Author/Editor | Rupnik, Maja; Pabst, Stefan; Rupnik, Marjan; von Eichel-Streiber, Christoph; Urlaub, Henning; Soeling, Hans-Dieter | |
Title | Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells | |
Type | članek | |
Source | Microbiology | |
Vol. and No. | Letnik 151 | |
Publication year | 2005 | |
Volume | str. 199-208 | |
Language | eng | |
Abstract | 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 intemalization, and the N-terminal catalytic (toxic) part. This study shows that TcdB is processed by a single proteolytic step which cleaves TcdB1063 between Leu5a3 and GIy544 and the naturally occurring variant TcdBsssa between Leu544 and GIy545. 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 [63121 Da (TcdB10463) and 62 761 Da (TcdB88s4)) 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)l 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, Gterminal, 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. | |
Descriptors | BACTERIAL PROTEINS BACTERIAL TOXINS PEPTIDE HYDROLASES LUNG CLOSTRIDIUM DIFFICILE AMINO ACID SEQUENCE VERO CELLS SUBCELLULAR FRACTIONS MOLECULAR SEQUENCE DATA HAMSTERS CELLS, CULTURED CRICETULUS FIBROBLASTS GLUCOSYLTRANSFERASES |