Search PubMed⌕ Search

Biomedical subjects

R A Dwek

Publications and source records attributed to R A Dwek.

At least 109 records · Page 6Linked to original sources
PubMed1996

Site-specific glycosylation of human immunoglobulin G is altered in four rheumatoid arthritis patients.

Alterations in the glycosylation of human IgG have been shown to occur in rheumatoid arthritis (RA). However, the precise nature and location of these changes have not been fully established. Therefore we carried out a detailed analysis of the oligosaccharides chemically released from intact human serum IgG and fragments of the molecule. Serum samples were from three healthy ('normal') individuals, and from four patients with RA. Site-specific glycolsylation of the glycoprotein was shown to occur, which extended to sites even within the Fab fragment. These were differences in galactosylation, sialylation and the presence of a bisecting N-acetylglucosomide. Disease related alterations were also shown to be site-specific. In particular, an increase in the proportion of agalactosylated oligosaccharides occurred on the Fc fragment in RA (P=0.057), but, in contrast to previous reports there was an increase on the light chain in the proportion of fully galactosylated, bisected and core fucosylated oligosaccharides (from 13% of total in normal to between 18 and 35% in RA, P=0.057)). There was also an Fab-specific increase in oligosaccharides bearing a bisecting N-acetylglucosamine and a core fucose (P=0.075) The site-specific glycosylation changes described in this paper reveal the complexity of the regulatory mechanism, perhaps reflecting the many levels at which regulation can occur.

Aged↗
PubMed1996

Effect of serine O-glycosylation on cis-trans proline isomerization.

In order to examine the ability of an O-glycosylated serine residue preceding proline to stabilize a cis amide conformation in a fashion similar to that observed with aromatic amino acid residues, we prepared a series of glycosylated analogs of a small linear peptide which we have previous reported to contain a cis conformation of an amide bond between tyrosine and proline. The glycopeptides were prepared by incorporating glycosylated N alpha- (fluoren-9-yl) methoxycarbonyl (Fmoc) amino acids into a standard solid phase peptide synthesis protocol. The peptides and glycopeptides were analyzed using 2-dimensional NMR spectroscopy. Unlike the case where the residue preceding proline was tyrosine, no signals corresponding to a cis proline conformation were detected in the spectra of the two glycopeptides containing serine O-glycosylated with either beta-linked N-acetyl glucosamine or alpha-linked N-acetyl galactosamine in the position preceding proline.

Acetylglucosamine↗
PubMed1996

The identification of abnormal glycoforms of serum transferrin in carbohydrate deficient glycoprotein syndrome type I by capillary zone electrophoresis.

One of the biochemical characteristics of carbohydrate deficient glycoprotein syndromes is the presence of abnormal glycoforms in serum transferrin. Both glycoform heterogeneity and variable site occupancy may, in principle, lead to the generation of a range of glycoforms which contain different numbers of sialic acid residues, and therefore variable amounts of negative charge. Capillary zone electrophoresis was used to resolve the glycoforms of normal human serum transferrin and also of a set of glycoforms which were prepared by digesting the sugars on the intact glycoprotein with sialidase. The sugars on the intact glycoprotein were also modified by a series of exoglycosidase enzymes to produce a series of neutral glycoforms which were-also analysed by capillary zone electrophoresis. The oligosaccharide population of human serum transferrin was analysed by a series of mixed exoglycosidase digests on the released glycan pool and quantified using a novel HPLC strategy. Transferrin was isolated from carbohydrate deficient glycoprotein syndromes type I serum and both the intact glycoforms and released sugars were resolved and quantified. The data presented here confirm the presence of a hexa-, penta- and tetra-sialoforms of human serum transferrin in both normal and carbohydrate deficient glycoprotein syndrome type I serum samples. Consistent with previous reports carbohydrate deficient glycoprotein syndrome type I transferrin also contained a di-sialoform, representing a glycoform in which one of the two N-glycosylation sites is unoccupied, and a non-glycosylated form where both remain unoccupied. This study demonstrates that capillary zone electrophoresis can be used to resolve quantitatively both sialylated and neutral complex type glycoforms, suggesting a rapid diagnostic test for the carbohydrate deficient glycoprotein syndromes group of diseases.

