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Biomedical subjects

R A Dwek

Publications and source records attributed to R A Dwek.

At least 19 recordsLinked to original sources
PubMed2003

Central nervous system inflammation is a hallmark of pathogenesis in mouse models of GM1 and GM2 gangliosidosis.

Mouse models of the GM2 gangliosidoses [Tay-Sachs, late onset Tay-Sachs (LOTS), Sandhoff] and GM1 gangliosidosis have been studied to determine whether there is a common neuro-inflammatory component to these disorders. During the disease course, we have: (i) examined the expression of a number of inflammatory markers in the CNS, including MHC class II, CD68, CD11b (CR3), 7/4, F4/80, nitrotyrosine, CD4 and CD8; (ii) profiled cytokine production [tumour necrosis factor alpha (TNF alpha), transforming growth factor (TGF beta 1) and interleukin 1 beta (IL1 beta)]; and (iii) studied blood-brain barrier (BBB) integrity. The kinetics of apoptosis and the expression of Fas and TNF-R1 were also assessed. In all symptomatic mouse models, a progressive increase in local microglial activation/expansion and infiltration of inflammatory cells was noted. Altered BBB permeability was evident in Sandhoff and GM1 mice, but absent in LOTS mice. Progressive CNS inflammation coincided with the onset of clinical signs in these mouse models. Substrate reduction therapy in the Sandhoff mouse model slowed the rate of accumulation of glycosphingolipids in the CNS, thus delaying the onset of the inflammatory process and disease pathogenesis. These data suggest that inflammation may play an important role in the pathogenesis of the gangliosidoses.

1-Deoxynojirimycin↗
PubMed2002

Glycosphingolipid lysosomal storage diseases: therapy and pathogenesis.

Paediatric neurodegenerative diseases are frequently caused by inborn errors in glycosphingolipid (GSL) catabolism and are collectively termed the glycosphingolipidoses. GSL catabolism occurs in the lysosome and a defect in an enzyme involved in GSL degradation leads to the lysosomal storage of its substrate(s). GSLs are abundantly expressed in the central nervous system (CNS) and the disorders frequently have a progressive neurodegenerative course. Our understanding of pathogenesis in these diseases is incomplete and currently few options exist for therapy. In this review we discuss how mouse models of these disorders are providing insights into pathogenesis and also leading to progress in evaluating experimental therapies.

1-Deoxynojirimycin↗
PubMed2001

High-performance liquid chromatography analysis of ganglioside carbohydrates at the picomole level after ceramide glycanase digestion and fluorescent labeling with 2-aminobenzamide.

The functional importance of glycolipids has emphasized the need for more sensitive methods of detection, characterization, and quantification than has often been possible using traditional thin-layer chromatographic techniques. We describe the use of ceramide glycanase and HPLC to identify and quantify gangliosides in which the carbohydrate is in Glcbeta1--> linkage with ceramide. Detection of released carbohydrate was by fluorescent labeling with 2-aminobenzamide at the reducing terminal prior to HPLC analysis. Under the conditions described, ceramide glycanase hydrolyzed all of the common gangliosides studied, offering a broad spectrum of specificity. Release and detection of carbohydrate were linear over a wide range (over two orders of magnitude) of micromolar glycolipid substrate concentrations. Use of an N-linked glycan as an internal standard allowed accurate quantification and a recovery of 93% was achieved. The method additionally maintained the sensitivity (chromatographic peaks containing 1 pmol were readily detected from tissue samples) and comparable resolution to related assays. This was shown by the separation, not only of isomeric carbohydrates from the "a" and "b" series, but also of ganglioside carbohydrate differing only by the presence of either N-acetyl- or N-glycolylneuraminic acid. Application of the method to neutral glycosphingolipids and to tissue samples, including 10-microl quantities of plasma, is illustrated. Glycan structures were confirmed by exoglycosidase digestion and/or matrix-assisted laser desorption/ionization mass spectrometry.

Aminopyridines↗
PubMed2001

Matrix remodelling enzymes, the protease cascade and glycosylation.

