28 results on '"Dwek, Ra"'
Search Results
2. EUROCarbDB: An open-access platform for glycoinformatics.
- Author
-
von der Lieth CW, Freire AA, Blank D, Campbell MP, Ceroni A, Damerell DR, Dell A, Dwek RA, Ernst B, Fogh R, Frank M, Geyer H, Geyer R, Harrison MJ, Henrick K, Herget S, Hull WE, Ionides J, Joshi HJ, Kamerling JP, Leeflang BR, Lütteke T, Lundborg M, Maass K, Merry A, Ranzinger R, Rosen J, Royle L, Rudd PM, Schloissnig S, Stenutz R, Vranken WF, Widmalm G, and Haslam SM more...
- Subjects
- Animals, Carbohydrate Conformation, Computational Biology, Glycomics, Humans, Models, Molecular, Molecular Weight, Online Systems, Carbohydrates chemistry, Databases as Topic, Software
- Abstract
The EUROCarbDB project is a design study for a technical framework, which provides sophisticated, freely accessible, open-source informatics tools and databases to support glycobiology and glycomic research. EUROCarbDB is a relational database containing glycan structures, their biological context and, when available, primary and interpreted analytical data from high-performance liquid chromatography, mass spectrometry and nuclear magnetic resonance experiments. Database content can be accessed via a web-based user interface. The database is complemented by a suite of glycoinformatics tools, specifically designed to assist the elucidation and submission of glycan structure and experimental data when used in conjunction with contemporary carbohydrate research workflows. All software tools and source code are licensed under the terms of the Lesser General Public License, and publicly contributed structures and data are freely accessible. The public test version of the web interface to the EUROCarbDB can be found at http://www.ebi.ac.uk/eurocarb. more...
- Published
- 2011
- Full Text
- View/download PDF
Catalog
3. Polysaccharide mimicry of the epitope of the broadly neutralizing anti-HIV antibody, 2G12, induces enhanced antibody responses to self oligomannose glycans.
- Author
-
Dunlop DC, Bonomelli C, Mansab F, Vasiljevic S, Doores KJ, Wormald MR, Palma AS, Feizi T, Harvey DJ, Dwek RA, Crispin M, and Scanlan CN
- Subjects
- Antibodies, Neutralizing, HIV Envelope Protein gp120 metabolism, Mannose metabolism, Molecular Mimicry immunology, Epitopes immunology, HIV Antibodies immunology, Mannose immunology
- Abstract
Immunologically, "self" carbohydrates protect the HIV-1 surface glycoprotein, gp120, from antibody recognition. However, one broadly neutralizing antibody, 2G12, neutralizes primary viral isolates by direct recognition of Manalpha1-->2Man motifs formed by the host-derived oligomannose glycans of the viral envelope. Immunogens, capable of eliciting antibodies of similar specificity to 2G12, are therefore candidates for HIV/AIDS vaccine development. In this context, it is known that the yeast mannan polysaccharides exhibit significant antigenic mimicry with the glycans of HIV-1. Here, we report that modulation of yeast polysaccharide biosynthesis directly controls the molecular specificity of cross-reactive antibodies to self oligomannose glycans. Saccharomyces cerevisiae mannans are typically terminated by alpha1-->3-linked mannoses that cap a Manalpha1-->2Man motif that otherwise closely resembles the part of the oligomannose epitope recognized by 2G12. Immunization with S. cerevisiae deficient for the alpha1-->3 mannosyltransferase gene (DeltaMnn1), but not with wild-type S. cerevisiae, reproducibly elicited antibodies to the self oligomannose glycans. Carbohydrate microarray analysis of DeltaMnn1 immune sera revealed fine carbohydrate specificity to Manalpha1-->2Man units, closely matching that of 2G12. These specificities were further corroborated by enzyme-linked immunosorbent assay with chemically defined glycoforms of gp120. These antibodies exhibited remarkable similarity in the carbohydrate specificity to 2G12 and displayed statistically significant, albeit extremely weak, neutralization of HIV-1 compared to control immune sera. These data confirm the Manalpha1-->2Man motif as the primary carbohydrate neutralization determinant of HIV-1 and show that the genetic modulation of microbial polysaccharides is a route towards immunogens capable of eliciting antibody responses to the glycans of HIV-1. more...
- Published
- 2010
- Full Text
- View/download PDF
4. A strategy to reveal potential glycan markers from serum glycoproteins associated with breast cancer progression.
- Author
-
Abd Hamid UM, Royle L, Saldova R, Radcliffe CM, Harvey DJ, Storr SJ, Pardo M, Antrobus R, Chapman CJ, Zitzmann N, Robertson JF, Dwek RA, and Rudd PM
- Subjects
- Adult, Aged, Chromatography, High Pressure Liquid, Disease Progression, Female, Humans, Middle Aged, Retrospective Studies, Biomarkers, Tumor blood, Breast Neoplasms blood, Breast Neoplasms pathology, Glycoproteins blood, Polysaccharides blood
- Abstract
Aberrant glycosylation on glycoproteins that are either presented on the surface or secreted by cancer cells is a potential source of disease biomarkers and provides insights into disease pathogenesis. N-Glycans of the total serum glycoproteins from advanced breast cancer patients and healthy individuals were sequenced by HPLC with fluorescence detection coupled with exoglycosidase digestions and mass spectrometry. We observed a significant increase in a trisialylated triantennary glycan containing alpha1,3-linked fucose which forms part of the sialyl Lewis x epitope. Following digestion of the total glycan pool with a combination of sialidase and beta-galactosidase, we segregated and quantified a digestion product, a monogalactosylated triantennary structure containing alpha1,3-linked fucose. We compared breast cancer patients and controls and detected a 2-fold increase in this glycan marker in patients. In 10 patients monitored longitudinally, we showed a positive correlation between this glycan marker and disease progression and also demonstrated its potential as a better indicator of metastasis compared to the currently used biomarkers, CA 15-3 and carcinoembryonic antigen (CEA). A pilot glycoproteomic study of advanced breast cancer serum highlighted acute-phase proteins alpha1-acid glycoprotein, alpha1-antichymotrypsin, and haptoglobin beta-chain as contributors to the increase in the glycan marker which, when quantified from each of these proteins, marked the onset of metastasis in advance of the CA 15-3 marker. These preliminary findings suggest that specific glycans and glycoforms of proteins may be candidates for improved markers in the monitoring of breast cancer progression. more...
- Published
- 2008
- Full Text
- View/download PDF
5. The O-linked glycosylation of secretory/shed MUC1 from an advanced breast cancer patient's serum.
- Author
-
Storr SJ, Royle L, Chapman CJ, Hamid UM, Robertson JF, Murray A, Dwek RA, and Rudd PM
- Subjects
- Breast Neoplasms metabolism, Carbohydrate Conformation, Cell Line, Female, Glucans biosynthesis, Glycosylation, Humans, Mucin-1 biosynthesis, Neoplasm Proteins biosynthesis, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Breast Neoplasms chemistry, Glucans chemistry, Mucin-1 chemistry, Neoplasm Proteins chemistry
- Abstract
MUC1 is a high molecular weight glycoprotein that is overexpressed in breast cancer. Aberrant O-linked glycosylation of MUC1 in cancer has been implicated in disease progression. We investigated the O-linked glycosylation of MUC1 purified from the serum of an advanced breast cancer patient. O-Glycans were released by hydrazinolysis and analyzed by liquid chromatography-electrospray ionization-mass spectrometry and by high performance liquid chromatography coupled with sequential exoglycosidase digestions. Core 1 type glycans (83%) dominated the profile which also confirmed high levels of sialylation: 80% of the glycans were mono-, di- or trisialylated. Core 2 type structures contributed approximately 17% of the assigned glycans and the oncofoetal Thomsen-Friedenreich (TF) antigen (Galbeta1-3GalNAc) accounted for 14% of the total glycans. Interestingly, two core 1 type glycans were identified that had sialic acid alpha2-8 linked to sialylated core 1 type structures (9% of the total glycan pool). This is the first O-glycan analysis of MUC1 from the serum of a breast cancer patient; the results suggest that amongst the cell lines commonly used to express recombinant MUC1 the T47D cell line processes glycans that are most similar to patient-derived material. more...
