Plasma glucosylceramide deficiency as potential risk factor for venous thrombosis and modulator of anticoagulant protein C pathway.
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To assess the relationship between venous thrombosis and plasma glucosylceramide (GlcCer) or phosphatidylethanolamine (PE), plasma levels of GlcCer and PE were determined for 70 venous thrombosis patients referred for evaluation and 70 healthy blood donors. The mean GlcCer level, but not the PE level, was lower in patients versus controls (4.9 vs 6.5 microg/mL [P =.0007] and 66 vs 71 microg/mL [P =.48], respectively). As a measure of relative risk, the odds ratio for deep vein thrombosis in subjects with GlcCer levels below the 10th percentile of controls was 5.7 (95% CI, 2.3-14). To assess the influence of glycolipids on anticoagulant response to activated protein C (APC):protein S in modified prothrombin time assays, the effects of depleting endogenous plasma GlcCer by glucocerebrosidase treatment or of adding exogenous purified GlcCer or other neutral glycolipids to plasma were tested. Glucocerebrosidase treatment reduced plasma sensitivity to APC:protein S in parallel with GlcCer reduction. Exogenously added GlcCer and the homologous Glc-containing globotriaosylceramide (Gb3Cer), but not galactosylceramide, dose-dependently prolonged clotting times of normal plasma in the presence, but not absence, of APC:protein S, which suggests that GlcCer or Gb3Cer can enhance protein C pathway anticoagulant activity. In studies using purified proteins, inactivation of factor Va by APC:protein S was enhanced by GlcCer alone and by GlcCer in multicomponent vesicles containing phosphatidylserine and phosphatidylcholine. These results suggest that the neutral glycolipids GlcCer and Gb3Cer may directly contribute to the anticoagulant activity of the protein C pathway and that deficiency of plasma GlcCer may be a risk factor for venous thrombosis. (Blood. 2001;97:1907-1914) (+info)
Inhibition of Vero cell cytotoxic activity in Escherichia coli O157:H7 lysates by globotriaosylceramide, Gb3, from bovine milk.
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In order to clarify the presence and verotoxin (VT) inhibitory activity of globotriaosylceramide (Gb3) in bovine milk, we analyzed neutral glycosphingolipids (GSLs) from bovine milk and investigated the inhibitory effect of bovine milk Gb3 on the cytotoxicity of VT2. Five species of neutral GSLs, designated as N-1, N-2, N-3, N-4, and N-5, were separated on thin-layer chromatography (TLC). N-1, N-2, and N-3 showed the same mobility as glucosylceramide, lactosylceramide, and Gb3 on the TLC plate, respectively. N-4 and N-5 GSLs migrated below globoside on the TLC plate. N-3 GSL having the same TLC mobility as Gb3 from bovine milk was immunologically identified as Gb3 by monoclonal antibody against Gb3, anti-CD77 monoclonal antibody. Furthermore, the effect of bovine milk Gb3 on VT2-induced cytotoxicity was investigated. We found that treatment of VT2 with bovine milk Gb3 can reduce the cytotoxic effect of VT2. (+info)
Safety and efficacy of recombinant human alpha-galactosidase A--replacement therapy in Fabry's disease.
