. 10786-10801 ISSN 0947-6539 Institutional support: RVO:61388971 Keywords : acceptor specificity * enzyme engineering * glycosylation Subject RIV: CE - Biochemistry Impact factor: 5.831, year: 2012

Glycosylation-mediated phenylpropanoid partitioning in Populus tremuloides cell cultures

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Babst Benjamin A

2009-12-01

Full Text Available Abstract Background Phenylpropanoid-derived phenolic glycosides (PGs and condensed tannins (CTs comprise large, multi-purpose non-structural carbon sinks in Populus. A negative correlation between PG and CT concentrations has been observed in several studies. However, the molecular mechanism underlying the relationship is not known. Results Populus cell cultures produce CTs but not PGs under normal conditions. Feeding salicyl alcohol resulted in accumulation of salicins, the simplest PG, in the cells, but not higher-order PGs. Salicin accrual reflected the stimulation of a glycosylation response which altered a number of metabolic activities. We utilized this suspension cell feeding system as a model for analyzing the possible role of glycosylation in regulating the metabolic competition between PG formation, CT synthesis and growth. Cells accumulated salicins in a dose-dependent manner following salicyl alcohol feeding. Higher feeding levels led to a decrease in cellular CT concentrations (at 5 or 10 mM, and a negative effect on cell growth (at 10 mM. The competition between salicin and CT formation was reciprocal, and depended on the metabolic status of the cells. We analyzed gene expression changes between controls and cells fed with 5 mM salicyl alcohol for 48 hr, a time point when salicin accumulation was near maximum and CT synthesis was reduced, with no effect on growth. Several stress-responsive genes were up-regulated, suggestive of a general stress response in the fed cells. Salicyl alcohol feeding also induced expression of genes associated with sucrose catabolism, glycolysis and the Krebs cycle. Transcript levels of phenylalanine ammonia lyase and most of the flavonoid pathway genes were reduced, consistent with down-regulated CT synthesis. Conclusions Exogenous salicyl alcohol was readily glycosylated in Populus cell cultures, a process that altered sugar utilization and phenolic partitioning in the cells. Using this system, we

Glycosyl-Nucleolipids as New Bioinspired Amphiphiles

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Philippe Barthélémy

2013-09-01

Full Text Available Four new Glycosyl-NucleoLipid (GNL analogs featuring either a single fluorocarbon or double hydrocarbon chains were synthesized in good yields from azido thymidine as starting material. Physicochemical studies (surface tension measurements, differential scanning calorimetry indicate that hydroxybutanamide-based GNLs feature endothermic phase transition temperatures like the previously reported double chain glycerol-based GNLs. The second generation of GNFs featuring a free nucleobase reported here presents a better surface activity (lower glim compared to the first generation of GNFs.

Molecular basis for the presence of glycosylated onco-foetal fibronectin in oral carcinomas

DEFF Research Database (Denmark)

Wandall, Hans H; Dabelsteen, Sally; Sørensen, Jens Ahm

2007-01-01

spliced IIICS region. Using cell culture experiments, immunohistochemical staining of primary tissue, and RT-PCR of tumour cells isolated by laser capture techniques we have examined the molecular basis for the production of GOF in oral carcinomas. Immuno-histochemical investigation confirmed the stromal......Glycosylated onco-foetal fibronectin (GOF) deposited in the stroma of oral squamous cell carcinomas correlates with survival. One of the two polypeptide GalNAc-transferases, GalNAc-T3 or GalNAc-T6, is required for the biosynthesis of GOF by the initiation of a unique O-glycan in the alternative...... deposition of GOF in oral carcinomas. However, neither GalNAc-T3 nor GalNAc-T6 could be detected in stromal fibroblasts. In contrast both transferases were present in the oral squamous carcinoma cells, suggesting that GOF is produced by the oral cancer cells and not only the stromal cells. RT-PCR analysis...

Muc1 based breast cancer vaccines: role of post translational modifications

International Nuclear Information System (INIS)

Begum, M.; Khurshid, R.; Nagra, S.A.

2008-01-01

Vaccine development is one of the most promising fields in cancer research. After autologous transplantation, due to low tumour burden, patients are more likely to respond immunologically to a cancer vaccine. MUC1 with its adhesive and anti adhesive functions, immunostimulatory and immunosuppressive activities, is therefore a good candidate for breast cancer vaccine. A structure-based insight into the immunogenicity of natural MUC1 glyco forms, of its sub-domains, motifs and post translational modification like glycosylation and myriostoylation may aid the design of tumour vaccines. Primary sequences of human MUC1 were retrieved from the SWISSPROT data bank. Protein pattern search: The primary sequence of Human MUC1 was searched at PROSITE (a dictionary of protein sites and patterns) database. Our study observes that post-translational modifications play an important role in presenting MUC1 as a candidate for breast cancer vaccine. It is found that the phosphorylation and glycosylation of important functional motifs of MUC1 may take part in the production of cytokines that may provide immunization. (author)

Fc-Glycosylation in Human IgG1 and IgG3 Is Similar for Both Total and Anti-Red-Blood Cell Anti-K Antibodies

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Myrthe E. Sonneveld

2018-01-01

Full Text Available After albumin, immunoglobulin G (IgG are the most abundant proteins in human serum, with IgG1 and IgG3 being the most abundant subclasses directed against protein antigens. The quality of the IgG-Fc-glycosylation has important functional consequences, which have been found to be skewed toward low fucosylation in some antigen-specific immune responses. This increases the affinity to IgG1-Fc-receptor (FcγRIIIa/b and thereby directly affects downstream effector functions and disease severity. To date, antigen-specific IgG-glycosylation have not been analyzed for IgG3. Here, we analyzed 30 pregnant women with anti-K alloantibodies from a prospective screening cohort and compared the type of Fc-tail glycosylation of total serum- and antigen-specific IgG1 and IgG3 using mass spectrometry. Total serum IgG1 and IgG3 Fc-glycoprofiles were highly similar. Fc glycosylation of antigen-specific IgG varied greatly between individuals, but correlated significantly with each other for IgG1 and IgG3, except for bisection. However, although the magnitude of changes in fucosylation and galactosylation were similar for both subclasses, this was not the case for sialylation levels, which were significantly higher for both total and anti-K IgG3. We found that the combination of relative IgG1 and IgG3 Fc-glycosylation levels did not improve the prediction of anti-K mediated disease over IgG1 alone. In conclusion, Fc-glycosylation profiles of serum- and antigen-specific IgG1 and IgG3 are highly similar.

Glycosylation in HIV-1 envelope glycoprotein and its biological implications

KAUST Repository

Ho, Yung Shwen; Saksena, Nitin K.

2013-01-01

architecture, also controls intra- and inter-clade genetic variations. Discerning intra- and inter-clade glycosylation variations could therefore yield important information for understanding the molecular and biological differences between HIV clades and may

UGT74AN1, a Permissive Glycosyltransferase from Asclepias curassavica for the Regiospecific Steroid 3-O-Glycosylation.

Science.gov (United States)

Wen, Chao; Huang, Wei; Zhu, Xue-Lin; Li, Xiao-San; Zhang, Fan; Jiang, Ren-Wang

2018-02-02

A permissive steroid glycosyltransferase (UGT74AN1) from Asclepias curassavica exhibited robust capabilities for the regiospecific C3 glycosylation of cardiotonic steroids and C 21 steroid precursors, and unprecedented promiscuity toward 53 structurally diverse natural and unnatural compounds to form O-, N-, and S-glycosides, along with the catalytic reversibility for a one-pot transglycosylation reaction. These findings highlight UGT74AN1 as the first regiospecific catalyst for cardiotonic steroid C3 glycosylation and exhibit significant potential for glycosylation of diverse bioactive molecules in drug discovery.

The interdomain flexible linker of the polypeptide GalNAc transferases dictates their long-range glycosylation preferences

DEFF Research Database (Denmark)

Rivas, Matilde De Las; Lira-Navarrete, Erandi; Daniel, Earnest James Paul

2017-01-01

The polypeptide GalNAc-transferases (GalNAc-Ts), that initiate mucin-type O-glycosylation, consist of a catalytic and a lectin domain connected by a flexible linker. In addition to recognizing polypeptide sequence, the GalNAc-Ts exhibit unique long-range N- A nd/or C-terminal prior glycosylation ...

Closely related glycosylation patterns of recombinant human IL-2 expressed in a CHO cell line and natural IL-2

International Nuclear Information System (INIS)

Vita, N.; Magazin, M.; Marchese, E.; Lupker, J.; Ferrara, P.

1990-01-01

We report here the study of the glycosylation pattern of human recombinant (r) IL2 expressed in a Chinese hamster ovary (CHO) cell line. The human rIL2 secreted by this high-producing recombinant CHO cell line was metabolically radiolabelled with [35S]-methionine, or with [3H]-glucosamine and [3H]-galactose, purified to homogeneity, and then characterized. The electrophoretic analysis of the [35S]-methionine-labelled proteins present in the culture medium of the CHO cell line showed that the rIL2 represents approximately 12% of the total secreted proteins. Furthermore, pulse-chase experiments showed that the glycosylated rIL2 is synthesized and secreted within 30 min. The point of attachment and the structure of the carbohydrate moiety of the rIL2 was determined by: amino-terminal sequencing and fingerprint analysis of the 3H-labelled rIL2, mass spectroscopy of the amino-terminal tryptic octapeptide, and carbohydrate analysis after enzymatic (Vibrio cholerae neuraminidase and Aspergillus oryzae beta-galactosidase) or sulfuric acid hydrolysis. The results indicate that the recombinant protein possesses a sugar moiety O-linked to the threonine residue at position 3 of the polypeptide chain, and that sialic acid, galactose and N-acetyl galactosamine are components of this carbohydrate moiety. Taken together these results suggest that the recombinant molecule is identical to natural IL2

Encoding asymmetry of the N-glycosylation motif facilitates glycoprotein evolution.

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Ryan Williams

Full Text Available Protein N-glycosylation is found in all domains of life and has a conserved role in glycoprotein folding and stability. In animals, glycoproteins transit through the Golgi where the N-glycans are trimmed and rebuilt with sequences that bind lectins, an innovation that greatly increases structural diversity and redundancy of glycoprotein-lectin interaction at the cell surface. Here we ask whether the natural tension between increasing diversity (glycan-protein interactions and site multiplicity (backup and status quo might be revealed by a phylogenic examination of glycoproteins and NXS/T(X ≠ P N-glycosylation sites. Site loss is more likely by mutation at Asn encoded by two adenosine (A-rich codons, while site gain is more probable by generating Ser or Thr downstream of an existing Asn. Thus mutations produce sites at novel positions more frequently than the reversal of recently lost sites, and therefore more paths though sequence space are made available to natural selection. An intra-species comparison of secretory and cytosolic proteins revealed a departure from equilibrium in sequences one-mutation-away from NXS/T and in (A content, indicating strong selective pressures and exploration of N-glycosylation positions during vertebrate evolution. Furthermore, secretory proteins have evolved at rates proportional to N-glycosylation site number, indicating adaptive interactions between the N-glycans and underlying protein. Given the topology of the genetic code, mutation of (A is more often nonsynonomous, and Lys, another target of many PTMs, is also encoded by two (A-rich codons. An examination of acetyl-Lys sites in proteins indicated similar evolutionary dynamics, consistent with asymmetry of the target and recognition portions of modified sites. Our results suggest that encoding asymmetry is an ancient mechanism of evolvability that increases diversity and experimentation with PTM site positions. Strong selective pressures on PTMs may have

Glycosylated yellow laccases of the basidiomycete Stropharia aeruginosa.

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Daroch, Maurycy; Houghton, Catharine A; Moore, Jonathan K; Wilkinson, Mark C; Carnell, Andrew J; Bates, Andrew D; Iwanejko, Lesley A

2014-05-10

Here we describe the identification, purification and characterisation of glycosylated yellow laccase proteins from the basidiomycete fungus Stropharia aeruginosa. Biochemical characterisation of two yellow laccases, Yel1p and Yel3p, show that they are both secreted, monomeric, N-glycosylated proteins of molecular weight around 55kDa with substrate specificities typical of laccases, but lacking the absorption band at 612nm typical of the blue laccase proteins. Low coverage, high throughput 454 transcriptome sequencing in combination with inverse-PCR was used to identify cDNA sequences. One of the cDNA sequences has been assigned to the Yel1p protein on the basis of identity between the translated protein sequence and the peptide data from the purified protein, and the full length gene sequence has been obtained. Biochemical properties, substrate specificities and protein sequence data have been used to discuss the unusual spectroscopic properties of S. aeruginosa proteins in the context of recent theories about the differences between yellow and blue laccases. Copyright © 2014 Elsevier Inc. All rights reserved.

N-glycosylation of the β2 adrenergic receptor regulates receptor function by modulating dimerization.

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Li, Xiaona; Zhou, Mang; Huang, Wei; Yang, Huaiyu

2017-07-01

N-glycosylation is a common post-translational modification of G-protein-coupled receptors (GPCRs). However, it remains unknown how N-glycosylation affects GPCR signaling. β 2 adrenergic receptor (β 2 AR) has three N-glycosylation sites: Asn6, Asn15 at the N-terminus, and Asn187 at the second extracellular loop (ECL2). Here, we show that deletion of the N-glycan did not affect receptor expression and ligand binding. Deletion of the N-glycan at the N-terminus rather than Asn187 showed decreased effects on isoproterenol-promoted G-protein-dependent signaling, β-arrestin2 recruitment, and receptor internalization. Both N6Q and N15Q showed decreased receptor dimerization, while N187Q did not influence receptor dimerization. As decreased β 2 AR homodimer accompanied with reduced efficiency for receptor function, we proposed that the N-glycosylation of β 2 AR regulated receptor function by influencing receptor dimerization. To verify this hypothesis, we further paid attention to the residues at the dimerization interface. Studies of Lys60 and Glu338, two residues at the receptor dimerization interface, exhibited that the K60A/E338A showed decreased β 2 AR dimerization and its effects on receptor signaling were similar to N6Q and N15Q, which further supported the importance of receptor dimerization for receptor function. This work provides new insights into the relationship among glycosylation, dimerization, and function of GPCRs. Peptide-N-glycosidase F (PNGase F, EC 3.2.2.11); endo-β-N-acetylglucosaminidase A (Endo-A, EC 3.2.1.96). © 2017 Federation of European Biochemical Societies.

DNA methylation in Cosmc promoter region and aberrantly glycosylated IgA1 associated with pediatric IgA nephropathy.

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Qiang Sun

Full Text Available IgA nephropathy (IgAN is one of the most common glomerular diseases leading to end-stage renal failure. Elevation of aberrantly glycosylated IgA1 is a key feature of it. The expression of the specific molecular chaperone of core1ß1, 3galactosyl transferase (Cosmc is known to be reduced in IgAN. We aimed to investigate whether the methylation of CpG islands of Cosmc gene promoter region could act as a possible mechanism responsible for down-regulation of Cosmc and related higher secretion of aberrantly glycosylated IgA1in lymphocytes from children with IgA nephropathy. Three groups were included: IgAN children (n = 26, other renal diseases (n = 11 and healthy children (n = 13. B-lymphocytes were isolated and cultured, treated or not with IL-4 or 5-Aza-2'-deoxycytidine (AZA. The levels of DNA methylation of Cosmc promotor region were not significantly different between the lymphocytes of the three children populations (P = 0.113, but there were significant differences between IgAN lymphocytes and lymphocytes of the other two children populations after IL-4 (P<0.0001 or AZA (P<0.0001. Cosmc mRNA expression was low in IgAN lymphocytes compared to the other two groups (P<0.0001. The level of aberrantly glycosylated IgA1 was markedly higher in IgAN group compared to the other groups (P<0.0001. After treatment with IL-4, the levels of Cosmc DNA methylation and aberrantly glycosylated IgA1 in IgAN lymphocytes were remarkably higher than the other two groups (P<0.0001 with more markedly decreased Cosmc mRNA content (P<0.0001. After treatment with AZA, the levels in IgAN lymphocytes were decreased, but was still remarkably higher than the other two groups (P<0.0001, while Cosmc mRNA content in IgAN lymphocytes were more markedly increased than the other two groups (P<0.0001. The alteration of DNA methylation by IL-4 or AZA specifically correlates in IgAN lymphocytes with alterations in Cosmc mRNA expression and with the level of aberrantly glycosylated

A new strategy for identification of N-glycosylated proteins and unambiguous assignment of their glycosylation sites using HILIC enrichment and partial deglycosylation

DEFF Research Database (Denmark)

Hägglund, Per; Bunkenborg, Jakob; Elortza, Felix

2004-01-01

remains linked to the asparagine residue. The removal of the major part of the glycan simplifies the MS/MS fragment ion spectra of glycopeptides, while the remaining GlcNAc residue enables unambiguous assignment of the glycosylation site together with the amino acid sequence. We first tested our approach...

[Proteins modified in the nonenzymatically glycosylation reaction (AGE-proteins)--new markers for diabetes?].

Science.gov (United States)

Zdrojewicz, Z; Januszewski, A; Kwiatkowska, D

1994-01-01

Paper present a recent review on the formation and clinical significance of advanced glycosylation end products, produced in nonenzymatically glycosylation, called Maillard reaction. The special attention was paid to AGEs role in diabetic and aging processes. Instant of occurring of AGEs in circulation or increase of AGE receptor concentration are many years faster than clinical pathology of vessels, nervous or kidneys connect with diabetes or aging. May be in the future it will be possible to decrease the consequence of Maillard reaction by using pharmacology drugs.

Glycosylation intermediates studied using low temperature ¹H- and ¹⁹F-DOSY NMR

DEFF Research Database (Denmark)

Qiao, Yan; Ge, Wenzhi; Jia, Lingyu

2016-01-01

Low temperature 1H- and 19F-DOSY have been used for analyzing reactive intermediates in glycosylation reactions, where a glycosyl trichloroacetimidate donor has been activated using different catalysts. The DOSY protocols have been optimized for low temperature experiments and provided new insight...

Glycosyl azide-a novel substrate for enzymatic transgycosylations

Czech Academy of Sciences Publication Activity Database

Fialová, Pavla; Carmona, A. T.; Robina, I.; Ettrich, R.; Sedmera, Petr; Přikrylová, Věra; Hušáková, Lucie; Křen, Vladimír

2005-01-01

Roč. 46, - (2005), s. 8715-8718 ISSN 0040-4039 R&D Projects: GA ČR GA203/05/0172; GA MŠk OC D25.002 Grant - others:GA KONTAKT 1862/04 Institutional research plan: CEZ:AV0Z50200510 Keywords : enzyme catalysis * glycosyl azide * molecular modelling Subject RIV: EE - Microbiology, Virology Impact factor: 2.477, year: 2005

Crystallization and preliminary crystallographic analysis of human glycosylated haemoglobin

International Nuclear Information System (INIS)

Syakhovich, Vitaly E.; Saraswathi, N. T.; Ruff, Marc; Bokut, Sergey B.; Moras, Dino

2006-01-01

Non enzymatic modification of haemoglobin by glucose plays an important role in diabetes pathogenesis. Here the purification, characterization and crystallization of human glycosylated haemoglobin are reported. Human glycosylated haemoglobin A 1C is a stable minor variant formed in vivo by post-translational modification of the main form of haemoglobin by glucose. Crystals of oxyHbA 1C were obtained using the hanging-drop vapour-diffusion method and PEG as precipitant. The diffraction pattern of the crystal extends to a resolution of 2.3 Å at 120 K. The crystals belong to space group C2, with unit-cell parameters a = 237.98, b = 59.27, c = 137.02 Å, α = 90.00, β = 125.40, γ = 90.00°. The presence of two and a half molecules per asymmetric unit gives a crystal volume per protein weight (V M ) of 9.70 Å 3 Da −1 and a solvent content of 49%

Crystallization and preliminary crystallographic analysis of human glycosylated haemoglobin

Energy Technology Data Exchange (ETDEWEB)

Syakhovich, Vitaly E. [Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus); Saraswathi, N. T.; Ruff, Marc, E-mail: ruff@igbmc.u-strasbg.fr [Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch (France); Bokut, Sergey B. [Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus); Moras, Dino [Département de Biologie et Génomique Structurales, Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 Rue Laurent Fries, BP 10142, 67404 Illkirch (France); Department of Biochemistry and Biophysics, International Sakharov Environmental University, Dolgobrodskaya St 23, 220009 Minsk (Belarus)

2006-02-01

Non enzymatic modification of haemoglobin by glucose plays an important role in diabetes pathogenesis. Here the purification, characterization and crystallization of human glycosylated haemoglobin are reported. Human glycosylated haemoglobin A{sub 1C} is a stable minor variant formed in vivo by post-translational modification of the main form of haemoglobin by glucose. Crystals of oxyHbA{sub 1C} were obtained using the hanging-drop vapour-diffusion method and PEG as precipitant. The diffraction pattern of the crystal extends to a resolution of 2.3 Å at 120 K. The crystals belong to space group C2, with unit-cell parameters a = 237.98, b = 59.27, c = 137.02 Å, α = 90.00, β = 125.40, γ = 90.00°. The presence of two and a half molecules per asymmetric unit gives a crystal volume per protein weight (V{sub M}) of 9.70 Å{sup 3} Da{sup −1} and a solvent content of 49%.

[Cancer-vitamins-minerals: Complex relation].

Science.gov (United States)

Adrianza de Baptista, Gertrudis; Murillo Melo, Carolain

2014-12-01

Since nutrition can influence the process of carcinogenesis, this study's objectives are to review the relationship between nutrition and cancer from the point of view of the role of micronutrients in the treatment of cancer patients, and to get to know the deficit relationship and the excess of micronutrients, with the etiology and cancer treatment. At the same time the patient's weight loss relates, among other things, to the type of cancerous tumor, its location, stage thereof, reason for which it may be associated with the deficiency of macro and micronutrients as from psychogenic, anorectics and mal-absorption effects or with mechanical effects as obstruction, among other toxic effects that are common in the treatment of cancer. Hence, the importance that the nutrition expert must have in making an adequate overall nutritional evaluation that allows the nutritional diagnosis, in studying the dietary patterns, to determine the toxic effects of the antineoplastic treatment in order to handle the treatment's timing excellence, symptoms and signs, and thus act effectively optiimizing the patient's life quality, and therewith his/her survival. There are controversies as to which specific dietary factors are related to cancer etiology and the results of studies on metabolic factors, and therefore, the relationship Cancer-Nutrition is quiet complex.

Detection of site specific glycosylation in proteins using flow cytometry†

Science.gov (United States)

Jayakumar, Deepak; Marathe, Dhananjay D.; Neelamegham, Sriram

2009-01-01

We tested the possibility that it is possible to express unique peptide probes on cell surfaces and detect site-specific glycosylation on these peptides using flow cytometry. Such development can enhance the application of flow cytometry to detect and quantify post-translational modifications in proteins. To this end, the N-terminal section of the human leukocyte glycoprotein PSGL-1 (P-selectin glycoprotein ligand-1) was modified to contain a poly-histidine tag followed by a proteolytic cleavage site. Amino acids preceding the cleavage site have a single O-linked glycosylation site. The recombinant protein called PSGL-1 (HT) was expressed on the surface of two mammalian cell lines, CHO and HL-60, using a lentiviral delivery approach. Results demonstrate that the N-terminal portion of PSGL-1 (HT) can be released from these cells by protease, and the resulting peptide can be readily captured and detected using cytometry-bead assays. Using this strategy, the peptide was immunoprecipitated onto beads bearing mAbs against either the poly-histidine sequence or the human PSGL-1. The carbohydrate epitope associated with the released peptide was detected using HECA-452 and CSLEX-1, monoclonal antibodies that recognize the sialyl Lewis-X epitope. Finally, the peptide released from cells could be separated and enriched using nickel chelate beads. Overall, such an approach that combines recombinant protein expression with flow cytometry, may be useful to quantify changes in site-specific glycosylation for basic science and clinical applications. PMID:19735085

[Cancer related fatigue in patients with breast cancer after chemotherapy and coping style].

Science.gov (United States)

Jiang, Pinglan; Wang, Shuhong; Jiang, Dongmei; Yu, Lingli

2011-04-01

To study the relevance between cancer related fatigue and coping styles in breast cancer patients after chemotherapy. A survey was conducted in 396 patients with breast cancer after chemotherapy on cancer related fatigue scale and Jalowiec coping scale, and the relation was analyzed. The rate of overall fatigue in breast cancer patients was 96.97%, mostly moderate fatigue. The rate of fatigue dimensions from high to low was physical fatigue, feeling fatigue and cognitive fatigue, respectively. The score of coping styles in patients with breast cancer after chemotherapy from high to low was optimistic coping, facing bravely, support seeking, self-reliance, emotional catharsis, avoidance, fatalism, and conservation. The most widely used coping style was optimistic coping style, and the least was emotional catharisis. There was a positive correlation between coping style of emotional catharsis and cancer related fatigue of all dimensions (Pself-reliance, or conservation (P<0.05). There was also a negative correlation between physical fatigue and optimistic or support seeking (P<0.05), but there was a positive correlation between avoidance or fatalism and the dimensions of general fatigue, physical fatigue, and cognitive fatigue (P<0.05). There is prevalent cancer related fatigue in patients with breast cancer after chemotherapy. We should guide the patients to more active coping styles, to enhance the ability of psychological adaption in patients, reduce cancer related fatigue, and improve the quality of life.

Catalytically Active Guanylyl Cyclase B Requires Endoplasmic Reticulum-mediated Glycosylation, and Mutations That Inhibit This Process Cause Dwarfism.

Science.gov (United States)

Dickey, Deborah M; Edmund, Aaron B; Otto, Neil M; Chaffee, Thomas S; Robinson, Jerid W; Potter, Lincoln R

2016-05-20

C-type natriuretic peptide activation of guanylyl cyclase B (GC-B), also known as natriuretic peptide receptor B or NPR2, stimulates long bone growth, and missense mutations in GC-B cause dwarfism. Four such mutants (L658F, Y708C, R776W, and G959A) bound (125)I-C-type natriuretic peptide on the surface of cells but failed to synthesize cGMP in membrane GC assays. Immunofluorescence microscopy also indicated that the mutant receptors were on the cell surface. All mutant proteins were dephosphorylated and incompletely glycosylated, but dephosphorylation did not explain the inactivation because the mutations inactivated a "constitutively phosphorylated" enzyme. Tunicamycin inhibition of glycosylation in the endoplasmic reticulum or mutation of the Asn-24 glycosylation site decreased GC activity, but neither inhibition of glycosylation in the Golgi by N-acetylglucosaminyltransferase I gene inactivation nor PNGase F deglycosylation of fully processed GC-B reduced GC activity. We conclude that endoplasmic reticulum-mediated glycosylation is required for the formation of an active catalytic, but not ligand-binding domain, and that mutations that inhibit this process cause dwarfism. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Introduction of a glycosylation site in the constant region decreases the aggregation of adalimumab Fab.

Science.gov (United States)

Nakamura, Hitomi; Oda-Ueda, Naoko; Ueda, Tadashi; Ohkuri, Takatoshi

2018-06-18

The production of therapeutic monoclonal antibodies is costly; therefore, antigen-binding fragments (Fabs) can be used instead. However, their tendency toward aggregation can reduce the half-life in the plasma and the therapeutic effectiveness. To examine the effect of glycosylation on the properties of the Fab of a therapeutic antibody, an N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis of L178 N (H:L178 N Fab), and then H:L178 N Fab was expressed in Pichia pastoris. SDS-PAGE analysis with treatment of N-glycosidase F or periodic acid-Schiff reagent showed that H:L178 N Fab contained a relatively low glycan level. Moreover, the H:L178 N mutation did not decrease the binding activity and thermal stability of Fab, and H:L178 N Fab was more resistant to protease digestion than wild-type Fab. The aggregation of Fab induced by pH-shift stress was measured by monitoring the optical density at 350 nm. Although the wild-type Fab showed a large increase in optical density with an increase of protein concentration, no such increase of turbidity during aggregation was found in H:L178 N Fab. These results demonstrated that glycosylation at position 178 of the H-chain constant region of adalimumab Fab can prevent protein aggregation, and therefore serve as a potentially effective platform for drug development. Copyright © 2018. Published by Elsevier Inc.

A Markov chain model for N-linked protein glycosylation – towards a low-parameter tool for model-driven glycoengineering

DEFF Research Database (Denmark)

Spahn, Philipp N.; Hansen, Anders Holmgaard; Hansen, Henning Gram

2016-01-01

Glycosylation is a critical quality attribute of most recombinant biotherapeutics. Consequently, drug development requires careful control of glycoforms to meet bioactivity and biosafety requirements. However, glycoengineering can be extraordinarily difficult given the complex reaction networks...... present a novel low-parameter approach to describe glycosylation using flux-balance and Markov chain modeling. The model recapitulates the biological complexity of glycosylation, but does not require user-provided kinetic information. We use this method to predict and experimentally validate glycoprofiles...

