The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium perfringens a-toxin

Clostridium perfringens a-toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. a-Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and H-NMR spectroscopy. C24:1-ceramide stimulated the toxin-induced hemolysis of saponin-pretreated sheep erythrocytes and increased the production of sphingosine 1-phosphate (S1P) in the cells, but N-lignoceroyl sphingosine did not. These events elicited by the toxin in the presence of C24:1ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C24:1-ceramide was highest among the ceramides with an unsaturated bond in the fatty acyl chain in the detergent-resistant membranes (DRMs). The toxin specifically bound to DRMs rich in cholesterol, resulting in the hydrolysis of N-nervonoic sphingomyelin (C24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the a-toxin-induced formation of C24:1-ceramide from C24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C24:1-SM to C24:1-ceramide, is controlled by PTsensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.—Oda, M., T. Matsuno, R. Shiihara, S. Ochi, R. Yamauchi, Y. Saito, H. Imagawa, M. Nagahama, M. Nishizawa, and J. Sakurai. The relationship between the metabolism of sphingomyelin species and the hemolysis of sheep erythrocytes induced by Clostridium per fringens a-toxin. J. Lipid Res. 2008. 49: 1039–1047. Supplementary key words C24:1-sphingomyelin & LC-MS/MS & detergentsoluble fractions Clostridium perfringens a-toxin is an important agent of gas gangrene (1–4). The toxin, which exhibits phospholipase C and sphingomyelinase (SMase) activities, causes hemolysis, necrosis, and death. It induces the hemolysis of various erythrocytes (5, 6). We reported that a-toxin induces the hemolysis of rabbit erythrocytes and the generation of superoxide anion in rabbit neutrophils through the activation of endogenous phospholipase C via a pertussis toxin (PT)-sensitive GTP binding protein, Gi (7, 8). We also reported that the toxin induces the hemolysis of sheep erythrocytes through the activation of endogenous SMase via Gi (9). The activation of the sphingomyelin (SM) cycle, analogous to the glycerophospholipid cycles, has been recognized as a key event in the signal transduction cascade involved in cellular proliferation, differentiation, and apoptosis (10). Ceramide causes the arrest of cell growth and apoptosis (11, 12). Sphingosine was found to be a potent inhibitor of protein kinase C (13) and to inhibit cell growth and induce apoptosis (11). Sphingosine 1-phosphate (S1P) has been reported to promote cell growth and inhibit apoptosis, in contrast with ceramides (14). Therefore, it appears that the dynamic balance among the intracellular levels of ceramide, sphingosine, and S1P is important in determining whether a cell survives or dies. The variation of SM molecular species is attributed to the Manuscript received 19 December 2007 and in revised form 4 February 2008. Published, JLR Papers in Press, February 8, 2008. DOI 10.1194/jlr.M700587-JLR200 Abbreviations: C16:0-ceramide, N-palmitoyl sphingosine (d18:1/ 16:0); C24:0-ceramide, N-lignoceroyl sphingosine (d18:1/24:0); C24:1ceramide, 15-N-nervonoyl sphingosine (d18:1/24:1); C24:2-ceramide, 15,18-N-tetracosadienoyl sphingosine (d18:1/24:2); C24:1-SM, N-nervonoic sphingomyelin; DGK, 1,2-diacylglycerol kinase; DHS, dihydrosphingosine; DRM, detergent-resistant membrane; FAB, fast atom bombardment; MbCD, methyl-b-cyclodextrin; N-OE, N-oleoylethanolamine; PT, pertussis toxin; S1P, sphingosine 1-phosphate; SM, sphingomyelin; SMase, sphingomyelinase. 1 To whom correspondence should be addressed. e-mail: [email protected] The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of three figures. Copyright D 2008 by the American Society for Biochemistry and Molecular Biology, Inc. This article is available online at http://www.jlr.org Journal of Lipid Research Volume 49, 2008 1039 by gest, on O cber 9, 2017 w w w .j.org D ow nladed fom 0.DC1.html http://www.jlr.org/content/suppl/2008/02/13/M700587-JLR20 Supplemental Material can be found at: