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R A Dwek

Publications and source records attributed to R A Dwek.

At least 127 records · Page 7Linked to original sources
PubMed1994

Agalactosyl glycoforms of IgG autoantibodies are pathogenic.

The glycosylation of IgG results in many different glycoforms. A large body of correlative data (including remission of arthritis during pregnancy) has suggested that IgG molecules lacking galactose were associated with rheumatoid arthritis. We now demonstrate that agalactosyl IgG glycoforms are directly associated with pathogenicity in murine collagen-induced arthritis. We show that passive transfer of an acute synovitis in T-cell-primed mice can be enhanced by using IgG containing autoantibodies to type II collagen when the antibodies are present as the agalactosyl glycoform. Thus, nonpathogenic doses of autoantibodies can be made pathogenic by altering their glycosylation state.

Animals↗
PubMed1994

Synthetic glycosylation of proteins using N-(beta-saccharide) iodoacetamides: applications in site-specific glycosylation and solid-phase enzymic oligosaccharide synthesis.

A simple and efficient synthetic glycosylation method suitable for use in solid-phase enzymic oligosaccharide synthesis and site-specific glycosylation of recombinant proteins to produce defined glycoforms is described. This strategy utilizes N-(beta-saccharide) haloacetamides for attaching oligosaccharides specifically to cysteine residues of proteins in solution to form neoglycoproteins. The alkylation reaction was tested using N-(beta-chitotriose) bromoacetamide and an unprotected synthetic hexapeptide containing a single cysteine residue. The glycosylated product was confirmed by amino acid and hexosamine analyses as well as laser desorption mass spectrometry. Similarly N-(beta-chitotriose) iodoacetamide was covalently linked to non-reduced BSA to produce a defined glycoform of this protein. The specific attachment of chitotriose at the single cysteine residue in non-reduced serum albumin was suggested by Ellman's assay for free thiols. This was verified by amino acid sequencing of tryptic glycopeptide derived from this neoglycoprotein. Multiple sugar attachment was accomplished using fully reduced serum albumin as demonstrated by the formation of two neoglycoproteins using iodoacetamide derivatives of galactose beta 1-3-N-acetylgalactosamine (Gal beta 1-3GalNAc) and the major xylose/fucose-class plant-type oligosaccharide of horseradish peroxidase. These two neoglycoproteins with an average of 18-21 sugar residues attached were assayed positively for binding to peanut agglutinin and a sugar-specific anti-(horseradish peroxidase) monoclonal antibody YZ1/2.23 respectively. Sialylation of the neoglycoprotein containing Gal beta 1-3GalNAc was accomplished using alpha-2,3-sialyltransferase and radiolabelled CMP-N-acetylneuraminic acid. Significantly, glycan attachment using this conjugation method is reversible as demonstrated by the release of oligosaccharides from these two neoglycoproteins using hydrazinolysis. Therefore this method could provide invaluable reagents for many glycobiological studies.

Alkylation↗
PubMed1994

Binding of sugar ligands to Ca(2+)-dependent animal lectins. I. Analysis of mannose binding by site-directed mutagenesis and NMR.

The Ca(2+)-dependent carbohydrate-recognition domain (CRD) of rat serum mannose-binding protein has been subjected to site-directed mutagenesis to determine the importance of individual residues in ligation of mannose and related sugars. The effects of the mutations were assessed by direct binding assays, competition binding studies, partial proteolysis, and NMR analysis of sugar-CRD titrations. As suggested by the crystal structure of the mannose-binding CRD complexed with oligosaccharide ligand, asparagine and glutamic acid residues that interact with hydroxyl groups 3 and 4 of the sugar, as well as with one of the two bound Ca2+, are critical for ligand binding. In addition, the beta-carbon of His189 contributes substantially to the binding affinity, apparently through a van der Waals contact with C-4 of the sugar ligand. van der Waals contacts between the imidazole ring of His189 and the 2 hydroxyl group of mannose, and between Ile207 and C-6 of mannose, observed in the crystal structure, contribute less to stability of the ligand complex. The effects of changes at positions 189 and 207 on the ability of the CRD to distinguish between alpha-and beta-methyl L-fucosides suggest that fucose may bind in an alternative orientation compared to the arrangement originally proposed based on the mannose-CRD complex.