Carbohydrates↗
PubMed1996

N-butyldeoxynojirimycin-mediated inhibition of human immunodeficiency virus entry correlates with changes in antibody recognition of the V1/V2 region of gp120.

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although NB-DNJ appears to inhibit HIV entry at the level of post-CD4 binding (P.B. Fischer, M. Collin, G.B. Karlsson, W. James, T.D. Butters, S.J. Davis, S. Gordon, R.A. Dwek, and F.M. Platt, J. Virol. 69:5791-5797, 1995), the exact mechanism of action remains to be established. In this study we have examined the effect of NB-DNJ on the structure of recombinant gp120 (rgpl20), expressed in CHO cells, by using a panel of 40 monoclonal antibodies. The levels of binding of antibodies to rgp120 produced in the presence [rgpl20(+)] and absence [rgpl20(-)] of NB-DNJ were compared by enzyme-linked immunosorbent assay and surface plasmon resonance (BIAcore; Pharmacia). The results showed an increase in the binding to rgp120(+) of antibodies directed against the C1 and C2 regions and a decrease in the binding of antibodies directed against the V1/V2 loops compared with antibody binding to rgpl20(-). A decrease in the binding to rgpl20(+) of antibodies directed against discontinuous epitopes was also observed. No differences were seen in the binding of antibodies directed against the crown of the V3 loop and the C4 region of gp120. Treatment of rgpl20 with alpha-glucosidases I and II had no effect on the differential binding observed, whereas treatment with sialidase abolished the differences seen in the binding of antibodies directed against the C1 and C2 regions of gp120. In addition to these findings, rgpl20(+) showed increased sensitivity to proteases released by CHO cells during expression, as well as to exogenous thrombin. Taken together, the data presented in this paper suggest that production of gp120 in the presence of NB-DNJ affects the conformation of the Vl/V2 loops of gpl20, as well as the overall charge of the C1 and C2 regions. These effects may play a role in the previously described NB-DNJ-mediated inhibition of HIV entry at the level of post-CD4 binding.

1-Deoxynojirimycin↗
PubMed1996

N-butyldeoxynojirimycin-mediated inhibition of human immunodeficiency virus entry correlates with impaired gp120 shedding and gp41 exposure.

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is an inhibitor of human immunodeficiency virus (HIV) replication and HIV-induced syncytium formation in vitro. Although an NB-DNJ-mediated change in viral envelope N-glycan composition inhibits HIV entry at the level of post-CD4 binding, the exact mechanism of inhibition remains to be established. In this study we have examined the effects of NB-DNJ on virion envelope composition and CD4-induced gp120 shedding and gp41 exposure. Virion composition analysis revealed an NB-DNJ-mediated reduction of 15% in overall virion envelope glycoprotein content and a reduction of 26% in the proteolytic maturation of virion gp160. Taken together, these two effects resulted in a reduction of approximately 40% in virion gp120 content. CD4-induced shedding of gp120 from the surfaces of envelope-transfected Cos cells was undetectable when gp120 was expressed in the presence of NB-DNJ. Similarly, the shedding of virion-associated gp120 was reduced 7.4-fold. CD4-induced exposure of cryptic gp41 epitopes on the surfaces of HIV-expressing ACH-2 cells was also greatly impaired, and the exposure of virion-associated gp41 epitopes was reduced 4.0-fold. Finally, CD4-induced increases in the binding of antibodies to the V3 loop of ACH-2-cell-expressed envelope glycoproteins were reduced 25-fold when the glycoproteins were expressed in the presence of NB-DNJ. These results suggest that the NB-DNJ-mediated retention of glycosylated N-glycans inhibits HIV entry by a combined effect of a reduction in virion gp120 content and a qualitative defect within the remaining gp120, preventing it from undergoing conformational changes after CD4 binding.

1-Deoxynojirimycin↗
PubMed1995

The effects of variable glycosylation on the functional activities of ribonuclease, plasminogen and tissue plasminogen activator.

