Avtor/Urednik		Rupnik, Maja; Pabst, Stefan; Rupnik, Marjan; von Eichel-Streiber, Christoph; Urlaub, Henning; Soeling, Hans-Dieter
Naslov		Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells
Tip		članek
Vir		Microbiology
Vol. in št.		Letnik 151
Leto izdaje		2005
Obseg		str. 199-208
Jezik		eng
Abstrakt		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.
Deskriptorji		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