Raymond Dwek - Academia.edu (original) (raw)

Papers by Raymond Dwek

Blood, 2001

Sandhoff disease is a lysosomal storage disorder characterized by G M2 ganglioside accumulation i... more Sandhoff disease is a lysosomal storage disorder characterized by G M2 ganglioside accumulation in the central nervous system (CNS) and periphery. It results from mutations in the HEXB gene, causing a deficiency in ␤-hexosaminidase. Bone marrow transplantation (BMT), which augments enzyme levels, and substrate deprivation (using the glycosphingolipid biosynthesis in-hibitor N-butyldeoxynojirimycin [NB-DNJ]) independently have been shown to extend life expectancy in a mouse model of Sandhoff disease. The efficacy of combining these 2 therapies was evaluated. Sandhoff disease mice treated with BMT and NB-DNJ survived significantly longer than those treated with BMT or NB-DNJ alone. When the mice were subdivided into 2 groups on the basis of their donor bone marrow-derived CNS enzyme levels, the high enzyme group exhibited a greater degree of synergy (25%) than the group as a whole (13%). Combination therapy may therefore be the strategy of choice for treating the infantile onset disease variants. (Blood. 2001;97:327-329)

J Virol, 2002

2G12 is a broadly neutralizing human monoclonal antibody against human immunodeficiency virus typ... more 2G12 is a broadly neutralizing human monoclonal antibody against human immunodeficiency virus type-1 (HIV-1) that has previously been shown to bind to a carbohydrate-dependent epitope on gp120. Here, sitedirected mutagenesis and carbohydrate analysis were used to define further the 2G12 epitope. Extensive alanine scanning mutagenesis showed that elimination of the N-linked carbohydrate attachment sequences associated with residues N295, N332, N339, N386, and N392 by N3A substitution produced significant decreases in 2G12 binding affinity to gp120 JR-CSF . Further mutagenesis suggested that the glycans at N339 and N386 were not critical for 2G12 binding to gp120 JR-CSF . Comparison of the sequences of isolates neutralized by 2G12 was also consistent with a lesser role for glycans attached at these positions. The mutagenesis studies provided no convincing evidence for the involvement of gp120 amino acid side chains in 2G12 binding. Antibody binding was inhibited when gp120 was treated with Aspergillus saitoi mannosidase, Jack Bean mannosidase, or endoglycosidase H, indicating that Man␣132Man-linked sugars of oligomannose glycans on gp120 are required for 2G12 binding. Consistent with this finding, the binding of 2G12 to gp120 could be inhibited by monomeric mannose but not by galactose, glucose, or N-acetylglucosamine. The inability of 2G12 to bind to gp120 produced in the presence of the glucose analogue N-butyl-deoxynojirimycin similarly implicated Man␣132Man-linked sugars in 2G12 binding. Competition experiments between 2G12 and the lectin cyanovirin for binding to gp120 showed that 2G12 only interacts with a subset of available Man␣132Manlinked sugars. Consideration of all the data, together with inspection of a molecular model of gp120, suggests that the most likely epitope for 2G12 is formed from mannose residues contributed by the glycans attached to N295 and N332, with the other glycans playing an indirect role in maintaining epitope conformation.

Biochemistry Usa, 1977

The Fv fragment (Hochman, J., Inbar, D., and Givol, D. (1 973), Biochemistry 12, I 130) derived f... more The Fv fragment (Hochman, J., Inbar, D., and Givol, D. (1 973), Biochemistry 12, I 130) derived from mouse myeloma protein 3 15, possessing anti-dinitrophenyl (Dnp)

Int J Biol Macromol, 1979

ABSTRACT A series of paramagnetic probes for Dnp binding antibodies has been developed by cupling... more ABSTRACT A series of paramagnetic probes for Dnp binding antibodies has been developed by cupling a metal chelating group onto the immunodominant Dnp-group. The approach is illustrated for protein 315. The haptens used in the present study are of the form N-Dnp-2-aminoethyl (phosphate)n and the paramagnetic ion chosen in Mn(II). The use of proton high resolution nuclear magnetic resonances (n.m.r.) difference spectroscopy allows quantitative measurements of the perturbation caused by the Mn(II) on several of the protein resonances. The sum of the distances between the metal and the ring centres of the two histidine residues, His 97L and His 102H, in the third hypervariable regions of the light (L) and heary (H) chain is found to be 20 ± 1 Å. This is in good agreement with the value of 19.4 a between the two histidines calculated from the coordinates of the predicted model of padlan et. al.4