Glycosylation influences the specific activities of serine proteases including tissue-type plasminogen activator and plasmin which act together in a ternary complex with fibrin. Serine proteases and matrix metalloproteinases (MMPs), including gelatinase B, participate in a protease cascade to remodel the extracellular matrix. In addition to the recognition and targeting functions of carbohydrates and the fact that they confer protease resistance on glycoproteins, oligosaccharides may extend particular protein domains of matrix remodelling enzymes and fine-control their activities within the context of the extracellular matrix. For example, the sialic acids of gelatinase B influence the catalytic activity of this enzyme in a complex with the tissue inhibitor of metalloproteinases-1 (TIMP-1).

Animals↗
PubMed2001

Antibodies inhibit prion propagation and clear cell cultures of prion infectivity.

Prions are the transmissible pathogenic agents responsible for diseases such as scrapie and bovine spongiform encephalopathy. In the favoured model of prion replication, direct interaction between the pathogenic prion protein (PrPSc) template and endogenous cellular prion protein (PrPC) is proposed to drive the formation of nascent infectious prions. Reagents specifically binding either prion-protein conformer may interrupt prion production by inhibiting this interaction. We examined the ability of several recombinant antibody antigen-binding fragments (Fabs) to inhibit prion propagation in cultured mouse neuroblastoma cells (ScN2a) infected with PrPSc. Here we show that antibodies binding cell-surface PrPC inhibit PrPSc formation in a dose-dependent manner. In cells treated with the most potent antibody, Fab D18, prion replication is abolished and pre-existing PrPSc is rapidly cleared, suggesting that this antibody may cure established infection. The potent activity of Fab D18 is associated with its ability to better recognize the total population of PrPC molecules on the cell surface, and with the location of its epitope on PrPC. Our observations support the use of antibodies in the prevention and treatment of prion diseases and identify a region of PrPC for drug targeting.

Animals↗
PubMed2001

Crystal structure of a neutralizing human IGG against HIV-1: a template for vaccine design.

We present the crystal structure at 2.7 angstrom resolution of the human antibody IgG1 b12. Antibody b12 recognizes the CD4-binding site of human immunodeficiency virus-1 (HIV-1) gp120 and is one of only two known antibodies against gp120 capable of broad and potent neutralization of primary HIV-1 isolates. A key feature of the antibody-combining site is the protruding, finger-like long CDR H3 that can penetrate the recessed CD4-binding site of gp120. A docking model of b12 and gp120 reveals severe structural constraints that explain the extraordinary challenge in eliciting effective neutralizing antibodies similar to b12. The structure, together with mutagenesis studies, provides a rationale for the extensive cross-reactivity of b12 and a valuable framework for the design of HIV-1 vaccines capable of eliciting b12-like activity.

AIDS Vaccines↗
PubMed2001

Prion glycoprotein: structure, dynamics, and roles for the sugars.

The prion protein contains two N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The large size of the N-linked sugars, together with their dynamic properties, enables them to shield two orthogonal faces of the protein almost completely. Thus, the sugars can protect large regions of the protein surface from proteases and from nonspecific protein-protein interactions. Immunoprecipitation of prion protein with calnexin suggests that in the ER the oligosaccharides may provide a route for protein folding via the calnexin pathway. Major questions relate to the relevance of the glycoform distribution (as defined by glycan site occupancy) to strain type and disease transmission. Glycan analysis has shown that prion protein contains at least 52 different sugars, that these consist of a subset of brain sugars, and that there is site specific glycan processing. PrP(Sc) from the brains of Syrian hamsters contains the same set of glycans as PrP(C), but a higher proportion of tri- and tetra-antennary sugars. This may be attributed to a decrease in the activity of GnTIII. The GPI anchor, which is modified with sialic acid, may allow the prion protein to be mobile in the lipid bilayer. Potentially, this provides a possible means for translocating the prions from one cell to another.

Animals↗
PubMed2001

Glycosylation and the immune system.