- Published
- 2008
- Full Text
- View/download PDF
6. Changes of serum glycans during sepsis and acute pancreatitis.
- Author
-
Gornik O, Royle L, Harvey DJ, Radcliffe CM, Saldova R, Dwek RA, Rudd P, and Lauc G
- Subjects
- Acute Disease, Anti-Inflammatory Agents pharmacology, Chromatography, Ion Exchange methods, Cytokines metabolism, Fucose chemistry, Glycosylation, Humans, Inflammation, Lipids chemistry, Mannose chemistry, Oligosaccharides metabolism, Pancreatitis metabolism, Pancreatitis pathology, Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase metabolism, Polysaccharides chemistry, Sepsis pathology, Sialyl Lewis X Antigen, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Pancreatitis blood, Polysaccharides blood, Polysaccharides metabolism, Sepsis blood
- Abstract
Acute inflammatory response is a complex process associated with the production of both pro- and anti-inflammatory mediators. Although it is generally considered to be a single homeostatic mechanism, there are differences associated with the nature and the site of inflammation. We examined the changes of N-linked glycans released from the serum of a patient with sepsis and a patient with acute pancreatitis during the first eight days of the disease. Sera were taken from patients at the time of reporting to hospital and then three more times. The blood from a healthy individual was drawn on one occasion only. Glycans were released using N-glycosidase F and were subjected to normal phase and weak anion exchange high-performance liquid chromatography, exoglycosidase digestions, and mass spectrometry. The levels of identified structures have been followed through the course of disease and compared to the control levels. Changes in serum glycans were found to occur very early in acute inflammation. The most prominent differences include the increase in ratio of outer arm to core fucose, increase in the amount of tetrasialylated structures, changes in the levels of mannose structures, and in the degree of branching. The relative proportions of different glycans changed daily and some differences were also observed between sepsis and pancreatitis, probably reflecting that in these two conditions, the acute phase response is triggered by a different stimulus that is associated with different patterns of production of cytokines. more...
- Published
- 2007
- Full Text
- View/download PDF
7. Ovarian cancer is associated with changes in glycosylation in both acute-phase proteins and IgG.
- Author
-
Saldova R, Royle L, Radcliffe CM, Abd Hamid UM, Evans R, Arnold JN, Banks RE, Hutson R, Harvey DJ, Antrobus R, Petrescu SM, Dwek RA, and Rudd PM
- Subjects
- Adult, Aged, Aged, 80 and over, Biomarkers chemistry, Carbohydrate Conformation, Female, Glycomics, Humans, Middle Aged, Ovarian Neoplasms blood, Polysaccharides chemistry, Sensitivity and Specificity, alpha 1-Antichymotrypsin chemistry, Acute-Phase Proteins chemistry, Gene Expression Regulation, Neoplastic, Glycosylation, Immunoglobulin G chemistry, Ovarian Neoplasms metabolism, Ovarian Neoplasms pathology
- Abstract
Ovarian cancer is the fourth most common cancer in women in the Western world. In a pilot scale study, we highlight changes in the total serum glycome of patients with advanced ovarian cancer that might shed insight into disease pathogenesis. These changes include increases in levels of core fucosylated, agalactosyl biantennary glycans (FA2) and sialyl Lewis x (SLe(x)). To investigate further which proteins contribute to these alterations, we developed technology to analyze simultaneously the glycosylation of protein glycoforms contained in single spots excised from a 2D gel (<1 ng protein). The acute-phase proteins, haptoglobin, alpha1-acid glycoprotein, and alpha1-antichymotrypsin from patients contained elevated levels of subsets of glycoforms containing SLe(x). We also established that IgG heavy chains from patients contained twice the level of FA2 compared with healthy controls. Serum CA125 is the only biomarker that is used routinely, and there is a need for complementary markers that will improve both sensitivity and specificity. There was some preliminary indication that combinations of changes in the serum glycome might improve the separation of ovarian cancer and benign tumors; however, a larger study using data receiver operating characteristic curves will be required to draw any firm conclusions. more...
- Published
- 2007
- Full Text
- View/download PDF
8. Glycosylation of serum ribonuclease 1 indicates a major endothelial origin and reveals an increase in core fucosylation in pancreatic cancer.
- Author
-
Barrabés S, Pagès-Pons L, Radcliffe CM, Tabarés G, Fort E, Royle L, Harvey DJ, Moenner M, Dwek RA, Rudd PM, De Llorens R, and Peracaula R
- Subjects
- Carbohydrate Sequence, Electrophoresis, Gel, Two-Dimensional, Enzyme-Linked Immunosorbent Assay, Glycoside Hydrolases metabolism, Glycosylation, Humans, Kinetics, Mass Spectrometry, Neuraminidase metabolism, Oligosaccharides chemistry, Oligosaccharides isolation & purification, Reference Values, Ribonuclease, Pancreatic chemistry, Ribonuclease, Pancreatic isolation & purification, Spectrometry, Mass, Electrospray Ionization, Endothelium, Vascular enzymology, Fucose metabolism, Pancreatic Neoplasms enzymology, Ribonuclease, Pancreatic blood
- Abstract
Human pancreatic ribonuclease 1 (RNase 1) is a glycoprotein expressed mainly by the pancreas and also found in endothelial cells. The diagnosis of pancreatic cancer (PaC) remains difficult and therefore the search for sensitive and specific markers is required. Previous studies showed that RNase 1 from human healthy pancreas contained only neutral glycans, whereas RNase 1 from PaC cell lines contained sialylated structures. To determine whether these glycan tumor cell-associated changes were also characteristic of serum RNase 1 and could be used as a marker of PaC, we have analyzed the glycosylation of serum RNase 1. The origin of serum RNase 1 was also investigated. Serum RNase 1 from two PaC patients and two controls was purified and the glycans analyzed by high-performance liquid chromatography (HPLC)-based sequencing and mass spectrometry. Although normal and tumor serum RNase 1 contained the same glycan structures, there was an increase of 40% in core fucosylation in the main sialylated biantennary glycans in the PaC serum RNase 1. This change in proportion would be indicative of a subset of tumor-associated glycoforms of RNase 1, which may provide a biomarker for PaC. Two-dimensional electrophoresis of the RNase 1 from several endothelial cell lines, EA.hy926, human umbilical vein endothelial cells (HUVEC), human mammary microvessel endothelial cells (HuMMEC), and human lung microvessel endothelial cells (HuLEC), showed basically the same pattern and was also very similar to that of serum RNase 1. RNase 1 from EA.hy926 was then purified and presented a glycosylation profile very similar to that from serum RNase 1, suggesting that endothelial cells are the main source of this enzyme. more...
