Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells.
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.
Rupnik
Maja
M
Max-Planck-Institute of Biophysical Chemistry, Department of Neurobiology, Göttingen, Germany.|Department of Biology, University of Ljubljana, Ljubljana, Slovenia.
Pabst
Stefan
S
Max-Planck-Institute of Biophysical Chemistry, Department of Neurobiology, Göttingen, Germany.
Rupnik
Marjan
M
European Neuroscience Institute, Göttingen, Germany.
von Eichel-Streiber
Christoph
C
Institute for Medical Microbiology and Hygiene, Johannes-Gutenberg-University, Mainz, Germany.
Urlaub
Henning
H
Max-Planck-Institute of Biophysical Chemistry, Department of Cellular Biochemistry, Göttingen, Germany.
Söling
Hans-Dieter
HD
Max-Planck-Institute of Biophysical Chemistry, Department of Neurobiology, Göttingen, Germany.
D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D016360:Clostridioides difficile / Q000472:pathogenicity; D006224:Cricetinae; D003412:Cricetulus; D005347:Fibroblasts; D005964:Glucosyltransferases; D008168:Lung / Q000166:cytology; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014709:Vero Cells
D001426:Bacterial Proteins; D001427:Bacterial Toxins; C031815:toxB protein, Clostridium difficile; D005964:Glucosyltransferases; D010447:Peptide Hydrolases