Amino Acid Sequence↗
PubMed1994

Structural analysis of the N-glycans from human immunoglobulin A1: comparison of normal human serum immunoglobulin A1 with that isolated from patients with rheumatoid arthritis.

The primary structures of the N-linked oligosaccharides from normal human serum IgA1 were determined by a combination of sequential exoglycosidase digestion, Bio-Gel P-4 chromatography, anion-exchange chromatography and one-dimensional n.m.r. spectroscopy. Three major N-linked disialylated biantennary-complex-type structures were found (55%). The remaining N-linked oligosaccharides consisted of at least nine further structures, some of which (7%) were of the triantennary type and included disialylated triantennary oligosaccharides with outer-arm fucose substitution [Fuc alpha 1-3(4)]. Compared with IgG, the N-glycan structures on IgA are more completely processed: the outer arms have a higher proportion of galactose and sialic acid, and only trace levels of incompletely galactosylated oligosaccharides, commonly found on IgG, were detected. Analysis of the sialylated O-glycans revealed that 64% were [NeuAc2 alpha 3(6)]2Gal beta 3GalNAc and 9% were [NeuAc2 alpha 3(6)]-Gal beta 4GlcNAc beta 6[NeuAc2 alpha 3(6)Gal beta 3]GalNAc, and 27% were monosialylated. The N-linked glycosylation of both serum IgA1 and IgG isolated from a group of six normal individuals was compared with that from ten patients with rheumatoid arthritis (RA). In contrast with the hypogalactosylation found in IgG from diseased sera, there was no evidence of an equivalent decrease in the galactosylation of the IgA1 oligosaccharides. In addition, the N-glycosylation of IgA1 was remarkably consistent within the group of normal individuals. These data suggest that incomplete galactosylation of N-linked glycans and its augmentation in RA does not extend to IgA1 and that the RA-associated galactosyltransferase deficiency may be restricted to cells producing gamma-chain.

Arthritis, Rheumatoid↗
PubMed1994

N-butyldeoxynojirimycin is a novel inhibitor of glycolipid biosynthesis.

The imino sugar deoxynojirimycin and its alkylated derivatives are inhibitors of the N-linked oligosaccharide processing enzymes alpha-glucosidase I and II. These compounds are glucose analogues and have the potential to inhibit both glucosidases and glucosyltransferases. However, to date there has been no report of deoxynojirimycin or similar analogues inhibiting a mammalian glucosyltransferase. We have investigated the effects of deoxynojirimycin and its alkylated derivatives on the biosynthesis of glycolipids in HL-60 cells. We have found that the N-butyl and N-hexyl derivatives of deoxynojirimycin, but not deoxynojirimycin itself, are novel inhibitors of the glucosyltransferase-catalyzed biosynthesis of glucosylceramide. This results in the inhibition of biosynthesis of all glucosylceramide-based glycosphingolipids. We have investigated the ability of one of these compounds, N-butyldeoxynojirimycin, to offset glucosylceramide accumulation in an in vitro Gaucher's disease model. This compound prevents lysosomal glycolipid storage and offers a novel therapeutic approach for the management of this and other glycolipid storage disorders.

1-Deoxynojirimycin↗
PubMed1994

Secretion of human hepatitis B virus is inhibited by the imino sugar N-butyldeoxynojirimycin.