- Published
- 2007
- Full Text
- View/download PDF
9. Comparison of the methods for profiling glycoprotein glycans--HUPO Human Disease Glycomics/Proteome Initiative multi-institutional study.
- Author
-
Wada Y, Azadi P, Costello CE, Dell A, Dwek RA, Geyer H, Geyer R, Kakehi K, Karlsson NG, Kato K, Kawasaki N, Khoo KH, Kim S, Kondo A, Lattova E, Mechref Y, Miyoshi E, Nakamura K, Narimatsu H, Novotny MV, Packer NH, Perreault H, Peter-Katalinic J, Pohlentz G, Reinhold VN, Rudd PM, Suzuki A, and Taniguchi N more...
- Subjects
- Carbohydrate Conformation, Glycopeptides chemistry, Glycoproteins chemistry, Humans, Mass Spectrometry, Models, Molecular, Oligosaccharides chemistry, Polysaccharides chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Gene Expression Profiling methods, Genetic Diseases, Inborn genetics, Glycoproteins genetics, Polysaccharides genetics, Proteome
- Abstract
Mass spectrometry (MS) of glycoproteins is an emerging field in proteomics, poised to meet the technical demand for elucidation of the structural complexity and functions of the oligosaccharide components of molecules. Considering the divergence of the mass spectrometric methods employed for oligosaccharide analysis in recent publications, it is necessary to establish technical standards and demonstrate capabilities. In the present study of the Human Proteome Organisation (HUPO) Human Disease Glycomics/Proteome Initiative (HGPI), the same samples of transferrin and immunoglobulin-G were analyzed for N-linked oligosaccharides and their relative abundances in 20 laboratories, and the chromatographic and mass spectrometric analysis results were evaluated. In general, matrix-assisted laser desorption/ionization (MALDI) time-of-flight MS of permethylated oligosaccharide mixtures carried out in six laboratories yielded good quantitation, and the results can be correlated to those of chromatography of reductive amination derivatives. For underivatized oligosaccharide alditols, graphitized carbon-liquid chromatography (LC)/electrospray ionization (ESI) MS detecting deprotonated molecules in the negative ion mode provided acceptable quantitation. The variance of the results among these three methods was small. Detailed analyses of tryptic glycopeptides employing either nano LC/ESI MS/MS or MALDI MS demonstrated excellent capability to determine site-specific or subclass-specific glycan profiles in these samples. Taking into account the variety of MS technologies and options for distinct protocols used in this study, the results of this multi-institutional study indicate that MS-based analysis appears as the efficient method for identification and quantitation of oligosaccharides in glycomic studies and endorse the power of MS for glycopeptide characterization with high sensitivity in proteomic programs. more...
- Published
- 2007
- Full Text
- View/download PDF
10. Oligosaccharides modulate the apoptotic activity of glycodelin.
- Author
-
Jayachandran R, Radcliffe CM, Royle L, Harvey DJ, Dwek RA, Rudd PM, and Karande AA
- Subjects
- Animals, Asparagine metabolism, Cell Line, Cell Proliferation drug effects, Cricetinae, Cricetulus, Female, Glycodelin, Glycoproteins genetics, Glycoproteins pharmacology, Glycosylation, Humans, Insecta, Jurkat Cells, Mutation, N-Acetylneuraminic Acid metabolism, Polysaccharides metabolism, Pregnancy Proteins genetics, Pregnancy Proteins pharmacology, Recombinant Proteins genetics, Recombinant Proteins pharmacology, Apoptosis, Glycoproteins physiology, Oligosaccharides pharmacology, Pregnancy Proteins physiology
- Abstract
GlycodelinA (GdA), a multifunctional glycoprotein secreted at high concentrations by the uterine endometrium during the early phases of pregnancy, carries glycan chains on asparagines at positions N28 and N63. GdA purified from amniotic fluid is known to be a suppressor of T-cell proliferation, an inducer of T-cell apoptosis, and an inhibitor of sperm-zona binding in contrast to its glycoform, glycodelinS (GdS), which is secreted by the seminal vesicles into the seminal plasma. The oligosaccharide chains of GdA terminate in sialic acid residues, whereas those of GdS are not sialylated but are heavily fucosylated. Our previous work has shown that the apoptogenic activity of GdA resides in the protein backbone, and we have also demonstrated the importance of sialylation for the manifestation of GdA-induced apoptosis. Recombinant glycodelin (Gd) expressed in the Sf21 insect cell line yielded an apoptotically active Gd; however, the same gene expressed in the insect cell line Tni produced apoptotically inactive Gd, as observed with the gene expressed in the Chinese hamster ovary (CHO) cell line and earlier in Pichia pastoris. Glycan analysis of the Tni and Sf21 cell line-expressed Gd proteins reveals differences in their glycan structures, which modulate the manifestation of apoptogenic activity of Gd. Through apoptotic assays carried out with the wild-type (WT) and glycosylation mutants of Gd expressed in Sf21 and Tni cells before and after mannosidase digestion, we conclude that the accessibility to the apoptogenic region of Gd is influenced by the size of the glycans. more...
- Published
- 2006
- Full Text
- View/download PDF
11. Inhibition of hybrid- and complex-type glycosylation reveals the presence of the GlcNAc transferase I-independent fucosylation pathway.
- Author
-
Crispin M, Harvey DJ, Chang VT, Yu C, Aricescu AR, Jones EY, Davis SJ, Dwek RA, and Rudd PM
- Subjects
- 1-Deoxynojirimycin analogs & derivatives, 1-Deoxynojirimycin pharmacology, Alkaloids pharmacology, Animals, CHO Cells, Cell Line, Cricetinae, DNA, Complementary, Enzyme Inhibitors pharmacology, Glycoproteins genetics, Glycoproteins metabolism, Glycosylation, Humans, N-Acetylglucosaminyltransferases genetics, Recombinant Proteins metabolism, Spectrometry, Mass, Electrospray Ionization, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Swainsonine pharmacology, alpha-Mannosidase antagonists & inhibitors, Fucose metabolism, N-Acetylglucosaminyltransferases metabolism
- Abstract
A mammalian N-acetylglucosamine (GlcNAc) transferase I (GnT I)-independent fucosylation pathway is revealed by the use of matrix-assisted laser desorption/ionization (MALDI) and negative-ion nano-electrospray ionization (ESI) mass spectrometry of N-linked glycans from natively folded recombinant glycoproteins, expressed in both human embryonic kidney (HEK) 293S and Chinese hamster ovary (CHO) Lec3.2.8.1 cells deficient in GnT I activity. The biosynthesis of core fucosylated Man5GlcNAc2 glycans was enhanced in CHO Lec3.2.8.1 cells by the alpha-glucosidase inhibitor, N-butyldeoxynojirimycin (NB-DNJ), leading to the increase in core fucosylated Man5GlcNAc2 glycans and the biosynthesis of a novel core fucosylated monoglucosylated oligomannose glycan, Glc1Man7GlcNAc2Fuc. Furthermore, no fucosylated Man9GlcNAc2 glycans were detected following inhibition of alpha-mannosidase I with kifunensine. Thus, core fucosylation is prevented by the presence of terminal alpha1-2 mannoses on the 6-antennae but not the 3-antennae of the trimannosyl core. Fucosylated Man5GlcNAc2 glycans were also detected on recombinant glycoprotein from HEK 293T cells following inhibition of Golgi alpha-mannosidase II with swainsonine. The paucity of fucosylated oligomannose glycans in wild-type mammalian cells is suggested to be due to kinetic properties of the pathway rather than the absence of the appropriate catalytic activity. The presence of the GnT I-independent fucosylation pathway is an important consideration when engineering mammalian glycosylation. more...