Journal of Virology, Jul 1, 2008

The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasi... more The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man 4 ) that corresponds to the D1 arm of Man 9 GlcNAc 2 inhibits 2G12 binding to gp120 as efficiently as Man 9 GlcNAc 2 itself, indicating the potential use of Man 4 as a building block for creating immunogens. Here, we describe the development of neoglycoconjugates displaying variable copy numbers of Man 4 on bovine serum albumin (BSA) molecules by conjugation to Lys residues. The increased valency enhances the apparent affinity of 2G12 for Man 4 up to a limit which is achieved at ϳ10 copies per BSA molecule, beyond which no further enhancement is observed. Immunization of rabbits with BSA-(Man 4 ) 14 elicits significant serum Ab titers to Man 4 . However, these Abs are unable to bind gp120. Further analysis reveals that the elicited Abs bind a variety of unbranched and, to a lesser extent, branched Man 9 derivatives but not natural N-linked oligomannose containing the chitobiose core. These results suggest that Abs can be readily elicited against the D1 arm; however, potential differences in the presentation of Man 4 on neoglycoconjugates, compared to glycoproteins, poses challenges for eliciting anti-mannose Abs capable of crossreacting with gp120 and HIV-1.

Molecular Physics, 1966

ABSTRACT The positive Overhauser enhancement observed for the tert-butyl groups of tri-tert-butyl... more ABSTRACT The positive Overhauser enhancement observed for the tert-butyl groups of tri-tert-butyl phenol in solutions of tri-tert-butyl phenoxyl radical has been shown to be associated with the proton exchange between the radical and phenol molecules. It is postulated that in the radical molecule the scalar coupling between the electron and the tert-butyl protons occurs at least in part by a hyperconjugative mechanism and therefore the coupling is modulated by random rotation of the tert-butyl groups. This leads to a positive polarization of the tert-butyl protons, which persists when the radical accepts a proton and becomes a tri-tert-butyl phenol molecule.

Biochemical Society Transactions, 1973

ABSTRACT

Journal of Anatomy, Oct 1, 1995

Immunoglobulin G (IgG) is glycosylated in both the Fc and the Fab regions of the protein with a h... more 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 'GO' glycoforms). The increased level of GO 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 GO oligosaccharides have an increased mobility resulting from the loss of binding between the GO 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 GO 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 GO glycoform in inflammation when the appropriate IgG glycoforms are clustered.

The Journal of Biological Chemistry, 1999

We previously reported that CR-Fc, an Fc chimeric protein containing the cysteine-rich (CR) domai... more We previously reported that CR-Fc, an Fc chimeric protein containing the cysteine-rich (CR) domain of the mannose receptor, binds to marginal zone metallophilic macrophages (Mo) and B cell areas in the spleen and to subcapsular sinus Mo in lymph nodes of naive mice (CR-Fc(+) cells). Several CR-Fc ligands were found in spleen and lymph node tissue lysates using ligand blots. In this paper we report the identification of two of these ligands as sialoadhesin (Sn), an Mo-specific membrane molecule, and the leukocyte common antigen, CD45. CR-Fc bound selectively to Sn purified from spleen and lymph nodes and to two low molecular weight isoforms of CD45 in a sugar-dependent manner. CR-Fc binding and non-binding forms of Sn, probably derived from CR-Fc(+) and CR-Fc(-) cells respectively, were selected from spleen lysates. Analysis of the glycan pool associated with the CR-Fc-binding form revealed the presence of charged structures resistant to sialidase, absent in the non-binding form, that could correspond to sulfated structures. These results confirm the identification of the CR region of the mannose receptor as a lectin. We also demonstrate that the same glycoprotein expressed in different cells of the same organ can display distinct sugar epitopes that determine its binding properties.