The imino sugar N-butyldeoxynojirimycin (NBDNJ) is a potent inhibitor of the oligosaccharide-trimming enzyme alpha-glucosidase I. Hepatitis B virus (HBV) contains three surface proteins (HBs proteins) of different sizes that are singly or doubly N-glycosylated and are essential for the formation of infectious virus. Therefore, the replication and secretion of HBV in the human hepatoma cell line HepG2 were studied in the presence of NBDNJ. In the stably HBV-transfected HepG 2.2.15 cells and in HBV-infected HepG2 cells, NBDNJ suppressed secretion of HBV particles and caused intracellular retention of HBV DNA. The secretion of subviral particles was less affected. These data suggest that inhibitors of oligosaccharide trimming may be useful for antiviral therapy of hepatitis B and for the study of the intracellular transport of the viral glycoproteins.

1-Deoxynojirimycin↗
PubMed1994

Glycoforms modify the dynamic stability and functional activity of an enzyme.

Glycoproteins generally consist of collections of glycosylated variants (glycoforms) in which an ensemble of different oligosaccharides is associated with each glycosylation site. Bovine pancreatic ribonuclease B occurs naturally as a mixture of five glycoforms in which the same polypeptide sequence is associated with a series of oligomannose sugars attached at the single N-glycosylation site. Individual glycoforms were prepared by exoglycosidase digestions of RNase B and analyzed directly at the protein level by capillary electrophoresis. For the first time, electrophoretically pure single glycoforms have been available to explore the possibility that different sugars might specifically modify the structure, dynamics, stability, and functional properties of the protein to which they are attached. Comparisons of the amide proton exchange rates for individual glycoforms of RNase B and unglycosylated RNase A showed that while the 3D structure was unaffected, glycosylation decreased dynamic fluctuations throughout the molecule. There was individual variation in the NH-ND exchange rates of the same protons in different glycoforms, demonstrating the effects of variable glycosylation on dynamic stability. Consistent with the overall decrease in flexibility, and with the possibility that all of the sugars may afford steric protection to susceptible sites, was the finding that each of the glycoforms tested showed increased resistance to Pronase compared with the unglycosylated protein. In a novel sensitive assay using double-stranded RNA substrate, the different glycoforms showed nearly a 4-fold variation in functional activity; molecular modeling suggested that steric factors may also play a role in modulating this interaction.

Amino Acid Sequence↗
PubMed1994

9-Fluorenylmethoxycarbonyl (Fmoc)-glycine coupling of saccharide beta-glycosylamines for the fractionation of oligosaccharides and the formation of neoglycoconjugates.

We have devised a new saccharide derivatization scheme to provide not only the temporary attachment of a chromophore for detecting and facilitating the chromatographic separation of carbohydrates, but also the intermediates for further derivatization to produce neoglycoconjugates. Several neutral unprotected saccharide beta-glycosylamines were formed by the direct condensation of the reducing saccharides with aqueous ammonium bicarbonate. The beta-glycosylamine derivatives of N-acetylglucosamine, di-N-acetylchitobiose, and asialo-, digalactosylated biantennary complex oligosaccharide were N-acylated separately with excess 9-fluorenylmethoxycarbonyl (Fmoc)-glycine. The Fmoc-glycinamido beta-derivatives of these unprotected saccharides were well separated by normal-phase high-performance liquid chromatography and detected by ultraviolet absorption. Similar derivatization and fractionation of a partial acid hydrolyzate of chitin were equally successful resulting in the separation of Fmoc-glycinamido derivatives of di-N-acetylchitobiose to hepta-N-acetylchitoheptaose in the hydrolyzate. The reversibility of the Fmoc derivatization was demonstrated by treating the Fmoc-glycinamido derivative of N-acetylglucosamine with piperidine to generate its 1-N-glycyl-beta-saccharide derivative. The structure and stereochemistry of this product was confirmed by proton nuclear magnetic resonance spectroscopy. The 1-N-glycyl-beta-saccharide derivatives are stable intermediates for the formation of asparagine-linked neoglycoconjugates.

Acetylglucosamine↗
PubMed1994

Analytical and preparative separation of anionic oligosaccharides by weak anion-exchange high-performance liquid chromatography on an inert polymer column.