Almost all of the key molecules involved in the innate and adaptive immune response are glycoproteins. In the cellular immune system, specific glycoforms are involved in the folding, quality control, and assembly of peptide-loaded major histocompatibility complex (MHC) antigens and the T cell receptor complex. Although some glycopeptide antigens are presented by the MHC, the generation of peptide antigens from glycoproteins may require enzymatic removal of sugars before the protein can be cleaved. Oligosaccharides attached to glycoproteins in the junction between T cells and antigen-presenting cells help to orient binding faces, provide protease protection, and restrict nonspecific lateral protein-protein interactions. In the humoral immune system, all of the immunoglobulins and most of the complement components are glycosylated. Although a major function for sugars is to contribute to the stability of the proteins to which they are attached, specific glycoforms are involved in recognition events. For example, in rheumatoid arthritis, an autoimmune disease, agalactosylated glycoforms of aggregated immunoglobulin G may induce association with the mannose-binding lectin and contribute to the pathology.

Animals↗
PubMed2001

Enhanced survival in Sandhoff disease mice receiving a combination of substrate deprivation therapy and bone marrow transplantation.

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 beta-hexosaminidase. Bone marrow transplantation (BMT), which augments enzyme levels, and substrate deprivation (using the glycosphingolipid biosynthesis inhibitor 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.

1-Deoxynojirimycin↗
PubMed2001

A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N-linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands.

The majority of biologically active proteins are glycosylated, therefore any approach to proteomics which fails to address the analysis of oligosaccharides is necessarily incomplete. To appreciate the structure of a glycoprotein fully, to understand the roles for the attached oligosaccharides and to monitor disease associated changes it is necessary to visualise the sugars as well as the protein. To achieve this aim when biological samples are available at the low microgram level or less has involved increasing the sensitivity of the technology for glycan analysis. Since one protein may have many different oligosaccharides attached to it (glycoforms) this is a major technical challenge. CD59, for example, has over 100 different sugars at one N-linked glycosylation site. Applications of recently developed technology suggest that it is now becoming realistic to extend the proteomics analysis of glycoproteins to include details of glycosylation. This is achieved by releasing the N-glycans from the protein in a gel by optimised peptide-N-glycosidase F digestion. The released glycans are then tagged with the fluorophore, 2-amino benzamide. The labelled glycan pools (containing 50-100 femtomoles of glycans) are resolved by predictive normal phase high performance liquid chromatography (HPLC) on an amide based column or by reverse phase HPLC on a C18 column. Preliminary structural assignments are confirmed by exoglycosidase array digestions of the entire glycan pool. Complementary matrix-assisted laser desorption/ionization-mass spectrometry, which requires 10-20 times as much sugar for a single run, can be used where there is sufficient material. This provides a composition analysis but not linkage information.

Amidohydrolases↗
PubMed2001

Inhibition of substrate synthesis as a strategy for glycolipid lysosomal storage disease therapy.

The glycosphingolipid (GSL) lysosomal storage diseases are caused by mutations in the genes encoding the glycohydrolases that catabolize GSLs within lysosomes. In these diseases the substrate for the defective enzyme accumulates in the lysosome and the stored GSL leads to cellular dysfunction and disease. The diseases frequently have a progressive neurodegenerative course. The therapeutic options for treating these diseases are relatively limited, and for the majority there are no effective therapies. The problem is further compounded by difficulties in delivering therapeutic agents to the brain. Most research effort to date has focused on strategies for augmenting enzyme levels to compensate for the underlying defect. These include bone marrow transplantation (BMT), enzyme replacement and gene therapy. An alternative strategy that we have been exploring is substrate deprivation. This approach aims to balance the rate of GSL synthesis with the impaired rate of GSL breakdown. The imino sugar N-butyldeoxynojirimycin (NB-DNJ) inhibits the first step in GSL biosynthesis and has been used to evaluate this approach. Studies in an asymptomatic mouse model of Tay-Sachs disease have shown that substrate deprivation prevents GSL storage in the CNS. In a severe neurodegenerative mouse model of Sandhoff disease, substrate deprivation delayed the onset of symptoms and disease progression and significantly increased life expectancy. Combining NB-DNJ and BMT was found to be synergistic in the Sandhoff mouse model. A clinical trial in type I Gaucher disease has been undertaken and has shown beneficial effects. Efficacy was demonstrated on the basis of significant decreases in liver and spleen volumes, gradual but significant improvement in haematological parameters and disease activity markers, together with diminished GSL biosynthesis and storage as determined by independent biochemical assays. Further trials in type I Gaucher disease are in progress; studies are planned in patients with GSL storage in the CNS.