Urological cancer related to familial syndromes

Directory of Open Access Journals (Sweden)

Walter Henriques da Costa

Full Text Available ABSTRACT Cancer related to hereditary syndromes corresponds to approximately 5-10% of all tumors. Among those from the genitourinary system, many tumors had been identified to be related to genetic syndromes in the last years with the advent of new molecular genetic tests. New entities were described or better characterized, especially in kidney cancer such as hereditary leiomyomatosis renal cell carcinoma (HLRCC, succinate dehydrogenase kidney cancer (SDH-RCC, and more recently BAP1 germline mutation related RCC. Among tumors from the bladder or renal pelvis, some studies had reinforced the role of germline mutations in mismatch repair (MMR genes, especially in young patients. In prostate adenocarcinoma, besides mutations in BRCA1 and BRCA2 genes that are known to increase the incidence of high-risk cancer in young patients, new studies have shown mutation in other gene such as HOXB13 and also polymorphisms in MYC, MSMB, KLK2 and KLK3 that can be related to hereditary prostate cancer. Finally, tumors from testis that showed an increased in 8 - 10-fold in siblings and 4 - 6-fold in sons of germ cell tumors (TGCT patients, have been related to alteration in X chromosome. Also genome wide association studies GWAS pointed new genes that can also be related to increase of this susceptibility.

Cancer in first-degree relatives and risk of testicular cancer in Denmark

Science.gov (United States)

Nordsborg, Rikke Baastrup; Meliker, Jaymie R.; Wohlfahrt, Jan; Melbye, Mads; Raaschou-Nielsen, Ole

2011-01-01

Familial aggregation of testicular cancer has been reported consistently, but it is less clear if there is any association between risk of testicular cancer and other cancers in the family. We conducted a population based case-control study to examine the relationship between risk of testicular cancer and 22 different cancers in first-degree relatives. We included 3297 cases of testicular cancer notified to the Danish Cancer Registry between 1991 and 2003. 6594 matched controls were selected from the Danish Civil Registration System, which also provided the identity of 40,104 first-degree relatives of case and controls. Familial cancer was identified by linkage to the Danish Cancer Registry, and we used conditional logistic regression to analyse whether cancer among first-degree relatives was associated with higher risk of testicular cancer. Rate ratio (RR) for testicular cancer was 4.63 (95% CI: 2.41–8.87) when a father, 8.30(95% CI: 3.81–18.10) when a brother and 5.23 (95% CI: 1.35–20.26) when a son had testicular cancer compared with no familial testicular cancer. Results were similar when analyses were stratified by histologic subtypes of testicular cancer. Familial Non-Hodgkin lymphoma and oesophageal cancer were associated with testicular cancer; however these may be chance findings. The familial aggregation of testicular and possibly other cancers may be explained by shared genes and/or shared environmental factors, but the mutual importance of each of these is difficult to determine. PMID:21207375

Engineering the yeast Yarrowia lipolytica for the production of therapeutic proteins homogeneously glycosylated with Man8GlcNAc2 and Man5GlcNAc2

Directory of Open Access Journals (Sweden)

De Pourcq Karen

2012-05-01

Full Text Available Abstract Background Protein-based therapeutics represent the fastest growing class of compounds in the pharmaceutical industry. This has created an increasing demand for powerful expression systems. Yeast systems are widely used, convenient and cost-effective. Yarrowia lipolytica is a suitable host that is generally regarded as safe (GRAS. Yeasts, however, modify their glycoproteins with heterogeneous glycans containing mainly mannoses, which complicates downstream processing and often interferes with protein function in man. Our aim was to glyco-engineer Y. lipolytica to abolish the heterogeneous, yeast-specific glycosylation and to obtain homogeneous human high-mannose type glycosylation. Results We engineered Y. lipolytica to produce homogeneous human-type terminal-mannose glycosylated proteins, i.e. glycosylated with Man8GlcNAc2 or Man5GlcNAc2. First, we inactivated the yeast-specific Golgi α-1,6-mannosyltransferases YlOch1p and YlMnn9p; the former inactivation yielded a strain producing homogeneous Man8GlcNAc2 glycoproteins. We tested this strain by expressing glucocerebrosidase and found that the hypermannosylation-related heterogeneity was eliminated. Furthermore, detailed analysis of N-glycans showed that YlOch1p and YlMnn9p, despite some initial uncertainty about their function, are most likely the α-1,6-mannosyltransferases responsible for the addition of the first and second mannose residue, respectively, to the glycan backbone. Second, introduction of an ER-retained α-1,2-mannosidase yielded a strain producing proteins homogeneously glycosylated with Man5GlcNAc2. The use of the endogenous LIP2pre signal sequence and codon optimization greatly improved the efficiency of this enzyme. Conclusions We generated a Y. lipolytica expression platform for the production of heterologous glycoproteins that are homogenously glycosylated with either Man8GlcNAc2 or Man5GlcNAc2 N-glycans. This platform expands the utility of Y. lipolytica as a

Mass spectrometry characterization for N-glycosylation of immunoglobulin Y from hen egg yolk.

Science.gov (United States)

Sheng, Long; He, Zhenjiao; Liu, Yaping; Ma, Meihu; Cai, Zhaoxia

2018-03-01

Immunoglobulin Y (IgY) is a new therapeutic antibody that exists in hen egg yolk. It is a glycoprotein, not much is known about its N-glycan structures, site occupancy and site-specific N-glycosylation. In this study, purified protein from hen egg yolk was identified as IgY based on SDS-PAGE and MALDI-TOF/TOF MS. N-glycan was released from IgY using peptide-N4-(N-acetyl-beta-glucosaminyl) asparagine-amidase treatment, and the molecular weight of IgY was calculated using the difference between the molecular weight of IgY and deglycosylated IgY. Two potential N-Glycosylation sites (ASN 308 and ASN 409 ) were detected on IgY by nanoLC-ESI MS. Sugar chains were separated using normal phase liquid chromatography after fluorescence labeling, and 17 N-glycan structures were confirmed using ESI-MS. The sugar chain pattern contained high-mannose oligosaccharide, hybrid oligosaccharide and complex oligosaccharide. These results could lead to other important information regarding IgY glycosylation. Copyright © 2017 Elsevier B.V. All rights reserved.

Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH.

Science.gov (United States)

Vallbracht, Melina; Rehwaldt, Sascha; Klupp, Barbara G; Mettenleiter, Thomas C; Fuchs, Walter

2018-05-01

Many viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reduced in vitro fusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in the Varicellovirus genus, resulted in enhanced in vitro fusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. IMPORTANCE Herpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide

Cancer-related identity and positive affect in survivors of prostate cancer.

Science.gov (United States)

Bellizzi, Keith M; Blank, Thomas O

2007-03-01

Despite a shift in the cancer culture and language used to describe individuals diagnosed with this disease, the extent to which individuals with cancer adopt a particular cancer-related identity and the impact of these identities in relation to their well-being is virtually unknown. Using a cross-sectional study design and a metropolitan tumor registry, a mail questionnaire to examine post-treatment quality of life was sent to prostate cancer (PCa) survivors. The sample consisted of 490 PCa survivors, ranging in age from 49-88 (M = 69.7; SD = 7.8), one to eight years after diagnosis. The outcome measure used in these analyses was the PANAS to assess positive and negative affect. The most frequently reported cancer-related identity was "someone who has had PCa" (57%). The least reported self view was "victim" (1%). Twenty-six percent of men self-identified as "survivors" while 6% thought of themselves as "cancer conquerors." Only 9% self-identified as a "patient." Multivariate analyses, adjusted for potential confounders, show respondents who identified themselves as "survivors" or "cancer conquerors" reported significantly higher scores on positive affect than men who self-identified as "patients" (p < .001). Although the majority of respondents identified themselves as "someone who has had cancer," identifying as a "survivor" or "someone who has conquered cancer" appears to have adaptive value for positive mood. Those who perceive themselves as survivors of prostate cancer may derive some benefit in well-being associated with this self assessment.

Biochemical characterization of individual human glycosylated pro-insulin-like growth factor (IGF)-II and big-IGF-II isoforms associated with cancer.

Science.gov (United States)

Greenall, Sameer A; Bentley, John D; Pearce, Lesley A; Scoble, Judith A; Sparrow, Lindsay G; Bartone, Nicola A; Xiao, Xiaowen; Baxter, Robert C; Cosgrove, Leah J; Adams, Timothy E

2013-01-04

Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed "pro" and "big" IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling.

Biochemical Characterization of Individual Human Glycosylated pro-Insulin-like Growth Factor (IGF)-II and big-IGF-II Isoforms Associated with Cancer

Science.gov (United States)

Greenall, Sameer A.; Bentley, John D.; Pearce, Lesley A.; Scoble, Judith A.; Sparrow, Lindsay G.; Bartone, Nicola A.; Xiao, Xiaowen; Baxter, Robert C.; Cosgrove, Leah J.; Adams, Timothy E.

2013-01-01

Insulin-like growth factor II (IGF-II) is a major embryonic growth factor belonging to the insulin-like growth factor family, which includes insulin and IGF-I. Its expression in humans is tightly controlled by maternal imprinting, a genetic restraint that is lost in many cancers, resulting in up-regulation of both mature IGF-II mRNA and protein expression. Additionally, increased expression of several longer isoforms of IGF-II, termed “pro” and “big” IGF-II, has been observed. To date, it is ambiguous as to what role these IGF-II isoforms have in initiating and sustaining tumorigenesis and whether they are bioavailable. We have expressed each individual IGF-II isoform in their proper O-glycosylated format and established that all bind to the IGF-I receptor and both insulin receptors A and B, resulting in their activation and subsequent stimulation of fibroblast proliferation. We also confirmed that all isoforms are able to be sequestered into binary complexes with several IGF-binding proteins (IGFBP-2, IGFBP-3, and IGFBP-5). In contrast to this, ternary complex formation with IGFBP-3 or IGFBP-5 and the auxillary protein, acid labile subunit, was severely diminished. Furthermore, big-IGF-II isoforms bound much more weakly to purified ectodomain of the natural IGF-II scavenging receptor, IGF-IIR. IGF-II isoforms thus possess unique biological properties that may enable them to escape normal sequestration avenues and remain bioavailable in vivo to sustain oncogenic signaling. PMID:23166326

Label-free electrochemical biosensing of small-molecule inhibition on O-GlcNAc glycosylation.

Science.gov (United States)

Yang, Yu; Gu, Yuxin; Wan, Bin; Ren, Xiaomin; Guo, Liang-Hong

2017-09-15

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) plays a critical role in modulating protein function in many cellular processes and human diseases such as Alzheimer's disease and type II diabetes, and has emerged as a promising new target. Specific inhibitors of OGT could be valuable tools to probe the biological functions of O-GlcNAcylation, but a lack of robust nonradiometric assay strategies to detect glycosylation, has impeded efforts to identify such compounds. Here we have developed a novel label-free electrochemical biosensor for the detection of peptide O-GlcNAcylation using protease-protection strategy and electrocatalytic oxidation of tyrosine mediated by osmium bipyridine as a signal reporter. There is a large difference in the abilities of proteolysis of the glycosylated and the unglycosylated peptides by protease, thus providing a sensing mechanism for OGT activity. When the O-GlcNAcylation is achieved, the glycosylated peptides cannot be cleaved by proteinase K and result in a high current response on indium tin oxide (ITO) electrode. However, when the O-GlcNAcylation is successfully inhibited using a small molecule, the unglycosylated peptides can be cleaved easily and lead to low current signal. Peptide O-GlcNAcylation reaction was performed in the presence of a well-defined small-molecule OGT inhibitor. The results indicated that the biosensor could be used to screen the OGT inhibitors effectively. Our label-free electrochemical method is a promising candidate for protein glycosylation pathway research in screening small-molecule inhibitors of OGT. Copyright © 2017 Elsevier B.V. All rights reserved.

Engineer Medium and Feed for Modulating N-Glycosylation of Recombinant Protein Production in CHO Cell Culture.

Science.gov (United States)

Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

2017-01-01

Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell. In this chapter, we will focus on how to carry out experiments for N-glycosylation modulation through medium and feed optimization. The workflow and typical methods involved in the experiment process will be presented.

Glycosylation at Asn91 of H1N1 haemagglutinin affects binding to glycan receptors.

Science.gov (United States)

Jayaraman, Akila; Koh, Xiaoying; Li, Jing; Raman, Rahul; Viswanathan, Karthik; Shriver, Zachary; Sasisekharan, Ram

2012-06-15

The glycoprotein HA (haemagglutinin) on the surface of influenza A virus plays a central role in recognition and binding to specific host cell-surface glycan receptors and in fusion of viral membrane to the host nuclear membrane during viral replication. Given the abundance of HA on the viral surface, this protein is also the primary target for host innate and adaptive immune responses. Although addition of glycosylation sites on HA are a part of viral evolution to evade the host immune responses, there are specific glycosylation sites that are conserved during most of the evolution of the virus. In the present study, it was demonstrated that one such conserved glycosylation site at Asn(91) in H1N1 HA critically governs the glycan receptor-binding specificity and hence would potentially impinge on the host adaptation of the virus.

Prepubertal growth in congenital disorder of glycosylation type Ia (CDG-Ia)

OpenAIRE

Kjaergaard, S; Muller, J; Skovby, F

2002-01-01

Aims: To delineate the pattern of growth in prepubertal children with congenital disorder of glycosylation type Ia (CDG-Ia) in order to identify critical period(s) and possible cause(s) of growth failure.

Anti-Podocalyxin Monoclonal Antibody 47-mG2a Detects Lung Cancers by Immunohistochemistry.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Kaneko, Mika K; Kato, Yukinari

2018-04-01

Lung cancer is one of the leading causes of cancer-related deaths in the world. Regardless of the advances in lung cancer treatments, the prognosis is still poor. Podocalyxin (PODXL) is a highly glycosylated type I transmembrane protein that is expressed in normal tissues, including the heart, pancreas, and breast. It is also found and used as a diagnostic marker in many cancers, such as renal, brain, breast, oral, and lung cancers. We previously developed specific and sensitive anti-PODXL monoclonal antibodies, PcMab-47 (mouse IgG 1 , kappa) and its mouse IgG 2a -type (47-mG 2a ), both of which were suitable for immunohistochemical analyses of oral cancers. In this study, we investigated the utility of PcMab-47 and 47-mG 2a for the immunohistochemical analyses of lung cancers. PcMab-47 stained 51/70 (72.9%) cases of lung cancer, whereas 47-mG 2a stained 59/70 (84.3%) cases, indicating that the latter antibody is more sensitive and is useful for detecting PODXL in lung cancers.

Predictive glycoengineering of biosimilars using a Markov chain glycosylation model

DEFF Research Database (Denmark)

Spahn, Philipp N.; Hansen, Anders Holmgaard; Kol, Stefan

2017-01-01

Biosimilar drugs must closely resemble the pharmacological attributes of innovator products to ensure safetyand efficacy to obtain regulatory approval. Glycosylation is one critical quality attribute that must be matched, but it is inherently difficult to control due to the complexity of its...

Aberrant O-GlcNAcylated proteins: New perspectives in breast and colorectal cancer

Directory of Open Access Journals (Sweden)

Parunya eChaiyawat

2014-11-01

Full Text Available Increasing glucose consumption is thought to provide an evolutionary advantage to cancer cells. Alteration of glucose metabolism in cancer influences various important metabolic pathways including the hexosamine biosynthesis pathway (HBP, a relatively minor branch of glycolysis. Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc, an end product of HBP, is a sugar substrate used for classical glycosylation and O-GlcNAcylation, a post-translational protein modification implicated in a wide range of effects on cellular functions. Emerging evidence reveals that certain cellular proteins are abnormally O-GlcNAc modified in many kinds of cancers, indicating O-GlcNAcylation is associated with malignancy. Since O-GlcNAc rapidly on and off modifies in a similar time scale as in phosphorylation and these modifications may occur on proteins at either on the same or adjacent sites, it suggests that both modifications can work to regulate the cellular signaling pathways. This review describes the metabolic shifts related to the HBP which are commonly found in most cancers. It also describes O-GlcNAc modified proteins identified in primary breast and colorectal cancer, as well as in the related cancer cell lines. Moreover, we also discuss the potential use of aberrant O-GlcNAcylated proteins as novel biomarkers of cancer. + P. Chaiyawat and P. Netsirisawan contributed equally to this study

Glycoprofiling of Early Gastric Cancer Using Lectin Microarray Technology.

Science.gov (United States)

Li, Taijie; Mo, Cuiju; Qin, Xue; Li, Shan; Liu, Yinkun; Liu, Zhiming

2018-01-01

Recently, studies have reported that protein glycosylation plays an important role in the occurrence and development of cancer. Gastric cancer is a common cancer with high morbidity and mortality owing to most gastric cancers are discovered only at an advanced stage. Here, we aim to discover novel specific serum glycanbased biomarkers for gastric cancer. A lectin microarray with 50 kinds of tumor-associated lectin was used to detect the glycan profiles of serum samples between early gastric cancer and healthy controls. Then lectin blot was performed to validate the differences. The result of the lectin microarray showed that the signal intensities of 13 lectins showed significant differences between the healthy controls and early gastric cancer. Compared to the healthy, the normalized fluorescent intensities of the lectins PWA, LEL, and STL were significantly increased, and it implied that their specifically recognized GlcNAc showed an especially elevated expression in early gastric cancer. Moreover, the binding affinity of the lectins EEL, RCA-II, RCA-I, VAL, DSA, PHA-L, UEA, and CAL were higher in the early gastric cancer than in healthy controls. These glycan structures containing GalNAc, terminal Galβ 1-4 GlcNAc, Tri/tetraantennary N-glycan, β-1, 6GlcNAc branching structure, α-linked fucose residues, and Tn antigen were elevated in gastric cancer. While the two lectins CFL GNL reduced their binding ability. In addition, their specifically recognized N-acetyl-D-galactosamine structure and (α-1,3) mannose residues were decreased in early gastric cancer. Furthermore, lectin blot results of LEL, STL, PHA-L, RCA-I were consistent with the results of the lectin microarray. The findings of our study clarify the specific alterations for glycosylation during the pathogenesis of gastric cancer. The specific high expression of GlcNAc structure may act as a potential early diagnostic marker for gastric cancer.

Perinatal and early infantile symptoms in congenital disorders of glycosylation

NARCIS (Netherlands)

Funke, S.; Gardeitchik, T.; Kouwenberg, D.; Mohamed, M.; Wortmann, S.B.; Korsch, E.; Adamowicz, M.; Al-Gazali, L.; Wevers, R.A.; Horvath, A.; Lefeber, D.J.; Morava, E.

2013-01-01

Congenital disorders of glycosylation (CDG) are a rapidly growing family of inborn errors. Screening for CDG in suspected cases is usually performed in the first year of life by serum transferrin isoelectric focusing or mass spectrometry. Based on the transferrin analysis patients can be

Glycosylation of inositol phosphorylceramide sphingolipids is required for normal growth and reproduction in Arabidopsis

Energy Technology Data Exchange (ETDEWEB)

Tartaglio, Virginia [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Rennie, Emilie A. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Cahoon, Rebecca [Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Wang, George [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Baidoo, Edward [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Mortimer, Jennifer C. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Cahoon, Edgar B. [Univ. of Nebraska, Lincoln, NE (United States). Center for Plant Science Innovation and Dept. of Biochemistry; Scheller, Henrik V. [Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biological Systems and Engineering Division; Univ. of California, Berkeley, CA (United States). Dept. of Plant and Microbial Biology

2016-09-19

Sphingolipids are a major component of plant plasma membranes and endomembranes, and mediate a diverse range of biological processes. Study of the highly glycosylated glycosyl inositol phosphorylceramide (GIPC) sphingolipids has been slow as a result of challenges associated with the extractability of GIPCs, and their functions in the plant remain poorly characterized. We recently discovered an Arabidopsis GIPC glucuronosyltransferase, INOSITOL PHOSPHORYLCERAMIDE GLUCURONOSYLTRANSFERASE 1 (IPUT1), which is the first enzyme in the GIPC glycosylation pathway. Plants homozygous for the iput1 loss-of-function mutation were unobtainable, and so the developmental effects of reduced GIPC glucuronosylation could not be analyzed in planta. Using a pollen-specific rescue construct, we have here isolated homozygous iput1 mutants. The iput1 mutants show severe dwarfism, compromised pollen tube guidance, and constitutive activation of salicyclic acid-mediated defense pathways. The mutants also possess reduced GIPCs, increased ceramides, and an increased incorporation of short-chain fatty acids and dihydroxylated bases into inositol phosphorylceramides and GIPCs. The assignment of a direct role for GIPC glycan head groups in the impaired processes in iput1 mutants is complicated by the vast compensatory changes in the sphingolipidome; however, our results reveal that the glycosylation steps of GIPC biosynthesis are important regulated components of sphingolipid metabolism. In conclusion, this study corroborates previously suggested roles for GIPC glycans in plant growth and defense, suggests important role s for them in reproduction and demonstrates that the entire sphingolipidome is sensitive to their status.

Controlling the Glycosylation Profile in mAbs Using Time-Dependent Media Supplementation

Directory of Open Access Journals (Sweden)

Devesh Radhakrishnan

2017-12-01

Full Text Available In order to meet desired drug product quality targets, the glycosylation profile of biotherapeutics such as monoclonal antibodies (mAbs must be maintained consistently during manufacturing. Achieving consistent glycan distribution profiles requires identifying factors that influence glycosylation, and manipulating them appropriately via well-designed control strategies. Now, the cell culture media supplement, MnCl2, is known to alter the glycosylation profile in mAbs generally, but its effect, particularly when introduced at different stages during cell growth, has yet to be investigated and quantified. In this study, we evaluate the effect of time-dependent addition of MnCl2 on the glycan profile quantitatively, using factorial design experiments. Our results show that MnCl2 addition during the lag and exponential phases affects the glycan profile significantly more than stationary phase supplementation does. Also, using a novel computational technique, we identify various combinations of glycan species that are affected by this dynamic media supplementation scheme, and quantify the effects mathematically. Our experiments demonstrate the importance of taking into consideration the time of addition of these trace supplements, not just their concentrations, and our computational analysis provides insight into what supplements to add, when, and how much, in order to induce desired changes.

25-Hydroxycholesterol Inhibition of Lassa Virus Infection through Aberrant GP1 Glycosylation

Directory of Open Access Journals (Sweden)

Punya Shrivastava-Ranjan

2016-12-01

Full Text Available Lassa virus (LASV infection is a major public health concern due to high fatality rates and limited effective treatment. The interferon-stimulated gene cholesterol 25-hydroxylase (CH25H encodes an enzyme that catalyzes the production of 25-hydroxycholesterol (25HC. 25HC is involved in regulating cholesterol biosynthesis and has recently been identified as a potent antiviral targeting enveloped virus entry. Here, we show a previously unrecognized role of CH25H in inhibiting LASV glycoprotein glycosylation and the production of infectious virus. Overexpression of CH25H or treatment with 25HC decreased LASV G1 glycoprotein N-glycan maturation and reduced the production of infectious LASV. Depletion of endogenous CH25H using small interfering RNA (siRNA enhanced the levels of fully glycosylated G1 and increased infectious LASV production. Finally, LASV particles produced from 25HC-treated cells were found to be less infectious, to incorporate aberrantly glycosylated GP1 species, and to be defective in binding alpha-dystroglycan, an attachment and entry receptor. Our findings identify a novel role for CH25H in controlling LASV propagation and indicate that manipulation of the expression of CH25H or the administration of 25HC may be a useful anti-LASV therapy.

Dynamic analysis of sexual organ weight and serum levels of glucose, glycosyl protein, testosterone in male rats with short-term diabetes

International Nuclear Information System (INIS)

Zou Donghui; Wang Zhongshan; Zhao Hui; Xu Zongge

2001-01-01

Objective: To study the effect of short-term diabetes on the changes of sexual organ weights and serum levels of testosterone in male rats. Methods: All rats were divided into control (C) and diabetes (D). Diabetes group was observed on 1 day, 3 days, 5 days, 7 days, 14 days after injection of streptozotocin. All rats were killed for measurement of serum levels of glucose, glycosyl protein, testosterone and weights of sexual organs (testis, epididymis, seminal vesicle and prostate). Results: The serum levels of glucose, glycosyl protein of diabetes group decreased significantly, compared with those of control group; the serum levels of T lowered remarkably compared with control levels after three days of injection of STZ. The weight of epididymis, seminal vesicle and prostate reduced remarkably, compared with control group. The weights of seminal vesicle, prostate negatively correlated with serum levels of glucose and glycosyl protein, and they positively correlated with serum levels of testosterone. Conclusion: The sexual organs (testis, epididymis, seminal vesicle and prostate) of male rats with short-term diabetes were damaged, and the changes of sexual organs closely related with the serum levels of testosterone besides irregular metabolism in diabetes

The Breast Cancer-Associated Glycoforms of MUC1, MUC1-Tn and sialyl-Tn, Are Expressed in COSMC Wild-Type Cells and Bind the C-Type Lectin MGL

DEFF Research Database (Denmark)

Beatson, Richard; Maurstad, Gjertrud; Picco, Gianfranco

2015-01-01

Aberrant glycosylation occurs in the majority of human cancers and changes in mucin-type O-glycosylation are key events that play a role in the induction of invasion and metastases. These changes generate novel cancer-specific glyco-antigens that can interact with cells of the immune system through...... carbohydrate binding lectins. Two glyco-epitopes that are found expressed by many carcinomas are Tn (GalNAc-Ser/Thr) and STn (NeuAcα2,6GalNAc-Ser/Thr). These glycans can be carried on many mucin-type glycoproteins including MUC1. We show that the majority of breast cancers carry Tn within the same cell...

Cancer-related fatigue: Mechanisms, risk factors, and treatments

Science.gov (United States)

Bower, Julienne E.

2015-01-01

Fatigue is one of the most common and distressing side effects of cancer and its treatment, and may persist for years after treatment completion in otherwise healthy survivors. Cancer-related fatigue causes disruption in all aspects of quality of life and may be a risk factor for reduced survival. The prevalence and course of fatigue in cancer patients has been well characterized, and there is growing understanding of underlying biological mechanisms. Inflammation has emerged as a key biological pathway for cancer-related fatigue, with studies documenting links between markers of inflammation and fatigue before, during, and particularly after treatment. There is considerable variability in the experience of cancer-related fatigue that is not explained by disease- or treatment-related characteristics, suggesting that host factors may play an important role in the development and persistence of this symptom. Indeed, longitudinal studies have begun to identify genetic, biological, psychosocial, and behavioral risk factors for cancer-related fatigue. Given the multi-factorial nature of cancer-related fatigue, a variety of intervention approaches have been examined in randomized controlled trials, including physical activity, psychosocial, mind-body, and pharmacological treatments. Although there is currently no gold standard for treating fatigue, several of these approaches have shown beneficial effects and can be recommended to patients. This report provides a state of the science review of mechanisms, risk factors, and interventions for cancer-related fatigue, with a focus on recent longitudinal studies and randomized trials that have targeted fatigued patients. PMID:25113839

N-Glycosylation of Human R-Spondin 1 Is Required for Efficient Secretion and Stability but Not for Its Heparin Binding Ability

Directory of Open Access Journals (Sweden)

Chiung-Fang Chang

2016-06-01

Full Text Available R-spondin 1 (Rspo1 plays an essential role in stem cell biology by potentiating Wnt signaling activity. Despite the fact that Rspo1 holds therapeutic potential for a number of diseases, its biogenesis is not fully elucidated. All Rspo proteins feature two amino-terminal furin-like repeats, which are responsible for Wnt signal potentiation, and a thrombospondin type 1 (TSR1 domain that can provide affinity towards heparan sulfate proteoglycans. Using chemical inhibitors, deglycosylase and site-directed mutagenesis, we found that human Rspo1 and Rspo3 are both N-glycosylated at N137, a site near the C-terminus of the furin repeat 2 domain, and Rspo2 is N-glycosylated at N160, a position near the N-terminus of TSR1 domain. Elimination of N-glycosylation at these sites affects their accumulation in media but have no effect on the ability towards heparin. Introduction of the N-glycosylation site to Rspo2 mutant at the position homologous to N137 in Rspo1 restored full glycosylation and rescued the accumulation defect of nonglycosylated Rspo2 mutant in media. Similar effect can be observed in the N137 Rspo1 or Rspo3 mutant engineered with Rspo2 N-glycosylation site. The results highlight the importance of N-glycosylation at these two positions in efficient folding and secretion of Rspo family. Finally, we further showed that human Rspo1 is subjected to endoplasmic reticulum (ER quality control in N-glycan-dependent manner. While N-glycan of Rspo1 plays a role in its intracellular stability, it had little effect on secreted Rspo1. Our findings provide evidence for the critical role of N-glycosylation in the biogenesis of Rspo1.

Resilience and positive affect contribute to lower cancer-related fatigue among Chinese patients with gastric cancer.

