Localization of the glucosyltransferase activity of Clostridium difficile toxin B to the N-terminal part of the holotoxin.
Clostridium difficile toxin B that is one of the largest cytotoxins (270 kDa) known acts on Rho subfamily proteins by monoglucosylation (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503). By deletion analysis we identified the enzyme and cytotoxic activity of the toxin to be located at the N terminus of the holotoxin. A 63-kDa fragment of toxin B covering the first 546 amino acid residues glucosylated Rho, Rac, and Cdc42, but not Ras, by using UDP-glucose as a cosubstrate. As known for the holotoxin, glucosylation by the toxin fragment was favored with the GDP-bound form of the low molecular mass GTPases. Microinjection of the toxin fragment into NIH-3T3 cells induced rounding up of cells and redistribution of the actin cytoskeleton. In contrast, a toxin fragment encompassing the first 516 amino acid residues was at least 1000-fold less active than toxin fragment 1-546 and cytotoxically inactive. The data give direct evidence for location of the enzyme activity of C. difficile toxin B at the N-terminal 546 amino acids residues and indicate a functionally and/or structurally important role of the region from amino acid residues 516 through 546 for enzyme and cytotoxic activities.
Hofmann
F
F
Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
Busch
C
C
Prepens
U
U
Just
I
I
Aktories
K
K
D016475:3T3 Cells; D000818:Animals; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000235:genetics / Q000378:metabolism*; D016360:Clostridioides difficile / Q000201:enzymology*; D003603:Cytotoxins / Q000235:genetics / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005798:Genes, Bacterial; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D051379:Mice; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D001426:Bacterial Proteins; D001427:Bacterial Toxins; D003603:Cytotoxins; D010446:Peptide Fragments; D011994:Recombinant Proteins; C031815:toxB protein, Clostridium difficile; D005964:Glucosyltransferases; D020558:GTP Phosphohydrolases