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BACKGROUND: Fabry's disease, lysosomal alpha-galactosidase A deficiency, results from the progressive accumulation of globotriaosylceramide and related glycosphingolipids. Affected patients have microvascular disease of the kidneys, heart, and brain. METHODS: We evaluated the safety and effectiveness of recombinant alpha-galactosidase A in a multicenter, randomized, placebo-controlled, double-blind study of 58 patients who were treated every 2 weeks for 20 weeks. Thereafter, all patients received recombinant alpha-galactosidase A in an open-label extension study. The primary efficacy end point was the percentage of patients in whom renal microvascular endothelial deposits of globotriaosylceramide were cleared (reduced to normal or near-normal levels). We also evaluated the histologic clearance of microvascular endothelial deposits of globotriaosylceramide in the endomyocardium and skin, as well as changes in the level of pain and the quality of life. RESULTS: In the double-blind study, 20 of the 29 patients in the recombinant alpha-galactosidase A group (69 percent) had no microvascular endothelial deposits of globotriaosylceramide after 20 weeks, as compared with none of the 29 patients in the placebo group (P<0.001). Patients in the recombinant alpha-galactosidase A group also had decreased microvascular endothelial deposits of globotriaosylceramide in the skin (P<0.001) and heart (P<0.001). Plasma levels of globotriaosylceramide were directly correlated with clearance of the microvascular deposits. After six months of open-label therapy, all patients in the former placebo group and 98 percent of patients in the former recombinant alpha-galactosidase A group who had biopsies had clearance of microvascular endothelial deposits of globotriaosylceramide. The incidence of most treatment-related adverse events was similar in the two groups, with the exception of mild-to-moderate infusion reactions (i.e., rigors and fever), which were more common in the recombinant alpha-galactosidase A group. IgG seroconversion occurred in 88 percent of patients who received recombinant alpha-galactosidase A. CONCLUSIONS: Recombinant alpha-galactosidase A replacement therapy cleared microvascular endothelial deposits of globotriaosylceramide from the kidneys, heart, and skin in patients with Fabry's disease, reversing the pathogenesis of the chief clinical manifestations of this disease. (+info)
Targeting of Shiga toxin B-subunit to retrograde transport route in association with detergent-resistant membranes.
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In HeLa cells, Shiga toxin B-subunit is transported from the plasma membrane to the endoplasmic reticulum, via early endosomes and the Golgi apparatus, circumventing the late endocytic pathway. We describe here that in cells derived from human monocytes, i.e., macrophages and dendritic cells, the B-subunit was internalized in a receptor-dependent manner, but retrograde transport to the biosynthetic/secretory pathway did not occur and part of the internalized protein was degraded in lysosomes. These differences correlated with the observation that the B-subunit associated with Triton X-100-resistant membranes in HeLa cells, but not in monocyte-derived cells, suggesting that retrograde targeting to the biosynthetic/secretory pathway required association with specialized microdomains of biological membranes. In agreement with this hypothesis we found that in HeLa cells, the B-subunit resisted extraction by Triton X-100 until its arrival in the target compartments of the retrograde pathway, i.e., the Golgi apparatus and the endoplasmic reticulum. Furthermore, destabilization of Triton X-100-resistant membranes by cholesterol extraction potently inhibited B-subunit transport from early endosomes to the trans-Golgi network, whereas under the same conditions, recycling of transferrin was not affected. Our data thus provide first evidence for a role of lipid asymmetry in membrane sorting at the interface between early endosomes and the trans-Golgi network. (+info)
Kinetic analysis of binding between Shiga toxin and receptor glycolipid Gb3Cer by surface plasmon resonance.
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Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surface and is responsible for hemolytic uremic syndrome. Stx has two isoforms, Stx1 and Stx2, and in clinical settings Stx2 is known to cause more severe symptoms, although the differences between the mechanisms of action of Stx1 and Stx2 are as yet unknown. In this study, the binding modes of these two isoforms to the receptor were investigated with a surface plasmon resonance analyzer to compare differences by real time receptor binding analysis. A sensor chip having a lipophilically modified dextran matrix or quasicrystalline hydrophobic layer was used to immobilize an amphipathic lipid layer that mimics the plasma membrane surface. Dose responsiveness was observed with both isoforms when either the toxin concentration or the Gb3Cer concentration was increased. In addition, this assay was shown to be specific, because neither Stx1 nor Stx2 bound to GM3, but both bound weakly to Gb4Cer. It was also shown that a number of fitting models can be used to analyze the sensorgrams obtained with different concentrations of the toxins, and the "bivalent analyte" model was found to best fit the interaction between Stxs and Gb3Cer. This shows that the interaction between Stxs and Gb3Cer in the lipid bilayer has a multivalent effect. The presence of cholesterol in the lipid bilayer significantly enhanced the binding of Stxs to Gb3Cer, although kinetics were unaffected. The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more slowly than Stx1 but, once bound, is difficult to dissociate. The data described herein clearly demonstrate differences between the binding properties of Stx1 and Stx2 and may facilitate understanding of the differences in clinical manifestations caused by these toxins. (+info)
Demonstration of the pH sensitive binding of multivalent carbohydrate ligands to immobilized Shiga-like toxin 1 B subunits.
