The Dramatic Modulatory Role of the 2'N Substitution of the Terminal Amino Hexose of Globotetraosylceramide in Determining Binding by Members of the Verotoxin Family

Although globotetraosylceramide (Gb4) is only recognized by a single member of the verotoxin family namely, the pig edema disease toxin (VT2e), removal of the acetyl group from the terminal N-acetyl hexosamine of Gb4 to generate the free amino sugar containing species (aminoGb4) results in the generation of a glycolipid preferentially recognized by all members of the verotoxin family (i.e., VT1, VT2, VT2c, and VT2e). GT3, a site-specific mutant of VT2e, in which Gb4 recognition is lost but Gb3 binding is retained, also binds aminoGb4. We have now compared the binding of VT1, VT2, VT2e, and GT3 to a series of aminoGb4 derivatives using a TLC overlay technique. DimethylaminoGb4 is bound by VT1 and VT2 but not VT2e or GT3; formylaminoGb4 binds all toxins but poorly to VT2 and preferentially VT2e; trifluoroacetylaminoGb4 binds only VT2e and GT3; isopropylaminoGb4 binds VT1 and poorly to VT2; benzylaminoGb4 binds all four toxins. Thus, there is a marked distinction between the permissible amino substitutions for VT1 and VT2e binding. GT3 is a hybrid between these in that, according to the substitution, it behaves similarly either to VT1 or to VT2e. For each species, GT3 does not however, show a hybrid binding between that of VT1 and VT2e. Analysis of the binding as a function of pH shows opposite effects for VT1 and VT2e: decreased pH increases VT1, but decreases VT2e receptor glycolipid binding

The Dramatic Modulatory Role of the 2'N Substitution of the Terminal Amino Hexose of Globotetraosylceramide in Determining Binding by Members of the Verotoxin Family

Although globotetraosylceramide (Gb4) is only recognized by a single member of the verotoxin family namely, the pig edema disease toxin (VT2e), removal of the acetyl group from the terminal N-acetyl hexosamine of Gb4 to generate the free amino sugar containing species (aminoGb4) results in the generation of a glycolipid preferentially recognized by all members of the verotoxin family (i.e., VT1, VT2, VT2c, and VT2e). GT3, a site-specific mutant of VT2e, in which Gb4 recognition is lost but Gb3 binding is retained, also binds aminoGb4. We have now compared the binding of VT1, VT2, VT2e, and GT3 to a series of aminoGb4 derivatives using a TLC overlay technique. DimethylaminoGb4 is bound by VT1 and VT2 but not VT2e or GT3; formylaminoGb4 binds all toxins but poorly to VT2 and preferentially VT2e; trifluoroacetylaminoGb4 binds only VT2e and GT3; isopropylaminoGb4 binds VT1 and poorly to VT2; benzylaminoGb4 binds all four toxins. Thus, there is a marked distinction between the permissible amino substitutions for VT1 and VT2e binding. GT3 is a hybrid between these in that, according to the substitution, it behaves similarly either to VT1 or to VT2e. For each species, GT3 does not however, show a hybrid binding between that of VT1 and VT2e. Analysis of the binding as a function of pH shows opposite effects for VT1 and VT2e: decreased pH increases VT1, but decreases VT2e receptor glycolipid binding

Mechanism of Small Molecule Inhibitors of Phagocytosis

Immune cytopenias occur when the body produces antibodies that target specific hematopoietic cells, inducing extravascular antibody-mediated phagocytosis by monocyte-macrophages in the spleen and/or liver through activation of Fcγ Receptors (FcγRs). Immune cytopenias include Immune Thrombocytopenia (ITP), Autoimmune Hemolytic Anemia (AIHA), Hemolytic Transfusion Reactions (HTR), Hemolytic Disease of the Fetus and Newborn (HDFN), and Autoimmune Neutropenia (AIN). Thus, novel therapeutics that inhibit phagocytosis would be useful, especially for short-term use while other therapies are being evaluated. In our earlier studies, we successfully identified two small-molecule drugs able to inhibit in vitro phagocytosis with a low IC50 concentration and negligible toxicity. These drugs, known as KB-151 and KB-208, have the potential to be utilized as lead compounds for further studies, once their mechanism of action is more clearly understood. In this regard, we have developed preliminary results that suggest that these small molecules may bind to the Fc receptors on monocyte macrophages and block the subsequent attachment of antibody-opsonized red blood cells to prevent phagocytosis

