1887

Abstract

toxin B (TcdB) is a single-stranded protein consisting of a C-terminal domain responsible for binding to the host cell membrane, a middle part involved in internalization, and the N-terminal catalytic (toxic) part. This study shows that TcdB is processed by a single proteolytic step which cleaves TcdB between Leu and Gly and the naturally occurring variant TcdB between Leu and Gly. The cleavage occurs at neutral pH and is catalysed by a pepstatin-sensitive protease localized in the cytoplasm and on the cytoplasmic face of intracellular membranes. The smaller N-terminal cleavage products [63 121 Da (TcdB) and 62 761 Da (TcdB)] harbour the cytotoxic and glucosyltransferase activities of the toxins. When microinjected into cultured Chinese hamster lung fibroblasts, the N-terminal cleavage fragment shows full cytotoxic activity shortly after injection whereas the holotoxin initially exhibits a very low activity which, however, increases with time. Twenty minutes after the start of internalization of TcdB, the larger cleavage products [206 609 Da (TcdB) and 206 245 Da (TcdB)] are found exclusively in a membrane fraction, whereas the N-terminal cleavage products appear mainly in the cytosol and associated with the membrane. This is in line with a proposed model according to which the longer, C-terminal, part of these toxins forms a channel allowing for the translocation of the toxic N-terminal part, which is subsequently cleaved off at the cytoplasmic face of an intracellular compartment, most likely endosomes.

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2005-01-01
2019-10-21
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Supplementary figures (PDF files): Processing of TcdB with cytosolic and membrane fraction by different cell lines. Time course of cleavage reactions - with Cy3-Tcd and Cy3-Tcd . Distribution of TcdB and its proteolytic fragments in DonWT cells after incubation with the toxin. Peptide mass fingerprint analysis of a 60 kDa fragment of TcdB . Peptide mass fingerprint analysis of a 60 kDa fragment of TcdB .

PDF

Supplementary figures (PDF files): Processing of TcdB with cytosolic and membrane fraction by different cell lines. Time course of cleavage reactions - with Cy3-Tcd and Cy3-Tcd . Distribution of TcdB and its proteolytic fragments in DonWT cells after incubation with the toxin. Peptide mass fingerprint analysis of a 60 kDa fragment of TcdB . Peptide mass fingerprint analysis of a 60 kDa fragment of TcdB .

PDF

Supplementary figures (PDF files): Processing of TcdB with cytosolic and membrane fraction by different cell lines. Time course of cleavage reactions - with Cy3-Tcd and Cy3-Tcd . Distribution of TcdB and its proteolytic fragments in DonWT cells after incubation with the toxin. Peptide mass fingerprint analysis of a 60 kDa fragment of TcdB . Peptide mass fingerprint analysis of a 60 kDa fragment of TcdB .

PDF

Supplementary figures (PDF files): Processing of TcdB with cytosolic and membrane fraction by different cell lines. Time course of cleavage reactions - with Cy3-Tcd and Cy3-Tcd . Distribution of TcdB and its proteolytic fragments in DonWT cells after incubation with the toxin. Peptide mass fingerprint analysis of a 60 kDa fragment of TcdB . Peptide mass fingerprint analysis of a 60 kDa fragment of TcdB .

PDF

Supplementary figures (PDF files): Processing of TcdB with cytosolic and membrane fraction by different cell lines. Time course of cleavage reactions - with Cy3-Tcd and Cy3-Tcd . Distribution of TcdB and its proteolytic fragments in DonWT cells after incubation with the toxin. Peptide mass fingerprint analysis of a 60 kDa fragment of TcdB . Peptide mass fingerprint analysis of a 60 kDa fragment of TcdB .

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