Science.gov (United States)

Zou, Guiyuan; Li, Ye; Xu, Ruicai; Li, Ping

2018-04-01

To investigate the prevalence of cancer-related fatigue and explore the relationship between resilience, positive affect, and fatigue among Chinese patients with gastric cancer. Cancer-related fatigue is the most distressing symptom reported frequently by cancer patients during both treatment and survival phases. Resilience and positive affect as vital protective factors against cancer-related fatigue have been examined, but the underlying psychological mechanisms are not well understood. A cross-sectional study. Two hundred and three gastric cancer patients were enrolled from three hospitals in China. The Cancer Fatigue Scale, the positive affect subscale of the Positive and Negative Affect Schedule and the Connor-Davidson Resilience Scale (CD-RISC10) were administered. Hierarchical linear regression modelling was conducted to examine the association between resilience and cancer-related fatigue, and the mediating effect of positive affect. The incidence of clinically relevant fatigue among patients with gastric cancer was 91.6%. Regression analysis showed that resilience was negatively associated with cancer-related fatigue, explaining 15.4% of variance in cancer-related fatigue. Mediation analysis showed that high resilience was associated with increased positive affect, which was associated with decreased cancer-related fatigue. Cancer-related fatigue is prevalent among patients with gastric cancer. Positive affect may mediate the relationship between resilience and cancer-related fatigue. Interventions that attend to resilience training and promotion of positive affect may be the focus for future clinical and research endeavours. © 2017 John Wiley & Sons Ltd.

Mapping N-linked Glycosylation Sites in the Secretome and Whole Cells of Aspergillus niger Using Hydrazide Chemistry and Mass Spectrometry

Energy Technology Data Exchange (ETDEWEB)

Wang, Lu; Aryal, Uma K.; Dai, Ziyu; Mason, Alisa C.; Monroe, Matthew E.; Tian, Zhixin; Zhou, Jianying; Su, Dian; Weitz, Karl K.; Liu, Tao; Camp, David G.; Smith, Richard D.; Baker, Scott E.; Qian, Weijun

2012-01-01

Protein glycosylation is known to play an essential role in both cellular functions and the secretory pathways; however, little information is available on the dynamics of glycosylated N-linked glycosites of fungi. Herein we present the first extensive mapping of glycosylated N-linked glycosites in industrial strain Aspergillus niger by applying an optimized solid phase enrichment of glycopeptide protocol using hydrazide modified magnetic beads. The enrichment protocol was initially optimized using mouse plasma and A. niger secretome samples, which was then applied to profile N-linked glycosites from both the secretome and whole cell lysates of A. niger. A total of 847 unique N-linked glycosites and 330 N-linked glycoproteins were confidently identified by LC-MS/MS. Based on gene ontology analysis, the identified N-linked glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, golgi apparatus, lysosome, and storage vacuoles. The identified N-linked glycoproteins are involved in a wide range of biological processes including gene regulation and signal transduction, protein folding and assembly, protein modification and carbohydrate metabolism. The extensive coverage of glycosylated N-linked glycosites along with identification of partial N-linked glycosylation in those enzymes involving in different biochemical pathways provide useful information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.

N-linked glycosylation at Asn152 on CD147 affects protein folding and stability: promoting tumour metastasis in hepatocellular carcinoma.

Science.gov (United States)

Li, Jiang-Hua; Huang, Wan; Lin, Peng; Wu, Bo; Fu, Zhi-Guang; Shen, Hao-Miao; Jing, Lin; Liu, Zhen-Yu; Zhou, Yang; Meng, Yao; Xu, Bao-Qing; Chen, Zhi-Nan; Jiang, Jian-Li

2016-11-21

Cluster of differentiation 147 (CD147), also known as extracellular matrix metalloproteinase inducer, is a transmembrane glycoprotein that mediates oncogenic processes partly through N-glycosylation modifications. N-glycosylation has been demonstrated to be instrumental for the regulation of CD147 function during malignant transformation. However, the role that site-specific glycosylation of CD147 plays in its defective function in hepatocellular carcinomacells needs to be determined. Here, we demonstrate that the modification of N-glycosylation at Asn152 on CD147 strongly promotes hepatocellular carcinoma (HCC) invasion and migration. After the removal of N-glycans at Asn152, CD147 was more susceptible to degradation by ER-localized ubiquitin ligase-mediated endoplasmic reticulum-associated degradation (ERAD). Furthermore, N-linked glycans at Asn152 were required for CD147 to acquire and maintain proper folding in the ER. Moreover, N-linked glycans at Asn152 functioned as a recognition motif that was directly mediated by the CNX quality control system. Two phases in the retention-based ER chaperones system drove ER-localized CD147 trafficking to degradation. Deletion of N-linked glycosylation at Asn152 on CD147 significantly suppressed in situ tumour metastasis. These data could potentially shed light on the molecular regulation of CD147 through glycosylation and provide a valuable means of developing drugs that target N-glycans at Asn152 on CD147.

Effect of Cola acuminate on Blood Glucose and Glycosylated ...

African Journals Online (AJOL)

The levels of blood glucose and glycosylated haemoglobin (GHB) were studied in 42 Wistar rats divided into three groups; controls, group A and group B. Control rats consumed only feeds, group A consumed 0.04g of Cola acuminate, while group B consumed 0.08g of Cola acuminate mixed with their feeds daily for six ...

Enzymatic Glycosylation of Phenolic Antioxidants: Phosphorylase-Mediated Synthesis and Characterization

Czech Academy of Sciences Publication Activity Database

De Winter, K.; Dewitte, W.; Dirks-Hofmeister, M. E.; De Laet, S.; Pelantová, Helena; Křen, Vladimír; Desmet, T.

2015-01-01

Roč. 63, č. 46 (2015), s. 10131-10139 ISSN 0021-8561 R&D Projects: GA MŠk(CZ) 7E11011; GA MŠk(CZ) LD13042 Institutional support: RVO:61388971 Keywords : glycosylation * antioxidant * ABTS Subject RIV: CE - Biochemistry Impact factor: 2.857, year: 2015

Cancer-related fatigue--mechanisms, risk factors, and treatments.

Science.gov (United States)

Bower, Julienne E

2014-10-01

Fatigue is one of the most common adverse effects of cancer that might persist for years after treatment completion in otherwise healthy survivors. Cancer-related fatigue causes disruption in all aspects of quality of life and might be a risk factor of reduced survival. The prevalence and course of fatigue in patients with cancer have been well characterized and there is growing understanding of the underlying biological mechanisms. Inflammation seems to have a key role in fatigue before, during, and after cancer-treatment. However, there is a considerable variability in the presentation of cancer-related fatigue, much of which is not explained by disease-related or treatment-related characteristics, suggesting that host factors might be important in the development and persistence of this symptom. Indeed, longitudinal studies have identified genetic, biological, psychosocial, and behavioural risk factors associated with cancer-related fatigue. Although no current gold-standard treatment for fatigue is available, a variety of intervention approaches have shown beneficial effects in randomized controlled trials, including physical activity, psychosocial, mind-body, and pharmacological treatments. This Review describes the mechanisms, risk factors, and possible interventions for cancer-related fatigue, focusing on recent longitudinal studies and randomized trials that have targeted fatigued patients.

High-Throughput Analysis of Plasma Hybrid Markers for Early Detection of Cancers

Directory of Open Access Journals (Sweden)

Jung-hyun Rho

2014-01-01

Full Text Available Biomarkers for the early detection of cancer in the general population have to perform with high sensitivity and specificity in order to prevent the costs associated with over-diagnosis. There are only a few current tissue or blood markers that are recommended for generalized cancer screening. Despite the recognition that combinations of multiple biomarkers will likely improve their utility, biomarker panels are usually limited to a single class of molecules. Tissues and body fluids including plasma and serum contain not only proteins, DNA and microRNAs that are differentially expressed in cancers but further cancer specific information might be gleaned by comparing different classes of biomolecules. For example, the level of a certain microRNA might be related to the level of a particular protein in a cancer specific manner. Proteins might have cancer-specific post-translational modifications (e.g., phosphorylation or glycosylation or lead to the generation of autoantibodies. Most currently approved biomarkers are glycoproteins. Autoantibodies can be produced as a host’s early surveillance response to cancer-specific proteins in pre-symptomatic and pre-diagnostic stages of cancer. Thus, measurement of the level of a protein, the level of its glycosylation or phosphorylation and whether autoantibodies are produced to it can yield multi-dimensional information on each protein. We consider specific proteins that show consistent cancer-specific changes in two or three of these measurements to be “hybrid markers”. We hypothesize these markers will suffer less variation between different individuals since one component can act to “standardize” the other measurement. As a proof of principle, a 180 plasma sample set consisting of 120 cases (60 colon cancers and 60 adenomas and 60 controls were analyzed using our high-density antibody array for changes in their protein, IgG-complex and sialyl-Lewis A (SLeA modified proteins. At p < 0

Association between cancer literacy and cancer-related behaviour: evidence from Ticino, Switzerland

Directory of Open Access Journals (Sweden)

Nicola Diviani

2014-07-01

Full Text Available Background. This paper details the role of different dimensions of health literacy in the relationship between health literacy and cancer-related health behaviours. In particular, Cancer Literacy is studied as an exemplar of a dimension of health literacy beyond basic reading and writing skills. The link between functional health literacy, Cancer Literacy and cancer-related health behaviours is investigated in a sample of Ticino (Switzerland residents (n=639. Design and methods. Detailed data is collected about respondents’ functional health literacy, Cancer Literacy, cancer information seeking behaviour, engagement in cancer preventive behaviours, participation to cancer screenings, and intention to adhere to current screening recommendations. Results. Results confirm the added value of Cancer Literacy – compared to functional health literacy – in explaining people’s cancer information seeking behaviour, their participation to several cancer screenings and their screening intention, underscoring the need to take into account dimensions of health literacy beyond basic functional skills. Conclusions. From a public health perspective, findings provide further evidence on the importance of adapting informational and educational communication intervention designed to improve cancer prevention and screening to different audiences.

Moisture-induced solid state instabilities in α-chymotrypsin and their reduction through chemical glycosylation

Directory of Open Access Journals (Sweden)

Solá Ricardo J

2010-08-01

Full Text Available Abstract Background Protein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state. Results First, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme α-chymotrypsin (α-CT under controlled humidity conditions. Results showed that α-CT aggregates and inactivates as a function of increased relative humidity (RH. Furthermore, α-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that α-CT structural dynamics increased at increasing RH. The magnitude of the structural changes in tendency parallels the solid-state instability data (i.e., formation of buffer-insoluble aggregates, inactivation, and loss of native conformation upon reconstitution. To determine if these moisture-induced instability issues could be ameliorated by chemical glycosylation we proceeded to modify our model protein with chemically activated glycans of differing lengths (lactose and dextran (10 kDa. The various glycoconjugates showed a marked decrease in aggregation and an increase in residual activity after incubation. These stabilization effects were found to be independent of the glycan size. Conclusion Water sorption leads to

Heterogeneity of rabbit endogenous pyrogens is not attributable to glycosylated variants of a single polypeptide chain.

OpenAIRE

Murphy, P A; Cebula, T A; Windle, B E

1981-01-01

Rabbit endogenous pyrogens were of about the same molecular size, but showed considerable heterogeneity of their isoelectric points. We attempted to show that this heterogeneity was attributable to variable glycosylation of a single polypeptide chain. When peritoneal exudate cells were stimulated to make pyrogens in the presence of 2-deoxy-D-glucose, there was a relatively trivial suppression of pyrogen release, and analysis by isoelectric focusing showed parallel inhibition of secretion of a...

Comparative Proteomics and Glycoproteomics Reveal Increased N-Linked Glycosylation and Relaxed Sequon Specificity in Campylobacter jejuni NCTC11168 O

DEFF Research Database (Denmark)

Scott, Nichollas E.; Marzook, N. Bishara; Cain, Joel A.

2014-01-01

Campylobacter jejuni is a major cause of bacterial gastroenteritis. C. jejuni encodes a protein glycosylation (Pgl) locus responsible for the N-glycosylation of membrane-associated proteins. We examined two variants of the genome sequenced strain NCTC11168: O, a representative of the original...

Relationship between cancer-related fatigue and physical activity in inpatient cancer rehabilitation.

Science.gov (United States)

Kummer, Franziska; Catuogno, Silvio; Perseus, Josef M; Bloch, Wilhelm; Baumann, Freerk T

2013-08-01

Fatigue is a serious problem for the majority of patients with cancer. In this context, several studies have shown benefits of physical activity during and following treatment. However, uncertainties remain regarding the optimal type and duration of physical activity. Therefore, this study examined the relationship between cancer-related fatigue and physical activity in the course of inpatient rehabilitation. Fatigue (Multidimensional Fatigue Inventory) and physical activity (Freiburg Questionnaire of Physical Activity) were assessed in a consecutive series of 35 patients with cancer attending oncological inpatient rehabilitation during a six-month study period. The three-week rehabilitation program included daily exercise therapy consisting of aerobic endurance training, moderate resistance training, coordination exercises, relaxation training and individual physiotherapy. At discharge, a significant improvement in each dimension of cancer-related fatigue (p=0.001-0.003) and a significant increase of physical activity levels (p=0.001) were observed. A small, but significant negative correlation was found between cancer-related fatigue and the level of physical activity (R=-0.438, p=0.004). The largest effects were associated with a weekly energy expenditure of 3000 kcal through physical activity. The results support a non-linear dose-response relationship between cancer-related fatigue and physical activity. Since this is the first study providing specific exercise recommendations for an effective treatment of cancer-related fatigue in the context of inpatient rehabilitation, further research is required to validate the observed trends.

Mutations altering the gammaretrovirus endoproteolytic motif affect glycosylation of the envelope glycoprotein and early events of the virus life cycle

Energy Technology Data Exchange (ETDEWEB)

Argaw, Takele; Wilson, Carolyn A., E-mail: carolyn.wilson@fda.hhs.gov

2015-01-15

Previously, we found that mutation of glutamine to proline in the endoproteolytic cleavage signal of the PERV-C envelope (RQKK to RPKK) resulted in non-infectious vectors. Here, we show that RPKK results in a non-infectious vector when placed in not only a PERV envelope, but also the envelope of a related gammaretrovirus, FeLV-B. The amino acid substitutions do not prevent envelope precursor cleavage, viral core and genome assembly, or receptor binding. Rather, the mutations result in the formation of hyperglycosylated glycoprotein and a reduction in the reverse transcribed minus strand synthesis and undetectable 2-LTR circular DNA in cells exposed to vectors with these mutated envelopes. Our findings suggest novel functions associated with the cleavage signal sequence that may affect trafficking through the glycosylation machinery of the cell. Further, the glycosylation status of the envelope appears to impact post-binding events of the viral life cycle, either membrane fusion, internalization, or reverse transcription. - Highlights: • Env cleavage signal impacts infectivity of gammaretroviruses. • Non-infectious mutants have hyper-glycosylated envelope that bind target cells. • Non-infectious mutants have defects in the formation of the double-stranded DNA. • Env cleavage motif has functions beyond cleavage of the env precursor.

Analytical tools for the study of cellular glycosylation in the immune system

Directory of Open Access Journals (Sweden)

Yvette eVan Kooyk

2013-12-01

Full Text Available It is becoming increasingly clear that glycosylation plays important role in intercellular communication within the immune system. Glycosylation-dependent interactions are crucial for the innate and adaptive immune system and regulate immune cell trafficking, synapse formation, activation, and survival. These functions take place by the cis or trans interaction of lectins with glycans. Classical immunological and biochemical methods have been used for the study of lectin function; however, the investigation of their counterparts, glycans, requires very specialized methodologies that have been extensively developed in the past decade within the Glycobiology scientific community. This Mini-Review intends to summarize the available technology for the study of glycan biosynthesis, its regulation and characterization for their application to the study of glycans in Immunology.

Chapter Three -- Glycosylation of Cellulases: Engineering Better Enzymes for Biofuels

Energy Technology Data Exchange (ETDEWEB)

Greene, Eric R. [Univ. of Colorado, Boulder, CO (United States). Dept. of Chemistry and Biochemistry and BioFrontiers Inst.; Himmel, Michael E. [National Renewable Energy Lab. (NREL), Golden, CO (United States). Biosciences Center; Beckham, Gregg T. [National Renewable Energy Lab. (NREL), Golden, CO (United States). National Bioenergy Center; Tan, Zhongping [Univ. of Colorado, Boulder, CO (United States). Dept. of Chemistry and Biochemistry and BioFrontiers Inst.

2015-10-24

Methods for the manipulation of glycan structures have been recently reported that employ genetic tuning of glycan-active enzymes expressed from homogeneous and heterologous fungal hosts. Taken together, these studies have enabled new strategies for the exploitation of protein glycosylation for the production of enhanced cellulases for biofuel production.

Site-specific protein O-glycosylation modulates proprotein processing - Deciphering specific functions of the large polypeptide GalNAc-transferase gene family

DEFF Research Database (Denmark)

Schjoldager, Katrine Ter-Borch Gram; Clausen, Henrik

2012-01-01

Posttranslational modifications (PTMs) greatly expand the function and regulation of proteins, and glycosylation is the most abundant and diverse PTM. Of the many different types of protein glycosylation, one is quite unique; GalNAc-type (or mucin-type) O-glycosylation, where biosynthesis...... and considerable redundancy. Recently we have begun to uncover human diseases associated with deficiencies in GalNAc-T genes (GALNTs). Thus deficiencies in individual GALNTs produce cell and protein specific effects and subtle distinct phenotypes such as hyperphosphatemia with hyperostosis (GALNT3...

GlcNAc-1-P-transferase–tunicamycin complex structure reveals basis for inhibition of N-glycosylation

Energy Technology Data Exchange (ETDEWEB)

Yoo, Jiho; Mashalidis, Ellene H.; Kuk, Alvin C. Y.; Yamamoto, Kazuki; Kaeser, Benjamin; Ichikawa, Satoshi; Lee, Seok-Yong

2018-02-19

N-linked glycosylation is a predominant post-translational modification of protein in eukaryotes, and its dysregulation is the etiology of several human disorders. The enzyme UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosaminephosphotransferase (GlcNAc-1-P-transferase or GPT) catalyzes the first and committed step of N-linked glycosylation in the endoplasmic reticulum membrane, and it is the target of the natural product tunicamycin. Tunicamycin has potent antibacterial activity, inhibiting the bacterial cell wall synthesis enzyme MraY, but its usefulness as an antibiotic is limited by off-target inhibition of human GPT. Our understanding of how tunicamycin inhibits N-linked glycosylation and efforts to selectively target MraY are hampered by a lack of structural information. Here we present crystal structures of human GPT in complex with tunicamycin. In conclusion, structural and functional analyses reveal the difference between GPT and MraY in their mechanisms of inhibition by tunicamycin. We demonstrate that this difference could be exploited to design MraY-specific inhibitors as potential antibiotics.

N-Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Science.gov (United States)

Niu, Canfang; Luo, Huiying; Shi, Pengjun; Huang, Huoqing; Wang, Yaru; Yang, Peilong

2015-01-01

N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed. PMID:26637601

Structural determination and gynecological tumor diagnosis using ...

African Journals Online (AJOL)

covalent bonding patterns and branched structures. Studies have shown that changes in protein glycosylation at the cell surface during tumorigenesis are closely related to the occurrence and development of some cancers, including pancreatic cancer, mammary cancer, prostatic cancer, colon cancer, and gastric cancer [1].

Characterization of the N-linked glycosylation site of recombinant pectate lyase

NARCIS (Netherlands)

Colangelo, J.; Licon, V.; Benen, J.A.E.; Visser, J.; Bergmann, C.; Orlando, R.

1999-01-01

Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for

Synergizing metabolic flux analysis and nucleotide sugar metabolism to understand the control of glycosylation of recombinant protein in CHO cells

LENUS (Irish Health Repository)

Burleigh, Susan C

2011-10-18

Abstract Background The glycosylation of recombinant proteins can be altered by a range of parameters including cellular metabolism, metabolic flux and the efficiency of the glycosylation process. We present an experimental set-up that allows determination of these key processes associated with the control of N-linked glycosylation of recombinant proteins. Results Chinese hamster ovary cells (CHO) were cultivated in shake flasks at 0 mM glutamine and displayed a reduced growth rate, glucose metabolism and a slower decrease in pH, when compared to other glutamine-supplemented cultures. The N-linked glycosylation of recombinant human chorionic gonadotrophin (HCG) was also altered under these conditions; the sialylation, fucosylation and antennarity decreased, while the proportion of neutral structures increased. A continuous culture set-up was subsequently used to understand the control of HCG glycosylation in the presence of varied glutamine concentrations; when glycolytic flux was reduced in the absence of glutamine, the glycosylation changes that were observed in shake flask culture were similarly detected. The intracellular content of UDP-GlcNAc was also reduced, which correlated with a decrease in sialylation and antennarity of the N-linked glycans attached to HCG. Conclusions The use of metabolic flux analysis illustrated a case of steady state multiplicity, where use of the same operating conditions at each steady state resulted in altered flux through glycolysis and the TCA cycle. This study clearly demonstrated that the control of glycoprotein microheterogeneity may be examined by use of a continuous culture system, metabolic flux analysis and assay of intracellular nucleotides. This system advances our knowledge of the relationship between metabolic flux and the glycosylation of biotherapeutics in CHO cells and will be of benefit to the bioprocessing industry.

The androgen receptor controls expression of the cancer-associated sTn antigen and cell adhesion through induction of ST6GalNAc1 in prostate cancer

Science.gov (United States)

Munkley, Jennifer; Oltean, Sebastian; Vodák, Daniel; Wilson, Brian T.; Livermore, Karen E.; Zhou, Yan; Star, Eleanor; Floros, Vasileios I.; Johannessen, Bjarne; Knight, Bridget; McCullagh, Paul; McGrath, John; Crundwell, Malcolm; Skotheim, Rolf I.; Robson, Craig N.; Leung, Hing Y.; Harries, Lorna W.; Rajan, Prabhakar; Mills, Ian G.; Elliott, David J.

2015-01-01

Patterns of glycosylation are important in cancer, but the molecular mechanisms that drive changes are often poorly understood. The androgen receptor drives prostate cancer (PCa) development and progression to lethal metastatic castration-resistant disease. Here we used RNA-Seq coupled with bioinformatic analyses of androgen-receptor (AR) binding sites and clinical PCa expression array data to identify ST6GalNAc1 as a direct and rapidly activated target gene of the AR in PCa cells. ST6GalNAc1 encodes a sialytransferase that catalyses formation of the cancer-associated sialyl-Tn antigen (sTn), which we find is also induced by androgen exposure. Androgens induce expression of a novel splice variant of the ST6GalNAc1 protein in PCa cells. This splice variant encodes a shorter protein isoform that is still fully functional as a sialyltransferase and able to induce expression of the sTn-antigen. Surprisingly, given its high expression in tumours, stable expression of ST6GalNAc1 in PCa cells reduced formation of stable tumours in mice, reduced cell adhesion and induced a switch towards a more mesenchymal-like cell phenotype in vitro. ST6GalNAc1 has a dynamic expression pattern in clinical datasets, being significantly up-regulated in primary prostate carcinoma but relatively down-regulated in established metastatic tissue. ST6GalNAc1 is frequently upregulated concurrently with another important glycosylation enzyme GCNT1 previously associated with prostate cancer progression and implicated in Sialyl Lewis X antigen synthesis. Together our data establishes an androgen-dependent mechanism for sTn antigen expression in PCa, and are consistent with a general role for the androgen receptor in driving important coordinate changes to the glycoproteome during PCa progression. PMID:26452038

Intolerance of uncertainty, cognitive complaints, and cancer-related distress in prostate cancer survivors.

Science.gov (United States)

Eisenberg, Stacy A; Kurita, Keiko; Taylor-Ford, Megan; Agus, David B; Gross, Mitchell E; Meyerowitz, Beth E

2015-02-01

Prostate cancer survivors have reported cognitive complaints following treatment, and these difficulties may be associated with survivors' ongoing cancer-related distress. Intolerance of uncertainty may exacerbate this hypothesized relationship by predisposing individuals to approach uncertain situations such as cancer survivorship in an inflexible and negative manner. We investigated whether greater cognitive complaints and higher intolerance of uncertainty would interact in their relation to more cancer-related distress symptoms. This cross-sectional, questionnaire-based study included 67 prostate cancer survivors who were 3 to 5 years post treatment. Hierarchical multiple regression analyses tested the extent to which intolerance of uncertainty, cognitive complaints, and their interaction were associated with cancer-related distress (measured with the Impact of Event Scale-Revised; IES-R) after adjusting for age, education, physical symptoms, and fear of cancer recurrence. Intolerance of uncertainty was positively associated with the IES-R avoidance and hyperarousal subscales. More cognitive complaints were associated with higher scores on the IES-R hyperarousal subscale. The interaction of intolerance of uncertainty and cognitive complaints was significantly associated with IES-R intrusion, such that greater cognitive complaints were associated with greater intrusive thoughts in survivors high in intolerance of uncertainty but not those low in it. Prostate cancer survivors who report cognitive difficulties or who find uncertainty uncomfortable and unacceptable may be at greater risk for cancer-related distress, even 3 to 5 years after completing treatment. It may be beneficial to address both cognitive complaints and intolerance of uncertainty in psychosocial interventions. Copyright © 2014 John Wiley & Sons, Ltd.

Pregnancy-related venous thromboembolism and risk of occult cancer

DEFF Research Database (Denmark)

Tarp Hansen, Anette; Veres, Katalin; Horváth-Puhó, Erzsébet

2017-01-01

The cancer risk during the first year after a pregnancy-related venous thromboembolism episode is higher than expected.An aggressive search for cancer in women with pregnancy-related venous thromboembolism is probably not warranted, due to low absolute risk.......The cancer risk during the first year after a pregnancy-related venous thromboembolism episode is higher than expected.An aggressive search for cancer in women with pregnancy-related venous thromboembolism is probably not warranted, due to low absolute risk....

Pregnancy-related venous thromboembolism and risk of occult cancer

DEFF Research Database (Denmark)

Hansen, Anette Tarp; Veres, Katalin; Horváth-Puhó, Erzsébet

2017-01-01

The cancer risk during the first year after a pregnancy-related venous thromboembolism episode is higher than expected. An aggressive search for cancer in women with pregnancy-related venous thromboembolism is probably not warranted, due to low absolute risk.......The cancer risk during the first year after a pregnancy-related venous thromboembolism episode is higher than expected. An aggressive search for cancer in women with pregnancy-related venous thromboembolism is probably not warranted, due to low absolute risk....

Coupled cell-free synthesis, segregation, and core glycosylation of a secretory protein.

Science.gov (United States)

Lingappa, V R; Lingappa, J R; Prasad, R; Ebner, K E; Blobel, G

1978-05-01

mRNA from rat mammary glands 13-15 days post partum was translated in a wheat germ cell-free system either in the absence or in the presence of ribosome-denuded membranes prepared from isolated rough microsomes of dog pancreas. Newly synthesized alpha-lactalbumin was identified by immunoprecipitation with a monospecific rabbit antiserum against rat alpha-lactalbumin and was characterized by partial amino-terminal sequence determination and by lectin affinity chromatography. In the absence of membranes a presumably unglycosylated form of alpha-lactalbumin was synthesized that bound neither to concanavalin A-Sepharose nor to Ricinus communis lectin-agarose and that contained an amino-terminal signal peptide region comprising 19 amino acid residues. In the presence of membranes a processed form was synthesized that lacked the signal peptide portion and that had an amino-terminal sequence identical to that of mature alpha-lactalbumin. Furthermore, this processed form was found to be segregated, presumably within the microsomal vesicles, because it was resistant to post-translational proteolysis. It was also found to be glycosylated, and because it bound to concanavalin A-Sepharose, from which it could be eluted specifically by alpha-methyl mannoside, but not to R. communis lectin-agarose, it was presumably core-glycosylated. Processing, segregation, and core glycosylation were observed to proceed only when membranes were present during translation and not when they were added after translation.

Charge and Polarity Preferences for N-Glycosylation: A Genome-Wide In Silico Study and Its Implications Regarding Constitutive Proliferation and Adhesion of Carcinoma Cells.

Science.gov (United States)

Manwar Hussain, Muhammad Ramzan; Iqbal, Zeeshan; Qazi, Wajahat M; Hoessli, Daniel C

2018-01-01

The structural and functional diversity of the human proteome is mediated by N - and O- linked glycosylations that define the individual properties of extracellular and membrane-associated proteins. In this study, we utilized different computational tools to perform in silico based genome-wide mapping of 1,117 human proteins and unravel the contribution of both penultimate and vicinal amino acids for the asparagine-based, site-specific N -glycosylation. Our results correlate the non-canonical involvement of charge and polarity environment of classified amino acids (designated as L, O, A, P, and N groups) in the N -glycosylation process, as validated by NetNGlyc predictions, and 130 literature-reported human proteins. From our results, particular charge and polarity combinations of non-polar aliphatic, acidic, basic, and aromatic polar side chain environment of both penultimate and vicinal amino acids were found to promote the N -glycosylation process. However, the alteration in side-chain charge and polarity environment of genetic variants, particularly in the vicinity of Asn-containing epitope, may induce constitutive glycosylation (e.g., aberrant glycosylation at preferred and non-preferred sites) of membrane proteins causing constitutive proliferation and triggering epithelial-to-mesenchymal transition. The current genome-wide mapping of 1,117 proteins (2,909 asparagine residues) was used to explore charge- and polarity-based mechanistic constraints in N -glycosylation, and discuss alterations of the neoplastic phenotype that can be ascribed to N -glycosylation at preferred and non-preferred sites.