Animals↗
PubMed2001

A family of novel, acidic N-glycans in Bowes melanoma tissue plasminogen activator have L2/HNK-1-bearing antennae, many with sulfation of the fucosylated chitobiose core.

A family of about 20 novel acidic bi- and tri-antennary N-glycans, amounting to almost half those expressed on Bowes melanoma tissue-plasminogen activator (t-PA) were found to possess Galbeta1-->4GlcNAcbeta1-->, sulfated and sialylated GalNAcbeta1-->4GlcNAcbeta1--> or sulfated GlcAbeta1--> 3Galbeta1-->4GlcNAcbeta1--> antennae, of which those containing sulfated GlcA, depicting the L2/HNK-1 carbohydrate epitope, were preferentially located on the 6 arm. A proportion of the glycans were highly charged, because of multiple and variously distributed sulfation, some of which was located on the fucosylated chitobiose core. Multiple expression of the L2/HNK-1 epitope on a single glycan was observed. The most abundant compound was a biantennary glycan carrying sulfated GlcA on the 6-branched antenna and an alpha2-->6 sialylated GalNAc on the other. The N-glycosylation sequon containing Asn448, which is known to express all of the sulfate-carrying N-glycans contains, unusually, an arginine residue. An electrostatic interaction between this cationic amino acid and the core-sulfate group of the N-glycan is proposed to reduce mobility of the carbohydrate in the region of the t-PA active site. Because of the 'brain-type' nature of the N-glycans described in this neuro-ectodermal cell line, the possibility of neural t-PA interacting with the L2/HNK-1-recognizing molecule, laminin, of the central nervous system extracellular matrix is discussed.

Antigens, Tumor-Associated, Carbohydrate↗
PubMed2001

Inhibition of hepatitis B virus DNA replication by imino sugars without the inhibition of the DNA polymerase: therapeutic implications.

Previously we have shown that the imino sugar inhibitor of N-linked glycan processing, N-nonyl-deoxynojirimycin (N-nonyl-DNJ), had antiviral activity in the woodchuck model of chronic hepatitis B virus (HBV) infection. In studying the mechanism of action of this compound, it was discovered that imino sugars could inhibit HBV secretion without inhibiting N-linked glycoprocessing. Although N-nonyl-DNJ is an inhibitor of the endoplasmic reticulum (ER) glucosidase, here it is shown that N-nonyl-DNJ retained antiviral activity at concentrations that had no significant impact on ER glucosidase function. Taken together, these results suggested that N-nonyl-DNJ possessed an antiviral activity attributable to a function other than an impact on glycoprocessing. This hypothesis was confirmed by experiments showing that N-nonyl-deoxygalactojirimycin (N-nonyl-DGJ), an alkyl derivative of galactose with no impact on glycoprocessing, retains anti-HBV activity. The data suggest that N-nonyl-DGJ exerts its antiviral action at a point before viral envelopment and may prevent the proper encapsidation of the HBV pregenomic RNA.

1-Deoxynojirimycin↗
PubMed2001

Hepatitis B virus MHBs antigen is selectively sensitive to glucosidase-mediated processing in the endoplasmic reticulum.