The use of ammonium formate buffers for analytical and preparative weak anion-exchange high-performance chromatography of anionic sugars is described. The method can be used for structures containing sulfate, phosphate, sialic acid, and uronic acid moieties. Excellent separation of the anionic sugars was achieved from pH 5.5 to 9. In contrast to silica matrix columns the polymer matrix column used can tolerate a wide range of pH values for the eluting buffer and it does not shed material into the eluent. The use of a single volatile buffer avoids additional purification steps to recover separated sugars suitable for further structural and functional analyses.

Anion Exchange Resins↗
PubMed1994

Suppression of allogeneic reactivity in vitro by the syncytiotrophoblast membrane glycocalyx of the human term placenta is carbohydrate dependent.

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.

Carbohydrate Metabolism↗
PubMed1994

A human T-cell receptor recognizes 'O'-linked sugars from the hinge region of human IgA1 and IgD.

A receptor which binds secretory IgA (sIgA) is expressed on human T cells from patients with systemic lupus erythematosus, rheumatoid arthritis, Behcet's syndrome and IgA nephropathy and on normal T cells following phytohaemagglutinin (PHA) stimulation. The specificity of this receptor was initially probed with a panel of normal serum immunoglobulins in competitive inhibition assays with sIgA using two-colour immunofluorescence. While the receptor showed the strongest affinity for IgA1 (IC(50)10(-6) M), IgD which has a similarly glycosylated hinge region to IgA1, also bound to the receptor (IC50 10(-5) M). IgA2, which lacks the 'O'-glycosylated hinge region, did not significantly inhibit the binding at these concentrations suggesting that the IgA determinants for this receptor might be the oligosaccharides present in the hinge region of IgA1. IgA1 has up to 10 'O'-linked oligosaccharides and four N-linked oligosaccharides per molecule. In order to probe the role of the 'O'-linked hinge sugars in the binding event, a sugar library was prepared from IgA1 by a procedure designed to release 'O'-linked oligosaccharides preferentially, and to retain them in the natural closed ring formation. The sugars were released by hydrazinolysis at 65 degrees and the resulting oligosaccharide library analysed by high voltage paper electrophoresis (HVE) and P4 gel permeation chromatography. Competitive inhibition studies demonstrated that both the library and the individual 'O'-linked sugars associated with IgA1 were implicated in the binding of IgA1 to this receptor (IC50 between 1 x 10(-5) M and 6 x 10(-5) M). Within this range the individual sugars showed small differences in their affinity for the receptor in the following order: Gal beta 3GalNAc = NeuNAc2 alpha 3(6)Gal beta 3GalNAc > NeuNAc2 alpha 3(6)Gal beta 3[NeuNAc2 alpha 6]GalNAc > or = GalNAc.

Binding, Competitive↗
PubMed1993

Analysis of carbohydrate-protein interactions with synthetic N-linked neoglycoconjugate probes.

Recently we have describe a simple efficient chemical method of generating an asparagine side-chain linker with beta-stereochemistry at the anomeric position of neutral oligosaccharides. We now report the 1-N-glycyl beta-derivatization of sialylated saccharides. Several neoglycoconjugates formed using these N-linked inter-mediates were investigated for their usefulness in probing carbohydrate-protein interactions. First, biotinyl derivatives of two xylose/fucose class plant-type oligosaccharides purified from horseradish peroxidase were effective in demonstrating the carbohydrate specificity of polyclonal anti-(horseradish peroxidase) antibodies. Secondly, a fluorescein-labelled asialo- and digalactosylated biantennary complex sugar was synthesized and shown to bind to a Ricinus communis agglutinin column. This galactose-specific recognition was abolished by treating this fluorescein-labelled oligosaccharide with jack bean beta-galactosidase. Finally, two 1-N-glycyl beta-saccharide derivatives were modified with thiophosgene to form their corresponding isothiocyanate derivatives. Coupling of these isothiocyanate derivatives of sugars to BSA, amino-derivatized polystyrene plates and glass-fibre discs resulted in multiple sugar presentation. The binding of an anti-N-acetylglucosamine monoclonal antibody to N,N'-diacetylchitobiose residues presented on BSA and solid supports was shown by e.l.i.s.a. Similarly the binding of concanavalin A to asialo-, agalactosylated biantennary complex oligosaccharide residues attached to BSA was demonstrated by a competitive e.l.i.s.a. Our results demonstrate that N-linked neoglycoconjugates could be made readily available and they are valuable tools for the detailed analyses of carbohydrates and carbohydrate-binding proteins.