Mechanism of increased efficacy of recombinant Fc‐μTP‐L309C compared to IVIg to ameliorate mouse immune thrombocytopenia

Abstract Recombinant Fc‐μTP‐L309C is more efficacious than intravenous immunoglobulin (IVIg) at ameliorating antibody‐mediated autoimmune diseases through its effects on Fcγ receptors (FcγRs). Fc‐μTP‐L309C inhibited in‐vitro FcγR‐mediated phagocytosis 104/105‐fold better than IVIg. Fc‐μTP‐L309C, given subcutaneously, recovered platelet counts in an immune thrombocytopenia (ITP) mouse model to a higher degree than IVIg at a 10‐fold lower dose. We show, using confocal microscopy, that Fc‐μTP‐L309C binds to monocyte‐macrophages and is rapidly internalized, whereas, IVIg remains on the cell surface. Western blotting showed that internalized FcγRIII is degraded through a lysosomal pathway, and this reduction of cell surface FcγRIII is likely responsible for the increased efficacy to ameliorate ITP

Interaction of the verotoxin 1B subunit with soluble aminodeoxy analogues of globotriaosyl ceramides.

Specific hydroxy groups of the terminal disaccharide unit of globotriaosyl ceramide (Gb(3)Cer) were identified from binding studies with deoxyGb(3)Cer and verotoxins (VTs) [Nyholm, Magnusson, Zheng, Norel, Binnington-Boyd and Lingwood (1996) Chem. Biol. 3, 263-275]. Four such hydroxy groups (2", 4", 6" and 6') were each substituted with an amino group and the corresponding deoxyamino globotrioses were conjugated to a ceramide-like aglycone which contained an adamantyl group instead of an acyl chain. Such aglycone modification significantly enhanced the water-solubility of the glycoconjugates [Mylvaganam and Lingwood (1999) Biochem. Biophys. Res. Commun. 257, 391-394]. The inhibitory potential of these soluble aminodeoxy conjugates on the binding of VT(1) to Gb(3)Cer immobilized on an ELISA plate was evaluated. Only the 2" and the 6' deoxyamino conjugates were effective inhibitors (IC(50) 10 microM); the 4" and 6" conjugates were ineffective up to 10 mM. To evaluate the importance of incorporating a rigid adamantyl hydrocarbon group into the ceramide aglycone, globotriaose was conjugated to a t- butylacetamido or an adamantaneacetamido aglycone. By similar ELISAs, only the adamantaneacetamido conjugate inhibited the binding of VT(1) to Gb(3)Cer. When deoxyamino conjugates were adsorbed to silica on TLC plates, only the 2" and 6" conjugates bound VT(1) and VT(2). By a similar TLC assay, acetamido derivatives of 2" and 6' deoxyamino conjugates showed less binding to VT(1) and VT(2). Neither the crystallographically determined structure of the VT(1)-globotriaose complex nor modelling studies fully explain the binding patterns shown by these deoxyamino glycoconjugates. Enhanced solvation of the ammonium group of the deoxyamino conjugate could enforce greater constraints in the binding interactions

Verotoxin A Subunit Protects Lymphocytes and T Cell Lines against X4 HIV Infection in Vitro

Our previous genetic, pharmacological and analogue protection studies identified the glycosphingolipid, Gb₃ (globotriaosylceramide, P^k blood group antigen) as a natural resistance factor for HIV infection. Gb₃ is a B cell marker (CD77), but a fraction of activated peripheral blood mononuclear cells (PBMCs) can also express Gb₃. Activated PBMCs predominantly comprise CD4⁺ T-cells, the primary HIV infection target. Gb₃ is the sole receptor for <i>Escherichia coli </i>verotoxins (VTs, Shiga toxins). VT1 contains a ribosome inactivating A subunit (VT1A) non-covalently associated with five smaller receptor-binding B subunits. The effect of VT on PHA/IL2-activated PBMC HIV susceptibility was determined. Following VT1 (or VT2) PBMC treatment during IL2/PHA activation, the small Gb₃⁺/CD4⁺ T-cell subset was eliminated but, surprisingly, remaining CD4⁺ T-cell HIV-1_IIIB (and HIV-1_Ba-L) susceptibility was significantly reduced. The Gb₃^-Jurkat T-cell line was similarly protected by brief VT exposure prior to HIV-1_IIIB infection. The efficacy of the VT1A subunit alone confirmed receptor independent protection. VT1 showed no binding or obvious Jurkat cell/PBMC effect. Protective VT1 concentrations reduced PBMC (but not Jurkat cell) proliferation by 50%. This may relate to the mechanism of action since HIV replication requires primary T-cell proliferation. Microarray analysis of VT1A-treated PBMCs indicated up regulation of 30 genes. Three of the top four were histone genes, suggesting HIV protection via reduced gene activation. VT blocked HDAC inhibitor enhancement of HIV infection, consistent with a histone-mediated mechanism. We speculate that VT1A may provide a benign approach to reduction of (X4 or R5) HIV cell susceptibility

Endoplasmic Reticulum-Targeted Subunit Toxins Provide a New Approach to Rescue Misfolded Mutant Proteins and Revert Cell Models of Genetic Diseases.