Molecular Immunology, Apr 1, 1984

A C1q binding assay is presented which is suitable for use in comparison of the binding ability o... more A C1q binding assay is presented which is suitable for use in comparison of the binding ability of different antibodies, and which allows the quantitative determination of their binding constants. The assay system uses IgG bound to a hapten-derivatized Affigel support. No non-specific binding is observed to a DNP-derivatized support, allowing the use of anti-DNP antibodies. With mouse anti-DNP hybridoma IgGs it was found that C1q binding followed the series IgG2a greater than IgG2b much greater than IgG1, in accordance with the complement fixing ability of these subclasses. Since it is relatively simple to couple any antigen to Affigel , this assay system should be generally applicable to any antibody-antigen system.

Biochem Biophys Res Commun, 1996

In order to examine the ability of an O-glycosylated serine residue preceding proline to stabiliz... more 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.

Crit Rev Biochem Molec Biol, 2002

Aβ 1-40 , amyloid-β peptide (1-40); ALS, amyotrophic lateral sclerosis; AP-1/2, activating protei... more Aβ 1-40 , amyloid-β peptide (1-40); ALS, amyotrophic lateral sclerosis; AP-1/2, activating protein-1/

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Nature Biotechnology, Jul 1, 2001

Glycoconjugate Journal, May 1, 1992

The development of methods to separate, analyse and monitor changes in glycoform populations is e... more The development of methods to separate, analyse and monitor changes in glycoform populations is essential if a more detailed understanding of the structure, function and processing of glycoproteins is to emerge. In this study, intact ribonuclease B was resolved by borate capillary electrophoresis into five populations according to the particular oligomannose structure associated with each glycoform. The relative proportions of these populations are correlated with the percentages obtained indirectly by analysis of the hydrazine released oligosaccharides using Bio-Gel P-4 gel filtration, matrix assisted laser desorption mass spectrometry and high performance anion exchange chromatography. Alterations in the composition of the glycoform populations during digestion of ribonuclease B with A. saitoi ~(1-2)mannosidase were monitored by capillary electrophoresis (CE). Digestion of the free oligosaccharides under the same conditions, monitored by anion exchange chromatography, revealed a difference in rate, allowing some insight into the role of the protein during oligosaccharide processing. In conjunction with other methods, this novel application of CE may prove a useful addition to the techniques available for the study of glycoform populations.

Biochemical and Biophysical Research Communications, Feb 8, 2002

Macrophages synthesize and secrete apolipoprotein E (apoE) constitutively. This process is upregu... more Macrophages synthesize and secrete apolipoprotein E (apoE) constitutively. This process is upregulated under conditions of cholesterol loading. The response to cholesterol is antiatherogenic as it is believed to promote cholesterol efflux from the artery wall. The concentration of lactosyl ceramide (LacCer), a glycosphingolipid recently discovered to regulate cellular signaling, proliferation, and expression of adhesion molecules, is also increased in atherosclerotic tissues. Here we have investigated the effect of exogenous LacCer on macrophage apoE levels. We show that increasing macrophage LacCer levels sevenfold led to reductions in cellular and secreted apoE (15 and 30%, respectively, over a 24-h period) as determined by enzyme-linked immunosorbent assay. A similar effect was also induced by glucosyl ceramide (GlcCer) but not by ganglioside species. When macrophages were converted to cholesterol-loaded foam cells by incubation with acetylated LDL, the resulting increase in cellular apoE levels was inhibited by 26% when the cells were subsequently enriched with LacCer. After metabolic labeling of cellular glycosphingolipids with [ 14 C]palmitate, we also discovered that high-density lipoprotein (HDL) stimulates the efflux of glycosphingolipids from foam cells. These data imply that LacCer and GlcCer may be proatherogenic due to the suppression of macrophage apoE production. Furthermore, the efflux of glycosphingolipids from macrophage foam cells to HDL could indicate a potential pathway for their removal from the artery wall and subsequent delivery to the liver. © 2002 Elsevier Science (USA)