Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells

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 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 TcdB 10463 between Leu 543 and Gly 544 and the naturally occurring variant TcdB 8864 between Leu 544 and Gly 545 . 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 10463 ) and 62 761 Da (TcdB 8864 )] 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 10463 ) and 206 245 Da (TcdB 8864 )] 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. Abbreviations: FBS, fetal bovine serum; LCT, large clostridial toxin; MALDI-TOF, matrix-assisted laser desorption ionization time of flight 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.