Antibodies↗
PubMed1993

Effects of glycosylation on protein structure and dynamics in ribonuclease B and some of its individual glycoforms.

In an attempt to elucidate the role of carbohydrates on protein structure and dynamics in glycoproteins, ribonuclease B (RNase B), containing a single glycosylation site at Asn34, has been investigated and compared with the enzyme in the unglycosylated form (RNase A). RNase B consists of five glycoforms: Man5GlcNAc2, Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2 and Man9GlcNAc2 (Man5-9GlcNAc2). The individual glycoforms Man1GlcNAc2 (synthetic) and Man5GlcNAc2 of RNase B have been studied to ascertain any specific effects of the different oligosaccharides. NMR measurement of amide-proton/deuterium exchange rates shows that glycosylation of the enzyme leads to the protection of amide-proton resonances from solvent exchange for a large number of residues, both in the vicinity of the glycosylation site (e.g. residues 29-34 and 35) and remote from it (e.g. residues 57-61 and 75-76). In addition, residues 10-13, 32, 34 and 35, which are observed to be protected from exchange as a result of glycosylation in the individual glycoforms Man1Glc-NAc2-RNase and Man5GlcNAc2-RNase (when compared with RNase A) are less protected in RNase B. This additional protection in the glycoforms Man1GlcNAc2-RNase and Man5GlcNAc2-RNase may arise from steric hindrance between the oligosaccharide and protein reducing solvent accessibility. The rates of solvent exchange of amide protons for residues 10-13, 32, 34 and 35 are dependent on the oligosaccharide moiety. The average amide-proton/deuterium exchange rate in Man6-9Glc-NAc2-ribonucleases for residues 10-13 and 35 is approximately three times greater than Man5Glc-NAc2-ribonuclease, while for residues 32 and 34 it is approximately 7-11 times greater. CD analysis of RNase A and RNase B revealed the carbohydrate moiety to have a small stabilizing effect (approximately 5 kJ/mol) on the protein.

Carbohydrate Sequence↗
PubMed1993

Glycosylation of CD4 and Thy-1.

The site-specific glycosylation of soluble recombinant variants of human and rat CD4 (sCD4) expressed in Chinese hamster ovary (CHO) cells has been characterized. The presence of identical oligosaccharides at the conserved glycosylation site in domain 3 of rat and human sCD4 and the greater abundance of oligomannose and hybrid type glycans at the non-conserved glycosylation site of rat sCD4 clearly indicate that the protein structure influences oligosaccharide processing. Comparisons of rat sCD4 glycopeptides with mutant molecules with only single glycosylation sites and with a truncated form containing only the two NH2-terminal domains, indicate that independent processing occurs at each glycosylation site and that domain interactions can also affect oligosaccharide processing. These and other analyses of sCD2 expressed in CHO cells and Thy-1 purified from various tissues suggest that the diversity of oligosaccharide structures on a protein is regulated by the location of the glycosylation sites and the nature of the target protein, cell and tissue. The functional significance of this control remains to be determined.

Animals↗
PubMed1993

Molecular characterization of Limulus polyphemus C-reactive protein. II. Asparagine-linked oligosaccharides.

