Inhibitory effect of ganglioside on mastoparan-induced cytotoxicity and degranulation in lipid raft of connective tissue type mast cell

Antihistamine, the most important drug for Hymenoptera stinging, cannot attenuate cytotoxicity and mast cell direct activation by mastoparan that is the most abundant polypeptides in the venoms of social wasps. The aim of this study was to investigate whether gangliosides inhibit the effect of mastoparan on mast cells activation. The degranulation and cytotoxicity in canine cutaneous mastocytoma cells (CM-MC) were done by measurement of β-hexosaminidase release and MTT assay. Lipid raft was isolated with discontinuous sucrose gradient centrifuge for the analysis of distribution of Gαq and Gαi protein by western blotting. We found that mastoparan induced the degranulation in (CM-MC) via direct activation of Gαi and Gαq with a decrease in their amount in lipid raft. Ganglioside GD1a(disialoganglioside) and GM1 (monosialoganglioside) strongly reduced the degranulation and cytotoxicity through stabilizing the structure of lipid raft domain. In addition, mastoparan generated intracellular reactive oxygen species (ROS) independently from cytotoxicity, through arachidonic cascade but not G-protein activations. Crude wasp venom showed cytotoxicity and induction of the release from CM-MC, which were potently reduced by gangliosides. We show here that mastoparan activates both Gαi and Gαq protein and that the exogenous ganglioside GD1a and GM1 inhibit the degranulation and cytotoxicity through stabilizing lipid raft. Gangliosides have potentials to be therapeutic tool or clinical prophylaxis for wasp stinging. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 25:158–168, 2011; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.20372