Binding of Clostridium botulinum C2 Toxin to Asparagine-linked Complex and Hybrid Carbohydrates

Eckhardt, Matthias; Barth, Holger; Blöcker, Dagmar; Aktories, Klaus

doi:10.1074/jbc.275.4.2328

Cited by 115 publications

(106 citation statements)

References 36 publications

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“…An ϳ20-kDa peptide is cleaved from the N terminus. The resulting C2IIa (ϳ60 kDa) forms ring-shaped heptamers, which assemble with C2I and bind to complex and hybrid carbohydrate structures on the cell surface (12). The C2 toxin-receptor complex is taken up via receptor-mediated endocytosis, and C2I translocates from the early acidic endosomal compartment into the cytosol (10).…”

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confidence: 99%

The Host Cell Chaperone Hsp90 Is Essential for Translocation of the Binary Clostridium botulinum C2 Toxin into the Cytosol

Haug

Leemhuis

Tiemann

et al. 2003

Journal of Biological Chemistry

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Clostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I. The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I. This toxin complex is taken up by receptor-mediated endocytosis. In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol. We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin. ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors. Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I. At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I. Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa. The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro. The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors. In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells. The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II.

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confidence: 99%

The Host Cell Chaperone Hsp90 Is Essential for Translocation of the Binary Clostridium botulinum C2 Toxin into the Cytosol

Haug

Leemhuis

Tiemann

et al. 2003

Journal of Biological Chemistry

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132

150

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“…Like PA, C2II has to be activated by proteolytic cleavage (18), and the resulting C2IIa forms ring-shaped heptamers (ϳ420 kDa), which have an outer diameter of ϳ15 nm and an inner diameter of ϳ1-2 nm (15). C2IIa binds to its receptor on target cells (19). C2I assembles with C2IIa, and the complex is taken up by receptor-mediated endocytosis (20).…”

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confidence: 99%

Clostridium botulinum C2 Toxin

Blöcker

Pohlmann

Haug

et al. 2003

Journal of Biological Chemistry

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The binary Clostridium botulinum C2 toxin consists of two individual proteins, the transport component C2II (80 kDa) and the enzyme component C2I, which ADPribosylates G-actin in the cytosol of cells. Trypsin-activated C2II (C2IIa) forms heptamers that bind to the cell receptor and mediate translocation of C2I from acidic endosomes into the cytosol of target cells. Here, we report that translocation of C2I across cell membranes is accompanied by pore formation of C2IIa. We used a radioactive rubidium release assay to detect C2IIa pores in the membranes of Chinese hamster ovary cells. Pore formation by C2IIa was dependent on the cellular C2 toxin receptor and an acidic pulse. Pores were formed when C2IIa was bound to cells at neutral pH and when cells were subsequently shifted to acidic medium (pH < 5.5), but no pores were detected when C2IIa was added to cells directly in acidic medium. Most likely, acidification induces a change from "pre-pore" to "pore" conformation of C2IIa, and formation of the pore conformation before membrane binding precludes insertion into membranes. When C2I was present during binding of C2IIa to cells prior to the acidification step, C2IIa-mediated rubidium release was decreased, suggesting that C2I interacted with the lumen of the C2IIa pore. A decrease of rubidium efflux was also detected when C2I was added to C2IIa-treated cells after the acidification step, suggesting that C2I interacted with C2IIa in its pore conformation. Moreover, C2I also interacted with C2IIa channels in artificial lipid membranes and blocked them partially. C2I was only translocated across the cell membrane when C2IIa plus C2I were bound to cells at neutral pH and subsequently shifted to acidic pH. When cell-bound C2IIa was exposed to acidic pH prior to C2I addition, only residual intoxication of cells was observed at high toxin concentrations, and binding of C2I to C2IIa was slightly decreased. Overall, C2IIa pores were essential but not sufficient for translocation of C2I. Intoxication of target cells with C2 toxin requires a strictly coordinated pH-dependent sequence of binding, pore formation by C2IIa, and translocation of C2I.

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“…Channel Formation of the C2II Mutants in Artificial Lipid Bilayer Membranes-A cellular receptor, a hybrid, and/or complex carbohydrate structure is involved in the binding of C2II on the surface of cells (23). A receptor is not required for formation of ion-permeable channels by activated C2II in black lipid bilayer membranes (28).…”

Section: Resultsmentioning

confidence: 99%

“…This cleavage generates a ϳ60-kDa fragment, which forms a ring-shaped heptamer (22), and a ϳ20-kDa fragment, which dissociates from C2II. The binding of the C2II heptamer depends on presence of asparagine-linked carbohydrates on the surface of target cells (23) and is also able to bind the enzymatic component C2I (24 -26). The complex is endocytosed into the cell.…”

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confidence: 99%