Biomedical subjects

F M Platt

Publications and source records attributed to F M Platt.

At least 37 records · Page 2Linked to original sources

PubMed1999

Imino sugars inhibit the formation and secretion of bovine viral diarrhea virus, a pestivirus model of hepatitis C virus: implications for the development of broad spectrum anti-hepatitis virus agents.

One function of N-linked glycans is to assist in the folding of glycoproteins by mediating interactions of the lectin-like chaperone proteins calnexin and calreticulin with nascent glycoproteins. These interactions can be prevented by inhibitors of the alpha-glucosidases, such as N-butyl-deoxynojirimycin (NB-DNJ) and N-nonyl-DNJ (NN-DNJ), and this causes some proteins to be misfolded and retained within the endoplasmic reticulum (ER). We have shown previously that the NN-DNJ-induced misfolding of one of the hepatitis B virus (HBV) envelope glycoproteins prevents the formation and secretion of virus in vitro and that this inhibitor alters glycosylation and reduces the viral levels in an animal model of chronic HBV infection. This led us to investigate the effect of glucosidase inhibitors on another ER-budding virus, bovine viral diarrhea virus, a tissue culture surrogate of human hepatitis C virus (HCV). Here we show that in MDBK cells alpha-glucosidase inhibitors prevented the formation and secretion of infectious bovine viral diarrhea virus. Data also are presented showing that NN-DNJ, compared with NB-DNJ, exhibits a prolonged retention in liver in vivo. Because viral secretion is selectively hypersensitive to glucosidase inhibition relative to the secretion of cellular proteins, the possibility that glucosidase inhibitors could be used as broad-based antiviral hepatitis agents is discussed. A single drug against HBV, HCV, and, possibly, HDV, which together chronically infect more than 400 million people worldwide, would be of great therapeutic value.

1-Deoxynojirimycin↗

PubMed1999

Tyrosinase folding and copper loading in vivo: a crucial role for calnexin and alpha-glucosidase II.

Tyrosinase is the key enzyme of melanin biosynthesis. It is a multiply glycosylated metalloenzyme, which has a long maturation time making it an ideal in vivo model system to probe protein folding and metal loading events. The use of NB-DNJ, an alpha-glucosidase I and II inhibitor has allowed us to dissect these processes. Here we show that tyrosinase folds through several inactive intermediates, at least two of which are recognised by the ER chaperone, calnexin. If the association with calnexin is prevented, more rapid folding occurs, the resulting protein fails to bind copper and is inactive. If dissociation from calnexin is inhibited, folding is prevented; the protein does not go through the normal secretory pathway and is targeted for degradation. Thus, tyrosinase folds off calnexin, giving alpha-glucosidase II a critical role, but the association with calnexin is essential to promote the correct folding which enables it to acquire copper.

Animals↗

PubMed1999

Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin.

Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the HEXB gene, which encodes the beta-subunit of beta-hexosaminidase. GM2 ganglioside fails to be degraded and accumulates within lysosomes in cells of the periphery and the central nervous system (CNS). There are currently no therapies for the glycosphingolipid lysosomal storage diseases that involve CNS pathology, including the GM2 gangliosidoses. One strategy for treating this and related diseases is substrate deprivation. This would utilize an inhibitor of glycosphingolipid biosynthesis to balance synthesis with the impaired rate of catabolism, thus preventing storage. One such inhibitor is N-butyldeoxynojirimycin, which currently is in clinical trials for the potential treatment of type 1 Gaucher disease, a related disease that involves glycosphingolipid storage in peripheral tissues, but not in the CNS. In this study, we have evaluated whether this drug also could be applied to the treatment of diseases with CNS storage and pathology. We therefore have treated a mouse model of Sandhoff disease with the inhibitor N-butyldeoxynojirimycin. The treated mice have delayed symptom onset, reduced storage in the brain and peripheral tissues, and increased life expectancy. Substrate deprivation therefore offers a potentially general therapy for this family of lysosomal storage diseases, including those with CNS disease.

1-Deoxynojirimycin↗

PubMed1998

New therapeutic prospects for the glycosphingolipid lysosomal storage diseases.

The glycosphingolipid (GSL) lysosomal storage diseases result from mutations in the genes that encode the enzymes required for glycosphingolipid catabolism within lysosomes. They are relatively rare diseases, but are frequently severe in terms of their pathology. Many involve progressive neurodegeneration, and in the most severe forms result in death in early infancy. The therapeutic options for treating these diseases are limited, and for the majority of these disorders there are currently no therapies available. To date, most research has focused on correcting the genetic lesion by gene therapy or by augmenting the enzyme activity deficient in these patients by introducing fully functional enzyme. This can be achieved by bone marrow transplantation or intravenous infusion of purified or recombinant enzyme (enzyme replacement). Gene therapy and enzyme replacement therapy are disease specific, and pharmacological approaches for the treatment of these disorders have not been fully explored. In this commentary, the problems associated with disease therapy are discussed, and a pharmacological agent (N-butyldeoxynojirimycin) is presented for the potential generic treatment of this family of disorders. Successful prevention of glycosphingolipid storage in a mouse model of Tay-Sachs disease suggests that this strategy merits clinical evaluation.

