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1 Department of Biology, University of Ljubljana, Ljubljana, Slovenia
2 Max-Planck-Institute of Biophysical Chemistry, Department of Neurobiology, Göttingen, Germany
3 European Neuroscience Institute, Göttingen, Germany
4 Institute for Medical Microbiology and Hygiene, Johannes-Gutenberg-University, Mainz, Germany
5 Max-Planck-Institute of Biophysical Chemistry, Department of Cellular Biochemistry, Göttingen, Germany
Correspondence
Maja Rupnik
maja.rupnik{at}bf.uni-lj.si
Hans-Dieter Söling
hsoelin{at}gwdg.de
Clostridium difficile 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 TcdB10463 between Leu543 and Gly544 and the naturally occurring variant TcdB8864 between Leu544 and Gly545. 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 (TcdB10463) and 62 761 Da (TcdB8864)] 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 (TcdB10463) and 206 245 Da (TcdB8864)] 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.
The online version of this paper contains five supplementary figures (Figs S1–S5).
Present address: Natural and Medical Sciences Institute at the University of Tübingen, Department of Biochemistry, Reutlingen, Germany.
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