- Published
- 2006
- Full Text
- View/download PDF
12. Different glycan structures in prostate-specific antigen from prostate cancer sera in relation to seminal plasma PSA.
- Author
-
Tabarés G, Radcliffe CM, Barrabés S, Ramírez M, Aleixandre RN, Hoesel W, Dwek RA, Rudd PM, Peracaula R, and de Llorens R
- Subjects
- Biomarkers analysis, Biomarkers blood, Fucose analysis, Humans, Male, N-Acetylneuraminic Acid analysis, Prostate-Specific Antigen blood, Polysaccharides analysis, Polysaccharides chemistry, Prostate-Specific Antigen analysis, Prostate-Specific Antigen chemistry, Prostatic Neoplasms blood, Semen chemistry
- Abstract
Prostate-specific antigen (PSA), the tumor marker currently used for prostate cancer (PCa), is not specific enough to distinguish between PCa and benign prostate hyperplasia (BPH). Glycan processing is normally perturbed in tumors, therefore we investigated whether changes in glycosylation of PSA could be useful diagnostic indicators. Previously we determined that the glycosylation of PSA secreted by the tumor prostate cell line LNCaP differs significantly from that of PSA from seminal plasma (normal control). We therefore undertook a detailed glycan analysis of PSA derived from sera from PCa patients and, importantly, established that the glycosylation of the PCa serum PSA was significantly different from the PSA from the LNCaP cell line. In comparison with seminal plasma PSA, the fucose content of PSA from the PCa patient serum was significantly lower and there was a decrease in alpha2,3-linked sialic acid. Differences in the glycosylation of PSA derived from PCa patients' sera, seminal plasma, and LNCaP cells were further established by lectin detection, glycosylation immunosorbent assay, and two-dimensional electrophoresis. We also investigated whether the impact of glycosylation changes initiated by the tumor was reflected in the serum glycome. By comparing the glycans released from the total glycoproteins in PCa patient serum with those of normal serum we found an increase in the proportion of sialyl-Lewis x structures. Further analysis of the glycosylation of PSA from PCa and BPH sera will be required in order to determine the utility of these glycan differences to discriminate specifically between benign and malignant prostate states. more...
- Published
- 2006
- Full Text
- View/download PDF
13. Imino sugar inhibitors for treating the lysosomal glycosphingolipidoses.
- Author
-
Butters TD, Dwek RA, and Platt FM
- Subjects
- Animals, Drug Evaluation, Preclinical, Gaucher Disease drug therapy, Glucosyltransferases antagonists & inhibitors, Glycosphingolipids antagonists & inhibitors, Glycosphingolipids biosynthesis, Humans, Sphingolipidoses enzymology, Enzyme Inhibitors therapeutic use, Glycosphingolipids metabolism, Imino Sugars therapeutic use, Sphingolipidoses drug therapy
- Abstract
The inherited metabolic disorders of glycosphingolipid (GSL) metabolism are a relatively rare group of diseases that have diverse and often neurodegenerative phenotypes. Typically, a deficiency in catabolic enzyme activity leads to lysosomal storage of GSL substrates and in many diseases, several other glycoconjugates. A novel generic approach to treating these diseases has been termed substrate reduction therapy (SRT), and the discovery and development of N-alkylated imino sugars as effective and approved drugs is discussed. An understanding of the molecular mechanism for the inhibition of the key enzyme in GSL biosynthesis, ceramide glucosyltransferase (CGT) by N-alkylated imino sugars, has also lead to compound design for improvements to inhibitory potency, bioavailability, enzyme selectivity, and biological safety. Following a successful clinical evaluation of one compound, N-butyl-deoxynojirimycin [(NB-DNJ), miglustat, Zavesca], for treating type I Gaucher disease, issues regarding the significance of side effects and CNS access have been addressed as exposure of drug to patients has increased. An alternative experimental approach to treat specific glycosphingolipid (GSL) lysosomal storage diseases is to use imino sugars as molecular chaperons that assist protein folding and stability of mutant enzymes. The principles of chaperon-mediated therapy (CMT) are described, and the potential efficacy and preclinical status of imino sugars is compared with substrate reduction therapy (SRT). The increasing use of imino sugars for clinical evaluation of a group of storage diseases that are complex and often intractable disorders to treat has considerable benefit. This is particularly so given the ability of small molecules to be orally available, penetrate the central nervous system (CNS), and have well-characterized biological and pharmacological properties. more...
- Published
- 2005
- Full Text
- View/download PDF
14. Structural characterization of the N-glycan moiety and site of glycosylation in vitellogenin from the decapod crustacean Cherax quadricarinatus.
- Author
-
Khalaila I, Peter-Katalinic J, Tsang C, Radcliffe CM, Aflalo ED, Harvey DJ, Dwek RA, Rudd PM, and Sagi A
- Subjects
- Animals, Blotting, Western, Crustacea, Electrophoresis, Polyacrylamide Gel, Glycosylation, Protein Conformation, Spectrometry, Mass, Electrospray Ionization, Vitellogenins metabolism, Polysaccharides chemistry, Vitellogenins chemistry
- Abstract
Glycosylation is of importance for the structure and function of proteins. In the case of vitellin (Vt), a ubiquitous protein accumulated into granules as the main yolk protein constituent of oocytes during oogenesis, glycosylation could be of importantance for the folding, processing and transport of the protein to the yolk and also provides a source of carbohydrate during embryogenesis. Vt from the crayfish Cherax quadricarinatus is synthesized as a precursor protein, vitellogenin (Vg), in the hepatopancreas, transferred to the hemolymph, and mobilized into the growing oocyte via receptor-mediated endocytosis. The gene sequence of C. quadricarinatus shows a 2584-amino-acid protein with 10 putative glycosylation sites. In this study a combined approach of lectin immunoblotting, in-gel deglycosylation, and mass spectrometry was used to identify the glycosylation sites and probe the structure of the glycan moieties using C. quadricarinatus Vg as a model system. Three of the consensus sites for N-glycosylation-namely, Asn(152), Asn(160) and Asn(2493)-were glycosylated with the high-mannose glycans, Man(5-9)GlcNAc(2), and the glucose-capped oligosaccharide Glc(1)Man(9)GlcNAc(2). more...
- Published
- 2004
- Full Text
- View/download PDF
15. Statistical analysis of the protein environment of N-glycosylation sites: implications for occupancy, structure, and folding.