Animals↗

PubMed1998

Inhibition of glycosphingolipid biosynthesis does not impair growth or morphogenesis of the postimplantation mouse embryo.

Whole embryo culture (WEC) of organogenesis-stage mouse embryos was adapted for glycosphingolipid (GSL) metabolic studies to evaluate the hypothesis that de novo GSL biosynthesis is a prerequisite for growth and morphogenesis of the early postimplantation embryo. WEC supports the growth and development of postimplantation mouse embryos to stages that are indistinguishable from those achieved in vivo. N-Butyldeoxygalactonojirimycin (NB-DGJ) is an N-alkylated imino sugar that specifically inhibits biosynthesis of all glucosylceramide-based GSLs. NB-DGJ inhibited glucosylceramide and lactosylceramide biosynthesis nearly completely and inhibited ganglioside biosynthesis approximately 90% in both the embryo and visceral yolk sac. NB-DGJ also significantly reduced total ganglioside content in both the embryo and visceral yolk sac as estimated by the cholera toxin immunooverlay technique. A shift in expression from the structurally simple to the structurally complex gangliosides was also observed in NB-DGJ-treated embryos and yolk sacs. Despite causing major changes in GSL biosynthesis and composition, NB-DGJ had no effect on embryo viability, growth, or morphology. The findings suggest that de novo GSL biosynthesis may not be a prerequisite for the growth and morphogenesis of the organogenesis-stage mouse embryo.

1-Deoxynojirimycin↗

PubMed1997

Extensive glycosphingolipid depletion in the liver and lymphoid organs of mice treated with N-butyldeoxynojirimycin.

The imino sugar N-butyldeoxynojirimycin is an inhibitor of the ceramide-specific glucosyltransferase that catalyzes the first step in glycosphingolipid biosynthesis. It results in extensive glycosphingolipid depletion in cells treated in vitro, without causing toxicity. However, we currently do not know the degree to which glycosphingolipids can be depleted in vivo in a mammalian species. We have therefore administered N-butyldeoxynojirimycin long term to young mice and have found that glycosphingolipid levels are reduced (50-70%) in all tissues examined, without resulting in any overt pathology. When the lymphoid tissues from these mice were examined, they were found to be 50% acellular relative to non-lymphoid tissues. These data implicate a role for glycosphingolipids in the biology of the immune system or indicate an additional as yet unknown activity of N-butyldeoxynojirimycin. Extensive glycosphingolipid depletion resulting from N-butyldeoxynojirimycin administration is therefore well tolerated in adult mice, and this compound may be in an invaluable tool for probing glycosphingolipid functions in vivo. In addition, this drug may be effective in clinical situations where glycosphingolipid depletion would be desirable, such as the in the treatment of the human glycosphingolipidoses.

1-Deoxynojirimycin↗

PubMed1997

The solution NMR structure of glucosylated N-glycans involved in the early stages of glycoprotein biosynthesis and folding.

Glucosylated oligomannose N-linked oligosaccharides (Glc(x)Man9GlcNAc2 where x = 1-3) are not normally found on mature glycoproteins but are involved in the early stages of glycoprotein biosynthesis and folding as (i) recognition elements during protein N-glycosylation and chaperone recognition and (ii) substrates in the initial steps of N-glycan processing. By inhibiting the first steps of glycan processing in CHO cells using the alpha-glucosidase inhibitor N-butyl-deoxynojirimycin, we have produced sufficient Glc3Man7GlcNAc2 for structural analysis by nuclear magnetic resonance (NMR) spectroscopy. Our results show the glucosyl cap to have a single, well-defined conformation independent of the rest of the saccharide. Comparison with the conformation of Man9GlcNAc2, previously determined by NMR and molecular dynamics, shows the mannose residues to be largely unaffected by the presence of the glucosyl cap. Sequential enzymatic cleavage of the glucose residues does not affect the conformation of the remaining saccharide. Modelling of the Glc3Man9GlcNAc2, Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2 conformations shows the glucose residues to be fully accessible for recognition. A more detailed analysis of the conformations allows potential recognition epitopes on the glycans to be identified and can form the basis for understanding the specificity of the glucosidases and chaperones (such as calnexin) that recognize these glycans, with implications for their mechanisms of action.

1-Deoxynojirimycin↗

PubMed1997

Inhibition of N-glycan processing in B16 melanoma cells results in inactivation of tyrosinase but does not prevent its transport to the melanosome.