Many germ line diseases stem from a relatively minor disturbance in mutant protein endoplasmic reticulum (ER) 3D assembly. Chaperones are recruited which, on failure to correct folding, sort the mutant for retrotranslocation and cytosolic proteasomal degradation (ER-associated degradation-ERAD), to initiate/exacerbate deficiency-disease symptoms. Several bacterial (and plant) subunit toxins, retrograde transport to the ER after initial cell surface receptor binding/internalization. The A subunit has evolved to mimic a misfolded protein and hijack the ERAD membrane translocon (dislocon), to effect cytosolic access and cytopathology. We show such toxins compete for ERAD to rescue endogenous misfolded proteins. Cholera toxin or verotoxin (Shiga toxin) containing genetically inactivated (± an N-terminal polyleucine tail) A subunit can, within 2-4 hrs, temporarily increase F508delCFTR protein, the major cystic fibrosis (CF) mutant (5-10x), F508delCFTR Golgi maturation (<10x), cell surface expression (20x) and chloride transport (2x) in F508del CFTR transfected cells and patient-derived F508delCFTR bronchiolar epithelia, without apparent cytopathology. These toxoids also increase glucocerobrosidase (GCC) in N370SGCC Gaucher Disease fibroblasts (3x), another ERAD-exacerbated misfiling disease. We identify a new, potentially benign approach to the treatment of certain genetic protein misfolding diseases

Verotoxin A Subunit Protects Lymphocytes and T Cell Lines against X4 HIV Infection in Vitro

toxin

A synthetic globotriaosylceramide analogue inhibits HIV-1 infection in vitro by two mechanisms

Previously, it was shown that the cell-membrane-expressed glycosphingolipid, globotriaosylceramide (Gb(3)/P-k/CD77), protects against HIV-1 infection and may be a newly described natural resistance factor against HIV infection. We have now investigated the potential of a novel, water soluble, non-toxic and completely synthetic analogue of Gb(3)/P-k (FSL-Gb(3)) to inhibit HIV-1 infection in vitro. A uniquely designed analogue, FSL-Gb(3), of the natural Gb(3)/P-k molecule was synthesized. HIV-1(IIIB) (X4 virus) and HIV-1(Ba-L) (R5 virus) infection of PHA/interleukin-2-activated, peripheral blood mononuclear cells (PBMCs) and Jurkat T cells in vitro was assessed, as well as infection of U87.CD4.CCR5 by various clinical R5 tropic viruses after treatment with FSL-Gb(3). We monitored Gb(3), CD4 and CXCR4 expression by fluorescent antibody cell sorting and viral replication by p24 (gag) ELISA. Total cellular Gb(3) was examined by glycosphingolipid extraction and thin layer chromatography. In vivo toxicity was monitored in mice by histological assessment of vital organs and lymphoid tissue. FSL-Gb(3) blocked X4 and R5 of both lab and clinical viral strains in activated PBMCs or the U87.CD4.CCR5 cell line with a 50% inhibitory concentration (IC50) of approximately 200-250 mu M. FACS and TLC overlay showed that FSL-Gb(3) can insert itself into cellular plasma membranes and that cellular membrane-absorbed FSL-Gb(3) is able to inhibit subsequent HIV-1 infection. There was no effect of FSL-Gb(3) on cell surface levels of CD4 or CXCR4. Thus, FSL-Gb(3) can inhibit HIV-1 by two mechanisms: direct inhibition of virus and inhibition of viral entry. Infusion of FSL-Gb(3) into laboratory mice at doses well in excess of theoretical therapeutic doses was tolerated with no untoward reactions. Our results demonstrate the potential utility of using a completely synthetic, water soluble globotriaosylceramide analogue, FSL-Gb(3), having low toxicity, for possible future use as a novel therapeutic approach for the systemic treatment of HIV/AIDS