TMEM199 Deficiency Is a Disorder of Golgi Homeostasis Characterized by Elevated Aminotransferases, Alkaline Phosphatase, and Cholesterol and Abnormal Glycosylation

NARCIS (Netherlands)

Jansen, Jos C.; Timal, Sharita; van Scherpenzeel, Monique; Michelakakis, Helen; Vicogne, Dorothée; Ashikov, Angel; Moraitou, Marina; Hoischen, Alexander; Huijben, Karin; Steenbergen, Gerry; van den Boogert, Marjolein A. W.; Porta, Francesco; Calvo, Pier Luigi; Mavrikou, Mersyni; Cenacchi, Giovanna; van den Bogaart, Geert; Salomon, Jody; Holleboom, Adriaan G.; Rodenburg, Richard J.; Drenth, Joost P. H.; Huynen, Martijn A.; Wevers, Ron A.; Morava, Eva; Foulquier, François; Veltman, Joris A.; Lefeber, Dirk J.

2016-01-01

Congenital disorders of glycosylation (CDGs) form a genetically and clinically heterogeneous group of diseases with aberrant protein glycosylation as a hallmark. A subgroup of CDGs can be attributed to disturbed Golgi homeostasis. However, identification of pathogenic variants is seriously

Is glycosylated haemoglobin a marker of fertility?

DEFF Research Database (Denmark)

Hjollund, N H; Jensen, T K; Bonde, J P

1999-01-01

We performed a follow-up study of time to pregnancy in a population of first-time pregnancy planners without previous reproductive experience. The objective of this paper is to report and discuss a finding of a strong relationship between glycosylated haemoglobin (HbA1C) and fertility. A total...... concentration of inhibin A. No association was found between HbA1C and psychosocial distress. The reduced fertility among women with high HbA1C may be due to an association with subclinical polycystic ovaries as indicated by the hormonal profile....

Inhibitory potential of pure isoflavonoids, red clover, and alfalfa extracts on hemoglobin glycosylation

Directory of Open Access Journals (Sweden)

Mohsen Hosseini

2015-03-01

Full Text Available BACKGROUND: Non-enzymatic glycosylation of hemoglobin is complications of diabetes. Antioxidant system imbalance can result in the emergence of free radicals’ destructive effects in the long-term. Red clover (Trifolium pratense L. and alfalfa (Medicago sativa L. contain isoflavonoids and have antioxidant activity. This experimental study evaluated the inhibitory activity of pure isoflavonoids (daidzein and genistein, red clover and alfalfa extracts on hemoglobin glycosylation. METHODS: This study was performed in Iran. Stock solution of hydroalcoholic extracts of red clover and alfalfa in concentrations of 1 and 10 g/100 ml and stock solution of daidzein and genistein in concentrations of 250 ng, 500 ng, 25 µg and 250 µg/100 ml were prepared as case groups. Control group was without hydroalcoholic extracts of plants and pure isoflavonoids. All experiments were performed in triplicate. Hemoglobin was prepared and antioxidant activities were investigated to estimate degree of nonenzymatic hemoglobin glycosylation. RESULTS: There was no significantly difference between used extracts (extract of red clover and alfalfa and control of the hemoglobin glycosylation but using daidzein (P = 0.046, 0.029 and 0.021, respectively and genistein (P = 0.034, 0.036 and 0.028 significantly inhibited (P < 0.050 this reaction in 25 µg/100 ml, 250 and 500 ng/100 ml concentrations when compared to control. in 25 µg/100 ml, 250 ng and 500 ng/100 ml concentrations percentage of inhibition were 32, 80 and 74.5% respectively with used of daidzein and were 21, 83 and 76% respectively with consumption of genistein. CONCLUSION: According to decrease of glycation of hemoglobin with isoflavonoids, two used plant in this study containing isoflavonoid may be useful on diabetes.   

Identification of α(1,6)fucosylated proteins differentially expressed in human colorectal cancer

International Nuclear Information System (INIS)

Muinelo-Romay, Laura; Villar-Portela, Susana; Cuevas, Elisa; Gil-Martín, Emilio; Fernández-Briera, Almudena

2011-01-01

A universal hallmark of cancer cells is the change in their glycosylation phenotype. One of the most frequent alterations in the normal glycosylation pattern observed during carcinogenesis is the enhancement of α(1,6)linked fucose residues of glycoproteins, due to the up-regulation of the α(1,6)fucosyltransferase activity. Our previous results demonstrated the specific alteration of this enzyme activity and expression in colorectal cancer, suggesting its implication in tumour development and progression. In the current work we combined a LCA-affinity chromatography with SDS-PAGE and mass spectrometry in order to identify α(1,6)fucosylated proteins differentially expressed in colorectal cancer. This strategy allowed the identification of a group of α(1,6)fucosylated proteins candidates to be involved in CRC malignancy. The majority of the identified proteins take part in cell signaling and interaction processes as well as in modulation of the immunological response. Likewise, we confirmed the increased expression of GRP94 in colorectal cancer tissue and the significant down-regulation of the IgGFcBP expression in tumour cells. All these results validate the importance of core-fucosylated proteins profile analysis to understand the mechanisms which promote cancer onset and progression and to discover new tumour markers or therapeutic targets

Unraveling the Molecular Complexity of O-Glycosylated Endogenous (N-Terminal) pro-B-Type Natriuretic Peptide Forms in Blood Plasma of Patients with Severe Heart Failure.

Science.gov (United States)

Halfinger, Bernhard; Hammerer-Lercher, Angelika; Amplatz, Benno; Sarg, Bettina; Kremser, Leopold; Lindner, Herbert H

2017-01-01

Currently, N-terminal pro-B-type natriuretic peptide (NT-proBNP) and its physiologically active counterpart, BNP, are most frequently used as biomarkers for diagnosis, prognosis, and disease monitoring of heart failure (HF). Commercial NT-proBNP and BNP immunoassays cross-react to varying degrees with unprocessed proBNP, which is also found in the circulation. ProBNP processing and immunoassay response are related to O-linked glycosylation of NT-proBNP and proBNP. There is a clear and urgent need to identify the glycosylation sites in the endogenously circulating peptides requested by the community to gain further insights into the different naturally occurring forms. The glycosylation sites of (NT-) proBNP (NT-proBNP and/or proBNP) were characterized in leftovers of heparinized plasma samples of severe HF patients (NT-proBNP: >10000 ng/L) by using tandem immunoaffinity purification, sequential exoglycosidase treatment for glycan trimming, β-elimination and Michael addition chemistry, as well as high-resolution nano-flow liquid chromatography electrospray multistage mass spectrometry. We describe 9 distinct glycosylation sites on circulating (NT-) proBNP in HF patients. Differentially glycosylated variants were detected based on highly accurate mass determination and multistage mass spectrometry. Remarkably, for each of the identified proteolytic glycopeptides, a nonglycosylated form also was detectable. Our results directly demonstrate for the first time a rather complex distribution of the endogenously circulating glycoforms by mass spectrometric analysis in HF patients, and show 9 glycosites in human (NT-) proBNP. This information may also have an impact on commercial immunoassays applying antibodies specific for the central region of (NT-) proBNP, which detect mostly nonglycosylated forms. © 2016 American Association for Clinical Chemistry.

Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability.

Science.gov (United States)

Colotta, Francesco; Allavena, Paola; Sica, Antonio; Garlanda, Cecilia; Mantovani, Alberto

2009-07-01

Inflammatory conditions in selected organs increase the risk of cancer. An inflammatory component is present also in the microenvironment of tumors that are not epidemiologically related to inflammation. Recent studies have begun to unravel molecular pathways linking inflammation and cancer. In the tumor microenvironment, smoldering inflammation contributes to proliferation and survival of malignant cells, angiogenesis, metastasis, subversion of adaptive immunity, reduced response to hormones and chemotherapeutic agents. Recent data suggest that an additional mechanism involved in cancer-related inflammation (CRI) is induction of genetic instability by inflammatory mediators, leading to accumulation of random genetic alterations in cancer cells. In a seminal contribution, Hanahan and Weinberg [(2000) Cell, 100, 57-70] identified the six hallmarks of cancer. We surmise that CRI represents the seventh hallmark.

Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates.

Science.gov (United States)

Bavaro, Teodora; Tengattini, Sara; Piubelli, Luciano; Mangione, Francesca; Bernardini, Roberta; Monzillo, Vincenzina; Calarota, Sandra; Marone, Piero; Amicosante, Massimo; Pollegioni, Loredano; Temporini, Caterina; Terreni, Marco

2017-06-29

Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy for developing efficient vaccines. In this work, we investigated the effect of the chemical glycosylation on two recombinant MTB proteins produced in E. coli with an additional seven-amino acid tag (recombinant Ag85B and TB10.4). Different semi-synthetic glycoconjugated derivatives were prepared, starting from mannose and two disaccharide analogs. The glycans were activated at the anomeric position with a thiocyanomethyl group, as required for protein glycosylation by selective reaction with lysines. The glycosylation sites and the ex vivo evaluation of the immunogenic activity of the different neo- glycoproteins were investigated. Glycosylation does not modify the immunological activity of the TB10.4 protein. Similarly, Ag85B maintains its B-cell activity after glycosylation while showing a significant reduction in the T-cell response. The results were correlated with the putative B- and T-cell epitopes, predicted using a combination of in silico systems. In the recombinant TB10.4, the unique lysine is not included in any T-cell epitope. Lys30 of Ag85B, identified as the main glycosylation site, proved to be the most important site involved in the formation of T-cell epitopes, reasonably explaining why its glycosylation strongly influenced the T-cell activity. Furthermore, additional lysines included in different epitopes (Lys103, -123 and -282) are also glycosylated. In contrast, B-cell epitopic lysines of Ag85B were found to be poorly glycosylated and, thus, the antibody interaction of Ag85B was only marginally affected after coupling with mono- or disaccharides.

High-throughput analysis of endogenous fruit glycosyl hydrolases using a novel chromogenic hydrogel substrate assay

DEFF Research Database (Denmark)

Schückel, Julia; Kracun, Stjepan Kresimir; Lausen, Thomas Frederik

2017-01-01

A broad range of enzyme activities can be found in a wide range of different fruits and fruiting bodies but there is a lack of methods where many samples can be handled in a high-throughput and efficient manner. In particular, plant polysaccharide degrading enzymes – glycosyl hydrolases (GHs) play...... led to a more profound understanding of the importance of GH activity and regulation, current methods for determining glycosyl hydrolase activity are lacking in throughput and fail to keep up with data output from transcriptome research. Here we present the use of a versatile, easy...

Chondrocyte secreted CRTAC1: a glycosylated extracellular matrix molecule of human articular cartilage.

Science.gov (United States)

Steck, Eric; Bräun, Jessica; Pelttari, Karoliina; Kadel, Stephanie; Kalbacher, Hubert; Richter, Wiltrud

2007-01-01

Cartilage acidic protein 1 (CRTAC1), a novel human marker which allowed discrimination of human chondrocytes from osteoblasts and mesenchymal stem cells in culture was so far studied only on the RNA-level. We here describe its genomic organisation and detect a new brain expressed (CRTAC1-B) isoform resulting from alternate last exon usage which is highly conserved in vertebrates. In humans, we identify an exon sharing process with the neighbouring tail-to-tail orientated gene leading to CRTAC1-A. This isoform is produced by cultured human chondrocytes, localized in the extracellular matrix of articular cartilage and its secretion can be stimulated by BMP4. Of five putative O-glycosylation motifs in the last exon of CRTAC1-A, the most C-terminal one is modified according to exposure of serial C-terminal deletion mutants to the O-glycosylation inhibitor Benzyl-alpha-GalNAc. Both isoforms contain four FG-GAP repeat domains and an RGD integrin binding motif, suggesting cell-cell or cell-matrix interaction potential. In summary, CRTAC1 acquired an alternate last exon from the tail-to-tail oriented neighbouring gene in humans resulting in the glycosylated isoform CRTAC1-A which represents a new extracellular matrix molecule of articular cartilage.

Phenylpropanoid Scent Compounds in Petunia x hybrida Are Glycosylated and Accumulate in Vacuoles

Science.gov (United States)

Cna'ani, Alon; Shavit, Reut; Ravid, Jasmin; Aravena-Calvo, Javiera; Skaliter, Oded; Masci, Tania; Vainstein, Alexander

2017-01-01

Floral scent has been studied extensively in the model plant Petunia. However, little is known about the intracellular fate of scent compounds. Here, we characterize the glycosylation of phenylpropanoid scent compounds in Petunia x hybrida. This modification reduces scent compounds' volatility, reactivity, and autotoxicity while increasing their water-solubility. Gas chromatography–mass spectrometry (GC–MS) analyses revealed that flowers of petunia cultivars accumulate substantial amounts of glycosylated scent compounds and that their increasing level parallels flower development. In contrast to the pool of accumulated aglycones, which drops considerably at the beginning of the light period, the collective pool of glycosides starts to increase at that time and does not decrease thereafter. The glycoside pool is dynamic and is generated or catabolized during peak scent emission, as inferred from phenylalanine isotope-feeding experiments. Using several approaches, we show that phenylpropanoid scent compounds are stored as glycosides in the vacuoles of petal cells: ectopic expression of Aspergillus niger β-glucosidase-1 targeted to the vacuole resulted in decreased glycoside accumulation; GC–MS analysis of intact vacuoles isolated from petal protoplasts revealed the presence of glycosylated scent compounds. Accumulation of glycosides in the vacuoles seems to be a common mechanism for phenylpropanoid metabolites. PMID:29163617

Neonatal severe intractable diarrhoea as the presenting manifestation of an unclassified congenital disorder of glycosylation (CDG-x)

OpenAIRE

Mention, K; Michaud, L; Dobbelaere, D; Guimber, D; Gottrand, F; Turck, D

2001-01-01

A case of severe and protracted diarrhoea is reported, which started in the neonatal period and progressively associated with neurological impairment, dysmorphy, hepatosplenomegaly, and hepatic insufficiency, from which the patient died at 2 years of age. Isoelectric focusing of serum transferrin showed a congenital disorder of glycosylation type I pattern but the basic defect could not be identified. This observation shows that congenital disorder of glycosylation is a cause of i...

Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation.

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Jeremy A Iwashkiw

Full Text Available Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening "superbugs" for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics.

Identification of a General O-linked Protein Glycosylation System in Acinetobacter baumannii and Its Role in Virulence and Biofilm Formation

Science.gov (United States)

Iwashkiw, Jeremy A.; Seper, Andrea; Weber, Brent S.; Scott, Nichollas E.; Vinogradov, Evgeny; Stratilo, Chad; Reiz, Bela; Cordwell, Stuart J.; Whittal, Randy; Schild, Stefan; Feldman, Mario F.

2012-01-01

Acinetobacter baumannii is an emerging cause of nosocomial infections. The isolation of strains resistant to multiple antibiotics is increasing at alarming rates. Although A. baumannii is considered as one of the more threatening “superbugs” for our healthcare system, little is known about the factors contributing to its pathogenesis. In this work we show that A. baumannii ATCC 17978 possesses an O-glycosylation system responsible for the glycosylation of multiple proteins. 2D-DIGE and mass spectrometry methods identified seven A. baumannii glycoproteins, of yet unknown function. The glycan structure was determined using a combination of MS and NMR techniques and consists of a branched pentasaccharide containing N-acetylgalactosamine, glucose, galactose, N-acetylglucosamine, and a derivative of glucuronic acid. A glycosylation deficient strain was generated by homologous recombination. This strain did not show any growth defects, but exhibited a severely diminished capacity to generate biofilms. Disruption of the glycosylation machinery also resulted in reduced virulence in two infection models, the amoebae Dictyostelium discoideum and the larvae of the insect Galleria mellonella, and reduced in vivo fitness in a mouse model of peritoneal sepsis. Despite A. baumannii genome plasticity, the O-glycosylation machinery appears to be present in all clinical isolates tested as well as in all of the genomes sequenced. This suggests the existence of a strong evolutionary pressure to retain this system. These results together indicate that O-glycosylation in A. baumannii is required for full virulence and therefore represents a novel target for the development of new antibiotics. PMID:22685409

Cysteine S-glycosylation, a new post-translational modification found in glycopeptide bacteriocins

Czech Academy of Sciences Publication Activity Database

Stepper, J.; Shastri, S.; Loo, T. S.; Preston, J. C.; Novák, Petr; Man, Petr; Moore, Ch. H.; Havlíček, Vladimír; Patchett, M. L.; Norris, G. E.

2011-01-01

Roč. 585, č. 4 (2011), s. 645-650 ISSN 0014-5793 Institutional research plan: CEZ:AV0Z50200510 Keywords : Post-translational modification * Glycosylation * Bacteriocin Subject RIV: CE - Biochemistry Impact factor: 3.538, year: 2011

Glycosylated hemoglobin as a forecast factor of progressing of diabetic nephropathy in patients with diabetes type 1

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Pertseva N.O.

2017-12-01

Full Text Available The aim of the study was to propose a mathematical model for prediction of development of diabetic nephropathy in patients with diabetes mellitus by determining the level of glycosylated hemoglobin - as a factor in the development and progression of diabetic nephropathy. A survey of 136 patients with type 1 diabetes was performed in the endocrinology department of the OSH «Clinic of the Medical Academy», Dnipro in 2016-2017. Clinical laboratory examination included: determination of the level of glycosylated hemoglobin (HbA1c, level of blood creatinine, level of albuminuria. The GFR was calculated by the formula CKD-EPI. The obtained results of the study, using methods of correlation and regression analysis, show a clear correlation between the GFR score in patients with diabetes mellitus and the level of glycosylated hemoglobin. Statistical methods of analysis have shown that the level of glycosylated hemoglobin can be considered as an early predictor of development of diabetic nephropathy. The mathematical equation of prognosis for the onset of diabetic nephropathy can be used to determine the prognosis for the development of diabetic nephropathy in diabetes mellitus patients in clinical practice for the timely inclusion of patients with a high prognostic risk in a group requiring more stringent glycemic control.

Linkage specific fucosylation of alpha-1-antitrypsin in liver cirrhosis and cancer patients: implications for a biomarker of hepatocellular carcinoma.

Directory of Open Access Journals (Sweden)

Mary Ann Comunale

2010-08-01

Full Text Available We previously reported increased levels of protein-linked fucosylation with the development of liver cancer and identified many of the proteins containing the altered glycan structures. One such protein is alpha-1-antitrypsin (A1AT. To advance these studies, we performed N-linked glycan analysis on the five major isoforms of A1AT and completed a comprehensive study of the glycosylation of A1AT found in healthy controls, patients with hepatitis C- (HCV induced liver cirrhosis, and in patients infected with HCV with a diagnosis of hepatocellular carcinoma (HCC.Patients with liver cirrhosis and liver cancer had increased levels of triantennary glycan-containing outer arm (alpha-1,3 fucosylation. Increases in core (alpha-1,6 fucosylation were observed only on A1AT from patients with cancer. We performed a lectin fluorophore-linked immunosorbent assay using Aleuria Aurantia lectin (AAL, specific for core and outer arm fucosylation in over 400 patients with liver disease. AAL-reactive A1AT was able to detect HCC with a sensitivity of 70% and a specificity of 86%, which was greater than that observed with the current marker of HCC, alpha-fetoprotein. Glycosylation analysis of the false positives was performed; results indicated that these patients had increases in outer arm fucosylation but not in core fucosylation, suggesting that core fucosylation is cancer specific.This report details the stepwise change in the glycosylation of A1AT with the progression from liver cirrhosis to cancer and identifies core fucosylation on A1AT as an HCC specific modification.

N-glycosylation increases the circulatory half-life of human growth hormone

DEFF Research Database (Denmark)

Flintegaard, Thomas V; Thygesen, Peter; Rahbek-Nielsen, Henrik

2010-01-01

Therapeutic use of recombinant GH typically involves daily sc injections. We examined the possibilities for prolonging the in vivo circulation of GH by introducing N-glycans. Human GH variants with a single potential N-glycosylation site (N-X-S/T) introduced by site-directed mutagenesis were expr...

[Cancer and family: tasks and stress of relatives].

Science.gov (United States)

Popek, V; Hönig, K

2015-03-01

Relatives are the primary and existential resource of cancer patients, while at the same time experiencing substantial distress themselves. This article presents a description of tasks, roles and distress factors, the prevalence of psychosocial distress, description of risk factors in families contributing to dysfunctional coping, options and empirical evidence for the efficacy of psychosocial support. Evaluation of registry data, analysis of case reports, discussion of basic research findings, meta-analyses and expert judgments. Psychosocial distress in relatives of cancer patients is comparable to the degree of distress experienced by the patients and is sometimes even higher. Distress in relatives is still underrecognized, underreported and undertreated. Hostile interaction patterns, low emotional expression and high conflict tendencies impair coping with cancer and its treatment. Psychosocial support for the family of cancer patients improves coping behavior and the quality of life both in relatives and patients. Professional and lay caregivers need to adopt a social perspective on cancer whereby participation and inclusion of relatives in the treatment, acknowledgment of their engagement and recognition of their distress is beneficial for both patients and their relatives. Screening for psychosocial distress in relatives is recommended, attention should be drawn to psychosocial support services and utilization should be encouraged.

Effects of yoga on cancer-related fatigue and global side-effect burden in older cancer survivors.

Science.gov (United States)

Sprod, Lisa K; Fernandez, Isabel D; Janelsins, Michelle C; Peppone, Luke J; Atkins, James N; Giguere, Jeffrey; Block, Robert; Mustian, Karen M

2015-01-01

Sixty percent of cancer survivors are 65years of age or older. Cancer and its treatments lead to cancer-related fatigue and many other side effects, in turn, creating substantial global side-effect burden (total burden from all side effects) which, ultimately, compromises functional independence and quality of life. Various modes of exercise, such as yoga, reduce cancer-related fatigue and global side-effect burden in younger cancer survivors, but no studies have specifically examined the effects of yoga on older cancer survivors. The purpose of this study was to assess the effects of a 4-week yoga intervention (Yoga for Cancer Survivors: YOCAS©®) on overall cancer-related fatigue, and due to its multidimensional nature, the subdomains of cancer-related fatigue (general, physical, emotional, and mental) and global side-effect burden in older cancer survivors. We conducted a secondary analysis on data from a multicenter phase III randomized controlled clinical trial with 2 arms (standard care and standard care plus a 4-week YOCAS©® intervention). The sample for this secondary analysis was 97 older cancer survivors (≥60years of age), between 2months and 2years post-treatment, who participated in the original trial. Participants in the YOCAS©® intervention arm reported significantly lower cancer-related fatigue, physical fatigue, mental fatigue, and global side-effect burden than participants in the standard care arm following the 4-week intervention period (peffective standardized yoga intervention for reducing cancer-related fatigue, physical fatigue, mental fatigue, and global side-effect burden among older cancer survivors. Copyright © 2014 Elsevier Inc. All rights reserved.

Glycosylation of Candida albicans cell wall proteins is critical for induction of innate immune responses and apoptosis of epithelial cells.

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Jeanette Wagener

Full Text Available C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo.

Storytelling as part of cancer rehabilitation to support cancer patients and their relatives.

Science.gov (United States)

la Cour, Karen; Ledderer, Loni; Hansen, Helle Ploug

2016-01-01

Previous research on psychosocial support for cancer-related concerns has primarily focused on either patients or their relatives, although limited research is available on how patients and their relatives can be supported together. The aim of this article is to explore the use of storytelling as a part of a residential cancer rehabilitation intervention for patients together with their relatives, with a specific focus on their management of cancer-related concerns. Ten pairs participated in the intervention and data were generated through ethnographic fieldwork, including participant observations, informal conversations and follow-up interviews conducted one month after completing the intervention. Analysis was performed drawing on narrative theory combined with social practice theory. The results demonstrate that the use of storytelling and metaphors intertwined with other course activities, such as dancing and arts & crafts, provided the patients and their relatives with strategies to manage cancer-related concerns, which they were later able to apply in their everyday lives. The study results may be useful to other professionals in clinical practice for rehabilitation purposes for addressing issues of fear and worry.

Production, crystallization and X-ray characterization of chemically glycosylated hen egg-white lysozyme

International Nuclear Information System (INIS)

López-Jaramillo, F. J.; Pérez-Banderas, F.; Hernández-Mateo, F.; Santoyo-González, F.

2005-01-01

The feasibility of glycosylation post-purification has been demonstrated by introducing glucose into the model protein lysozyme via a novel reaction that is compatible with biological samples. The crystallization of glycoproteins is one of the challenges to be confronted by the crystallographic community in the frame of what is known as glycobiology. The state of the art for the crystallization of glycoproteins is not promising and removal of the carbohydrate chains is generally suggested since they are flexible and a source of heterogeneity. In this paper, the feasibility of introducing glucose into the model protein hen egg-white lysozyme via a post-purification glycosylation reaction that may turn any protein into a model glycoprotein whose carbohydrate fraction can be manipulated is demonstrated

Coding completeness and quality of relative survival-related variables in the National Program of Cancer Registries Cancer Surveillance System, 1995-2008.

Science.gov (United States)

Wilson, Reda J; O'Neil, M E; Ntekop, E; Zhang, Kevin; Ren, Y

2014-01-01

Calculating accurate estimates of cancer survival is important for various analyses of cancer patient care and prognosis. Current US survival rates are estimated based on data from the National Cancer Institute's (NCI's) Surveillance, Epidemiology, and End RESULTS (SEER) program, covering approximately 28 percent of the US population. The National Program of Cancer Registries (NPCR) covers about 96 percent of the US population. Using a population-based database with greater US population coverage to calculate survival rates at the national, state, and regional levels can further enhance the effective monitoring of cancer patient care and prognosis in the United States. The first step is to establish the coding completeness and coding quality of the NPCR data needed for calculating survival rates and conducting related validation analyses. Using data from the NPCR-Cancer Surveillance System (CSS) from 1995 through 2008, we assessed coding completeness and quality on 26 data elements that are needed to calculate cancer relative survival estimates and conduct related analyses. Data elements evaluated consisted of demographic, follow-up, prognostic, and cancer identification variables. Analyses were performed showing trends of these variables by diagnostic year, state of residence at diagnosis, and cancer site. Mean overall percent coding completeness by each NPCR central cancer registry averaged across all data elements and diagnosis years ranged from 92.3 percent to 100 percent. RESULTS showing the mean percent coding completeness for the relative survival-related variables in NPCR data are presented. All data elements but 1 have a mean coding completeness greater than 90 percent as was the mean completeness by data item group type. Statistically significant differences in coding completeness were found in the ICD revision number, cause of death, vital status, and date of last contact variables when comparing diagnosis years. The majority of data items had a coding

Role of Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer Cells

National Research Council Canada - National Science Library

Zolov, Sergey N

2006-01-01

...: protein glycosylation and its sorting. For analysis of COG complex function we utilized RNA interference assay to knockdown COG3p subunit of COG complex in normal and breast cancer cells and other tumor cell lines...

Glycomic Analysis of Prostate Cancer

Science.gov (United States)

2012-07-01

A, Mucci LA, Ekbom A, et al. The risk of liver and bile duct cancer in patients with chronic viral hepatitis, alcoholism, or cirrhosis. Hepatology...the AA and EA men in both GSE6956 and GSE17356 datasets. Table 5. Glycogenes selected from Table 2. N-glycans in liver -secreted and immunoglogulin...January 2012 Available online 3 February 2012 N-glycosylation of proteins provides a rich source of information on liver disease progression

Identification of a protein glycosylation operon from Campylobacter jejuni JCM 2013 and its heterologous expression in Escherichia coli.

