Clostridium novyi alpha-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins.
The lethal and edema-inducing alpha-toxin from Clostridium novyi causes rounding up of cultured cell lines by redistribution of the actin cytoskeleton. alpha-Toxin belongs to the family of large clostridial cytotoxins that encompasses Clostridium difficile toxin A and B and the lethal toxin from Clostridium sordellii. Toxin A and toxin B have been recently identified as monoglucosyltransferases to modify the low molecular mass GTPases of the Rho subfamily (Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) Nature 375, 500-503 and Just, I., Wilm, M., Selzer, J., Rex, G., Von Eichel-Streiber, C., Mann, M., and Aktories, K. (1995) J. Biol. Chem. 270, 13932-13936). We report here the identification of the alpha-toxin-catalyzed modification of Rho. Using electrospray mass spectrometry, the mass of the modification was determined as 203 Da, consistent with a N-acetyl-hexosamine moiety. UDP-N-acetyl-glucosamine selectively served as cosubstrate for alpha-toxin-catalyzed modification into the Rho subfamily proteins Rho, Rac, Cdc42, and RhoG. The acceptor amino acid of N-acetyl-glucosaminylation was identified by mutagenesis as Thr-37 in Rho (equivalent to Thr-35 in Rac/Cdc42), which is located in the effector domain of the GTPases. C. novyi alpha-toxin seems to mediate its cytotoxic effects on cells by mimicking endogenous post-translational modification of cellular proteins.
Selzer
J
J
Institut für Pharmakologie und Toxikologie der Universität Freiburg, D-79104 Freiburg, Federal Republic of Germany.
Hofmann
F
F
Rex
G
G
Wilm
M
M
Mann
M
M
Just
I
I
Aktories
K
K
D016475:3T3 Cells; D000117:Acetylglucosamine / Q000378:metabolism; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001427:Bacterial Toxins / Q000302:isolation & purification / Q000633:toxicity*; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006031:Glycosylation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D013379:Substrate Specificity; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism
D001427:Bacterial Toxins; C075216:Clostridium novyi alpha-toxin; D006153:Guanosine Diphosphate; D000246:Adenosine Diphosphate Ribose; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D014537:Uridine Diphosphate N-Acetylglucosamine; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins; D000117:Acetylglucosamine