- Author
-
Petrescu AJ, Milac AL, Petrescu SM, Dwek RA, and Wormald MR
- Subjects
- Amino Acids analysis, Binding Sites, Crystallography, X-Ray, Databases, Protein, Glycoproteins chemistry, Glycoproteins metabolism, Models, Statistical, Polysaccharides chemistry, Polysaccharides metabolism, Protein Folding, Protein Structure, Quaternary, Protein Structure, Secondary, Proteins chemistry, Structure-Activity Relationship, Torsion Abnormality, Glycosylation, Proteins metabolism
- Abstract
We recently reported statistical analysis of structural data on glycosidic linkages. Here we extend this analysis to the glycan-protein linkage, and the peptide primary, secondary, and tertiary structures around N-glycosylation sites. We surveyed 506 glycoproteins in the Protein Data Bank crystallographic database, giving 2592 glycosylation sequons (1683 occupied) and generated a database of 626 nonredundant sequons with 386 occupied. Deviations in the expected amino acid composition were seen around occupied asparagines, particularly an increased occurrence of aromatic residues before the asparagine and threonine at position +2. Glycosylation alters the asparagine side chain torsion angle distribution and reduces its flexibility. There is an elevated probability of finding glycosylation sites in which secondary structure changes. An 11-class taxonomy was developed to describe protein surface geometry around glycosylation sites. Thirty-three percent of the occupied sites are on exposed convex surfaces, 10% in deep recesses and 20% on the edge of grooves with the glycan filling the cleft. A surprisingly large number of glycosylated asparagine residues have a low accessibility. The incidence of aromatic amino acids brought into close contact with the glycan by the folding process is higher than their normal levels on the surface or in the protein core. These data have significant implications for control of sequon occupancy and evolutionary selection of glycosylation sites and are discussed in relation to mechanisms of protein fold stabilization and regional quality control of protein folding. Hydrophobic protein-glycan interactions and the low accessibility of glycosylation sites in folded proteins are common features and may be critical in mediating these functions. more...
- Published
- 2004
- Full Text
- View/download PDF
16. Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis.
- Author
-
Butler M, Quelhas D, Critchley AJ, Carchon H, Hebestreit HF, Hibbert RG, Vilarinho L, Teles E, Matthijs G, Schollen E, Argibay P, Harvey DJ, Dwek RA, Jaeken J, and Rudd PM
- Subjects
- Carbohydrate Metabolism, Inborn Errors enzymology, Carbohydrate Metabolism, Inborn Errors genetics, Chromatography, High Pressure Liquid, Electrophoresis, Polyacrylamide Gel, Glycosylation, Humans, Hydrogen-Ion Concentration, Immunoglobulin G chemistry, Molecular Structure, N-Acetylglucosaminyltransferases genetics, N-Acetylglucosaminyltransferases metabolism, Protein Isoforms, Proteome analysis, Proteome chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Transferrin chemistry, Carbohydrate Metabolism, Inborn Errors blood, Carbohydrate Metabolism, Inborn Errors metabolism, Glycoproteins blood, Glycoproteins chemistry, Polysaccharides analysis
- Abstract
The fundamental importance of correct protein glycosylation is abundantly clear in a group of diseases known as congenital disorders of glycosylation (CDGs). In these diseases, many biological functions are compromised, giving rise to a wide range of severe clinical conditions. By performing detailed analyses of the total serum glycoproteins as well as isolated transferrin and IgG, we have directly correlated aberrant glycosylation with a faulty glycosylation processing step. In one patient the complete absence of complex type sugars was consistent with ablation of GlcNAcTase II activity. In another CDG type II patient, the identification of specific hybrid sugars suggested that the defective processing step was cell type-specific and involved the mannosidase III pathway. In each case, complementary serum proteome analyses revealed significant changes in some 31 glycoproteins, including components of the complement system. This biochemical approach to charting diseases that involve alterations in glycan processing provides a rapid indicator of the nature, severity, and cell type specificity of the suboptimal glycan processing steps; allows links to genetic mutations; indicates the expression levels of proteins; and gives insight into the pathways affected in the disease process. more...
- Published
- 2003
- Full Text
- View/download PDF
17. Altered glycosylation pattern allows the distinction between prostate-specific antigen (PSA) from normal and tumor origins.
- Author
-
Peracaula R, Tabarés G, Royle L, Harvey DJ, Dwek RA, Rudd PM, and de Llorens R
- Subjects
- Animals, Carbohydrate Sequence, Cell Line, Tumor, Chromatography, High Pressure Liquid, Enzyme-Linked Immunosorbent Assay, Gene Expression, Glycosylation, Humans, Male, Mice, Molecular Sequence Data, Prostate-Specific Antigen metabolism, Sequence Analysis, Protein, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Oligosaccharides analysis, Prostate-Specific Antigen analysis, Prostate-Specific Antigen chemistry, Prostatic Neoplasms chemistry, Semen chemistry
- Abstract
Prostate-specific antigen (PSA) is a glycoprotein secreted by prostate epithelial cells. PSA is currently used as a marker of prostate carcinoma because high levels of PSA are indicative of a tumor situation. However, PSA tests still suffer from a lack of specificity to distinguish between benign prostate hyperplasia and prostate cancer. To determine whether PSA glycosylation could provide a means of differentiating between PSA from normal and tumor origins, N-glycan characterization of PSA from seminal fluid and prostate cancer cells (LNCaP cell line) by sequencing analysis and mass spectrometry was carried out. Glycans from normal PSA (that correspond to low and high pI PSA fractions) were sialylated biantennary complex structures, half of them being disialylated in the low pI PSA fraction and mostly monosialylated in the high pI PSA. PSA from LNCaP cells was purified to homogeneity, and its glycan analysis showed a significantly different pattern, especially in the outer ends of the biantennary complex structures. In contrast to normal PSA glycans, which were sialylated, LNCaP PSA oligosaccharides were all neutral and contained a higher fucose content. In 10-15% of the structures fucose was linked alpha1-2 to galactose, forming the H2 epitope absent in normal PSA. GalNAc was increased in LNCaP glycans to 65%, whereas in normal PSA it was only present in 25% of the structures. These carbohydrate differences allow a distinction to be made between PSA from normal and tumor origins and suggest a valuable biochemical tool for diagnosis and follow-up purposes. more...
- Published
- 2003
- Full Text
- View/download PDF
18. Glycosylation of human pancreatic ribonuclease: differences between normal and tumor states.
- Author
-
Peracaula R, Royle L, Tabares G, Mallorqui-Fernández G, Barrabés S, Harvey DJ, Dwek RA, Rudd PM, and de Llorens R
- Subjects
- Adenocarcinoma pathology, Blotting, Western, Carbohydrates analysis, Cell Line, Tumor, Chromatography, High Pressure Liquid, Electrophoresis, Polyacrylamide Gel, Enzyme-Linked Immunosorbent Assay, Glycosylation, Humans, Oligosaccharides chemistry, Oligosaccharides isolation & purification, Oligosaccharides metabolism, Pancreatic Neoplasms pathology, Ribonuclease, Pancreatic isolation & purification, Ribonuclease, Pancreatic metabolism, Spectrometry, Mass, Electrospray Ionization methods, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization methods, Adenocarcinoma enzymology, Pancreas enzymology, Pancreatic Neoplasms enzymology, Ribonuclease, Pancreatic chemistry
- Abstract
Characterization of the N-glycans from human pancreatic ribonuclease (RNase 1) isolated from healthy pancreas and from pancreatic adenocarcinoma tumor cells (Capan-1 and MDAPanc-3) revealed completely different glycosylation patterns. RNase 1 from healthy cells contained neutral complex biantennary structures, with smaller amounts of tri- and tetraantennary compounds, and glycans with poly-N-acetyllactosamine extensions, all extensively fucosylated. In contrast, RNase 1 glycans from tumor cells (Capan-1) were fucosylated hybrid and complex biantennary glycans with GalNAc-GlcNAc antennae. RNase 1 glycans from Capan-1 and MDAPanc-3 cells also contained sialylated structures completely absent in the healthy pancreas. Some of these features provide distinct epitopes that were clearly detected using monoclonal antibodies against carbohydrate antigens. Thus monoclonal antibodies to Lewis(y) reacted only with normal pancreatic RNase 1, whereas, in contrast, monoclonal antibodies to sialyl-Lewis(x) and sialyl-Lewis(a) reacted only with RNase 1 secreted from the tumor cells. These glycosylation changes in a tumor-secreted protein, which reflect fundamental changes in the enzymes involved in the glycosylation pathway, open up the possibility of using serum RNase 1 as a tumor marker of pancreatic adenocarcinoma. more...
