Inhibition of FcRI-mediated Activation of Rat Basophilic Leukemia Cells by Clostridium difficile Toxin B (Monoglucosyltransferase)

doi:10.1074/jbc.271.13.7324

Inhibition of FcRI-mediated Activation of Rat Basophilic Leukemia Cells by Clostridium difficile Toxin B (Monoglucosyltransferase) (*)

From the Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Strasse 5, D-79104 Freiburg
Institut für Medizinische Mikrobiologie und Hygiene der Johannes-Gutenberg-Universität, D-55101 Mainz, Federal Republic of Germany

^§To whom correspondence should be addressed:
Institut für Pharmakologie und Toxikologie, Hermann-Herder-Str. 5, D-79104 Freiburg, Germany.
Tel.: 49-761-2035301; Fax: 49-761-2035311.

Abstract

Treatment of rat basophilic leukemia (RBL) 2H3-hm1 cells with Clostridium difficile toxin B (2 ng/ml), which reportedly depolymerizes the actin cytoskeleton, blocked [³H]serotonin release induced by 2,4-dinitrophenyl-bovine serum albumin, carbachol, mastoparan, and reduced ionophore A23187-stimulated degranulation by about 55-60%. In lysates of RBL cells, toxin B ¹⁴C-glucosylated two major and one minor protein. By using two-dimensional gel electrophoresis and immunoblotting, RhoA and Cdc42 were identified as protein substrates of toxin B. In contrast to toxin B, Clostridium botulinum transferase C3 that selectively inactivates RhoA by ADP-ribosylation did not inhibit degranulation up to a concentration of 150 μg/ml. Antigen-stimulated tyrosine phosphorylation of a 110-kDa protein was inhibited by toxin B as well as by the phosphatidylinositol 3-kinase inhibitor wortmannin. Depolymerization of the microfilament cytoskeleton of RBL cells by C. botulinum C2 toxin or cytochalasin D resulted in an increased [³H]serotonin release induced by antigen, carbachol, mastoparan, or by calcium ionophore A23187, but without affecting toxin B-induced inhibition of degranulation. The data indicate that toxin B inhibits activation of RBL cells by glucosylation of low molecular mass GTP-binding proteins of the Rho subfamily (most likely Cdc42) by a mechanism not involving the actin cytoskeleton.

Footnotes

↵* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 246 project B10, DFG Ju 231/3-1 and Ei 206/3-1) and by the Fonds der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

RBL

rat basophilic leukemia

RBL 2H3-hm1

2H3-hm1 subline of rat basophilic leukemia cells

A23187

calcium ionophore A23187

C2 toxin

C. botulinum C2 toxin

C2I

enzyme component of C. botulinum C2 toxin

C2II

binding component of C. botulinum C2 toxin

C3

ADP-ribosyltransferase from C. botulinum

DNP-BSA

dinitrophenyl-conjugated bovine serum albumin

FcRI

high affinity receptor for IgE

G-protein

GTP-binding protein

IgE

immunoglobulin E

PAGE

polyacrylamide gel electrophoresis

toxin B

C. difficile toxin B

GTPS

guanosine 5′-O-(thiotriphosphate).
- Received November 1, 1995.
- Revision received December 27, 1995.