The relatively large size and dynamics of oligosaccharides can result in substantial shielding of functionally important areas of proteins to which they are attached, modulate the interactions of glycoconjugates with other molecules and affect the rate of processes which involve conformational changes. This review focuses on the occupancy of N-linked glycosylation sites on three enzymes, ribonuclease, plasminogen and tissue plasminogen activator. Each of these proteins occurs naturally as two populations of molecules, distinguished from each other only by the presence or absence of an oligosaccharide at one glycosylation site. The presence of an oligomannose sugar on ribonuclease (at Asn-34) alters its overall dynamics, increases its stability towards proteinases and decreases its functional activity towards double-stranded RNA. The N-linked sugar on plasminogen (at Asn-288) within kringle 3 reduces the rate of the beta- to alpha-conformational change, modulates the transport of plasminogen into the extravascular compartment, decreases plasminogen binding to U937 cells and downregulates the activation of plasminogen by both urokinase and tissue plasminogen activator. Additionally, in fibrinolysis, within a ternary complex of fibrin, plasminogen and tissue plasminogen activator, the N-linked sugar of plasminogen hinders the initial interaction with tissue plasminogen activator (i.e., it alters Km). The presence of an N-linked glycan (at Asn-184) in the kringle 2 domain of tissue plasminogen activator hinders the rearrangement of this ternary complex, decreasing the turnover rate (Kcat).

Animals↗
PubMed1995

The activation of type 1 and type 2 plasminogen by type I and type II tissue plasminogen activator.

Tissue plasminogen activator (tPA) was fractionated using lysine-Sepharose affinity chromatography. Type I, type II, and a minor peak with high affinity for lysine (designated type D) tPA were recovered. In an indirect amidolytic assay involving native human Glu-plasminogen and fibrin, type II tPA showed a 2-fold higher activity than type I. To explore the combinatorial effect of the variable glycosylation status of both tPA and plasminogen, kinetic constants for fibrin-dependent plasminogen activation were determined for combinations of type I, II, and D tPA with type 1 and 2 plasminogen. Within a 4-fold range, the fastest rate was achieved from the combination of type D (type II + D) tPA and type 2 plasminogen. N-Glycosylation of plasminogen increased the Km value for activation by all tPA variants; N-glycosylation of type I tPA at Asn184 decreased the kcat (turnover) values for the fibrin-dependent activation of plasminogen over type II tPA, while type D tPA showed the highest turnover rate. In the presence of fibrinogen fragments, N-glycosylation of plasminogen at site 289 modulates the kinetics of association of enzyme and substrate, while N-glycosylation at site 184 on tPA modulates the turnover rate of the enzyme.

Amino Acid Sequence↗
PubMed1995

Ligand binding by the immunoglobulin superfamily recognition molecule CD2 is glycosylation-independent.

The evolutionary success of the immunoglobulin superfamily (IgSF) is thought to reflect the ability of IgSF protein domains to form stable structural units. The role of glycosylation in stabilizing these domains is controversial, however. In this study a systematic analysis of the effect of glycosylation on the ligand-binding properties of the cell-cell recognition molecule CD2, which consists of two IgSF domains, was undertaken. A form of human soluble CD2 (hsCD2) with single N-acetylglucosamine residues at each glycosylation site was produced by inhibiting glucosidase I with N-butyldeoxynojirimycin during expression in Chinese hamster ovary cells and digesting the expressed hsCD2 with endoglycosidase H. The ligand and antibody binding properties of this form of hsCD2 were indistinguishable from those of fully glycosylated hsCD2 as determined by surface plasmon resonance analyses. The protein also formed diffraction quality crystals and analysis of the 2.5-A resolution crystal structure indicated that the single N-acetylglucosamine residue present on domain 1 is unlikely to stabilize the ligand binding face of hsCD2. A second, fully deglycosylated form of hsCD2 also bound the ligand and antibodies although this form of the protein tended to aggregate. In contrast to the results of previous studies, the current data indicate that the structural integrity and ligand binding function of human CD2 are glycosylation-independent.

1-Deoxynojirimycin↗
PubMed1995

Peptide anchor residue glycosylation: effect on class I major histocompatibility complex binding and cytotoxic T lymphocyte recognition.