Science.gov (United States)

Srichaisupakit, Akkaraphol; Ohashi, Takao; Fujiyama, Kazuhito

2014-09-01

Campylobacter jejuni is a human enteropathogenic bacterium possessing an N-glycosylation system. In this work, a protein glycosylation (pgl) operon conferring prokaryotic N-glycosylation in C. jejuni JCM 2013 was cloned and identified. Fourteen open reading frames (ORFs) were found in the pgl operon. The operon organization was similar to that of C. jejuni NCTC 11168, with 98% and 99% identities in overall nucleotide sequence and amino acid sequence, respectively. The pgl operon was heterologously co-expressed with model protein CmeA in the Escherichia coli BL21 ΔwaaL mutant. The immuno- and lectin-blotting analysis indicated the protein glycosylation on the recombinant CmeA. In addition, to analyze the glycan composition, the recombinant CmeA was purified and subjected to in-gel trypsin digestion followed by mass spectrometry analysis. The mass spectrometry analysis showed the presence of the N-acetylhexosamine residue at the reducing end but not the predicted di-N-acetylbacillosamine (diNAcBac) residue. Further glycan structural study using the conventional fluorophore-labeling method revealed the GalNAcα-GalNAcα-(Hex-)HexNAc-HexNAc-HexNAc-HexNAc structure. Transcriptional analysis showed that UDP-diNAcBac synthases and diNAcBac transferase are transcribed but might not function in the constructed system. In conclusion, a pgl operon from C. jejuni JCM 2013 successfully functioned in E. coli, resulting in the observed prokaryotic glycosylation. Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Expression of Iron-Related Proteins Differentiate Non-Cancerous and Cancerous Breast Tumors

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Sara Pizzamiglio

2017-02-01

Full Text Available We have previously reported hepcidin and ferritin increases in the plasma of breast cancer patients, but not in patients with benign breast disease. We hypothesized that these differences in systemic iron homeostasis may reflect alterations in different iron-related proteins also play a key biochemical and regulatory role in breast cancer. Thus, here we explored the expression of a bundle of molecules involved in both iron homeostasis and tumorigenesis in tissue samples. Enzyme-linked immunosorbent assay (ELISA or reverse-phase protein array (RPPA, were used to measure the expression of 20 proteins linked to iron processes in 24 non-cancerous, and 56 cancerous, breast tumors. We found that cancerous tissues had higher level of hepcidin than benign lesions (p = 0.012. The univariate analysis of RPPA data highlighted the following seven proteins differentially expressed between non-cancerous and cancerous breast tissue: signal transducer and transcriptional activator 5 (STAT5, signal transducer and activator of transcription 3 (STAT3, bone morphogenetic protein 6 (BMP6, cluster of differentiation 74 (CD74, transferrin receptor (TFRC, inhibin alpha (INHA, and STAT5_pY694. These findings were confirmed for STAT5, STAT3, BMP6, CD74 and INHA when adjusting for age. The multivariate statistical analysis indicated an iron-related 10-protein panel effective in separating non-cancerous from cancerous lesions including STAT5, STAT5_pY694, myeloid differentiation factor 88 (MYD88, CD74, iron exporter ferroportin (FPN, high mobility group box 1 (HMGB1, STAT3_pS727, TFRC, ferritin heavy chain (FTH, and ferritin light chain (FTL. Our results showed an association between some iron-related proteins and the type of tumor tissue, which may provide insight in strategies for using iron chelators to treat breast cancer.

Epitopes of MUC1 Tandem Repeats in Cancer as Revealed by Antibody Crystallography: Toward Glycopeptide Signature-Guided Therapy

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Dapeng Zhou

2018-05-01

Full Text Available Abnormally O-glycosylated MUC1 tandem repeat glycopeptide epitopes expressed by multiple types of cancer have long been attractive targets for therapy in the race against genetic mutations of tumor cells. Glycopeptide signature-guided therapy might be a more promising avenue than mutation signature-guided therapy. Three O-glycosylated peptide motifs, PDTR, GSTA, and GVTS, exist in a tandem repeat HGVTSAPDTRPAPGSTAPPA, containing five O-glycosylation sites. The exact peptide and sugar residues involved in antibody binding are poorly defined. Co-crystal structures of glycopeptides and respective monoclonal antibodies are very few. Here we review 3 groups of monoclonal antibodies: antibodies which only bind to peptide portion, antibodies which only bind to sugar portion, and antibodies which bind to both peptide and sugar portions. The antigenicity of peptide and sugar portions of glyco-MUC1 tandem repeat were analyzed according to available biochemical and structural data, especially the GSTA and GVTS motifs independent from the most studied PDTR. Tn is focused as a peptide-modifying residue in vaccine design, to induce glycopeptide-binding antibodies with cross reactivity to Tn-related tumor glycans, but not glycans of healthy cells. The unique requirement for the designs of antibody in antibody-drug conjugate, bi-specific antibodies, and chimeric antigen receptors are also discussed.

Exploratory study of impact of cancer-related posttraumatic stress symptoms on diabetes self-management among cancer survivors.

Science.gov (United States)

James, Janey; Harris, Yael T; Kronish, Ian M; Wisnivesky, Juan P; Lin, Jenny J

2018-02-01

Posttraumatic stress symptoms (PTSS) can be triggered by a diagnosis of a potentially life-threatening illness such as cancer. Little is known about the impact of cancer-related PTSS symptoms on self-management behaviors for comorbid chronic medical conditions such as diabetes mellitus (DM). We recruited patients with DM and a recent diagnosis of early-stage cancer from 2 medical centers in New York City. Cancer-related PTSS were assessed using the Impact of Events Scale (score ≥ 26). DM self-management behaviors (medication adherence, exercise, healthy diet, and glucose testing) were measured 3 months later. Logistic regression was used to assess the association between cancer-related PTSS symptoms and DM self-management behaviors, adjusting for gender, marital status, and anxiety symptoms. Of 56 participants recruited, 33% reported cancer-related PTSS symptoms. Elevated cancer-related PTSS symptoms were associated with lack of healthy diet (odds ratio: 0.08, 95% confidence interval: 0.01-0.62). Early-stage cancer survivors with cancer-related PTSS symptoms were less likely to adhere to some DM self-management behaviors. Providers should recognize the impact of cancer-related PTSS symptoms to better support comorbid disease management in cancer survivors. Copyright © 2017 John Wiley & Sons, Ltd.

Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation

DEFF Research Database (Denmark)

Pinto, Rita; Carvalho, Ana S; Conze, Tim

2012-01-01

Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these b......Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification...... of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA......) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Le(a) (SLe(a) ) and Sialyl-Le(x) (SLe(x) ) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla...

Neonatal severe intractable diarrhoea as the presenting manifestation of an unclassified congenital disorder of glycosylation (CDG-x)

Science.gov (United States)

Mention, K; Michaud, L; Dobbelaere, D; Guimber, D; Gottrand, F; Turck, D

2001-01-01

A case of severe and protracted diarrhoea is reported, which started in the neonatal period and progressively associated with neurological impairment, dysmorphy, hepatosplenomegaly, and hepatic insufficiency, from which the patient died at 2 years of age. Isoelectric focusing of serum transferrin showed a congenital disorder of glycosylation type I pattern but the basic defect could not be identified. This observation shows that congenital disorder of glycosylation is a cause of intractable diarrhoea in neonates.

 PMID:11668168

Rapid and individual-specific glycoprofiling of the low abundance N-glycosylated protein tissue inhibitor of metalloproteinases-1

DEFF Research Database (Denmark)

Thaysen-Andersen, Morten; Thøgersen, Ida B.; Nielsen, Hans Jørgen

2007-01-01

A gel-based method for a mass spectrometric site-specific glycoanalysis was developed using a recombinant glycoprotein expressed in two different cell lines. Hydrophilic interaction liquid chromatography at nanoscale level was used to enrich for glycopeptides prior to MS. The glycoprofiling...... glycoprofiling of a low abundance glycoprotein performed in an individual-specific manner allows for future studies of glycosylated biomarkers for person-specific detection of altered glycosylation and may thus allow early detection and monitoring of diseases....

Cell culture media supplementation of infrequently used sugars for the targeted shifting of protein glycosylation profiles.

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Hossler, Patrick; Racicot, Christopher; Chumsae, Christopher; McDermott, Sean; Cochran, Keith

2017-03-01

Mammalian cells in culture rely on sources of carbohydrates to supply the energy requirements for proliferation. In addition, carbohydrates provide a large source of the carbon supply for supporting various other metabolic activities, including the intermediates involved in the protein glycosylation pathway. Glucose and galactose, in particular, are commonly used sugars in culture media for these purposes. However, there exists a very large repertoire of other sugars in nature, and many that have been chemically synthesized. These sugars are particularly interesting because they can be utilized by cells in culture in distinct ways. In the present work it has been found that many infrequently used sugars, and the corresponding cellular response towards them as substrates, led to differences in the protein N-glycosylation profile of a recombinant glycoprotein. The selective media supplementation of raffinose, trehalose, turanose, palatinose, melezitose, psicose, lactose, lactulose, and mannose were found to be capable of redirecting N-glycan oligosaccharide profiles. Despite this shifting of protein glycosylation, there were no other adverse changes in culture performance, including both cell growth and cellular productivity over a wide range of supplemented sugar concentrations. The approach presented highlights a potential means towards both the targeted shifting of protein glycosylation profiles and ensuring recombinant protein comparability, which up to this point in time has remained under-appreciated for these under-utilized compounds. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:511-522, 2017. © 2017 American Institute of Chemical Engineers.

Overelaborated synaptic architecture and reduced synaptomatrix glycosylation in a Drosophila classic galactosemia disease model

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Patricia Jumbo-Lucioni

2014-12-01

Full Text Available Classic galactosemia (CG is an autosomal recessive disorder resulting from loss of galactose-1-phosphate uridyltransferase (GALT, which catalyzes conversion of galactose-1-phosphate and uridine diphosphate (UDP-glucose to glucose-1-phosphate and UDP-galactose, immediately upstream of UDP–N-acetylgalactosamine and UDP–N-acetylglucosamine synthesis. These four UDP-sugars are essential donors for driving the synthesis of glycoproteins and glycolipids, which heavily decorate cell surfaces and extracellular spaces. In addition to acute, potentially lethal neonatal symptoms, maturing individuals with CG develop striking neurodevelopmental, motor and cognitive impairments. Previous studies suggest that neurological symptoms are associated with glycosylation defects, with CG recently being described as a congenital disorder of glycosylation (CDG, showing defects in both N- and O-linked glycans. Here, we characterize behavioral traits, synaptic development and glycosylated synaptomatrix formation in a GALT-deficient Drosophila disease model. Loss of Drosophila GALT (dGALT greatly impairs coordinated movement and results in structural overelaboration and architectural abnormalities at the neuromuscular junction (NMJ. Dietary galactose and mutation of galactokinase (dGALK or UDP-glucose dehydrogenase (sugarless genes are identified, respectively, as critical environmental and genetic modifiers of behavioral and cellular defects. Assaying the NMJ extracellular synaptomatrix with a broad panel of lectin probes reveals profound alterations in dGALT mutants, including depletion of galactosyl, N-acetylgalactosamine and fucosylated horseradish peroxidase (HRP moieties, which are differentially corrected by dGALK co-removal and sugarless overexpression. Synaptogenesis relies on trans-synaptic signals modulated by this synaptomatrix carbohydrate environment, and dGALT-null NMJs display striking changes in heparan sulfate proteoglycan (HSPG co-receptor and Wnt

EMMPRIN in gynecologic cancers: pathologic and therapeutic aspects.

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Liu, Dan-tong

2015-07-01

The highly glycosylated transmembrane protein extracellular matrix metalloproteinase inducer (EMMPRIN) is associated with several pathological conditions, including various types of cancers. In different gynecological malignancies, such as ovarian, cervical, and endometrial cancers, EMMPRIN plays significant roles in cell adhesion modulation, tumor growth, invasion, angiogenesis, and metastasis by inducing the production of various molecules, including matrix metalloproteinases and vascular endothelial growth factor. Because of its high level of expression, EMMPRIN can possibly be used as a diagnostic marker of gynecological cancers. Recent studies have showed that targeting EMMPRIN, especially by RNA interference (RNAi) technology, has promising therapeutic potential in basic research on gynecological cancer treatments, which make a platform for the future clinical success. This review study focused on the association of EMMPRIN in gynecological cancers in the perspectives of pathogenesis, diagnosis, and therapeutics.

Glycosylation of voltage-gated calcium channels in health and disease

Czech Academy of Sciences Publication Activity Database

Lazniewska, Joanna; Weiss, Norbert

2017-01-01

Roč. 1859, č. 5 (2017), s. 662-668 ISSN 0005-2736 R&D Projects: GA ČR GA15-13556S; GA MŠk 7AMB15FR015 Institutional support: RVO:61388963 Keywords : calcium channels * voltage-gated calcium channels * N-glycosylation * ancillary subunit * trafficking * stability Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 3.498, year: 2016

Engineer medium and feed for modulating N-glycosylation of recombinant protein production in CHO cell culture

DEFF Research Database (Denmark)

Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

2017-01-01

Chinese hamster ovary (CHO) cells have become the primary expression system for the production of complex recombinant proteins due to their long-term success in industrial scale production and generating appropriate protein N-glycans similar to that of humans. Control and optimization of protein N......-glycosylation is crucial, as the structure of N-glycans can largely influence both biological and physicochemical properties of recombinant proteins. Protein N-glycosylation in CHO cell culture can be controlled and tuned by engineering medium, feed, culture process, as well as genetic elements of the cell...

Cancer survivors. Work related issues.

Science.gov (United States)

Schultz, Pamela N; Beck, Martha L; Stava, Charles; Sellin, Rena V

2002-05-01

New and more effective treatments for cancer have resulted in individuals living longer with a better quality of life. Many more survivors are employed in the workplace. Cancer is no longer only an issue for survivors and their families; it has become an issue for the employer and the workplace. This article describes survey results of 4,364 long term cancer survivors in which they were asked to respond to items describing their ability to work, job discrimination, and quality of life. Thirty-five percent of survivors were working at the time they completed the survey, and 8.5% considered themselves unable to work. This research has shown that age, gender, ethnic group, and cancer type affected the working status of the survivors. Of survivors continuing to work, 7.3% indicated they had experienced job discrimination. The results indicate most cancer survivors do not perceive employment related problems, and are readily assimilated into the work force. Job discrimination and the ability to work is a quality of life issue.

Towards the implementation of quality by design to the production of therapeutic monoclonal antibodies with desired glycosylation patterns.

Science.gov (United States)

del Val, Ioscani Jimenez; Kontoravdi, Cleo; Nagy, Judit M

2010-01-01

Quality by design (QbD) is a scheme for the development, manufacture, and approval of pharmaceutical products. The end goal of QbD is to ensure product quality by building it into the manufacturing process. The main regulatory bodies are encouraging its implementation to the manufacture of all new pharmaceuticals including biological products. Monoclonal antibodies (mAbs) are currently the leading products of the biopharmaceutical industry. It has been widely reported that glycosylation directly influences the therapeutic mechanisms by which mAbs function in vivo. In addition, glycosylation has been identified as one of the main sources of monoclonal antibody heterogeneity, and thus, a critical parameter to follow during mAb manufacture. This article reviews the research on glycosylation of mAbs over the past 2 decades under the QbD scope. The categories presented under this scope are: (a) definition of the desired clinical effects of mAbs, (b) definition of the glycosylation-associated critical quality attributes (glycCQAs) of mAbs, (c) assessment of process parameters that pose a risk for mAb glycCQAs, and (d) methods for accurately quantifying glycCQAs of mAbs. The information available in all four areas leads us to conclude that implementation of QbD to the manufacture of mAbs with specific glycosylation patterns will be a reality in the near future. We also foresee that the implementation of QbD will lead to the development of more robust and efficient manufacturing processes and to a new generation of mAbs with increased clinical efficacy. Copyright © 2010 American Institute of Chemical Engineers (AIChE).

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus*

Science.gov (United States)

Bagdonaite, Ieva; Nordén, Rickard; Joshi, Hiren J.; King, Sarah L.; Vakhrushev, Sergey Y.; Olofsson, Sigvard; Wandall, Hans H.

2016-01-01

Herpesviruses are among the most complex and widespread viruses, infection and propagation of which depend on envelope proteins. These proteins serve as mediators of cell entry as well as modulators of the immune response and are attractive vaccine targets. Although envelope proteins are known to carry glycans, little is known about the distribution, nature, and functions of these modifications. This is particularly true for O-glycans; thus we have recently developed a “bottom up” mass spectrometry-based technique for mapping O-glycosylation sites on herpes simplex virus type 1. We found wide distribution of O-glycans on herpes simplex virus type 1 glycoproteins and demonstrated that elongated O-glycans were essential for the propagation of the virus. Here, we applied our proteome-wide discovery platform for mapping O-glycosites on representative and clinically significant members of the herpesvirus family: varicella zoster virus, human cytomegalovirus, and Epstein-Barr virus. We identified a large number of O-glycosites distributed on most envelope proteins in all viruses and further demonstrated conserved patterns of O-glycans on distinct homologous proteins. Because glycosylation is highly dependent on the host cell, we tested varicella zoster virus-infected cell lysates and clinically isolated virus and found evidence of consistent O-glycosites. These results present a comprehensive view of herpesvirus O-glycosylation and point to the widespread occurrence of O-glycans in regions of envelope proteins important for virus entry, formation, and recognition by the host immune system. This knowledge enables dissection of specific functional roles of individual glycosites and, moreover, provides a framework for design of glycoprotein vaccines with representative glycosylation. PMID:27129252

Rapid chemical de-N-glycosylation and derivatization for liquid chromatography of immunoglobulin N-linked glycans.

Directory of Open Access Journals (Sweden)

Akihiko Kameyama

Full Text Available Glycan analysis may result in exploitation of glycan biomarkers and evaluation of heterogeneity of glycosylation of biopharmaceuticals. For N-linked glycan analysis, we investigated alkaline hydrolysis of the asparagine glycosyl carboxamide of glycoproteins as a deglycosylation reaction. By adding hydroxylamine into alkaline de-N-glycosylation, we suppressed the degradation of released glycans and obtained a mixture of oximes, free glycans, and glycosylamines. The reaction was completed within 1 h, and the mixture containing oximes was easily tagged with 2-aminobenzamide by reductive amination. Here, we demonstrated N-linked glycan analysis using this method for a monoclonal antibody, and examined whether this method could liberate glycans without degradation from apo-transferrin containing NeuAc and NeuGc and horseradish peroxidase containing Fuc α1-3 GlcNAc at the reducing end. Furthermore, we compared glycan recoveries between conventional enzymatic glycan release and this method. Increasing the reaction temperature and reaction duration led to degradation, whereas decreasing these parameters resulted in lower release. Considering this balance, we proposed to carry out the reaction at 80°C for 1 h for asialo glycoproteins from mammals and at 50°C for 1 h for sialoglycoproteins.

Dependency of the regio- and stereoselectivity of intramolecular, ring-closing glycosylations upon the ring size

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Patrick Claude

2011-12-01

Full Text Available Phenyl 3,4,6-tri-O-benzyl-2-O-(3-carboxypropionyl-1-thio-β-D-galactopyranoside (1 was condensed via its pentafluorophenyl ester 2 with 5-aminopentyl (4a, 4-aminobutyl (4b, 3-aminopropyl (4c and 2-aminoethyl 4,6-O-benzylidene-β-D-glucopyranoside (4d, prepared from the corresponding N-Cbz protected glucosides 3a–d, to give the corresponding 2-[3-(alkylcarbamoylpropionyl] tethered saccharides 5a–d. Intramolecular, ring closing glycosylation of the saccharides with NIS and TMSOTf afforded the tethered β(1→3 linked disaccharides 6a–c, the α(1→3 linked disaccharides 7a–d and the α(1→2 linked disaccharide 8d in ratios depending upon the ring size formed during glycosylation. No β(1→2 linked disaccharides were formed. Molecular modeling of saccharides 6–8 revealed that a strong aromatic stacking interaction between the aromatic parts of the benzyl and benzylidene protecting groups in the galactosyl and glucosyl moieties was mainly responsible for the observed regioselectivity and anomeric selectivity of the ring-closing glycosylation step.

Cancer-Related Post-traumatic Stress (PDQ®)—Patient Version

Science.gov (United States)

Cancer-related post-traumatic stress can occur any time from diagnosis to after treatment ends. Shock, fear, helplessness, or horror can be felt by cancer patients and lead to cancer-related post-traumatic stress. Learn about the causes and ways doctors can help manage these symptoms of distress in this expert-reviewed summary.

Relative Risks for Lethal Prostate Cancer Based on Complete Family History of Prostate Cancer Death.

Science.gov (United States)

Albright, Frederick S; Stephenson, Robert A; Agarwal, Neeraj; Cannon-Albright, Lisa A

2017-01-01

There are few published familial relative risks (RR) for lethal prostate cancer. This study estimates RRs for lethal prostate cancer based on comprehensive family history data, with the goal of improving identification of those men at highest risk of dying from prostate cancer. We used a population-based genealogical resource linked to a statewide electronic SEER cancer registry and death certificates to estimate relative risks (RR) for death from prostate cancer based upon family history. Over 600,000 male probands were analyzed, representing a variety of family history constellations of lethal prostate cancer. RR estimates were based on the ratio of the observed to the expected number of lethal prostate cancer cases using internal rates. RRs for lethal prostate cancer based on the number of affected first-degree relatives (FDR) ranged from 2.49 (95% CI: 2.27, 2.73) for exactly 1 FDR to 5.30 (2.13, 10.93) for ≥3 affected FDRs. In an absence of affected FDRs, increased risk was also significant for increasing numbers of affected second-degree or third degree relatives. Equivalent risks were observed for similar maternal and paternal family history. This study provides population-based estimates of lethal prostate cancer risk based on lethal prostate cancer family history. Many family history constellations associated with two to greater than five times increased risk for lethal prostate cancer were identified. These lethal prostate cancer risk estimates hold potential for use in identification, screening, early diagnosis, and treatment of men at high risk for death from prostate cancer. Prostate77:41-48, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Gender differences in associations between cancer-related problems and relationship dissolution among cancer survivors.

Science.gov (United States)

Stephens, Cristina; Westmaas, J Lee; Kim, Jihye; Cannady, Rachel; Stein, Kevin

2016-10-01

Research suggests that a cancer diagnosis predicts marital dissolution more strongly for women survivors than men, but there is a paucity of research on potential processes underlying this vulnerability. The present cross-sectional study examined whether specific cancer-related problems were associated with the odds of relationship breakup following diagnosis and whether these relationships differed between male and female cancer survivors. A national cross-sectional quality of life study assessed self-reported cancer-related problems and relationship change among survivors who were either 2, 6, or 10 years post-diagnosis (n = 6099). Bivariate analyses indicated that cancer-related problems (e.g., emotional distress) were greater for divorced/separated survivors compared to those with intact relationships and were greater for women versus men. Logistic regressions indicated that for both male and female survivors, lower income, younger age, and longer time since diagnosis were associated with greater odds of divorce or separation after diagnosis (ORs > 2.14, p emotional distress (OR = 1.14, p divorce or separation. For men only, fear of cancer recurrence was associated with greater odds of divorce or separation (OR = 1.32, p emotional or financial/employment problems attributed to the cancer diagnosis were associated with the likelihood of reporting relationship dissolution. Although directions of causality could not be ascertained, results suggest the possibility that helping male and female cancer survivors cope with specific cancer-related problems may benefit the quality and stability of their relationships with significant others following diagnosis.

Effects of laser acupoint irradiation on blood glucose and glycosylated hemoglobin in type 2 diabetes mellitus

Science.gov (United States)

Hui-Hui, Liu; Guo-Xin, Xiong; Li-Ping, Zhang

2016-06-01

To investigate the effects of semiconductor laser acupoint irradiation on blood glucose, glycosylated hemoglobin and physical fitness in type 2 diabetes mellitus, 44 cases of type 2 diabetic patients were randomly divided into a control group and a treatment group. All patients in both groups were given a drug treatment. The Hegu, Quchi and Zusanli acupoints of patients in the treatment group were then irradiated daily for 15 d with a 10 MW semiconductor laser. Before and after treatment, patients in both groups underwent a variety of tests and measurements: a two-hour postprandial blood glucose test; a glycosylated hemoglobin test and body mass index (BMI), waist-to-hip ratio (WHR) and body fat percentage (BFP) measurements. The data detected after treatment greatly decreased in the treatment group and was significantly different from that in the control group. It is shown that the acupoint irradiation with a semiconductor laser can improve two-hour postprandial blood glucose, glycosylated hemoglobin and some physical fitness measurements in type 2 diabetes mellitus patients.

Chemokines in cancer related inflammation

Energy Technology Data Exchange (ETDEWEB)

Allavena, Paola; Germano, Giovanni; Marchesi, Federica [Department of Immunology and Inflammation, IRCCS Humanitas Clinical Institute, Via Manzoni 56, 20089, Rozzano, Milan (Italy); Mantovani, Alberto, E-mail: alberto.mantovani@humanitasresearch.it [Department of Immunology and Inflammation, IRCCS Humanitas Clinical Institute, Via Manzoni 56, 20089, Rozzano, Milan (Italy); Department of Translational Medicine, University of Milan (Italy)

2011-03-10

Chemokines are key players of the cancer-related inflammation. Chemokine ligands and receptors are downstream of genetic events that cause neoplastic transformation and are abundantly expressed in chronic inflammatory conditions which predispose to cancer. Components of the chemokine system affect multiple pathways of tumor progression including: leukocyte recruitment, neo-angiogenesis, tumor cell proliferation and survival, invasion and metastasis. Evidence in pre-clinical and clinical settings suggests that the chemokine system represents a valuable target for the development of innovative therapeutic strategies.

XMRV Discovery and Prostate Cancer-Related Research

Directory of Open Access Journals (Sweden)

David E. Kang

2011-01-01

Full Text Available Xenotropic murine leukemia virus-related virus (XMRV was first reported in 2006 in a study of human prostate cancer patients with genetic variants of the antiviral enzyme, RNase L. Subsequent investigations in North America, Europe, Asia, and Africa have either observed or failed to detect XMRV in patients (prostate cancer, chronic fatigue syndrome-myalgic encephalomyelitis (CFS-ME, and immunosuppressed with respiratory tract infections or normal, healthy, control individuals. The principal confounding factors are the near ubiquitous presence of mouse-derived reagents, antibodies and cells, and often XMRV itself, in laboratories. XMRV infects and replicates well in many human cell lines, but especially in certain prostate cancer cell lines. XMRV also traffics to prostate in a nonhuman primate model of infection. Here, we will review the discovery of XMRV and then focus on prostate cancer-related research involving this intriguing virus.

Stability of Curcuma longa rhizome lectin: Role of N-linked glycosylation.

Science.gov (United States)

Biswas, Himadri; Chattopadhyaya, Rajagopal

2016-04-01

Curcuma longa rhizome lectin, a mannose-binding protein of non-seed portions of turmeric, is known to have antifungal, antibacterial and α-glucosidase inhibitory activities. We studied the role of complex-type glycans attached to asparagine (Asn) 66 and Asn 110 to elucidate the role of carbohydrates in lectin activity and stability. Apart from the native lectin, the characteristics of a deglycosylated Escherichia coli expressed lectin, high-mannose oligosaccharides at both asparagines and its glycosylation mutants N66Q and N110Q expressed in Pichia pastoris, were compared to understand the relationship between glycosylation and activity. Far UV circular dichroism (CD) spectra, fluorescence emission maximum, hemagglutination assay show no change in secondary or tertiary structures or sugar-binding properties between wild-type and aforementioned recombinant lectins under physiological pH. But reduced agglutination activity and loss of tertiary structure are observed in the acidic pH range for the deglycosylated and the N110Q protein. In thermal and guanidine hydrochloride (GdnCl)-induced unfolding, the wild-type and high-mannose lectins possess higher stability compared with the deglycosylated recombinant lectin and both mutants, as measured by a higher Tm of denaturation or a greater free energy change, respectively. Reversibility experiments after thermal denaturation reveal that deglycosylated proteins tend to aggregate during thermal inactivation but the wild type shows a much greater recovery to the native state upon refolding. These results suggest that N-glycosylation in turmeric lectin is important for the maintenance of its proper folding upon changes in pH, and that the oligosaccharides help in maintaining the active conformation and prevent aggregation in unfolded or partially folded molecules. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

MALDI-TOF MS applied to apoC-III glycoforms of patients with congenital disorders affecting O-glycosylation. Comparison with two-dimensional electrophoresis

NARCIS (Netherlands)

Yen-Nicolay, S.; Boursier, C.; Rio, M. del; Lefeber, D.J.; Pilon, A.; Seta, N.; Bruneel, A.

2015-01-01

PURPOSE: The O-glycan abnormalities accompanying some congenital disorders of glycosylation, namely conserved oligomeric Golgi-congenital disorders of glycosylation (COG-CDGs) and ATP6V0A2-CDGs, are mainly detected using electrophoresis methods applied to circulating apolipoprotein C-III. The

Splitting of α-Helical Structure as Molecular Basis for Abolishing an Amyloid Formation by Multiple Glycosylation: A Molecular Dynamics Simulation Study

Energy Technology Data Exchange (ETDEWEB)

Choi, Youngjin [Hoseo University, Asan (Korea, Republic of); Cho, Eunae; Jung, Seunho [Konkuk University, Seoul (Korea, Republic of)

2016-07-15

Molecular details played by glycosylation are complicated by the subtle nature of variations in the glycan structure, and this complexity is one of the research barriers to establish structure-function relationship on the protein modification. This is particularly true for understanding the exact structural consequence of the glycosylation of the biological proteins. The present MD simulation revealed molecular-level mechanism of the glycosylation effect on the peptide to understand the experimentally observed phenomenon for inhibiting amyloid formation in the model peptide. The galactose residue on the Ser17 undermined the helical integrity of main protein region by enhancing sugar–amino acid interaction and perturbing natural interactions between amino acid residues.

