Delineation of the catalytic domain of Clostridium difficile toxin B-10463 to an enzymatically active N-terminal 467 amino acid fragment

被引:14
作者
WagenknechtWiesner, A [1 ]
Weidmann, M [1 ]
Braun, V [1 ]
Leukel, P [1 ]
Moos, M [1 ]
vonEichelStreiber, C [1 ]
机构
[1] UNIV MAINZ,INST MED MIKROBIOL & HYG,D-55101 MAINZ,GERMANY
关键词
Clostridium difficile; glucosyltransferase; toxic domain; large clostridial cytotoxin; small GTP-binding cytotoxin; Rho; Rac; Cdc42;
D O I
10.1016/S0378-1097(97)00189-4
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
In an attempt to directly approach the postulated toxic domain of Clostridium difficile's TcdB-10463, eight subclones of different size and locations in the N-terminal third of the toxin were generated. Expression of these toxin fragments was checked in Western blots and the enzymatic activity of the expressed proteins was analyzed by glucosylating Ras related small GTP-binding proteins. Two polypeptides of 875 aa (TcdBcl-3) and 557 aa (TcdBcl-H) glucosylated their targets Rho, Rac and Cdc42 with the same activity and specificity as the holotoxin. In comparison 516 aa (TcdBcl-N) and 467 aa (TcdBcl-A) protein fragments exhibited highly reduced activity, while Tcdcl and TcdB2-3 (aa 1-243 and 244-890, respectively) were enzymatically inactive. Our results indicate that all structures involved in the catalysis are located at several different sites within the 557 aa fully active fragment. The shortest enzymatically still active protein covers aa 1-467 and obviously fulfils all minimal requirements for glucosylation. The data support the postulated three domain model of 'large clostridial cytotoxins'.
引用
收藏
页码:109 / 116
页数:8
相关论文
共 22 条
[1]   STRUCTURE OF EXOTOXIN-A OF PSEUDOMONAS-AERUGINOSA AT 3.0-ANGSTROM RESOLUTION [J].
ALLURED, VS ;
COLLIER, RJ ;
CARROLL, SF ;
MCKAY, DB .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1986, 83 (05) :1320-1324
[2]   MUTAGENESIS OF THE CLOSTRIDIUM-DIFFICILE TOXIN-B GENE AND EFFECT ON CYTOTOXIC ACTIVITY [J].
BARROSO, LA ;
MONCRIEF, JS ;
LYERLY, DM ;
WILKINS, TD .
MICROBIAL PATHOGENESIS, 1994, 16 (04) :297-303
[3]   ACTIVE-SITE MUTATIONS OF DIPHTHERIA-TOXIN - ROLE OF TYROSINE-65 IN NAD BINDING AND ADP-RIBOSYLATION [J].
BLANKE, SR ;
HUANG, K ;
COLLIER, RJ .
BIOCHEMISTRY, 1994, 33 (51) :15494-15500
[4]   THE CRYSTAL-STRUCTURE OF DIPHTHERIA-TOXIN [J].
CHOE, S ;
BENNETT, MJ ;
FUJII, G ;
CURMI, PMG ;
KANTARDJIEFF, KA ;
COLLIER, RJ ;
EISENBERG, D .
NATURE, 1992, 357 (6375) :216-222
[5]   COMMON FEATURES OF THE NAD-BINDING AND CATALYTIC SITE OF ADP-RIBOSYLATING TOXINS [J].
DOMENIGHINI, M ;
MAGAGNOLI, C ;
PIZZA, M ;
RAPPUOLI, R .
MOLECULAR MICROBIOLOGY, 1994, 14 (01) :41-50
[6]  
HENRIQUES B, 1986, MICROB PATHOG, V2, P455
[7]   THE ENTEROTOXIN FROM CLOSTRIDIUM-DIFFICILE (TOXA) MONOGLUCOSYLATES THE RHO-PROTEINS [J].
JUST, I ;
WILM, M ;
SELZER, J ;
REX, G ;
VONEICHELSTREIBER, C ;
MANN, M ;
AKTORIES, K .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1995, 270 (23) :13932-13936
[8]   GLUCOSYLATION OF RHO-PROTEINS BY CLOSTRIDIUM-DIFFICILE TOXIN-B [J].
JUST, I ;
SELZER, J ;
WILM, M ;
VONEICHELSTREIBER, C ;
MANN, M ;
AKTORIES, K .
NATURE, 1995, 375 (6531) :500-503
[9]   CLOSTRIDIUM-DIFFICILE - CLINICAL-DISEASE AND DIAGNOSIS [J].
KNOOP, FC ;
OWENS, M ;
CROCKER, IC .
CLINICAL MICROBIOLOGY REVIEWS, 1993, 6 (03) :251-265
[10]   Ras, Rap, and Rac small GTP-binding proteins are targets for Clostridium sordellii lethal toxin glucosylation [J].
Popoff, MR ;
ChavesOlarte, E ;
Lemichez, E ;
vonEichelStreiber, C ;
Thelestam, M ;
Chardin, P ;
Cussac, D ;
Antonny, B ;
Chavrier, P ;
Flatau, G ;
Giry, M ;
deGunzburg, J ;
Boquet, P .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1996, 271 (17) :10217-10224