This study extends our previous observation that glycopeptides bind to class I major histocompatibility complex (MHC) molecules and elicit carbohydrate-specific CTL responses. The Sendai virus nucleoprotein wild-type (WT) peptide (FAPGNYPAL) binds H-2Db using the P5-Asn as an anchor. The peptide K2 carrying a P5 serine substitution did not bind Db. Surprisingly, glycosylation of the serine (K2-O-GlcNAc) with N-acetylglucosamine (GlcNAc), a novel cytosolic O-linked glycosylation, partially restored peptide binding to Db. We argue that the N-acetyl group of GlcNAc may fulfil the hydrogen bonding requirements of the Db pocket which normally accomodates P5-Asn. Glycosylation of the P5-Asn residue itself abrogated binding similar to K2, probably for steric reasons. The peptide K2-O-GlcNAc readily elicited Db-restricted cytotoxic T lymphocytes (CTL), which did not cross-react with K2 or WT. However, all Db-restricted CTL raised against K2-O-GlcNAc cross-reacted strongly with another glycopeptide, K3-O-GlcNAc, where the GlcNAc substitution is on a neighboring P4-Ser. Furthermore, Db-restricted CTL clones raised against K2-O-GlcNAc or K3-O-GlcNAc displayed a striking TCR conservation. Our interpretation is that the carbohydrate of K2-O-GlcNAc not only mediates binding to Db, but also interacts with the TCR in such a way as to mimic K3-O-GlcNAc. This unusual example of molecular mimicry extends the known effects of peptide glycosylation from what we and others have previously reported: glycosylation may create a T cell neo-epitope, or, conversely, abrogate recognition. Alternatively, glycosylation may block peptide binding to MHC class I and finally, as reported here, restore binding, presumably through direct interaction of the carbohydrate with the MHC molecule.

Acetylglucosamine↗
PubMed1995

Glycosylation changes of IgG associated with rheumatoid arthritis can activate complement via the mannose-binding protein.

The glycosylation of the circulating immunoglobulin-gamma (IgG) antibody molecules changes in rheumatoid arthritis. The extent of the changes correlates with the disease severity and reverses in remission. We demonstrate here that the alteration in glycosylation associated with rheumatoid arthritis can create a new mode for the interaction of IgG with complement through binding to the collagenous lectin mannose-binding protein (MBP). Rheumatoid arthritis is associated with a marked increases in IgG glycoforms that lack galactose (referred to as G0 glycoforms) in the Fc region of the molecule and that terminate in N-acetyl glucosamine (GlcNAc). We show, using nuclear magnetic resonance (NMR) and X-ray data, that these terminal GlcNAc residues become accessible for MBP binding. We further demonstrate that multiple presentation of IgG-G0 glycoforms to MBP results in activation of the complement. This suggests that a contribution to the chronic inflammation of the synovial membrane could arise from the localization of the IgG-G0 glycoforms in the affected joint and from resulting activation of complement.

Arthritis, Rheumatoid↗
PubMed1995

Cells regulate the activities of cytokines by glycosylation.

Cytokines, including the interferons, interleukins, colony stimulating factors, and chemokines, are first-line, hormone-like defense molecules that orchestrate immune functions in a nonspecific, i.e., antigen-independent, way. The multiplicity of cytokines has a genetic basis, but oligomerization and especially glycosylation add to the heterogeneity and signaling functions of the cytokines. It is theorized that the cell uses glycosylation of cytokines to alter its functions. This is achieved by changing the specific biological activities, by altering diffusability, tissue distribution, and pharmacokinetics, and by targeting different populations of responsive cells. One interesting possibility is that multiple glycosylation forms compete for receptor binding resulting in a natural selection process or possibly antagonistic effects. Cytokines as ligands often are multimers and cytokine receptors are almost without exception heterodimers, which within receptor families share common subunits. Hence, carbohydrate-lectin interactions might contribute to ligand dimerization and receptor recognition through multivalent bindings.

Animals↗
PubMed1995

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin inhibits human immunodeficiency virus entry at the level of post-CD4 binding.

The alpha-glucosidase inhibitor N-butyldeoxynojirimycin (NB-DNJ) is a potent inhibitor of human immunodeficiency virus (HIV) replication and syncytium formation in vitro. However, the exact mechanism of action of NB-DNJ remains to be determined. In this study we have examined the impairment of HIV infectivity mediated by NB-DNJ. By two independent HIV entry assays [PCR-based HIV entry assay and entry of Cocal(HIV) pseudotypes], the reduction in infectivity was found to be due to an impairment of viral entry. No effect of NB-DNJ treatment was seen on the kinetics of the interaction between gp120 and CD4 (surface plasmon resonance; BIAcore) or on the binding of virus particles to H9 cells (using radiolabeled virions). We therefore conclude that a major mechanism of action of NB-DNJ as an inhibitor of HIV replication is the impairment of viral entry at the level of post-CD4 binding, due to an effect on viral envelope components.