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The cytotoxic effects of Shiga-like toxins from enterohemorrhagic Escherichia coli O157:H7 depend on the recognition of carbohydrate determinants by B subunits. As a specific carbohydrate ligand, globotriaosylceramide has been characterized. We developed an alternative binding assay using multivalent carbohydrate ligands. We prepared globotriose-conjugated poly-lysine, and measured their binding to immobilized recombinant B subunits by an ELISA format. The signals representing ligand binding were dependent on the amount of immobilized B subunits as well as on the concentration of the ligands. The ligand binding activity was lost in an acidic environment, in which changes in the local conformation of the B subunits have been reported. Furthermore, pH dependent dissociation of the ligands from the B subunits was observed. We also demonstrate that antiserum from mice immunized with the B subunits specifically interferes with ligand binding. This suggests further potential for an assay to screen for blocking antibodies that could inhibit toxin internalization into host cells. (+info)
A mutational analysis of the globotriaosylceramide-binding sites of verotoxin VT1.
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Escherichia coli verotoxin, also known as Shiga-like toxin, binds to eukaryotic cell membranes via the glycolipid Gb(3) receptors which present the P(k) trisaccharide Galalpha(1-4)Galbeta(1-4)Glcbeta. Crystallographic studies have identified three P(k) trisaccharide (P(k)-glycoside) binding sites per verotoxin 1B subunit (VT1B) monomer while NMR studies have identified binding of P(k)-glycoside only at site 2. To understand the basis for this difference, we studied binding of wild type VT1B and VT1B mutants, defective at one or more of the three sites, to P(k)-glycoside and pentavalent P(k) trisaccharide (pentaSTARFISH) in solution and Gb(3) presented on liposomal membranes using surface plasmon resonance. Site 2 was the key site in terms of free trisaccharide binding since mutants altered at sites 1 and 3 bound this ligand with wild type affinity. However, effective binding of the pentaSTARFISH molecule also required a functional site 3, suggesting that site 3 promotes pentavalent binding of linked trisaccharides at site 1 and site 2. Optimal binding to membrane-associated Gb(3) involved all three sites. Binding of all single site mutants to liposomal Gb(3) was weaker than wild type VT1B binding. Site 3 mutants behaved as if they had reduced ability to enter into high avidity interactions with Gb(3) in the membrane context. Double mutants at site 1/site 3 and site 2/site 3 were completely inactive in terms of binding to liposomal Gb(3,) even though the site 1/site 3 mutant bound trisaccharide with almost wild type affinity. Thus site 2 alone is not sufficient to confer high avidity binding to membrane-localized Gb(3). Cytotoxic activity paralleled membrane glycolipid binding. Our data show that the interaction of verotoxin with the Gb(3) trisaccharide is highly context dependent and that a membrane environment is required for biologically relevant studies of the interaction. (+info)
Role of lipid rafts in Shiga toxin 1 interaction with the apical surface of Caco-2 cells.
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Enterohemorrhagic Escherichia coli producing Shiga toxins 1 and/or 2 have become major foodborne pathogens. The specific binding of Shiga toxin 1 B-subunit to its receptor, a neutral glycolipid globotriaosylceramide Gb(3), on the apical surface of colonic epithelium followed by toxin entry into cells are the initial steps of the process, which can result in toxin transcytosis and systemic effects of infection including hemolytic uremic syndrome. Understanding the complex mechanisms of Shiga toxin 1 binding and internalization may help to develop new strategies directed at preventing toxin internalization. Fluorescence resonance energy transfer microscopy revealed the clustering of Shiga toxin receptors Gb(3) in lipid rafts with another glycosphingolipid G(M1) on the apical surface of highly polarized intestinal epithelial Caco-2 cells. Lipid rafts disruption significantly decreased internalization of Shiga toxin 1 B-subunit. Although disruption of lipid rafts by cholesterol depletion did not affect the amount of bound Shiga toxin 1 B-subunit, lipid rafts are necessary for toxin uptake across the apical membrane of Caco-2 cells. (+info)