Glycosylation of type II collagen is of major importance for T cell tolerance and pathology in collagen-induced arthritis

DEFF Research Database (Denmark)

Bäcklund, Johan; Treschow, Alexandra; Bockermann, Robert

2002-01-01

Type II collagen (CII) is a candidate cartilage-specific autoantigen, which can become post-translationally modified by hydroxylation and glycosylation. T cell recognition of CII is essential for the development of murine collagen-induced arthritis (CIA) and also occurs in rheumatoid arthritis (RA......). The common denominator of murine CIA and human RA is the presentation of an immunodominant CII-derived glycosylated peptide on murine Aq and human DR4 molecules, respectively. To investigate the importance of T cell recognition of glycosylated CII in CIA development after immunization with heterologous CII......, we treated neonatal mice with different heterologous CII-peptides (non-modified, hydroxylated and galactosylated). Treatment with the galactosylated peptide (galactose at position 264) was superior in protecting mice from CIA. Protection was accompanied by a reduced antibody response to CII...

Self-regulating insulin delivery systems I. Synthesis and characterization of glycosylated insulin

NARCIS (Netherlands)

Jeong, Seo Young; Kim, Sung Wan; Eenink, Martinus J.D.; Feijen, Jan

1984-01-01

A design for a self-regulating insulin delivery system based on the competitive binding of glucose and glycosylated insulin to the lectin Concanavalin A is proposed. A differnt approach to diabetes therapy is the attempt to effect a permanent cure of the disease by supplementing the patient's

Pre-column derivatisation method for the measurement of glycosylated hydroxylysines of collagenous proteins

NARCIS (Netherlands)

Bank, R.A.; Beekman, B.; Tenni, R.; Tekoppele, J.M.

1997-01-01

Measurement of the glycosylated hydroxylysines galactosyl- and glucosylgalactosylhydroxylysine (GH and GGH) in combination with other amino acids has been based on ion-exchange chromatography followed by reaction with ninhydrin. Here, a rapid and sensitive high-performance liquid chromatographic

Site-specific O-glycosylation of members of the low-density lipoprotein receptor superfamily enhances ligand interactions

DEFF Research Database (Denmark)

Wang, Shengjun; Mao, Yang; Narimatsu, Yoshiki

2018-01-01

in the LA linker regions of VLDLR, LRP1, and LRP2 (Megalin) from both cell lines and rat organs. Using a panel of gene-edited isogenic cell line models, we demonstrate that GalNAc-T11-mediated LDLR and VLDLR O-glycosylation is not required for transport and cell surface expression and stability...... of these receptors, but markedly enhances LDL and VLDL binding and uptake. Direct ELISA-based binding assays with truncated LDLR constructs revealed that O-glycosylation increased affinity for LDL by approximately 5-fold. The molecular basis for this observation is currently unknown, but these findings open up new...

Cell culture media supplementation of uncommonly used sugars sucrose and tagatose for the targeted shifting of protein glycosylation profiles of recombinant protein therapeutics.

Science.gov (United States)

Hossler, Patrick; McDermott, Sean; Racicot, Christopher; Chumsae, Christopher; Raharimampionona, Haly; Zhou, Yu; Ouellette, David; Matuck, Joseph; Correia, Ivan; Fann, John; Li, Jianmin

2014-01-01

Protein glycosylation is an important post-translational modification toward the structure and function of recombinant therapeutics. The addition of oligosaccharides to recombinant proteins has been shown to greatly influence the overall physiochemical attributes of many proteins. It is for this reason that protein glycosylation is monitored by the developer of a recombinant protein therapeutic, and why protein glycosylation is typically considered a critical quality attribute. In this work, we highlight a systematic study toward the supplementation of sucrose and tagatose into cell culture media for the targeted modulation of protein glycosylation profiles on recombinant proteins. Both sugars were found to affect oligosaccharide maturation resulting in an increase in the percentage of high mannose N-glycan species, as well as a concomitant reduction in fucosylation. The latter effect was demonstrated to increase antibody-dependent cell-mediated cytotoxicity for a recombinant antibody. These aforementioned results were found to be reproducible at different scales, and across different Chinese hamster ovary cell lines. Through the selective supplementation of these described sugars, the targeted modulation of protein glycosylation profiles is demonstrated, as well as yet another tool in the cell culture toolbox for ensuring product comparability. © 2014 American Institute of Chemical Engineers.

Posttraumatic Stress and Attentional Bias towards Cancer-Related Stimuli in Parents of Children Recently Diagnosed with Cancer.

Directory of Open Access Journals (Sweden)

Martin Cernvall

Full Text Available To investigate whether posttraumatic stress symptoms (PTSS are related to attentional bias towards cancer-related stimuli among parents of children recently diagnosed with cancer.Sixty-two parents completed questionnaires measuring PTSS, depression, and anxiety and the emotional Stroop task via the Internet. The emotional Stroop task included cancer-related words, cardiovascular disease-related words, and neutral words.Participants were split in two groups based on the median of PTSS: High-PTSS and Low-PTSS. There was a significant interaction between word-type and group and a planned contrast test of this interaction indicated that the High-PTSS group had longer response latencies on cancer-related words compared to the other word-type and group combinations.Findings suggest that PTSS are related to attentional bias towards cancer-related stimuli among parents of children recently diagnosed with cancer. Implications of this finding for the understanding of PTSS in this population, future research, and clinical practice are discussed.

Role of Conserved Oligomeric Golgi Complex in the Abnormalities of Glycoprotein Processing in Breast Cancer Cells

Science.gov (United States)

2006-05-01

of COG complex function we utilized RNA interference assay to knockdown COG3p subunit of COG complex in normal and breast cancer cells and other tumor...protein trafficking, but the role of the COG complex in the abnormal glycosylation and secretion of tumor markers in breast cancer cells remains... COG complex in breast cancer cells MCF7 had been elevated 2-4 times in comparison to HB2 cells (Figure 5 A). The expression of HeLa COG3 CD44 ab

2,4-dimethoxybenzyl: An amide protecting group for 2-acetamido glycosyl donors

DEFF Research Database (Denmark)

Kelly, N.M.; Jensen, Knud Jørgen

2001-01-01

2,4-Dimethoxybenzyl (Dmob) was used as an amide protecting group for 2-acetamido glycosyl donors. The N-Dmob group was introduced by imine formation between 2,4-dimethoxybenzaldehyde and d-glucosamine, followed by per-O-acylation, reduction to form the amine, and finally N-acetylation to give 1...

[Effect of high-intensity interval training on the reduction of glycosylated hemoglobin in type-2 diabetic adult patients].

Science.gov (United States)

Aguilera Eguía, Raúl Alberto; Russell Guzmán, Javier Antonio; Soto Muñoz, Marcelo Enrique; Villegas González, Bastián Eduardo; Poblete Aro, Carlos Emilio; Ibacache Palma, Alejandro

2015-03-05

Type 2 diabetes mellitus is one of the major non-communicable chronic diseases in the world. Its prevalence in Chile is significant, and complications associated with this disease involve great costs, which is why prevention and treatment of this condition are essential. Physical exercise is an effective means for prevention and treatment of type 2 diabetes mellitus. The emergence of new forms of physical training, such as "high intensity interval training", presents novel therapeutic alternatives for patients and health care professionals. To assess the validity and applicability of the results regarding the effectiveness of high intensity interval training in reducing glycosylated hemoglobin in adult patients with type 2 diabetes mellitus and answer the following question: In subjects with type 2 diabetes, can the method of high intensity interval training compared to moderate intensity exercise decrease glycosylated hemoglobin? We performed a critical analysis of the article "Feasibility and preliminary effectiveness of high intensity interval training in type 2 diabetes". We found no significant differences in the amount of glycosylated hemoglobin between groups of high intensity interval training and moderate-intensity exercise upon completion of the study (p>0.05). In adult patients with type 2 diabetes mellitus, high intensity interval training does not significantly improve glycosylated hemoglobin levels. Despite this, the high intensity interval training method shows as much improvement in body composition and physical condition as the moderate intensity exercise program.

Lysosomal enzyme delivery by ICAM-1-targeted nanocarriers bypassing glycosylation- and clathrin-dependent endocytosis.

Science.gov (United States)

Muro, Silvia; Schuchman, Edward H; Muzykantov, Vladimir R

2006-01-01

Enzyme replacement therapy, a state-of-the-art treatment for many lysosomal storage disorders, relies on carbohydrate-mediated binding of recombinant enzymes to receptors that mediate lysosomal delivery via clathrin-dependent endocytosis. Suboptimal glycosylation of recombinant enzymes and deficiency of clathrin-mediated endocytosis in some lysosomal enzyme-deficient cells limit delivery and efficacy of enzyme replacement therapy for lysosomal disorders. We explored a novel delivery strategy utilizing nanocarriers targeted to a glycosylation- and clathrin-independent receptor, intercellular adhesion molecule (ICAM)-1, a glycoprotein expressed on diverse cell types, up-regulated and functionally involved in inflammation, a hallmark of many lysosomal disorders. We targeted recombinant human acid sphingomyelinase (ASM), deficient in types A and B Niemann-Pick disease, to ICAM-1 by loading this enzyme to nanocarriers coated with anti-ICAM. Anti-ICAM/ASM nanocarriers, but not control ASM or ASM nanocarriers, bound to ICAM-1-positive cells (activated endothelial cells and Niemann-Pick disease patient fibroblasts) via ICAM-1, in a glycosylation-independent manner. Anti-ICAM/ASM nanocarriers entered cells via CAM-mediated endocytosis, bypassing the clathrin-dependent pathway, and trafficked to lysosomes, where delivered ASM displayed stable activity and alleviated lysosomal lipid accumulation. Therefore, lysosomal enzyme targeting using nanocarriers targeted to ICAM-1 bypasses defunct pathways and may improve the efficacy of enzyme replacement therapy for lysosomal disorders, such as Niemann-Pick disease.

Amphiphilic glycosylated block copolypeptides as macromolecular surfactants in the emulsion polymerization of styrene

NARCIS (Netherlands)

Jacobs, Jaco; Gathergood, N.; Heuts, J.P.A.; Heise, A.

2015-01-01

Diblock copolymers consisting of poly(L-phenyl alanine) and poly(benzyl-L-glutamate) or poly(CBZ-L-lysine), respectively, were synthesized via sequential NCA polymerization. After deprotection, subsequent partial glycosylation of the glutamic acid and lysine units with galactosamine hydrochloride or

Mucin-type O-glycosylation and its potential use in drug and vaccine development

DEFF Research Database (Denmark)

Tarp, Mads Agervig; Clausen, Henrik

2007-01-01

decade an increasing number of GalNAc-transferase isoforms have been cloned and their substrate-specificities partly characterized. These differences in substrate specificities have been exploited for in vitro site-directed O-glycosylation. In GlycoPEGylation, polyehylene glycol (PEG) is transferred...

Therapeutic monoclonal antibody N-glycosylation - Structure, function and therapeutic potential.

Science.gov (United States)

Cymer, Florian; Beck, Hermann; Rohde, Adelheid; Reusch, Dietmar

2018-03-01

Therapeutic antibodies (IgG-type) contain several post-translational modifications (PTMs) whereby introducing a large heterogeneity, both structural and functional, into this class of therapeutics. Of these modifications, glycosylation in the fragment crystallizable (Fc) region is the most heterogeneous PTM, which can affect the stability of the molecule and interactions with Fc-receptors in vivo. Hence, the glycoform distribution can affect the mode of action and have implications for bioactivity, safety and efficacy of the drug. Main topics of the manuscript include: What factors influence the (Fc) glycan pattern in therapeutic antibodies and how can these glycans be characterized? How does structure of the Fc-glycan relate to function and what methods are available to characterize those functions? Although heterogeneous in their scope, the different sections are intended to combine current knowledge on structure-function correlations of IgG glycan structures with regard to Fc (effector) functions, as well as basic aspects and methodologies for their assessment. Copyright © 2017. Published by Elsevier Ltd.

Stemness-related markers in cancer

Directory of Open Access Journals (Sweden)

Wenxiu Zhao

2017-01-01

Full Text Available Cancer stem cells (CSCs, with their self-renewal ability and multilineage differentiation potential, are a critical subpopulation of tumor cells that can drive tumor initiation, growth, and resistance to therapy. Like embryonic and adult stem cells, CSCs express markers that are not expressed in normal somatic cells and are thus thought to contribute toward a “stemness” phenotype. This review summarizes the current knowledge of stemness-related markers in human cancers, with a particular focus on important transcription factors, protein surface markers, and signaling pathways.

NAD+ Deficits in Age-Related Diseases and Cancer.

Science.gov (United States)

Garrido, Amanda; Djouder, Nabil

2017-08-01

The phenomenon of aging has gained widespread attention in recent times. Although significant advances have been made to better understand aging and its related pathologies including cancer, there is not yet a clear mechanism explaining why diseases and cancer are inherent parts of the aging process. Finding a unifying equation that could bridge aging and its related diseases would allow therapeutic development and solve an immense human health problem to live longer and better. In this review, we discuss NAD + reduction as the central mechanism that may connect aging to its related pathologies and cancer. NAD + boosters would ensure and ameliorate health quality during aging. Copyright © 2017 Elsevier Inc. All rights reserved.

Cancer-Related Stress and Complementary and Alternative Medicine: A Review

Directory of Open Access Journals (Sweden)

Kavita D. Chandwani

2012-01-01

Full Text Available A cancer diagnosis elicits strong psychophysiological reactions that characterize stress. Stress is experienced by all patients but is usually not discussed during patient-healthcare professional interaction; thus underdiagnosed, very few are referred to support services. The prevalence of CAM use in patients with history of cancer is growing. The purpose of the paper is to review the aspects of cancer-related stress and interventions of commonly used complementary and alternative techniques/products for amelioration of cancer-related stress. Feasibility of intervention of several CAM techniques and products commonly used by cancer patients and survivors has been established in some cancer populations. Efficacy of some CAM techniques and products in reducing stress has been documented as well as stress-related symptoms in patients with cancer such as mindfulness-based stress reduction, yoga, Tai Chi Chuan, acupuncture, energy-based techniques, and physical activity. Much of the research limitations include small study samples and variety of intervention length and content. Efficacy and safety of many CAM techniques and some herbs and vitamin B and D supplements need to be confirmed in further studies using scientific methodology. Several complementary and alternative medicine therapies could be integrated into standard cancer care to ameliorate cancer-related stress.

An enzymatic glycosylation of nucleoside analogues using beta-galactosidase from Escherichia coli

Czech Academy of Sciences Publication Activity Database

Blažek, Jiří; Jansa, Petr; Baszczyňski, Ondřej; Kaiser, Martin Maxmilian; Otmar, Miroslav; Krečmerová, Marcela; Dračínský, Martin; Holý, Antonín; Králová, B.

2012-01-01

Roč. 20, č. 9 (2012), s. 3111-3118 ISSN 0968-0896 R&D Projects: GA MŠk 1M0508 Institutional research plan: CEZ:AV0Z40550506 Keywords : glycosylation * galactosylation * beta-galactosidase * enzymatic synthesis * nucleoside * acyclic nucleoside analogues Subject RIV: CC - Organic Chemistry Impact factor: 2.903, year: 2012

TNF-α levels in cancer patients relate to social variables

Science.gov (United States)

Marucha, Phillip T.; Crespin, Timothy R.; Shelby, Rebecca A.; Andersen, Barbara L.

2008-01-01

Tumor necrosis factor-α (TNF-α) is an important cytokine associated with tumor regression and increased survival time for cancer patients. Research evidence relates immune factors (e.g., natural killer (NK) cell counts, NK cell lysis, lymphocyte profile, and lymphocyte proliferation) to the frequency and quality of social relations among cancer patients. We hypothesized that disruptions in social relations would be associated with lower TNF-α responses, and conversely, that reports of positive changes in social relations correlate with stronger responses. A prospective design measured changes in social activity and relationship satisfaction with a partner in 44 breast cancer patients at the time of cancer diagnosis, and initial surgery and 12 months later. Results indicated that patients reporting increased social activities or satisfaction exhibited stronger stimulated TNF-α responses. This is the first study to link changes in patient social relations with a cancer-relevant immune variable. PMID:15890493

How new subscribers use cancer-related online mailing lists.

Science.gov (United States)

Rimer, Barbara K; Lyons, Elizabeth J; Ribisl, Kurt M; Bowling, J Michael; Golin, Carol E; Forlenza, Michael J; Meier, Andrea

2005-07-01

Online cancer-related support is an under-studied resource that may serve an important function in the information seeking, care, and support of cancer patients and their families. With over 9.8 million cancer survivors (defined as anyone living with cancer) in the United States alone and the number growing worldwide, it is important to understand how they seek and use online resources to obtain the information they need, when they need it, and in a form and manner appropriate to them. These are stated cancer communication goals of the US National Cancer Institute. Our purposes are to (1) present background information about online mailing lists and electronic support groups, (2) describe the rationale and methodology for the Health eCommunities (HeC) study, and (3) present preliminary baseline data on new subscribers to cancer-related mailing lists. In particular, we describe subscribers' use of mailing lists, their reasons for using them, and their reactions to participating shortly after joining the lists. From April to August 2004, we invited all new subscribers to 10 Association of Cancer Online Resources mailing lists to complete Web-based surveys. We analyzed baseline data from the respondents to examine their perceptions about cancer-related mailing lists and to describe how cancer patients and survivors used these lists in the period shortly after joining them. Cumulative email invitations were sent to 1368 new mailing list subscribers; 293 Web surveys were completed within the allotted time frame (21.4% response rate). Most respondents were over age 50 (n = 203, 72%), white (n = 286, 98%), college graduates (n = 161, 55%), and had health insurance (n = 283, 97%). About 41% (n = 116) of new subscribers reported spending 1 to 3 hours per day reading and responding to list messages. They used the mailing lists for several reasons. Among the most frequently reported, 62% (n = 179) strongly agreed they used mailing lists to obtain information on how to deal

Topological studies of hSVCT1, the human sodium-dependent vitamin C transporter and the influence of N-glycosylation on its intracellular targeting

Energy Technology Data Exchange (ETDEWEB)

Velho, Albertina M. [Department of Biosciences University of Kent, CT2 7NJ (United Kingdom); Jarvis, Simon M., E-mail: S.M.Jarvis@westminster.ac.uk [Department of Biosciences University of Kent, CT2 7NJ (United Kingdom); University of Westminster, School of Biosciences, London W1W 6UW (United Kingdom)

2009-08-01

The Na{sup +}-dependent transporters, hSVCT1 and hSVCT2, were assessed in COS-1 cells for their membrane topology. Antibodies to N- and C-termini of hSVCT1 and C-terminus of hSVCT2 identified positive immunofluorescence only after permeabilisation, suggesting these regions are intracellular. PNGase F treatment confirmed that WT hSVCT1 ({approx} 70-100 kDa) is glycosylated and site-directed mutagenesis of the three putative N-glycosylation sites, Asn138, Asn144, Asn230, demonstrated that mutants N138Q and N144Q were glycosylated ({approx} 68-90 kDa) with only 31-65% of WT L-ascorbic acid (AA) uptake while the glycosylation profile of N230Q remained unaltered ({approx} 98% of WT activity). However, the N138Q/N144Q double mutant displayed barely detectable membrane expression at {approx} 65 kDa, no apparent glycosylation and minimal AA uptake (< 10%) with no discernible improvement in expression or activity when cultured at 28 {sup o}C or 37 {sup o}C. Marker protein immunocytochemistry with N138Q/N144Q identified intracellular aggregates with hSVCT1 localised at the nuclear membrane but absent at the plasma membrane thus implicating its role as a possible intracellular transporter and suggesting N-glycosylation is required for hSVCT1 membrane targeting. Also, Lys242 on the same putative hydrophilic loop as Asn230 after biotinylation was inaccessible from the extracellular side when analysed by MALDI-TOF MS. A new hSVCT1 secondary structure model supporting these findings is proposed.

The management of breast cancer-related lymphoedema

African Journals Online (AJOL)

Definition. Breast cancer-related lymphoedema (BCRL) is a secondary form of lymphoedema and a major complication of post-breast cancer surgery and radiotherapy. It occurs when the network of lymphatic vessels and lymph nodes is damaged by disease, surgery or radiotherapy. The lymph, a protein-rich fluid, then ...

Management of Dysgeusia related to Cancer

Directory of Open Access Journals (Sweden)

DURGESHWORI MUNANKARMI

2017-05-01

Full Text Available Introduction: Taste change due to cancer is a notorious side effect, adversely affecting appetite and weight. Even though taste change or dysgeusia is one of the major causes of poor nutritional status in cancer survivors, it is not addressed as a significant problem and is often left untreated. The main purpose of this review is to explore current knowledge of pharmacological and behavioral interventions for the treatment of cancer related taste change. Methods: This systematic review was conducted in accordance with PRISMA guidelines to identify original articles on taste change. Multiple databases including; Scopus, Medline, EMBASE, CINAHL, and all databases via Pro quest were searched for original articles or studies related to taste change caused by cancer or its treatment. Relevant articles were subjected to a full text evaluation and assessed by Critical appraisal skills program (CASP guidelines and the Effective public health practice project (EPHPP instrument. Results: The search revealed 12 eligible studies, six of which were randomized controlled trials. Most of the studies used a standardized validated tool to measure taste change. Dysgeusia is common in cancer, 14 to 100% cancer patient report it. Pharmacological management with zinc remains inconclusive as one study reports it as beneficial and two other studies reported null effect. Few studies suggested dietary modifications such as use of sugary, salty foods that are helpful to reduce the effect of dysgeusia. Conclusion: Dietary counseling and informing the patient well about self-care strategies before treatment has consistently shown positive results on taste change, with strong statistical power. Other potential treatments for dysgeusia such as zinc, amifostine, and megestrol acetate gave inconsistent results.

Electrospray Ionization Mass Spectrometric Analysis of Highly Reactive Glycosyl Halides

Directory of Open Access Journals (Sweden)

Lajos Kovács

2012-07-01

Full Text Available Highly reactive glycosyl chlorides and bromides have been analysed by a routine mass spectrometric method using electrospray ionization and lithium salt adduct-forming agents in anhydrous acetonitrile solution, providing salient lithiated molecular ions [M+Li]⁺, [2M+Li]⁺ etc. The role of other adduct-forming salts has also been evaluated. The lithium salt method is useful for accurate mass determination of these highly sensitive compounds.

Silyl-protective groups influencing the reactivity and selectivity in glycosylations

DEFF Research Database (Denmark)

Bols, Mikael; Pedersen, Christian Marcus

2017-01-01

Silyl groups such as TBDPS, TBDMS, TIPS or TMS are well-known and widely used alcohol protective groups in organic chemistry. Cyclic silylene protective groups are also becoming increasingly popular. In carbohydrate chemistry silyl protective groups have frequently been used primarily as an ortho...... protected. Within the last decade polysilylated glycosyl donors have been found to have unusual properties such as high (or low) reactivity or high stereoselectivity. This mini review will summarize these findings...

Classification of treatment-related mortality in children with cancer

DEFF Research Database (Denmark)

Alexander, Sarah; Pole, Jason D; Gibson, Paul

2015-01-01

Treatment-related mortality is an important outcome in paediatric cancer clinical trials. An international group of experts in supportive care in paediatric cancer developed a consensus-based definition of treatment-related mortality and a cause-of-death attribution system. The reliability and va...

Ultrasound-Assisted Synthesis of 1-N-glycosyl-1H-1,2,3-Triazole Derivatives and their Anti-inflammatory Activity

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Gilson Bezerra Silva

2012-06-01

Full Text Available We have been synthesized various glycosyl triazoles from the reaction between glycosyl azide (1 and terminal alkynes (2a-g. The glycopiranosyl 1,2,3-triazoles (3-9 have been obtained in moderate-to-excellent yields (63-99% through the copper (I-catalyst 1,3-dipolar cycloaddition reaction at room temperature using ultrasound irradiation. In addition, preliminary anti-inflammatory tests have been performed in the compounds conjugates with benzoheterocycles (3-7 moieties that shown moderate activity

Relative risks of radiation-associated cancer: comparison of second cancer in therapeutically irradiated populations with the Japanese atomic bomb survivors

International Nuclear Information System (INIS)

Little, M.P.; Muirhead, C.R.; Haylock, R.G.E.; Thomas, J.M.

1999-01-01

In this paper the radiation-associated relative risks of second primary cancer incidence in groups treated for first primary cancer by radiotherapy are compared with radiation-associated relative risk estimates in the Japanese atomic bomb survivor cancer incidence data. For four cancer sites, namely lung cancer, bone cancer, ovarian cancer and leukaemia, the relative risks in the comparable (age at exposure, time since exposure, sex matched) subsets of the Japanese data are significantly greater than those in the majority of second cancer studies. Even when the differences between the relative risks in the Japanese atomic bomb survivors and the medical series do not approach conventional levels of statistical significance, relative risks tend to be higher in the Japanese data than in the second cancer studies. At least for leukaemia, the discrepancy between the Japanese and second cancer risks can be largely explained by cell- sterilisation effects. There are few indications of modification of radiation-associated second cancer relative risk among those treated with adjuvant chemotherapy, nor are there strong indications of modification of radiation- associated relative risk by heritable genetic factors. If anything, there is evidence that second cancer relative excess risks are lower among those patients with cancer-prone disorders than among non-susceptible patients. However, the higher underlying cancer risk in some of these medically exposed populations should also be considered, in particular for those with cancer-prone conditions, so that the absolute excess risk is sometimes higher than in the Japanese data. (orig.)

Temporal expression of HIV-1 envelope proteins in baculovirus-infected insect cells: Implications for glycosylation and CD4 binding

International Nuclear Information System (INIS)

Murphy, C.I.; Lennick, M.; Lehar, S.M.; Beltz, G.A.; Young, E.

1990-01-01

Three different human immunodeficiency virus type I (HIV-1) envelope derived recombinant proteins and the full length human CD4 polypeptide were expressed in Spodoptera frugiperda (Sf9) cells. DNA constructs encoding CD4, gp120, gp160, and gp160 delta were cloned into the baculovirus expression vector pVL941 or a derivative and used to generate recombinant viruses in a cotransfection with DNA from Autographa californica nuclear polyhedrosis virus (AcMNPV). Western blotting of cell extracts of the recombinant HIV-1 proteins showed that for each construct two major bands specifically reacted with anti-HIV-1 envelope antiserum. These bands corresponded to glycosylated and nonglycosylated versions of the HIV proteins as determined by 3H-mannose labeling and tunicamycin treatment of infected cells. A time course of HIV envelope expression revealed that at early times post-infection (24 hours) the proteins were fully glycosylated and soluble in nonionic detergents. However, at later times postinfection (48 hours), expression levels of recombinant protein reached a maximum but most of the increase was due to a rise in the level of the nonglycosylated species, which was largely insoluble in nonionic detergents. Thus, it appears that Sf9 cells cannot process large amounts of glycosylated recombinant proteins efficiently. As a measure of biological activity, the CD4 binding ability of both glycosylated and nonglycosylated recombinant HIV envelope proteins was tested in a coimmunoprecipitation assay. The results showed that CD4 and the glycosylated versions of recombinant gp120 or gp160 delta specifically associated with one another in this analysis. Nonglycosylated gp120 or gp160 delta proteins from tunicamycin-treated cultures did immunoprecipitate with anti-HIV-1 antiserum but did not interact with CD4

Relatives' level of satisfaction with advanced cancer care in Greenland

DEFF Research Database (Denmark)

Augustussen, Mikaela; Hounsgaard, Lise; Pedersen, Michael Lynge

2017-01-01

Palliative cancer care in Greenland is provided by health professionals at local level, the national Queen Ingrid's Hospital and at Rigshospitalet in Denmark. To improve and develop care for relatives of patients with advanced cancer, we conducted a mixed method study examining relatives' level...... of satisfaction with care and treatment and their current main concerns. The aim was to investigate relatives' level of satisfaction with advanced cancer care and bring to light their current main concerns. The FAMCARE-20 questionnaire was translated to Greenlandic and pilot tested. The questionnaire...... (66%) and relatives were the most dissatisfied with the lack of inclusion in decision making related to treatment and care (71%) and the length of time required to diagnose cancer (70%). Responses to the open-ended questions revealed that relatives faced challenges in gaining access to information...

Structure/functional aspects of the human riboflavin transporter-3 (SLC52A3): role of the predicted glycosylation and substrate-interacting sites.

Science.gov (United States)

Subramanian, Veedamali S; Sabui, Subrata; Teafatiller, Trevor; Bohl, Jennifer A; Said, Hamid M

2017-08-01

The human riboflavin (RF) transporter-3 (hRFVT-3; product of the SLC52A3 gene) plays an essential role in the intestinal RF absorption process and is expressed exclusively at the apical membrane domain of polarized enterocytes. Previous studies have characterized different physiological/biological aspects of this transporter, but nothing is known about the glycosylation status of the hRFVT-3 protein and role of this modification in its physiology/biology. Additionally, little is known about the residues in the hRFVT-3 protein that interact with the ligand, RF. We addressed these issues using appropriate biochemical/molecular approaches, a protein-docking model, and established intestinal/renal epithelial cells. Our results showed that the hRFVT-3 protein is glycosylated and that glycosylation is important for its function. Mutating the predicted N -glycosylation sites at Asn 94 and Asn 168 led to a significant decrease in RF uptake; it also led to a marked intracellular (in the endoplasmic reticulum, ER) retention of the mutated proteins as shown by live-cell confocal imaging studies. The protein-docking model used in this study has identified a number of putative substrate-interacting sites: Ser 16 , Ile 20 , Trp 24 , Phe 142 , Thr 314 , and Asn 315 Mutating these potential interacting sites was indeed found to lead to a significant inhibition in RF uptake and to intracellular (ER) retention of the mutated proteins (except for the Phe 142 mutant). These results demonstrate that the hRFVT-3 protein is glycosylated and this glycosylation is important for its function and cell surface expression. This study also identified a number of residues in the hRFVT-3 polypeptide that are important for its function/cell surface expression.