Previous studies have shown that hepatitis B virus (HBV) secretion from HepG 2.2.15 cells is prevented by inhibitors of the endoplasmic reticulum (ER) glucosidase under conditions where secretion of cellular glycoproteins are not detectably affected. The 2.2.15 cells are derived from HepG2 and contain intact dimers of the viral genome. They produce and secrete infectious HBV. The secretion of the viral envelope polypeptide, MHBs, was selectively and quantitatively reduced from 2.2.15 cells in which glucosidase was inhibited, whereas the envelope polypeptide, SHBs, was relatively insensitive, being as resistant as were most host glycoproteins. Because 2.2.15 cells express all HBV ORFs, it seemed possible that the sensitivity of MHBs secretion involved its interaction with the viral nucleocapsid or other viral gene products. The work reported here showed that MHBs secretion from HepG2 cells transfected with a plasmid that expresses only the MHBs polypeptide was as sensitive to glucosidase inhibitors as it was from 2.2.15 cells. These data show that the sensitivity of the MHBs polypeptide secretion to glucosidase inhibitors is entirely encrypted within its structural gene. The reasons the MHBs polypeptide, but not SHBs, is so sensitive to glucosidase processing are discussed.

Cell Line↗
PubMed2001

Study of the mechanism of antiviral action of iminosugar derivatives against bovine viral diarrhea virus.

The glucose-derived iminosugar derivatives N-butyl- and N-nonyl-deoxynojirimycin (DNJ) have an antiviral effect against a broad spectrum of viruses including Bovine viral diarrhea virus (BVDV). For BVDV, this effect has been attributed to the reduction of viral secretion due to an impairment of viral morphogenesis caused by the ability of DNJ-based iminosugar derivatives to inhibit ER alpha-glucosidases (N. Zitzmann, A. S. Mehta, S. Carrouée, T. D. Butters, F. M. Platt, J. McCauley, B. S. Blumberg, R. A. Dwek, and T. M. Block, Proc. Natl. Acad. Sci. USA 96:11878-11882, 1999). Here we present the antiviral features of newly designed DNJ derivatives and report for the first time the antiviral activity of long-alkyl-chain derivatives of deoxygalactonojirimycin (DGJ), a class of iminosugars derived from galactose which does not inhibit endoplasmic reticulum (ER) alpha-glucosidases. We demonstrate the lack of correlation between the ability of long-alkyl-chain DNJ derivatives to inhibit ER alpha-glucosidases and their antiviral effect, ruling out ER alpha-glucosidase inhibition as the sole mechanism responsible. Using short- and long-alkyl-chain DNJ and DGJ derivatives, we investigated the mechanisms of action of these drugs. First, we excluded their potential action at the level of the replication, protein synthesis, and protein processing. Second, we demonstrated that DNJ derivatives cause both a reduction in viral secretion and a reduction in the infectivity of newly released viral particles. Long-alkyl-chain DGJ derivatives exert their antiviral effect solely via the production of viral particles with reduced infectivity. We demonstrate that long-alkyl-chain DNJ and DGJ derivatives induce an increase in the quantity of E2-E2 dimers accumulated within the ER. The subsequent enrichment of these homodimers in secreted virus particles correlates with their reduced infectivity.

1-Deoxynojirimycin↗
PubMed2001

Antiviral effect of N-butyldeoxynojirimycin against bovine viral diarrhea virus correlates with misfolding of E2 envelope proteins and impairment of their association into E1-E2 heterodimers.

The iminosugar N-butyldeoxynojirimycin (NB-DNJ), an endoplasmic reticulum alpha-glucosidase inhibitor, has an antiviral effect against bovine viral diarrhea virus (BVDV). In this report, we investigate the molecular mechanism of this inhibition by studying the folding pathway of BVDV envelope glycoproteins in the presence and absence of NB-DNJ. Our results show that, while the disulfide-dependent folding of E2 glycoprotein occurs rapidly (2.5 min), the folding of E1 occurs slowly (30 min). Both BVDV envelope glycoproteins associate rapidly with calnexin and dissociate with different kinetics. The release of E1 from the interaction with calnexin coincides with the beginning of E1 and E2 association into disulfide-linked heterodimers. In the presence of NB-DNJ, the interaction of E1 and E2 with calnexin is prevented, leading to misfolding of the envelope glycoproteins and inefficient formation of E1-E2 heterodimers. The degree of misfolding and the lack of association of E1 and E2 into disulfide-linked complexes in the presence of NB-DNJ correlate with the dose-dependent antiviral effect observed for this iminosugar.

1-Deoxynojirimycin↗