Hofmann F, Busch C, Prepens U, Just I, Aktories K
Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, D-79104 Freiburg, Germany.
J Biol Chem. 1997 Apr 25;272(17):11074-8. doi: 10.1074/jbc.272.17.11074.
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.
艰难梭菌毒素B是已知最大的细胞毒素之一（270 kDa），通过单糖基化作用于Rho亚家族蛋白（Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500 - 503）。通过缺失分析，我们确定毒素的酶活性和细胞毒性活性位于全毒素的N末端。毒素B的一个63 kDa片段，覆盖前546个氨基酸残基，以UDP-葡萄糖作为共底物，可使Rho、Rac和Cdc42发生糖基化，但不能使Ras发生糖基化。正如全毒素的情况一样，毒素片段的糖基化作用更倾向于低分子量GTP酶的GDP结合形式。将毒素片段显微注射到NIH-3T3细胞中会导致细胞变圆以及肌动蛋白细胞骨架的重新分布。相比之下，一个包含前516个氨基酸残基的毒素片段的活性至少比毒素片段1 - 546低1000倍，且无细胞毒性活性。这些数据直接证明了艰难梭菌毒素B的酶活性位于N末端的546个氨基酸残基处，并表明从氨基酸残基516到546的区域对于酶活性和细胞毒性活性具有功能和/或结构上的重要作用。