Relatives' level of satisfaction with advanced cancer care in Greenland

DEFF Research Database (Denmark)

Augustussen, Mikaela; Hounsgaard, Lise; Pedersen, Michael Lynge

2017-01-01

from health professionals. They experienced a lack of security, worries about the future and a lack of support at home. The study showed a substantial level of dissatisfaction among relatives of patients with advanced cancer. We strongly recommend a focus on psychosocial care, more access......Palliative cancer care in Greenland is provided by health professionals at local level, the national Queen Ingrid's Hospital and at Rigshospitalet in Denmark. To improve and develop care for relatives of patients with advanced cancer, we conducted a mixed method study examining relatives' level...... was supplemented by open-ended questions about relative's current main concerns and analyzed with a phenomenological hermeneutical approach. Greenlandic patients with advanced cancer who were previously participating in a prospective study were asked if their closest adult relative would participate in the study...

The Arabidopsis SKU5 gene encodes an extracellular glycosyl phosphatidylinositol-anchored glycoprotein involved in directional root growth

Science.gov (United States)

Sedbrook, John C.; Carroll, Kathleen L.; Hung, Kai F.; Masson, Patrick H.; Somerville, Chris R.

2002-01-01

To investigate how roots respond to directional cues, we characterized a T-DNA-tagged Arabidopsis mutant named sku5 in which the roots skewed and looped away from the normal downward direction of growth on inclined agar surfaces. sku5 roots and etiolated hypocotyls were slightly shorter than normal and exhibited a counterclockwise (left-handed) axial rotation bias. The surface-dependent skewing phenotype disappeared when the roots penetrated the agar surface, but the axial rotation defect persisted, revealing that these two directional growth processes are separable. The SKU5 gene belongs to a 19-member gene family designated SKS (SKU5 Similar) that is related structurally to the multiple-copper oxidases ascorbate oxidase and laccase. However, the SKS proteins lack several of the conserved copper binding motifs characteristic of copper oxidases, and no enzymatic function could be assigned to the SKU5 protein. Analysis of plants expressing SKU5 reporter constructs and protein gel blot analysis showed that SKU5 was expressed most strongly in expanding tissues. SKU5 was glycosylated and modified by glycosyl phosphatidylinositol and localized to both the plasma membrane and the cell wall. Our observations suggest that SKU5 affects two directional growth processes, possibly by participating in cell wall expansion.

Cancer-related symptoms predict psychological wellbeing among prostate cancer survivors: results from the PiCTure study.

Science.gov (United States)

Sharp, Linda; O'Leary, Eamonn; Kinnear, Heather; Gavin, Anna; Drummond, Frances J

2016-03-01

Prostate cancer treatments are associated with a range of symptoms and physical side-effects. Cancer can also adversely impact on psychological wellbeing. Because many prostate cancer-related symptoms and side-effects are potentially modifiable, we investigated associations between symptoms and psychological wellbeing among prostate cancer survivors. Postal questionnaires were distributed to men diagnosed with prostate cancer 2-18 years previously identified through cancer registries. General and prostate cancer-specific symptoms were assessed using the EORTC QLQ-C30 and QLQ-PR25, with higher symptom scores indicating more/worse symptomatology. Psychological wellbeing was assessed by the DASS-21. Associations between symptoms and each outcome were investigated using multivariate logistic regression, controlling for socio-demographic and clinical factors. A total 3348 men participated (response rate = 54%). Seventeen percent (95%CI 15.2%-17.9%), 16% (95%CI 15.1%-17.8%) and 11% (95%CI 9.5%-11.8%) of survivors scored in the range for depression, anxiety and distress on the DASS scales, respectively. In multivariate models, risk of depression on the DASS scale was significantly higher in men with higher urinary and androgen deprivation therapy (ADT)-related symptoms, and higher scores for fatigue, insomnia and financial difficulties. Risk of anxiety on the DASS scale was higher in men with higher scores for urinary, bowel and ADT-related symptoms and fatigue, dyspnoea and financial difficulties. Risk of distress on the DASS scale was positively associated with urinary, bowel and ADT-related symptoms, fatigue, insomnia and financial difficulties. Cancer-related symptoms significantly predict psychological wellbeing among prostate cancer survivors. Greater use of interventions and medications and to alleviate symptoms might improve psychological wellbeing of prostate cancer survivors. Copyright © 2015 John Wiley & Sons, Ltd.

Trends and approaches in N-Glycosylation engineering in Chinese hamster ovary cell culture

DEFF Research Database (Denmark)

Fan, Yuzhou; Kildegaard, Helene Faustrup; Andersen, Mikael Rørdam

will summarize a group of recent strategies andapproaches and come up with case studies for N-glycosylation engineering in CHO cells and show several examples of relevantstudy cases from our research: 1) media and feed design, 2) culture process optimization, 3) substrate addition, 4) geneticengineering, 5...

Pre-diagnosis employment status and financial circumstances predict cancer-related financial stress and strain among breast and prostate cancer survivors.

Science.gov (United States)

Sharp, Linda; Timmons, Aileen

2016-02-01

Cancer may have a significant financial impact on patients, but the characteristics that predispose patients to cancer-related financial hardship are poorly understood. We investigated factors associated with cancer-related financial stress and strain in breast and prostate cancer survivors in Ireland, which has a complex mixed public-private healthcare system. Postal questionnaires were distributed to 1373 people diagnosed with cancer 3-24 months previously identified from the National Cancer Registry Ireland. Outcomes were cancer-related financial stress (impact of cancer diagnosis on household ability to make ends meet) and financial strain (concerns about household financial situation since cancer diagnosis). Modified Poisson regression was used to estimate relative risks (RR) for factors associated with cancer-related financial stress and strain. Seven hundred forty survivors participated (response rate = 54 %). Of the respondents, 48 % reported cancer-related financial stress and 32 % cancer-related financial strain. Compared to those employed at diagnosis, risk of cancer-related financial stress was significantly lower in those not working (RR = 0.71, 95 % CI 0.58-0.86) or retired (RR = 0.48, 95 % CI 0.34-0.68). It was significantly higher in those who had dependents; experienced financial stress pre-diagnosis; had a mortgage/personal loans; had higher direct medical out-of-pocket costs; and had increased household bills post-diagnosis. For cancer-related financial strain, significant associations were found with dependents, pre-diagnosis employment status and pre-diagnosis financial stress; risk was lower in those with higher direct medical out-of-pocket costs. Cancer-related financial stress and strain are common. Pre-diagnosis employment status and financial circumstances are important predictors of post-diagnosis financial wellbeing. These findings could inform development of tools to identify patients/survivors most in need of financial

Glycosylation of DMP1 Is Essential for Chondrogenesis of Condylar Cartilage.

Science.gov (United States)

Weng, Y; Liu, Y; Du, H; Li, L; Jing, B; Zhang, Q; Wang, X; Wang, Z; Sun, Y

2017-12-01

The mandibular condylar cartilage (MCC) shoulders force for the subchondral bone during mastication. The cartilage matrix contains various large molecules, such as type I, II, and X collagens and proteoglycans (PGs), which jointly play essential roles in maintaining cartilage characteristics. PGs play key roles in maintaining the elasticity of cartilage and providing a cushion against mastication forces. In addition to the well-known PGs, DMP1-PG, which is the PG form of dentin matrix protein 1 (DMP1), is a newly identified PG. DMP1 is proteolytically processed in vivo, and the N-terminus is glycosylated into its PG form-that is, DMP1-PG, which is highly expressed not only in tooth and bone but also in the matrix of the MCC. However, the specific functions of DMP1-PG in the MCC remain unclear. In human temporomandibular joint osteoarthritis and hyperocclusion model rat specimens, PGs are significantly downregulated, and DMP1-PG is the most prominently affected PG. To further investigate the role of DMP1-PG in condylar chondrogenesis, a glycosylation site mutant (S 89 -G 89 ) mouse model was established with knock-in methods. In the MCC of the S89G-DMP1 mice, the glycosylation level of DMP1 was significantly downregulated, and a series of abnormal developmental and pathologic changes could be observed. The morphologic changes included thinner cartilage layers, deformations of the MCC, and disordered arrangements of the chondrocytes, and an earlier onset of temporomandibular joint osteoarthritis-like changes was observed. In addition, markers of chondrogenesis were downregulated, and the matrix of the MCC displayed OA phenotypes in the S89G-DMP1 mice. Further investigations showed that the transforming growth factor β signaling molecules were affected in the MCC after the loss of DMP1-PG. In addition, the loss of DMP1-PG significantly accelerated the progression of cartilage injuries in the hyperocclusion models. Given these findings, we investigated the significant

Effect of aerobic exercise on cancer-related fatigue

Directory of Open Access Journals (Sweden)

Jaivika Govindbhai Patel

2017-01-01

Full Text Available Background: Fatigue is the most common side effect of cancer treatment with chemotherapy and/or radiation therapy, selected biologic response modifiers. The main purpose of this study is to evaluate the effects of aerobic exercise on cancer-related fatigue in patients of the solid tumor after chemotherapy and radiotherapy. Methods: After screening for cancer-related fatigue, 34 patients fulfilled the inclusive criteria and were assigned into two groups (n = 17 recruited in the intervention group and n = 17 in control group. The intervention group received aerobic exercise program which included treadmill walking with low to moderate intensity (50%–70% of maximum heart rate, for 20–40 min/day for 5 days/week. Control group were taught stretching exercises of hamstrings, gastrocnemius, and soleus (to be done at home and were encouraged to remain active. Outcome measures such as brief fatigue inventory (BFI, 6-min walk test, and functional assessment of cancer therapy-general (FACT-G were taken at baseline and after 6-weeks. Results: The data were analyzed using the Wilcoxon matched-pairs signed rank test for within group and Mann–Whitney U-test for between group comparisons. The results of this study showed that there was a significant reduction in cancer-related fatigue BFI score (P < 0.0001,, also there was significant improvement in the physical performance as in 6-min walk distance (P < 0.0001 and quality of life, FACT-G score (P = 0.0001. Conclusion: Aerobic exercise for 6 weeks has beneficial effects on cancer-related fatigue in patients with solid tumor after chemotherapy and/or radiotherapy.

Microwave Effect for Glycosylation Promoted by Solid Super Acid in Supercritical Carbon Dioxide

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Takahiko Maeda

2009-12-01

Full Text Available The effects of microwave irradiation (2.45 GHz, 200 W on glycosylation promoted by a solid super acid in supercritical carbon dioxide was investigated with particular attention paid to the structure of the acceptor substrate. Because of the symmetrical structure and high diffusive property of supercritical carbon dioxide, microwave irradiation did not alter the temperature of the reaction solution, but enhanced reaction yield when aliphatic acceptors are employed. Interestingly, the use of a phenolic acceptor under the same reaction conditions did not show these promoting effects due to microwave irradiation. In the case of aliphatic diol acceptors, the yield seemed to be dependent on the symmetrical properties of the acceptors. The results suggest that microwave irradiation do not affect the reactivity of the donor nor promoter independently. We conclude that the effect of acceptor structure on glycosylation yield is due to electric delocalization of hydroxyl group and dielectrically symmetric structure of whole molecule.

Tobacco and Alcohol in Relation to Male Breast Cancer

DEFF Research Database (Denmark)

Cook, Michael B; Guénel, Pascal; Gapstur, Susan M

2015-01-01

BACKGROUND: The etiology of male breast cancer is poorly understood, partly due to its relative rarity. Although tobacco and alcohol exposures are known carcinogens, their association with male breast cancer risk remains ill-defined. METHODS: The Male Breast Cancer Pooling Project consortium prov...

Associations of Filaggrin Gene Loss-of-Function Variants and Human Papillomavirus-Related Cancer and PreCancer in Danish Adults

DEFF Research Database (Denmark)

Skaaby, Tea; Husemoen, Lise Lotte N; Jørgensen, Torben

2014-01-01

to human papillomavirus (HPV) infection and thus a higher risk of HPV-related cancer and pre-cancer. We investigated the association of the FLG genotype with incidence of HPV-related cancer of cervix, vagina, vulva, penis, anus and head and neck, and pre-cancer of the cervix. METHODS: We included 13......,376 persons from four population-based studies conducted in the same background population in Copenhagen, Denmark. Participants were genotyped for the most common FLG mutations in Europeans. Information on cancer was obtained from The Danish Cancer Registry until 11 July 2011. RESULTS: There were 489 cases...... of prevalent and 97 cases of incident HPV-related cancer and pre-cancer (median follow-up 11.5 years). There was a statistically significant association between FLG genotype and incident HPV-related cancer and pre-cancer with a hazard ratio, HR = 2.1 (95% confidence intervals, CI: 1.2, 3.7) for FLG mutation...

Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation

DEFF Research Database (Denmark)

Fan, Yuzhou; Jimenez Del Val, Ioscani; Müller, Christian

2015-01-01

optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylationrelated product quality. In this work, different fed-batch processes with two chemically defined proprietary media......Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer, and N-glycosylation patterns are always the major concerns during upstream process...... and glutamine concentrations and uptake rates were positively correlated with intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality....

Abnormalities in alternative splicing of angiogenesis-related genes and their role in HIV-related cancers

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Mthembu NN

2017-03-01

Full Text Available Nonkululeko N Mthembu,1 Zukile Mbita,2 Rodney Hull,1 Zodwa Dlamini1 1Research, Innovation and Engagements, Mangosuthu University of Technology, Durban, 2Department of Biochemistry, Microbiology and Biotechnology, University of Limpopo, Sovenga, South Africa Abstract: Alternative splicing of mRNA leads to an increase in proteome biodiversity by allowing the generation of multiple mRNAs, coding for multiple protein isoforms of various structural and functional properties from a single primary pre-mRNA transcript. The protein isoforms produced are tightly regulated in normal development but are mostly deregulated in various cancers. In HIV-infected individuals with AIDS, there is an increase in aberrant alternative splicing, resulting in an increase in HIV/AIDS-related cancers, such as Kaposi’s sarcoma, non-Hodgkin’s lymphoma, and cervical cancer. This aberrant splicing leads to abnormal production of protein and is caused by mutations in cis-acting elements or trans-acting factors in angiogenesis-related genes. Restoring the normal regulation of alternative splicing of angiogenic genes would alter the expression of protein isoforms and may confer normal cell physiology in patients with these cancers. This review highlights the abnormalities in alternative splicing of angiogenesis-related genes and their implication in HIV/AIDS-related cancers. This allows us to gain an insight into the pathogenesis of HIV/AIDS-related cancer and in turn elucidate the therapeutic potential of alternatively spliced genes in HIV/AIDS-related malignancies. Keywords: vascular endothelial growth factor, oncogenic viruses, hypoxia induced factor 1, Kaposi’s sarcoma, non-Hodgkin’s lymphoma, therapies targeting alternative splicing

Analysis of factors related to arm weakness in patients with breast cancer-related lymphedema.

Science.gov (United States)

Lee, Daegu; Hwang, Ji Hye; Chu, Inho; Chang, Hyun Ju; Shim, Young Hun; Kim, Jung Hyun

2015-08-01

The aim of this study was to evaluate the ratio of significant weakness in the affected arm of breast cancer-related lymphedema patients to their unaffected side. Another purpose was to identify factors related to arm weakness and physical function in patients with breast cancer-related lymphedema. Consecutive patients (n = 80) attended a single evaluation session following their outpatient lymphedema clinic visit. Possible independent factors (i.e., lymphedema, pain, psychological, educational, and behavioral) were evaluated. Handgrip strength was used to assess upper extremity muscle strength and the disabilities of arm, shoulder, and hand (DASH) questionnaire was used to assess upper extremity physical function. Multivariate logistic regression was performed using factors that had significant differences between the handgrip weakness and non-weakness groups. Out of the 80 patients with breast cancer-related lymphedema, 29 patients (36.3 %) had significant weakness in the affected arm. Weakness of the arm with lymphedema was not related to lymphedema itself, but was related to the fear of using the affected limb (odds ratio = 1.76, 95 % confidence interval = 1.30-2.37). Fears of using the affected limb and depression significantly contributed to the variance in DASH scores. Appropriate physical and psychological interventions, including providing accurate information and reassurance of physical activity safety, are necessary to prevent arm weakness and physical dysfunction in patients with breast cancer-related lymphedema.

Benefits of marriage on relative and conditional relative cancer survival differ between males and females in the USA.

Science.gov (United States)

Merrill, Ray M; Johnson, Erin

2017-10-01

The purpose of the paper is to assess the influence of marital status on conditional relative survival of cancer according to sex. Analyses involved 779,978 males and 1,032,868 females diagnosed with 1 of 13 cancer types between 2000 and 2008, and followed through 2013. Data are from the Surveillance, Epidemiology, and End Results (SEER) Program. Regression models were adjusted for age, sex, race, and tumor stage. Five-year relative survival conditional on years already survived is higher among married patients with less lethal cancers (oral cavity and pharynx, colon and rectum, breast, urinary bladder, kidney and renal pelvis, melanoma of the skin, thyroid, lymphoma). For more lethal cancers, married patients have similar (liver, lung and bronchus, pancreas, leukemia) or poorer (brain and other nervous system) cancer survival. Separated/divorced or widowed patients have the lowest conditional relative survival rates. For most cancers, 5-year cancer relative survival rates conditional on time already survived through 5 years approach 70 to 90% of that for the general population. The beneficial effect of marriage on survival decreases with years already survived. Superior conditional relative survival rates in females decrease with time already survived and are less pronounced in married patients. Five-year relative survival rates improve with time already survived. The benefits of marriage on conditional relative survival are greater for less lethal cancers. Greater 5-year conditional relative survival rates in females narrow with time already survived and are less pronounced in married patients. Conditional relative survival rates of cancer can lead to more informed decisions and understanding regarding treatment and prognosis.

Adherence to Guidelines for Cancer Survivors and Health-Related Quality of Life among Korean Breast Cancer Survivors

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Sihan Song

2015-12-01

Full Text Available There is limited evidence on the association between adherence to guidelines for cancer survivors and health-related quality of life (HRQoL. In a cross-sectional study of Korean breast cancer survivors, we examined whether adherence to the guidelines of the American Cancer Society (ACS and World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR for cancer survivors was related to levels of HRQoL, assessed by the Korean version of Core 30 (C30 and Breast cancer module 23 (BR23 of the European Organization for Research and Treatment of Cancer-Quality of Life Questionnaire (EORTC-QLQ. We included a total of 160 women aged 21 to 79 years who had been diagnosed with breast cancer according to American Joint Committee on Cancer (AJCC stages I to III and had breast cancer surgery at least six months before the interview. Increasing adherence to ACS guidelines was associated with higher scores of social functioning (p for trend = 0.05, whereas increasing adherence to WCRF/AICR recommendations was associated with higher scores of arm symptoms (p for trend = 0.01. These associations were limited to those with stage II or III cancer. Diet may be an important factor in relation to quality of life among Korean breast cancer survivors, however our findings warrant further prospective studies to evaluate whether healthy diet improves survivors’ quality of life.

Aids-Related Cancers in Africa

Science.gov (United States)

Mbulaiteye, Sam M.

2014-07-01

Thank you Professor Zichichi for inviting me to give a talk about AIDS-related cancers in Africa. Let me begin by congratulating the team that organized the 46th Session of the Erice International Seminar Series, whose theme is THE ROLE OF SCIENCE IN THE THIRD MILLENIUM. I also congratulate the scientists from 38 countries who are attending these seminars. They are perpetuating the principle of SCIENCE WITHOUT SECRETS in the true spirit espoused by Archimedes, Galileo, and Fermi. It is a wonderful honor for me to be here to shed some light on the health impacts of the HIV epidemic in the area of cancer...

Comparison of transferrin isoform analysis by capillary electrophoresis and HPLC for screening congenital disorders of glycosylation.

Science.gov (United States)

Dave, Mihika B; Dherai, Alpa J; Udani, Vrajesh P; Hegde, Anaita U; Desai, Neelu A; Ashavaid, Tester F

2018-01-01

Transferrin, a major glycoprotein has different isoforms depending on the number of sialic acid residues present on its oligosaccharide chain. Genetic variants of transferrin as well as the primary (CDG) & secondary glycosylation defects lead to an altered transferrin pattern. Isoform analysis methods are based on charge/mass variations. We aimed to compare the performance of commercially available capillary electrophoresis CDT kit for diagnosing congenital disorders of glycosylation with our in-house optimized HPLC method for transferrin isoform analysis. The isoform pattern of 30 healthy controls & 50 CDG-suspected patients was determined by CE using a Carbohydrate-Deficient Transferrin kit. The results were compared with in-house HPLC-based assay for transferrin isoforms. Transferrin isoform pattern for healthy individuals showed a predominant tetrasialo transferrin fraction followed by pentasialo, trisialo, and disialotransferrin. Two of 50 CDG-suspected patients showed the presence of asialylated isoforms. The results were comparable with isoform pattern obtained by HPLC. The commercial controls showed a <20% CV for each isoform. Bland Altman plot showed the difference plot to be within +1.96 with no systemic bias in the test results by HPLC & CE. The CE method is rapid, reproducible and comparable with HPLC and can be used for screening Glycosylation defects. © 2017 Wiley Periodicals, Inc.

The role of glycosylation and domain interactions in the thermal stability of human angiotensin-converting enzyme.

Science.gov (United States)

O'Neill, Hester G; Redelinghuys, Pierre; Schwager, Sylva L U; Sturrock, Edward D

2008-09-01

The N and C domains of somatic angiotensin-converting enzyme (sACE) differ in terms of their substrate specificity, inhibitor profiling, chloride dependency and thermal stability. The C domain is thermally less stable than sACE or the N domain. Since both domains are heavily glycosylated, the effect of glycosylation on their thermal stability was investigated by assessing their catalytic and physicochemical properties. Testis ACE (tACE) expressed in mammalian cells, mammalian cells in the presence of a glucosidase inhibitor and insect cells yielded proteins with altered catalytic and physicochemical properties, indicating that the more complex glycans confer greater thermal stabilization. Furthermore, a decrease in tACE and N-domain N-glycans using site-directed mutagenesis decreased their thermal stability, suggesting that certain N-glycans have an important effect on the protein's thermodynamic properties. Evaluation of the thermal stability of sACE domain swopover and domain duplication mutants, together with sACE expressed in insect cells, showed that the C domain contained in sACE is less dependent on glycosylation for thermal stabilization than a single C domain, indicating that stabilizing interactions between the two domains contribute to the thermal stability of sACE and are decreased in a C-domain-duplicating mutant.

Model-based investigation of intracellular processes determining antibody Fc-glycosylation under mild hypothermia.

Science.gov (United States)

Sou, Si Nga; Jedrzejewski, Philip M; Lee, Ken; Sellick, Christopher; Polizzi, Karen M; Kontoravdi, Cleo

2017-07-01

Despite the positive effects of mild hypothermic conditions on monoclonal antibody (mAb) productivity (q mAb ) during mammalian cell culture, the impact of reduced culture temperature on mAb Fc-glycosylation and the mechanism behind changes in the glycan composition are not fully established. The lack of knowledge about the regulation of dynamic intracellular processes under mild hypothermia restricts bioprocess optimization. To address this issue, a mathematical model that quantitatively describes Chinese hamster ovary (CHO) cell behavior and metabolism, mAb synthesis and mAb N-linked glycosylation profile before and after the induction of mild hypothermia is constructed. Results from this study show that the model is capable of representing experimental results well in all of the aspects mentioned above, including the N-linked glycosylation profile of mAb produced under mild hypothermia. Most importantly, comparison between model simulation results for different culture temperatures suggests the reduced rates of nucleotide sugar donor production and galactosyltransferase (GalT) expression to be critical contributing factors that determine the variation in Fc-glycan profiles between physiological and mild hypothermic conditions in stable CHO transfectants. This is then confirmed using experimental measurements of GalT expression levels, thereby closing the loop between the experimental and the computational system. The identification of bottlenecks within CHO cell metabolism under mild hypothermic conditions will aid bioprocess optimization, for example, by tailoring feeding strategies to improve NSD production, or manipulating the expression of specific glycosyltransferases through cell line engineering. Biotechnol. Bioeng. 2017;114: 1570-1582. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals Inc.

Cancer immunotherapy and immune-related response assessment: The role of radiologists in the new arena of cancer treatment

Energy Technology Data Exchange (ETDEWEB)

Nishino, Mizuki, E-mail: Mizuki_Nishino@DFCI.HARVARD.EDU [Department of Radiology, Brigham and Women' s Hospital and Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215 (United States); Tirumani, Sree H.; Ramaiya, Nikhil H. [Department of Radiology, Brigham and Women' s Hospital and Dana-Farber Cancer Institute, 450 Brookline Avenue, Boston, MA 02215 (United States); Hodi, F. Stephen [Department of Medical Oncology and Department of Medicine, Dana-Farber Cancer Institute and Brigham and Women' s Hospital, 450 Brookline Ave., Boston, MA 02215 (United States)

2015-07-15

Highlights: • The successful clinical application of cancer immunotherapy has opened a new arena for the treatment of advanced cancers. • Cancer immunotherapy is associated with a variety of important radiographic features in the assessments of tumor response and immune-related adverse events. • The state-of-the art knowledge of immunotherapy and the related radiologic manifestations are essential for radiologists. - Abstract: The recent advances in the clinical application of anti-cancer immunotherapeutic agents have opened a new arena for the treatment of advanced cancers. Cancer immunotherapy is associated with a variety of important radiographic features in the assessments of tumor response and immune-related adverse events, which calls for radiologists’ awareness and in-depth knowledge on the topic. This article will provide the state-of-the art review and perspectives of cancer immunotherapy, including its molecular mechanisms, the strategies for immune-related response assessment on imaging and their pitfalls, and the emerging knowledge of radiologic manifestations of immune-related adverse events. The cutting edge clinical and radiologic investigations are presented to provide future directions.

Cancer immunotherapy and immune-related response assessment: The role of radiologists in the new arena of cancer treatment

International Nuclear Information System (INIS)

Nishino, Mizuki; Tirumani, Sree H.; Ramaiya, Nikhil H.; Hodi, F. Stephen

2015-01-01

Highlights: • The successful clinical application of cancer immunotherapy has opened a new arena for the treatment of advanced cancers. • Cancer immunotherapy is associated with a variety of important radiographic features in the assessments of tumor response and immune-related adverse events. • The state-of-the art knowledge of immunotherapy and the related radiologic manifestations are essential for radiologists. - Abstract: The recent advances in the clinical application of anti-cancer immunotherapeutic agents have opened a new arena for the treatment of advanced cancers. Cancer immunotherapy is associated with a variety of important radiographic features in the assessments of tumor response and immune-related adverse events, which calls for radiologists’ awareness and in-depth knowledge on the topic. This article will provide the state-of-the art review and perspectives of cancer immunotherapy, including its molecular mechanisms, the strategies for immune-related response assessment on imaging and their pitfalls, and the emerging knowledge of radiologic manifestations of immune-related adverse events. The cutting edge clinical and radiologic investigations are presented to provide future directions

Basic research on cancer related to radiation associated medical researches

International Nuclear Information System (INIS)

Lee, Jong In; Hwang, Dae Yong; Bang, Ho Yoon

2000-12-01

Basic Research on Cancer related to Radiation Associated Medical Researches including 1. Establishment of animal model of colorectal cancer liver metastasis and measurement of angiogenesis, 2. Tissue expression of Tie-1 and Tie-2 in human colorectal cancer, 3. Enhancement of G2/Mphase Cell Fraction by Adenovirus-mediated p53 Gene Transfer in Ovarian Cancer Cell Lines, 4. Clinical Characteristics of the patients with Non-B Non-C Hepatocellular Carcinoma and Frequency of HBV, HCV and TTV Viremia in these Patients, 5. Significance of serum iron and ferritin in patients with stomach cancer, 6. Telomerase assay for early detection of lung cancer, 7. Study on the Usefulness of Aldehyde dehydrogenase-2 Genotyping for Risk Group of Alcohol-related Cancer Screening, 8. Gene therapy using hepatoma specific promoter, 9. Study on the Influence of DNA repair gene, XRCC1 Genotypes on the Risk of Head and Neck Cancer were performed

Basic research on cancer related to radiation associated medical researches

Energy Technology Data Exchange (ETDEWEB)

Lee, Jong In; Hwang, Dae Yong; Bang, Ho Yoon [and others

2000-12-01

Basic Research on Cancer related to Radiation Associated Medical Researches including 1. Establishment of animal model of colorectal cancer liver metastasis and measurement of angiogenesis, 2. Tissue expression of Tie-1 and Tie-2 in human colorectal cancer, 3. Enhancement of G2/Mphase Cell Fraction by Adenovirus-mediated p53 Gene Transfer in Ovarian Cancer Cell Lines, 4. Clinical Characteristics of the patients with Non-B Non-C Hepatocellular Carcinoma and Frequency of HBV, HCV and TTV Viremia in these Patients, 5. Significance of serum iron and ferritin in patients with stomach cancer, 6. Telomerase assay for early detection of lung cancer, 7. Study on the Usefulness of Aldehyde dehydrogenase-2 Genotyping for Risk Group of Alcohol-related Cancer Screening, 8. Gene therapy using hepatoma specific promoter, 9. Study on the Influence of DNA repair gene, XRCC1 Genotypes on the Risk of Head and Neck Cancer were performed.