Tyrosinase is the key enzyme in melanin biosynthesis, catalyzing multiple steps in this pathway. The mature glycoprotein is transported from the Golgi to the melanosome where melanin biosynthesis occurs. In this study, we have investigated the effects of inhibitors of N-glycan processing on the synthesis, transport, and catalytic activity of tyrosinase. When B16 mouse melanoma cells were cultured in the presence of N-butyldeoxynojirimycin, an inhibitor of the endoplasmic reticulum-processing enzymes alpha-glucosidases I and II, the enzyme was synthesized and transported to the melanosome but almost completely lacked catalytic activity. The cells contained only 2% of the melanin found in untreated cells. Structural analysis of the N-glycans from N-butyldeoxynojirimycin-treated B16 cells demonstrated that three oligosaccharide structures (Glc3Man7-9) predominated. Removal of the glucose residues with alpha-glucosidases I and II failed to restore enzymatic activity, suggesting that the glucosylated N-glycans do not sterically interfere with the enzyme's active sites. The mannosidase inhibitor deoxymannojirimycin had no effect on catalytic activity suggesting that the retention of glucosylated N-glycans results in the inactivation of this enzyme. The retention of glucosylated N-glycans does not therefore result in misfolding and degradation of the glycoprotein, as the enzyme is transported to the melanosome, but may cause conformational changes in its catalytic domains.

1-Deoxynojirimycin↗

PubMed1997

Prevention of lysosomal storage in Tay-Sachs mice treated with N-butyldeoxynojirimycin.

The glycosphingolipid (GSL) lysosomal storage diseases result from the inheritance of defects in the genes encoding the enzymes required for catabolism of GSLs within lysosomes. A strategy for the treatment of these diseases, based on an inhibitor of GSL biosynthesis N-butyldeoxynojirimycin, was evaluated in a mouse model of Tay-Sachs disease. When Tay-Sachs mice were treated with N-butyldeoxynojirimycin, the accumulation of GM2 in the brain was prevented, with the number of storage neurons and the quantity of ganglioside stored per cell markedly reduced. Thus, limiting the biosynthesis of the substrate (GM2) for the defective enzyme (beta-hexosaminidase A) prevents GSL accumulation and the neuropathology associated with its lysosomal storage.

1-Deoxynojirimycin↗

PubMed1997

Ultrastructural changes in the Golgi apparatus and secretory granules of HL-60 cells treated with the imino sugar N-butyldeoxynojirimycin.

The imino sugar N-butyldeoxynojirimycin inhibits the N-linked oligosaccharide processing enzymes alpha-glucosidases I and II, and the ceramide specific glucosyltransferase which catalyses the first step in glucosphingolipid biosynthesis. We have studied the effects of this compound on the ultrastructure of HL-60 cells to identify novel activities of this compound. Treatment of HL-60 cells with this imino sugar results in several morphological changes within the cell, none of which result in cytotoxicity. The plasma membrane stains heavily with potassium ferrocyanide within 30 min following addition of the compound to the medium, and there is then a time dependent involvement of all other intracellular membranes. Secretory granules become enlarged and lose their dense core morphology and appear either empty and vacuolated or have low density contents. However, the most striking effect of NB-DNJ treatment is on the Golgi apparatus. The Golgi exhibits a time-dependent change from typical Golgi morphology to a structure almost completely devoid of cisternae and consisting predominantly of vesicles. All the observed changes are fully reversible on withdrawal of the compound.

1-Deoxynojirimycin↗

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 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↗

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

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↗

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↗

PubMed1993

Aberrant control of galactosyltransferase in peripheral B lymphocytes and Epstein-Barr virus transformed B lymphoblasts from patients with rheumatoid arthritis.

It is now well established that hypogalactosylation of IgG is a molecular marker for rheumatoid arthritis (RA). However, the mechanism for the alteration of the galactosylation status has not been resolved. We compared the galactosyltransferase activities of anti-CD19 selected peripheral B lymphocytes of healthy subjects and patients with RA using ovalbumin as the acceptor substrate. In addition, certain samples of lymphocytes were assayed after Epstein-Barr virus (EBV) transformation and, also, the ability of bovine milk galactosyltransferase to galactosylate IgG in vitro was examined. Our results indicate that there is a significant difference between the galactosyltransferase activities of rheumatoid and control peripheral B lymphocytes and that EBV transformation causes a variable increase (15-1225%) in galactosyltransferase activity, over that present in the peripheral B lymphocytes from which the transformed cells were derived. Also the ubiquitous "lactose synthetase" type galactosyltransferase (EC 2.4.1.38) will galactosylate normal native IgG at concentrations of 500 mU/ml in vitro. We conclude that there is no evidence from our study for an IgG specific galactosyltransferase and that galactosyltransferase is an enzyme that is aberrantly modulated in peripheral B lymphocytes and EBV transformed B lymphoblasts derived from patients with RA.

Adult↗