The N-linked oligosaccharides of C-reactive protein (CRP) from the arachnid Limulus polyphemus, the horseshoe crab, were characterized after their release by hydrazinolysis, re-N-acetylation, and reduction with NaB3H4. High-voltage paper electrophoresis of the reduced oligosaccharides revealed only neutral species. Gel-permeation chromatography on Bio-Gel P4 yielded five fractions. The oligosaccharide fractions were further fractionated using high-voltage borate paper electrophoresis and Dionex BioLC ion-exchange chromatography. The oligosaccharides were structurally characterized by sequential exoglycosidase digestion, fragmentation by acetolysis and methylation analysis. Three major structures were found, of which two were the biantennary oligomannose type with compositions Man5GlcNAc2 (B-1), Man4GlcNAc2 (C-3) and one was the monoantennary structure Man3GlcNAc2 (D-1). The biantennary oligomannose structures B-1 and C-3 contained the structural unit Man alpha 6Man alpha 6R. This unusual arrangement of mannose linkages suggests a biosynthetic pathway in Limulus which differs from that reported in mammals, plants and the parasitic protozoa.

Animals↗
PubMed1993

Site-specific glycosylation of recombinant rat and human soluble CD4 variants expressed in Chinese hamster ovary cells.

The rat and human forms of the T-cell surface glycoprotein CD4 share a common glycosylation site at the Asn270/271 position but differ with respect to the locations of the second glycosylation sites at Asn159 (rat) and Asn300 (human). The glycosylation of soluble recombinant forms of human and rat CD4 (sCD4) expressed in Chinese hamster ovary cells has been characterized. The most obvious differences between the rat and human sCD4 oligosaccharides were the greater abundance of oligomannose and hybrid oligosaccharides on rat sCD4 and the presence of oligosaccharides carrying a terminal alpha-galactose residue on human sCD4. This is the first report of the occurrence of alpha-galactose residues on a glycoprotein expressed in Chinese hamster ovary cells. Comparison of mutant rat sCD4 molecules with single glycosylation sites and glycopeptides indicated that site-specific and independent processing occurred at each glycosylation site. The glycosylation at the conserved site at Asn270 of rat sCD4 was identical to that seen for the equivalent site in human sCD4, and the oligomannose and hybrid structures were restricted to the nonconserved site at Asn159 in rat sCD4. However, there was more oligosaccharide processing at this site in a truncated form of rat sCD4 consisting of the two NH2-terminal domains. These results indicate that not only the local three-dimensional structure but also domain interactions can influence the processing at individual glycosylation sites.

Amino Acid Sequence↗
PubMed1993

Effects of the imino sugar N-butyldeoxynojirimycin on the N-glycosylation of recombinant gp120.

The imino sugar N-butyldeoxynojirimycin (NB-DNJ) exhibits anti-HIV activity in vitro and inhibits the purified glycoprocessing enzyme alpha 1,2-glucosidase I. It has been speculated that the anti-viral activity of this compound may result from inhibition of HIV envelope glycoprotein processing. However, structural evidence that glucosidase inhibition takes place in intact cells at the anti-viral concentration (0.5 mM) is lacking. In this study, N-linked glycosylation of recombinant gp120 expressed in Chinese hamster ovary cells cultured in the presence or absence of NB-DNJ has been characterized. Immunoprecipitation, in conjunction with endoglycosidase H (endo H) digestion and SDS-polyacrylamide gel electrophoresis analysis, revealed that the glycosylation of gp120 was profoundly altered in the presence of NB-DNJ. The majority of the gp120 oligosaccharides from untreated cells were resistant to endo H. However, nearly complete endo H sensitivity was observed following treatment with 0.5 mM NB-DNJ indicating that gp120 expressed in treated cells carries immature, high mannose type oligosaccharides. In addition, using metabolic labeling with [3H]mannose, gel filtration chromatography, and digestion with highly purified glucosidases I and II, we provide the first definitive evidence that glucosidase I inhibition occurs at the anti-viral concentration of NB-DNJ. These data indicate that glucosidase inhibition is a candidate mechanism for the anti-viral activity of this compound.

1-Deoxynojirimycin↗