Characterization of the oligosaccharide structure of human glycosylated prolactin (G-hPRL) native and recombinant

International Nuclear Information System (INIS)

Marcos Vinicius Nucci Capone

2013-01-01

Human prolactin (hPRL) is a polypeptide hormone secreted by the anterior pituitary under the regulation of the hypothalamus, involved in a variety of biological processes such as mammary gland development and lactation. The recombinant product is important in medical diagnosis and treatment of failure of lactation. This hormone may occur in the form of non-glycosylated protein (NGhPRL) and glycosylated (G-hPRL) with molecular weights of approximately 23 and 25 kilodalton (kDa), respectively; has a single N-glycosylation site located at asparagine (Asn) position 31, which is partially occupied, thus being a particularly interesting model of glycosylation. The biological activity of G-hPRL is lower compared to NG-hPRL (~4 times) and its physiological function is not well defined: the portion of carbohydrate appears to have an important role in the hormone biosynthesis, secretion, biological activity, and plasma survival of the hormone. The main objective of this study was to compare the structures of N-glycans present in glycosylated pituitary prolactin (G-hPRL-NHPP) with those present in the recombinant. To obtain the recombinant G-hPRL the production was performed in laboratory scale from Chinese hamster ovary cells (CHO), genetically modified and adapted to growth in suspension. Cycloheximide (CHX), whose main effect was to increase the ratio G-hPRL/NG-hPRL from 5% to 38% was added to the culture medium, thereby facilitating the purification of G-hPRL. The G-hPRL was purified in two steps, a cation exchanger followed by a purification by reversed-phase high performance liquid chromatography (RP-HPLC) which demonstrated the efficient separation of the two isoforms of hPRL. Recombinant G-hPRL-IPEN was well characterized by several techniques confirming its purity and biological activity, including comparisons with other reference preparation of pituitary origin purchased from the N ational Hormone & Peptide Program (NHPPU. S.) . The composition of N-glycans present

Scanning the available Dictyostelium discoideum proteome for O-linked GlcNAc glycosylation sitesusing neural networks

DEFF Research Database (Denmark)

Gupta, Ramneek; Jung, Eva; Gooley, Andrew A

1999-01-01

Dictyostelium discoideum has been suggested as a eukaryotic model organism for glycobiology studies. Presently, the characteristics of acceptor sites for the N-acetylglucosaminyl-transferases in Dictyostelium discoideum, which link GlcNAc in an alpha linkage to hydroxyl residues, are largely...... unknown. This motivates the development of a species specific method for prediction of O-linked GlcNAc glycosylation sites in secreted and membrane proteins of D. discoideum. The method presented here employs a jury of artificial neural networks. These networks were trained to recognize the sequence...... context and protein surface accessibility in 39 experimentally determined O-alpha-GlcNAc sites found in D. discoideum glycoproteins expressed in vivo. Cross-validation of the data revealed a correlation in which 97% of the glycosylated and nonglycosylated sites were correctly identified. Based...

Cigarette smoking and radiation exposure in relation to cancer mortality, Hiroshima and Nagasaki

International Nuclear Information System (INIS)

Prentice, R.L.; Yoshimoto, Yasuhiko; Mason, M.W.

1983-05-01

Cancer mortality among 40,498 Hiroshima and Nagasaki residents was examined in relation to cigarette smoking habits and estimated atomic bomb radiation exposure. Relative risk models that are either multiplicative or additive in the two exposures (smoking radiation) were emphasized. Most analyses were directed toward all nonhematologic cancer, stomach cancer, lung cancer, or digestive cancer other than stomach, for which there were, respectively, 1,725, 658, 281, and 338 deaths in the follow-up period of this study. Persons heavily exposed to both cigarette smoke and radiation were found to have significantly lower cancer mortality than multiplcative relative risk models would suggest for all nonhematologic cancer, stomach cancer, and digestive cancer other than stomach. Surprisingly, the relative risk function appeared not only to be submultiplicative for these cancer sites, but to be subadditive as well. The lung cancer relative risk function could not be distinguished from either a multiplicative or an additive form. The number of deaths was sufficient to permit some more detailed study of all nonhematologic cancer mortality: Relative risk functions appeared to be consistent between males and females though a paucity of heavy smoking females limits the precision of this comparison. (author)

Associations of filaggrin gene loss-of-function variants and human papillomavirus-related cancer and pre-cancer in Danish adults.

Directory of Open Access Journals (Sweden)

Tea Skaaby

Full Text Available Filaggrin proteins are expressed in the skin, oral cavity, oesophagus, and cervical mucose. Loss-of-function mutations in the filaggrin gene (FLG reduce filaggrin expression and cause an impaired skin barrier function. We hypothesized that FLG mutation carriers would be more susceptible to human papillomavirus (HPV infection and thus a higher risk of HPV-related cancer and pre-cancer. We investigated the association of the FLG genotype with incidence of HPV-related cancer of cervix, vagina, vulva, penis, anus and head and neck, and pre-cancer of the cervix.We included 13,376 persons from four population-based studies conducted in the same background population in Copenhagen, Denmark. Participants were genotyped for the most common FLG mutations in Europeans. Information on cancer was obtained from The Danish Cancer Registry until 11 July 2011.There were 489 cases of prevalent and 97 cases of incident HPV-related cancer and pre-cancer (median follow-up 11.5 years. There was a statistically significant association between FLG genotype and incident HPV-related cancer and pre-cancer with a hazard ratio, HR = 2.1 (95% confidence intervals, CI: 1.2, 3.7 for FLG mutation carriers vs. wild types.FLG loss-of-function mutations were associated with higher incidence of HPV-related cancers and pre-cancers that are potentially screening and vaccine preventable.

A computerized Stroop task to assess cancer-related cognitive biases.

Science.gov (United States)

DiBonaventura, Marco DaCosta; Erblich, Joel; Sloan, Richard P; Bovbjerg, Dana H

2010-01-01

Biases in processing information related to sources of stress have widely been demonstrated with the use of Stroop emotional color word tasks. One study reported such biases among women with histories of breast cancer in a first-degree relative (FH+) who were given a Stroop cancer word task. This study aimed to replicate and extend these findings with a computerized version of the task. Response latencies and errors were recorded during administration of the task to FH+ and FH- women. A cancer list and 5 comparison lists were administered. Results indicated that FH+ women exhibited longer response latencies for cancer words than did FH- women (p Stroop cancer word task.

Cancer incidence in relatives of British Fanconi Anaemia patients

Directory of Open Access Journals (Sweden)

Hodgson Shirley V

2008-09-01

Full Text Available Abstract Background Fanconi anemia (FA is an autosomal recessive DNA repair disorder with affected individuals having a high risk of developing acute myeloid leukaemia and certain solid tumours. Thirteen complementation groups have been identified and the genes for all of these are known (FANCA, B, C, D1/BRCA2, D2, E, F, G, I, J/BRIP1, L, M and N/PALB2. Previous studies of cancer incidence in relatives of Fanconi anemia cases have produced conflicting results. A study of British FA families was therefore carried out to investigate this question, since increases in cancer risk in FA heterozygotes would have implications for counselling FA family members, and possibly also for the implementation of preventative screening measures in FA heterozygotes. Methods Thirty-six families took part and data was collected on 575 individuals (276 males, 299 females, representing 18,136 person years. In this cohort, 25 males and 30 females were reported with cancer under the age of 85 years, and 36 cancers (65% could be confirmed from death certificates, cancer registries or clinical records. Results A total of 55 cancers were reported in the FA families compared to an estimated incidence of 56.95 in a comparable general population cohort, and the relative risk of cancer was 0.97 (95% C.I. = 0.71–1.23, p = 0.62 for FA family members. Analysis of relative risk for individual cancer types in each carrier probability group did not reveal any significant differences with the possible exception of prostate cancer (RR = 3.089 (95% C.I. = 1.09 – 8.78; Χ2 = 4.767, p = 0.029. Conclusion This study has not shown a significant difference in overall cancer risk in FA families.

Gene identification in the congenital disorders of glycosylation type I by whole-exome sequencing

NARCIS (Netherlands)

Timal, Sharita; Hoischen, Alexander; Lehle, Ludwig; Adamowicz, Maciej; Huijben, Karin; Sykut-Cegielska, Jolanta; Paprocka, Justyna; Jamroz, Ewa; van Spronsen, Francjan J.; Koerner, Christian; Gilissen, Christian; Rodenburg, Richard J.; Eidhof, Ilse; Van den Heuvel, Lambert; Thiel, Christian; Wevers, Ron A.; Morava, Eva; Veltman, Joris; Lefeber, Dirk J.

2012-01-01

Congenital disorders of glycosylation type I (CDG-I) form a growing group of recessive neurometabolic diseases. Identification of disease genes is compromised by the enormous heterogeneity in clinical symptoms and the large number of potential genes involved. Until now, gene identification included

A zebrafish model of congenital disorders of glycosylation with phosphomannose isomerase deficiency reveals an early opportunity for corrective mannose supplementation

Directory of Open Access Journals (Sweden)

Jaime Chu

2013-01-01

Individuals with congenital disorders of glycosylation (CDG have recessive mutations in genes required for protein N-glycosylation, resulting in multi-systemic disease. Despite the well-characterized biochemical consequences in these individuals, the underlying cellular defects that contribute to CDG are not well understood. Synthesis of the lipid-linked oligosaccharide (LLO, which serves as the sugar donor for the N-glycosylation of secretory proteins, requires conversion of fructose-6-phosphate to mannose-6-phosphate via the phosphomannose isomerase (MPI enzyme. Individuals who are deficient in MPI present with bleeding, diarrhea, edema, gastrointestinal bleeding and liver fibrosis. MPI-CDG patients can be treated with oral mannose supplements, which is converted to mannose-6-phosphate through a minor complementary metabolic pathway, restoring protein glycosylation and ameliorating most symptoms, although liver disease continues to progress. Because Mpi deletion in mice causes early embryonic lethality and thus is difficult to study, we used zebrafish to establish a model of MPI-CDG. We used a morpholino to block mpi mRNA translation and established a concentration that consistently yielded 13% residual Mpi enzyme activity at 4 days post-fertilization (dpf, which is within the range of MPI activity detected in fibroblasts from MPI-CDG patients. Fluorophore-assisted carbohydrate electrophoresis detected decreased LLO and N-glycans in mpi morphants. These deficiencies resulted in 50% embryonic lethality by 4 dpf. Multi-systemic abnormalities, including small eyes, dysmorphic jaws, pericardial edema, a small liver and curled tails, occurred in 82% of the surviving larvae. Importantly, these phenotypes could be rescued with mannose supplementation. Thus, parallel processes in fish and humans contribute to the phenotypes caused by Mpi depletion. Interestingly, mannose was only effective if provided prior to 24 hpf. These data provide insight into treatment efficacy

Effects of an expressive writing intervention on cancer-related distress in Danish breast cancer survivors

DEFF Research Database (Denmark)

Jensen-Johansen, Mikael Birkelund; Christensen, Søren; Valdimarsdottir, Heiddis

2013-01-01

Objective: To examine the effects of an expressive writing intervention (EWI) on cancer-related distress, depressive symptoms, and mood in women treated for early stage breast cancer. Methods: A nationwide sample of 507 Danish women who had recently completed treatment for primary breast cancer...... were randomly assigned to three 20-min home-based writing exercises, one week apart, focusing on either emotional disclosure (EWI group) or a non-emotional topic (control group). Cancer-related distress [Impact of Event Scale (IES)], depressive symptoms (Beck Depression Inventory—Short Form......), and negative (37-item Profile of Moods State) and positive mood (Passive Positive Mood Scale) were assessed at baseline and at 3 and 9 months post-intervention. Choice of writing topic (cancer versus other), alexithymia (20-item Toronto Alexithymia Scale), and social constraints (Social Constraints Scale) were...

Posttraumatic Stress Disorder Among Bereaved Relatives of Cancer Patients

DEFF Research Database (Denmark)

Elklit, Ask; Reinholt, N.; Nielsen, Louise Hjort

2010-01-01

The aim of this study was to assess post-traumatic stress disorder (PTSD) and predictors of PTSD in individuals who experienced the loss of a close relative to cancer. A total of 251 bereaved relatives ages 14 to 76 (M = 41.3, SD = 16.8) were recruited at a counseling service for cancer patients...

Meat-related compounds and colorectal cancer risk by anatomical subsite.

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Miller, Paige E; Lazarus, Philip; Lesko, Samuel M; Cross, Amanda J; Sinha, Rashmi; Laio, Jason; Zhu, Jay; Harper, Gregory; Muscat, Joshua E; Hartman, Terryl J

2013-01-01

Since meat may be involved in the etiology of colorectal cancer, associations between meat-related compounds were examined to elucidate underlying mechanisms in a population-based case-control study. Participants (989 cases/1,033 healthy controls) completed a food frequency questionnaire with a meat-specific module. Multivariable logistic regression was used to examine associations between meat variables and colorectal cancer; polytomous logistic regression was used for subsite-specific analyses. The following significant positive associations were observed for meat-related compounds: 2-amino-3,4,8-trimethylimidazo[4,5-f]quinoxaline (DiMeIQx) and colorectal, distal colon, and rectal tumors; 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and colorectal and colon cancer tumors; nitrites/nitrates and proximal colon cancer; 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and rectal cancer; and benzo[a]pyrene and rectal cancer (P-trends cancer and pan-fried red meat and colorectal cancer were found (P-trends cancer; and well-done/charred poultry and colorectal, colon, and proximal colon tumors (P-trends nitrates may be involved in colorectal cancer etiology. Further examination into the unexpected inverse associations between poultry and colorectal cancer is warranted.

Chemoenzymatically synthesized multimeric Tn/STn MUC1 glycopeptides elicit cancer-specific anti-MUC1 antibody responses and override tolerance

DEFF Research Database (Denmark)

Sørensen, Anne Louise; Reis, Celso A; Tarp, Mads A

2005-01-01

The MUC1 mucin represents a prime target antigen for cancer immunotherapy because it is abundantly expressed and aberrantly glycosylated in carcinomas. Attempts to generate strong humoral immunity to MUC1 by immunization with peptides have generally failed partly because of tolerance. In this stu...

Morphology, histochemistry and glycosylation of the placenta and associated tissues in the European hedgehog (Erinaceus europaeus)

DEFF Research Database (Denmark)

Jones, Carolyn J P; Carter, A M; Allen, W R

2016-01-01

glycosylated. Yolk sac inner and outer endoderm expressed similar glycans except for N-acetylgalactosamine residues in endodermal acini. DISCUSSION: New features of near-term hedgehog placenta and associated tissues are presented, including their glycosylation, and novel yolk sac acinar structures......INTRODUCTION: There are few descriptions of the placenta and associated tissues of the European hedgehog (Erinaceus europaeus) and here we present findings on a near-term pregnant specimen. METHODS: Tissues were examined grossly and then formalin fixed and wax-embedded for histology...... and immunocytochemistry (cytokeratin) and resin embedded for lectin histochemistry. RESULTS: Each of four well-developed and near term hoglets displayed a discoid, haemochorial placenta with typical labyrinth and spongy zones. In addition there was a paraplacenta incorporating Reichert's membrane and a largely detached...

Risk of cancer in relatives of patients with myotonic dystrophy

DEFF Research Database (Denmark)

Lund, M; Diaz, L J; Gørtz, S

2014-01-01

BACKGROUND AND PURPOSE: Myotonic dystrophies (DM) are autosomal dominantly inherited neuromuscular disorders caused by unstable nucleotide repeat expansions. DM and cancer have been associated, but the pathogenesis behind the association remains unclear. It could relate to derived effects of the DM...... genotype in which case non-DM relatives of DM patients would not be expected to be at increased risk of cancer. To elucidate this, a population-based cohort study investigating risk of cancer in relatives of DM patients was conducted. METHODS: DM was identified using the National Danish Patient Registry...

Recent declines in cancer incidence: related to the Great Recession?

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Gomez, Scarlett Lin; Canchola, Alison J; Nelson, David O; Keegan, Theresa H M; Clarke, Christina A; Cheng, Iona; Shariff-Marco, Salma; DeRouen, Mindy; Catalano, Ralph; Satariano, William A; Davidson-Allen, Kathleen; Glaser, Sally L

2017-02-01

In recent years, cancer case counts in the U.S. underwent a large, rapid decline-an unexpected change given population growth for older persons at highest cancer risk. As these declines coincided with the Great Recession, we examined whether they were related to economic conditions. Using California Cancer Registry data from California's 30 most populous counties, we analyzed trends in cancer incidence during pre-recession (1996-2007) and recession/recovery (2008-2012) periods for all cancers combined and the ten most common sites. We evaluated the recession's association with rates using a multifactorial index that measured recession impact, and modeled associations between case counts and county-level unemployment rates using Poisson regression. Yearly cancer incidence rate declines were greater during the recession/recovery (3.3% among males, 1.4% among females) than before (0.7 and 0.5%, respectively), particularly for prostate, lung, and colorectal cancers. Lower case counts, especially for prostate and liver cancer among males and breast cancer, melanoma, and ovarian cancer among females, were associated with higher unemployment rates, irrespective of time period, but independent of secular effects. The associations for melanoma translated up to a 3.6% decrease in cases with each 1% increase in unemployment. Incidence declines were not greater in counties with higher recession impact index. Although recent declines in incidence of certain cancers are not differentially impacted by economic conditions related to the Great Recession relative to pre-recession conditions, the large recent absolute declines in the case counts of some cancer may be attributable to the large declines in unemployment in the recessionary period. This may occur through decreased engagement in preventive health behaviors, particularly for clinically less urgent cancers. Continued monitoring of trends is important to detect any rises in incidence rates as deferred diagnoses come to

The glycosylation status of the murine hepatitis coronavirus M protein affects the interferogenic capacity of the virus in vitro and its ability to replicate in the liver but not the brain

International Nuclear Information System (INIS)

Haan, Cornelis A.M. de; Wit, Marel de; Kuo, Lili; Montalto-Morrison, Cynthia; Haagmans, Bart L.; Weiss, Susan R.; Masters, Paul S.; Rottier, Peter J.M.

2003-01-01

The coronavirus M protein, the most abundant coronaviral envelope component, is invariably glycosylated, which provides the virion with a diffuse, hydrophilic cover on its outer surface. Remarkably, while the group 1 and group 3 coronaviruses all have M proteins with N-linked sugars, the M proteins of the group 2 coronaviruses [e.g., mouse hepatitis virus (MHV)] are O-glycosylated. The conservation of the N- and O-glycosylation motifs suggests that each of these types of carbohydrate modifications is beneficial to their respective virus. Since glycosylation of the M protein is not required for virus assembly, the oligosaccharides are likely to be involved in the virus-host interaction. In order to investigate the role of the M protein glycosylation in the host, two genetically modified MHVs were generated by using targeted RNA recombination. The recombinant viruses carried M proteins that were either N-glycosylated or not glycosylated at all, and these were compared with the parental, O-glycosylated, virus. The M protein glycosylation state did not influence the tissue culture growth characteristics of the recombinant viruses. However, it affected their interferogenic capacity as measured using fixed, virus-infected cells. Viruses containing M proteins with N-linked sugars induced type I interferons to higher levels than viruses carrying M proteins with O-linked sugars. MHV with unglycosylated M proteins appeared to be a poor interferon inducer. In mice, the recombinant viruses differed in their ability to replicate in the liver, but not in the brain, whereas their in vivo interferogenic capacity did not appear to be affected by their glycosylation status. Strikingly, their abilities to replicate in the liver correlated with their in vitro interferogenic capacity. This apparent correlation might be explained by the functioning of lectins, such as the mannose receptor, which are abundantly expressed in the liver but also play a role in the induction of interferon

Chiral reagents in glycosylation and modification of carbohydrates.

Science.gov (United States)

Wang, Hao-Yuan; Blaszczyk, Stephanie A; Xiao, Guozhi; Tang, Weiping

2018-02-05

Carbohydrates play a significant role in numerous biological events, and the chemical synthesis of carbohydrates is vital for further studies to understand their various biological functions. Due to the structural complexity of carbohydrates, the stereoselective formation of glycosidic linkages and the site-selective modification of hydroxyl groups are very challenging and at the same time extremely important. In recent years, the rapid development of chiral reagents including both chiral auxiliaries and chiral catalysts has significantly improved the stereoselectivity for glycosylation reactions and the site-selectivity for the modification of carbohydrates. These new tools will greatly facilitate the efficient synthesis of oligosaccharides, polysaccharides, and glycoconjugates. In this tutorial review, we will summarize these advances and highlight the most recent examples.

Revealing the mechanisms of protein disorder and N-glycosylation in CD44-hyaluronan binding using molecular simulation

Directory of Open Access Journals (Sweden)

Olgun eGuvench

2015-06-01

Full Text Available The extracellular N-terminal hyaluronan binding domain (HABD of CD44 is a small globular domain that confers hyaluronan (HA binding functionality to this large transmembrane glycoprotein. When recombinantly expressed by itself, HABD exists as a globular water-soluble protein that retains the capacity to bind HA. This has enabled atomic-resolution structural biology experiments that have revealed the structure of HABD and its binding mode with oligomeric HA. Such experiments have also pointed to an order-to-disorder transition in HABD that is associated with HA binding. However, it had remained unclear how this structural transition was involved in binding since it occurs in a region of HABD distant from the HA-binding site. Furthermore, HABD is known to be N-glycosylated, and such glycosylation can diminish HA binding when the associated N-glycans are capped with sialic acid residues. The intrinsic flexibility of disordered proteins and of N-glycans makes it difficult to apply experimental structural biology approaches to probe the molecular mechanisms of how the order-to-disorder transition and N-glycosylation can modulate HA binding by HABD. We review recent results from molecular dynamics simulations that provide atomic-resolution mechanistic understanding of such modulation to help bridge gaps between existing experimental binding and structural biology data. Findings from these simulations include: Tyr42 may function as a molecular switch that converts the HA binding site from a low affinity to a high affinity state; in the partially-disordered form of HABD, basic amino acids in the C-terminal region can gain sufficient mobility to form direct contacts with bound HA to further stabilize binding; and terminal sialic acids on covalently-attached N-glycans can form charge-paired hydrogen bonding interactions with basic amino acids that could otherwise bind to HA, thereby blocking HA binding to glycosylated CD44 HABD.

Involvement of Relatives in Cancer Rehabilitation

DEFF Research Database (Denmark)

Ledderer, Loni; Madsen, Biddy; Mogensen, Ole

2010-01-01

using validated questionnaires including EORTC QLQ-C30, POMS, WHO-5 well-being index and supplemented with ad hoc questions. The rehabilitation needs are furthermore explored in qualitative interviews and participant-observations with pairs from both the intervention and the control group. Result...... with lung cancer or gynecological cancer are included in the study together with a relative by patient’s choice both giving informed consent (N=120 pairs). The intervention group (60 pairs) receives three supportive conversations with a trained nurse initiated from the admission date and completed within...

Is there an increased risk of cancer among spouses of patients with an HPV-related cancer: A systematic review.

Science.gov (United States)

Mirghani, Haitham; Sturgis, Erich M; Aupérin, Anne; Monsonego, Joseph; Blanchard, Pierre

2017-04-01

High-risk human papillomaviruses (HR-HPV) are the cause of most ano-genital cancers and a fast growing subset of oropharyngeal cancer. As these malignancies occur as a result of an HPV- infection transmitted through intimate contact, many patients with HPV- induced cancer and their partners are concerned about HPV-transmission and the potential partners' cancer risk. Few studies have addressed this issue and whether the HPV-related cancer risk of partners of patients with HPV-related cancers is comparable to or greater than that of the general population. We performed a systematic review of the published literature addressing this issue. Out of 1055 references screened, 53 articles were found eligible for inclusion. Regarding the issue of coincidence of HPV-induced oropharyngeal and/or anogenital cancers in couples, 13 case-reports or case-series were reported and 9 larger studies based on population-registries. Four of these registry studies showed an increased risk of cervical cancer in the partner while four did not. Among the four positive studies, odds ratios for the development of HPV-related cancer among spouses were between 2.6 and 6.7. One study showed an increased risk of tongue or tonsil cancer among husbands of women with cervical dysplasia or cancer. Overall the absolute risk increase in all these studies was small, on the order of 1-3%, although potentially underestimated. Indeed, all these studies have assessed partner's cancer risk at only one anatomical site whereas HPV- related malignancies can affect different locations. This systematic review suggests a small trend of increase risk in HPV-associated cancers among spouses of patients with HPV-related cancer. Copyright © 2017 Elsevier Ltd. All rights reserved.

A Crude 1-DNJ Extract from Home Made Bombyx Batryticatus Inhibits Diabetic Cardiomyopathy-Associated Fibrosis in db/db Mice and Reduces Protein N-Glycosylation Levels

Directory of Open Access Journals (Sweden)

Qing Zhao

2018-06-01

Full Text Available The traditional Chinese drug Bombyx Batryticatus (BB, which is also named the white stiff silkworm, has been widely used in Chinese clinics for thousands of years. It is famous for its antispasmodic and blood circulation-promoting effects. Cardiomyocyte hypertrophy, interstitial cell hyperplasia, and myocardial fibrosis are closely related to the N-glycosylation of key proteins. To examine the alterations of N-glycosylation that occur in diabetic myocardium during the early stage of the disease, and to clarify the therapeutic effect of 1-Deoxynojirimycin (1-DNJ extracted from BB, we used the db/db (diabetic mouse model and an approach based on hydrophilic chromatography solid-phase extraction integrated with an liquid Chromatograph Mass Spectrometer (LC-MS identification strategy to perform a site-specific N-glycosylation analysis of left ventricular cardiomyocyte proteins. Advanced glycation end products (AGEs, hydroxyproline, connective tissue growth factor (CTGF, and other serum biochemical indicators were measured with enzyme-linked immunosorbent assays (ELISA. In addition, the α-1,6-fucosylation of N-glycans was profiled with lens culinaris agglutinin (LCA lectin blots and fluorescein isothiocyanate (FITC-labelled lectin affinity histochemistry. The results indicated that 1-DNJ administration obviously downregulated myocardium protein N-glycosylation in db/db mice. The expression levels of serum indicators and fibrosis-related cytokines were reduced significantly by 1-DNJ in a dose-dependent manner. The glycan α-1,6-fucosylation level of the db/db mouse myocardium was elevated, and the intervention effect of 1-DNJ administration on N-glycan α-1,6-fucosylation was significant. To verify this result, the well-known transforming growth factor-β (TGF-β/Smad2/3 pathway was selected, and core α-1,6-fucosylated TGF-β receptor II (TGFR-βII was analysed semi-quantitatively with western blotting. The result supported the conclusions obtained

Concordance with World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) guidelines for cancer prevention and obesity-related cancer risk in the Framingham Offspring cohort (1991-2008).

Science.gov (United States)

Makarem, Nour; Lin, Yong; Bandera, Elisa V; Jacques, Paul F; Parekh, Niyati

2015-02-01

This prospective cohort study evaluates associations between healthful behaviors consistent with WCRF/AICR cancer prevention guidelines and obesity-related cancer risk, as a third of cancers are estimated to be preventable. The study sample consisted of adults from the Framingham Offspring cohort (n = 2,983). From 1991 to 2008, 480 incident doctor-diagnosed obesity-related cancers were identified. Data on diet, measured by a food frequency questionnaire, anthropometric measures, and self-reported physical activity, collected in 1991 was used to construct a 7-component score based on recommendations for body fatness, physical activity, foods that promote weight gain, plant foods, animal foods, alcohol, and food preservation, processing, and preparation. Multivariable Cox regression models were used to estimate associations between the computed score, its components, and subcomponents in relation to obesity-related cancer risk. The overall score was not associated with obesity-related cancer risk after adjusting for age, sex, smoking, energy, and preexisting conditions (HR 0.94, 95 % CI 0.86-1.02). When score components were evaluated separately, for every unit increment in the alcohol score, there was 29 % lower risk of obesity-related cancers (HR 0.71, 95 % CI 0.51-0.99) and 49-71 % reduced risk of breast, prostate, and colorectal cancers. Every unit increment in the subcomponent score for non-starchy plant foods (fruits, vegetables, and legumes) among participants who consume starchy vegetables was associated with 66 % reduced risk of colorectal cancer (HR 0.44, 95 % CI 0.22-0.88). Lower alcohol consumption and a plant-based diet consistent with the cancer prevention guidelines were associated with reduced risk of obesity-related cancers in this population.