Abstract
Using large clostridial cytotoxins as tools, the role of Rho GTPases in activation of RBL 2H3 hm1 cells was studied. Clostridium difficile toxin B, which glucosylates Rho, Rac, and Cdc42 and Clostridium sordellii lethal toxin, which glucosylates Rac and Cdc42 but not Rho, inhibited the release of hexosaminidase from RBL cells mediated by the high affinity antigen receptor (FcepsilonRI). Additionally, toxin B and lethal toxin inhibited the intracellular Ca(2+) mobilization induced by FcepsilonRI-stimulation and thapsigargin, mainly by reducing the influx of extracellular Ca(2+). In patch clamp recordings, toxin B and lethal toxin inhibited the calcium release-activated calcium current by about 45%. Calcium release-activated calcium current, the receptor-stimulated Ca(2+) influx, and secretion were inhibited neither by the Rho-ADP-ribosylating C3-fusion toxin C2IN-C3 nor by the actin-ADP-ribosylating Clostridium botulinum C2 toxin. The data indicate that Rac and Cdc42 but not Rho are not only involved in late exocytosis events but are also involved in Ca(2+) mobilization most likely by regulating the Ca(2+) influx through calcium release-activated calcium channels activated via FcepsilonRI receptor in RBL cells.
Highlights
Cross-linking of the high affinity IgE receptor (Fc⑀RI)1 by antigen-binding induces a cascade of morphological and biochemical reactions, resulting in degranulation [1]
1 The abbreviations used are: Fc⑀RI, high affinity receptor for IgE; C2 toxin, C. botulinum C2 toxin consisting of the enzyme component C2I and the binding component C2II; C2IN-C3, C3 fusion toxin consisting of C3 ADP-ribosyltransferase and the N-terminal part of component I of C. botulinum C2 toxin; [Ca2ϩ]i, cytoplasmic free calcium; [Ca2ϩ]o, extracellular free calcium; TNP-OVA, trinitrophenyl-conjugated ovalbumin; ICRAC, calcium release-activated calcium current; IgE, immunoglobulin E; IP3, inositol 1,4,5-triphosphate; lethal toxin, C. sordellii lethal toxin; PAGE, polyacrylamide gel electrophoresis; F, farad(s); PBS, phosphate-buffered saline
To obtain information about the involvement of Rho GTPases in RBL cell activation, we applied the glucosylating C. difficile toxin B and C. sordellii lethal toxin, which differ in substrate specificity
Summary
Fc⑀RI, high affinity receptor for IgE; C2 toxin, C. botulinum C2 toxin consisting of the enzyme component C2I and the binding component C2II; C2IN-C3, C3 fusion toxin consisting of C3 ADP-ribosyltransferase and the N-terminal part of component I of C. botulinum C2 toxin; [Ca2ϩ]i, cytoplasmic free calcium; [Ca2ϩ]o, extracellular free calcium; TNP-OVA, trinitrophenyl-conjugated ovalbumin; ICRAC, calcium release-activated calcium current; IgE, immunoglobulin E; IP3, inositol 1,4,5-triphosphate; lethal toxin, C. sordellii lethal toxin; PAGE, polyacrylamide gel electrophoresis; F, farad(s); PBS, phosphate-buffered saline. The low molecular mass GTP-binding proteins of the Rho family (Rho, Rac, Cdc42) appear to be involved in activation of mast cells and RBL cells [20]. We report that toxin B and lethal toxin but not the Rho-modifying chimeric toxin C2IN-C3 inhibit secretion and increase of [Ca2ϩ]i by the Fc⑀RI receptor in RBL cells. The toxins inhibit thapsigargin-induced Ca2ϩ mobilization and the activation of ICRAC by depletion of intracellular Ca2ϩ stores, indicating that Rac/ Cdc but not Rho participates in regulation of capacitative Ca2ϩ entry.
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