- Published
- 2003
- Full Text
- View/download PDF
19. Structural determination of the N-glycans of a lepidopteran arylphorin reveals the presence of a monoglucosylated oligosaccharide in the storage protein.
- Author
-
Kim S, Hwang SK, Dwek RA, Rudd PM, Ahn YH, Kim EH, Cheong C, Kim SI, Park NS, and Lee SM
- Subjects
- Amidohydrolases metabolism, Animals, Bombyx, Carbohydrate Conformation, Carbohydrate Sequence, Chromatography, High Pressure Liquid, Glycoproteins metabolism, Hemolymph chemistry, Insect Proteins metabolism, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Oligosaccharides isolation & purification, Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase, Glucose metabolism, Glycoproteins chemistry, Insect Proteins chemistry, Lepidoptera chemistry, Oligosaccharides chemistry, Oligosaccharides metabolism
- Abstract
The structures of the oligosaccharides attached to arylphorin from Chinese oak silkworm, Antheraea pernyi, have been determined. Arylphorin, a storage protein present in fifth larval hemolymph, contained 4.8% (w/w) of carbohydrate that was composed of Fuc:GlcNAc:Glc:Man=0.2:4.0:1.4:13.6 moles per mole protein. Four moles of GlcNAc in oligomannose-type oligosaccharides strongly suggest that the protein contains two N-glycosylation sites. Normal-phase HPLC and mass spectrometry oligosaccharide profiles confirmed that arylphorin contained mainly oligomannose-type glycans as well as truncated mannose-type structures with or without fucosylation. Interestingly, the most abundant oligosaccharide was monoglucosylated Man9-GlcNAc2, which was characterized by normal-phase HPLC, mass spectrometry, Aspergillus saitoi alpha-mannosidase digestion, and 1H 600 MHz NMR spectrometry. This glycan structure is not normally present in secreted mammalian glycoproteins; however, it has been identified in avian species. The Glc1Man9GlcNAc2 structure was present only in arylphorin, whereas other hemolymph proteins contained only oligomannose and truncated oligosaccharides. The oligosaccharide was also detected in the arylphorin of another silkworm, Bombyx mori, suggesting a specific function for the Glc1Man9GlcNAc2 glycan. There were no processed glucosylated oligosaccharides such as Glc1Man5-8GlcNAc2. Furthermore, Glc1Man9GlcNAc2 was not released from arylophorin by PNGase F under nondenaturing conditions, suggesting that the N-glycosidic linkage to Asn is protected by the protein. Glc1Man9GlcNAc2 may play a role in the folding of arylphorin or in the assembly of hexamers. more...
- Published
- 2003
- Full Text
- View/download PDF
20. Comparison of the N-linked glycans from soluble and GPI-anchored CD59 expressed in CHO cells.
- Author
-
Wheeler SF, Rudd PM, Davis SJ, Dwek RA, and Harvey DJ
- Subjects
- Amino Acid Sequence, Animals, CHO Cells, Chromatography, High Pressure Liquid, Cricetinae, Electrophoresis, Polyacrylamide Gel, Molecular Sequence Data, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, CD59 Antigens chemistry, Glycosylphosphatidylinositols chemistry, Polysaccharides chemistry
- Abstract
The N-linked glycosylation of recombinant human CD59, expressed in Chinese hamster ovary (CHO) cells with and without a membrane anchor, was compared to examine the effect of the anchor on glycan processing. N-Linked glycans were released with peptide-N-glycosidase F (PNGase F) within gel from SDS-PAGE-isolated soluble and glycosylphosphatidylinositol (GPI)-anchored human CD59 expressed in CHO cells. The anchored form contained core-fucosylated neutral and sialylated bi-, tri-, and tetraantennary glycans with up to four N-acetyllactosamine extensions. Exoglycosidase digestions and analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry were used to define the relative amounts of the bi-, tri-, and tetraantennary glycans and to investigate the distribution of N-acetyllactosamine extensions between their antennae. Biantennary structures accounted for about 60% of the glycans, 30% of the triantennary structures, and about 10% of the tetraantennary structures. For tri- and tetraantennary glycans, those with extended antennae were found to be more abundant than those without extensions. The soluble form of CD59, expressed in CHO cells without the GPI anchor signal sequence, consisted almost entirely (97%) of biantennary glycans, of which 81% were unmodified, 17% contained one N-acetyllactosamine extension, and 2% contained two extensions. No compounds with longer extensions were found. A MALDI spectrum of the intact glycoprotein showed a distribution of glycans that matched those released with PNGase F. In addition, the protein was substituted with several small glycans, such as HexNAc, HexNAc-->Fuc, and HexNAc-->HexNAc, probably as the result of degradation of the mature N-linked glycans. The results show that the presence of the anchor increases the extent of glycan processing, possibly as the result of longer exposure to the glycosyltransferases or to a closer proximity of the protein to these enzymes. more...
- Published
- 2002
- Full Text
- View/download PDF
21. Localization and characterization of polysialic acid-containing N-linked glycans from bovine NCAM.
- Author
-
von Der Ohe M, Wheeler SF, Wuhrer M, Harvey DJ, Liedtke S, Mühlenhoff M, Gerardy-Schahn R, Geyer H, Dwek RA, Geyer R, Wing DR, and Schachner M
- Subjects
- Animals, Cattle, Neural Cell Adhesion Molecules chemistry, Polysaccharides chemistry, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Neural Cell Adhesion Molecules metabolism, Polysaccharides metabolism, Sialic Acids metabolism
- Abstract
The neural cell adhesion molecule (NCAM) plays important roles during development, plasticity, and regeneration in the adult nervous system. Its function is strongly influenced by attachment of the unusual alpha 2-8-linked polysialic acid (PSA). Here we analyzed the N-glycosylation pattern of polysialylated NCAM from brains of newborn calves. Purified PSA-NCAM glycoprotein was digested with trypsin, and PSA-glycopeptides were separated by immunoaffinity chromatography. For determining the N-glycosylation sites, PNGase F-treated glycopeptides were analyzed by Edman degradation and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). They were found to be exclusively linked to the fifth (Asn 439) and sixth (Asn 468) N-glycosylation sites in the fifth immunoglobulin-like domain of NCAM. The chain length of PSA consisted of at least 30 sialic acid residues, as shown by anion exchange chromatography. For analysis of the core structures, endoneuraminidase N-treated PSA-NCAM was separated by SDS-PAGE and digested with PNGase F. The core structures of polysialylated glycans were characterized by MALDI-MS combined with exoglycosidase digestions and chromatographic fractionation. They include hybrid, di-, tri-, and small amounts of tetraantennary carbohydrates, which were all fucosylated at the innermost N-acetylglucosamine. For the triantennary glycans, the "2,6" arm was preferred in polysialylated structures. High levels of sulfated groups were found on polysialylated structures and to a lower extent also on nonpolysialylated glycans. In addition, high-mannose-type glycans could be detected on PSA-NCAM glycoforms ranging from (GlcNAc)(2)(Man)(5) up to (GlcNAc)(2)(Man)(9). In conclusion, we observed a structural variability and high regional selectivity for the PSA-glycans attached to the NCAM molecule that are most likely influencing its biological functions. more...