1-Deoxynojirimycin↗
PubMed1995

Glycobiology: 'the function of sugar in the IgG molecule'.

Immunoglobulin G (IgG) is glycosylated in both the Fc and the Fab regions of the protein with a heterogeneous ensemble of structures (glycoforms) that is both highly reproducible (i.e. nonrandom) and site specific. In normal IgG, the 2 highly conserved oligosaccharides of the Fc region are found buried between the CH2 domains, forming specific protein-saccharide interactions with the Fc protein surface. One of the functions attributed to the Fc oligosaccharides of normal IgG is to maintain the conformational arrangements of the Fc domains as well as the hinge regions. These structural features are necessary for Fc effector functions such as Clq and monocyte binding. A hallmark of rheumatoid arthritis (RA) patients is a dramatic increase in the presence of serum IgG containing Fc oligosaccharides lacking an outer arm galactose residue (termed 'G0' glycoforms). The increased level of G0 has been shown to be directly related to the pathogenesis of RA. Nuclear magnetic resonance relaxation studies of the Fc region from normal and RA IgG, as well as examination of x-ray structures, show that the G0 oligosaccharides have an increased mobility resulting from the loss of binding between the G0 oligosaccharide and the Fc protein surface. From these observations it follows that regions of the protein surface that are normally covered by the oligosaccharide are revealed. The newly accessible protein surface could have lectin-like activity and also be inherently antigenic. In addition, the more mobile G0 oligosaccharide can be recognised by mannose binding protein. As the mannose binding protein can activate complement, and the Fc oligosaccharide would not normally be accessible to protein recognition, this finding might suggest a specific role for the G0 glycoform in inflammation when the appropriate IgG glycoforms are clustered.

Acute-Phase Proteins↗
PubMed1994

N-butyldeoxygalactonojirimycin inhibits glycolipid biosynthesis but does not affect N-linked oligosaccharide processing.

We have previously reported that the imino sugar N-butyldeoxynojirimycin (NB-DNJ) inhibits glycolipid biosynthesis, in addition to its known activity as an inhibitor of the N-linked oligosaccharide processing enzyme alpha-glucosidase I. In an attempt to dissociate these two activities and identify an inhibitor which was more selective for the glycolipid biosynthetic pathway, several imino sugars have been N-alkylated and tested for inhibitory activity. The galactose analogue N-butyldeoxygalactonojirimycin (NB-DGJ) was found to be a potent inhibitor of glycolipid biosynthesis but in contrast to NB-DNJ had no effect on the maturation of N-linked oligosaccharides or on lysosomal glucocerebrosidase. The effect of increasing N-alkyl chain length on glycolipid inhibition was investigated. Nonalkylated DGJ, the N-methyl and N-ethyl derivatives, were noninhibitory. However, N-propylation resulted in partial inhibition while the N-butyl and N-hexyl derivatives resulted in maximal inhibition. Increasing alkyl chain length also resulted in increased potency of glucosyltransferase inhibition. In an in vitro Gaucher's disease model NB-DGJ was as effective as NB-DNJ in preventing glycolipid storage and may represent a more selective potential therapeutic agent than NB-DNJ for the management of this and other glycosphingolipidoses.

1-Deoxynojirimycin↗
PubMed1994

Recognition of carbohydrate by major histocompatibility complex class I-restricted, glycopeptide-specific cytotoxic T lymphocytes.

Cytotoxic T cells (CTL) recognize short peptide epitopes presented by class I glycoproteins encoded by the major histocompatibility complex (MHC). It is not yet known whether peptides containing posttranslationally modified amino acids can also be recognized by CTL. To address this issue, we have studied the immunogenicity and recognition of a glycopeptide carrying an O-linked N-acetylglucosamine (GlcNAc) monosaccharide-substituted serine residue. This posttranslational modification is catalyzed by a recently described cytosolic glycosyltransferase. We show that glycosylation does not affect peptide binding to MHC class I and that glycopeptides can elicit a strong CTL response that is glycopeptide specific. Furthermore, glycopeptide recognition by cytotoxic T cells is dependent on the chemical structure of the glycan as well as its position within the peptide.

Amino Acid Sequence↗