- Published
- 2002
- Full Text
- View/download PDF
22. Imino sugar therapy for type 1 Gaucher disease.
- Author
-
Priestman DA, Platt FM, Dwek RA, and Butters TD
- Subjects
- 1-Deoxynojirimycin administration & dosage, 1-Deoxynojirimycin therapeutic use, Animals, Enzyme Inhibitors administration & dosage, Gaucher Disease metabolism, Glucosylceramidase administration & dosage, Glucosylceramidase blood, Glycosphingolipids metabolism, Humans, Mice, 1-Deoxynojirimycin analogs & derivatives, Enzyme Inhibitors therapeutic use, Gaucher Disease drug therapy, Glycoside Hydrolase Inhibitors
- Published
- 2000
23. The glycan processing and site occupancy of recombinant Thy-1 is markedly affected by the presence of a glycosylphosphatidylinositol anchor.
- Author
-
Devasahayam M, Catalino PD, Rudd PM, Dwek RA, and Barclay AN
- Subjects
- Animals, Binding Sites, CHO Cells, Cricetinae, Fucose analysis, Glycosylation, Glycosylphosphatidylinositols genetics, Mannose analysis, N-Acetylneuraminic Acid analysis, Rats, Recombinant Proteins metabolism, Solubility, Thy-1 Antigens chemistry, Thy-1 Antigens genetics, Tunicamycin pharmacology, Glycosylphosphatidylinositols metabolism, Thy-1 Antigens metabolism
- Abstract
Thy-1 is a cell surface glycoprotein containing three N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The effect of the anchor on its N-linked glyco-sylation was investigated by comparing the glycosylation of soluble recombinant Thy-1 (sThy-1) with that of recombinant GPI anchored Thy-1, both expressed in Chinese hamster ovary cells. The sThy-1 was produced in a variety of isoforms including some which lacked carbohydrate on all three sequons whereas the GPI anchored form appeared fully glycosylated like native Thy-1. This was surprising as the attachment of N-linked sugars occurs cotranslationally and it was not expected that the presence of a C-terminal GPI anchor signal sequence would affect sequon occupancy. Furthermore sThy-1 lacking glycosylation could be produced with the inhibitor tunicamycin but in contrast cell surface expression of unglycosylated GPI anchored Thy-1 could not be obtained. The GPI anchored form appeared less processed with almost 4-fold more oligo-mannose oligosaccharides than in sThy-1 and also with less sialylated and core fucosylated biantennary glycans. Possible mechanisms whereby the anchor or the anchor signal sequence, control site occupancy and maturation are discussed. more...
- Published
- 1999
- Full Text
- View/download PDF
24. Glycosylation of a CNS-specific extracellular matrix glycoprotein, tenascin-R, is dominated by O-linked sialylated glycans and "brain-type" neutral N-glycans.
- Author
-
Zamze S, Harvey DJ, Pesheva P, Mattu TS, Schachner M, Dwek RA, and Wing DR
- Subjects
- Animals, Carbohydrate Conformation, Carbohydrate Sequence, Chromatography, Gel, Chromatography, High Pressure Liquid, Extracellular Matrix Proteins isolation & purification, Extracellular Matrix Proteins metabolism, Glycoside Hydrolases, Glycosylation, Mice, Molecular Sequence Data, Oligosaccharides isolation & purification, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Tenascin isolation & purification, Tenascin metabolism, Brain Chemistry, Extracellular Matrix Proteins chemistry, Oligosaccharides chemistry, Polysaccharides chemistry, Tenascin chemistry
- Abstract
As a member of the tenascin family of extracellular matrix glycoproteins, tenascin-R is located exclusively in the CNS. It is believed to play a role in myelination and axonal stabilization and, through repulsive properties, may contribute to the lack of regeneration of CNS axons following damage. The contrary functions of the tenascins have been localized to the different structural domains of the protein. However, little is known concerning the influence of the carbohydrate conjugated to the many potential sites for N - and O -glycosylation (10-20% by weight). As a first analytical requirement, we show that >80% of the N -glycans in tenascin-R are neutral and dominated by complex biantennary structures. These display the "brain-type" characteristics of outer-arm- and core-fucosylation, a bisecting N -acetylglucosamine and, significantly, an abundance of antennae truncation. In some structures, truncation resulted in only a single mannose residue remaining on the 3-arm, a particularly unusual consequence of the N -glycan processing pathway. In contrast to brain tissue, hybrid and oligomannosidic N -glycans were either absent or in low abundance. A high relative abundance of O -linked sialylated glycans was found. This was associated with a significant potential for O -linked glycosylation sites and multivalent display of the sialic acid residues. These O -glycans were dominated by the disialylated structure, NeuAcalpha2-3Galbeta1-3(NeuAcalpha2-6)GalNAc. The possibility that these O -glycans enable tenascin-R to interact in the CNS either with the myelin associated glycoprotein or with sialoadhesin on activated microglia is discussed. more...
- Published
- 1999
- Full Text
- View/download PDF
25. Oligosaccharide analysis and molecular modeling of soluble forms of glycoproteins belonging to the Ly-6, scavenger receptor, and immunoglobulin superfamilies expressed in Chinese hamster ovary cells.
- Author
-
Rudd PM, Wormald MR, Harvey DJ, Devasahayam M, McAlister MS, Brown MH, Davis SJ, Barclay AN, and Dwek RA
- Subjects
- Animals, Antigens, CD chemistry, Antigens, Differentiation metabolism, Antigens, Ly chemistry, CD2 Antigens chemistry, CD4 Antigens chemistry, CD48 Antigen, CHO Cells, Carbohydrate Conformation, Carbohydrate Sequence, Cricetinae, Glycoproteins metabolism, Glycosylation, Humans, Molecular Sequence Data, Protein Conformation, Protein Processing, Post-Translational, Protein Structure, Secondary, Rats, Receptors, Immunologic chemistry, Receptors, Scavenger, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Scavenger Receptors, Class B, Thy-1 Antigens chemistry, Antigens, Differentiation chemistry, Glycoproteins chemistry, Membrane Proteins, Models, Molecular, Oligosaccharides analysis, Receptors, Lipoprotein
- Abstract
Most cell surface molecules are glycoproteins consisting of linear arrays of globular domains containing stretches of amino acid sequence with similarities to regions in other proteins. These conserved regions form the basis for the classification of proteins into superfamilies. Recombinant soluble forms of six leukocyte antigens belonging to the Ly-6 (CD59), scavenger receptor (CD5), and immunoglobulin (CD2, CD48, CD4, and Thy-1) superfamilies were expressed in the same Chinese hamster ovary cell line, thus providing an opportunity to examine the extent to which N-linked oligosaccharide processing might vary in a superfamily-, domain-, or protein-dependent manner in a given cell. While we found no evidence for superfamily-specific modifications of the glycans, marked differences were seen in the types of oligosaccharides attached to individual proteins within a given superfamily. The relative importance of local protein surface properties versus the overall tertiary structure of the molecules in directing this protein-specific variation was examined in the context of molecular models. These were constructed using the 3D structures of the proteins, glycan data from this study, and an oligosaccharide structural database. The results indicated that both the overall organization of the domains and the local protein structure can have a large bearing on site-specific glycan modification of cells in stasis. This level of control ensures that the surface of a single cell will display a diverse repertoire of glycans and precludes the presentation of multiple copies of a single oligosaccharide on the cell surface. The glycans invariably shield large regions of the protein surfaces although, for the glycoproteins examined here, these did not hinder the known active sites of the molecules. The models also indicated that sugars are likely to play a role in the packing of the native cell surface glycoproteins and to limit nonspecific protein-protein interactions. In addition, glycans located close to the cell membrane are likely to affect crucially the orientation of the glycoproteins to which they are attached. more...
- Published
- 1999
- Full Text
- View/download PDF
26. A statistical analysis of N- and O-glycan linkage conformations from crystallographic data.
- Author
-
Petrescu AJ, Petrescu SM, Dwek RA, and Wormald MR
- Subjects
- Carbohydrate Conformation, Carbohydrate Sequence, Crystallography, X-Ray, Data Interpretation, Statistical, Databases, Factual, Glycoproteins chemistry, Models, Molecular, Oligosaccharides chemistry, Polysaccharides chemistry
- Abstract
We have generated a database of 639 glycosidic linkage structures by an exhaustive survey of the available crystallographic data for isolated oligosaccharides, glycoproteins, and glycan-binding proteins. For isolated oligosaccharides there is relatively little crystallographic data available. A much larger number of glycoprotein and glycan-binding protein structures have now been solved in which two or more linked monosaccharides can be resolved. In the majority of these cases, only a few residues can be seen. Using the 639 glycosidic linkage structures, we have identified one or more distinct conformers for all the linkages. The O5-C1-O-C(x)' torsion angles for all these distinct conformers appear to be determined chiefly by the exo-anomeric effect. The Manalpha1-6Man linkage appears to be less restrained than the others, showing a wide degree of dispersion outside the ranges of the defined conformers. The identification of distinct conformers for glyco-sidic linkages allows "average" glycan structures to be modeled and also allows the easy identification of distorted glycosidic linkages. Such an analysis shows that the interactions between IgG Fc and its own N-linked glycan result in severe distortion of the terminal Galbeta1-4GlcNAc linkage only, indicating the strong interactions that must be present between the Gal residue and the protein surface. The applicability of this crystallographic based analysis to glycan structures in solution is discussed. This database of linkagestructures should be a very useful reference tool in three-dimensional structure determinations. more...
- Published
- 1999
- Full Text
- View/download PDF
27. Suppression of allogeneic reactivity in vitro by the syncytiotrophoblast membrane glycocalyx of the human term placenta is carbohydrate dependent.
- Author
-
Arkwright PD, Rademacher TW, Boutignon F, Dwek RA, and Redman CW
- Subjects
- Carbohydrate Metabolism, Cell Division, Female, Glycoproteins metabolism, Humans, Leukocytes, Mononuclear cytology, Membrane Glycoproteins metabolism, Membrane Lipids immunology, Microvilli immunology, Microvilli metabolism, Polysaccharides metabolism, Pregnancy, Pronase, Trophoblasts metabolism, Carbohydrates immunology, Glycoproteins immunology, Immune Tolerance physiology, Membrane Glycoproteins immunology, Polysaccharides immunology, Trophoblasts immunology
- Abstract
Immunosuppressive factors isolated from trophoblast are known to block both innate and major histocompatibility complex (MHC)-dependent cell-mediated immune responses in vitro and, in some cases, in vivo. We investigated the biochemical nature of these factors, which is presently unknown. Immunosuppressive activity, assessed by inhibition of two-way MLR, was extracted from term syncytiotrophoblast microvilli using 3 M KCl. The activity resisted both extensive pronase digestion and heating to 90 degrees C for 1 h, demonstrating that intact membrane proteins were not required. Although purified protein-linked oligosaccharides released by hydrazinolysis from the syncytiotrophoblast membrane were themselves inactive, they blocked the immunosuppressive activity of the KCl extract. After pronase digestion, the activity could be fractionated by TSK 55S gel filtration, followed by C18 reverse-phase chromatography. Sequential exoglycosidase digestion of hydrazine-released sugars of the active fraction demonstrated that it contained neutral N-linked oligomannose and hybrid oligosaccharides, which normally make up < 3% of the total syncytiotrophoblast-derived protein glycan library. These glycopeptides of the active fraction were associated with membrane phospholipid micelles. The possible mechanism by which incompletely processed N-linked oligosaccharides expressed by a variety of syncytiotrophoblast membrane glycoproteins may block allogeneic reactivity when presented as polyvalent sugar groups is discussed. more...
- Published
- 1994
- Full Text
- View/download PDF
28. Comparative analysis of the N-glycans of rat, mouse and human Thy-1. Site-specific oligosaccharide patterns of neural Thy-1, a member of the immunoglobulin superfamily.
- Author
-
Williams AF, Parekh RB, Wing DR, Willis AC, Barclay AN, Dalchau R, Fabre JW, Dwek RA, and Rademacher TW
- Subjects
- Amino Acid Sequence, Animals, Antigens, Surface classification, Brain Chemistry, Glycopeptides chemistry, Glycosylation, Humans, Membrane Glycoproteins classification, Mice, Molecular Sequence Data, Multigene Family, N-Acetylneuraminic Acid, Nerve Tissue Proteins classification, Oligosaccharides chemistry, Oligosaccharides classification, Peptide Fragments chemistry, Polysaccharides classification, Protein Conformation, Rats, Regulatory Sequences, Nucleic Acid, Sequence Homology, Amino Acid, Sialic Acids chemistry, Thy-1 Antigens, Antigens, Surface chemistry, Membrane Glycoproteins chemistry, Nerve Tissue Proteins chemistry, Polysaccharides chemistry
- Abstract
Protein structure and tissue type are known to influence glycosylation of proteins. We have previously investigated the N-glycans at each of the three glycosylation sites of the cell surface glycoprotein Thy-1 when isolated from rat brain and thymocytes. Here we report a comparative analysis of the site-specific N-glycosylation patterns from rat (Asn 23, 74, 98), mouse (Asn 23, 75, 99) and human (Asn 23, 60, 100) neural Thy-1. Despite considerable differences in amino acid sequence, the results show a remarkable conservation of the pattern of N-glycans at corresponding sites between the three species, as judged by chromatographic comparisons and glycosidase susceptibility. This is particularly marked for sites at Asn 74/75 in rat/mouse and the equivalent site at 60 in human Thy-1, as well as for sites at Asn 98/99 and 100, respectively. The sites at Asn 23 in rat/mouse also contained almost identical glycosylation patterns, but at this site human Thy-1 showed significantly different glycosylation patterns. These site glycosylation patterns are discussed in relation to the likely accessibility of the oligosaccharides for processing. It is known that within a species, the glycosylation of Thy-1 is tissue specific; therefore, this degree of conservation of glycosylation of Thy-1 expressed in the same tissue in different species is all the more striking, given the known variation between species in the amino acid sequence of Thy-1. It is therefore proposed that neural cells have a particular requirement for specific surface carbohydrates and that the Thy-1 polypeptide serves as an appropriate carrier for these structures. more...
- Published
- 1993
- Full Text
- View/download PDF
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.