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Papers In Press, published online ahead of print May 1, 2008
J. Lipid Res., doi:10.1194/jlr.M700587-JLR200

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Journal of Lipid Research, Vol. 49, 1039-1047, May 2008
Copyright © 2008 by American Society for Biochemistry and Molecular Biology

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

Masataka Oda^*, Takayuki Matsuno^*, Ryouta Shiihara^*, Sadayuki Ochi, Rieko Yamauchi, Yuki Saito^*, Hiroshi Imagawa, Masahiro Nagahama^*, Mugio Nishizawa and Jun Sakurai^1,*

^* Department of Microbiology, Faculty of Pharmaceutical Science, Tokushima Bunri University, Tokushima, Japan
School of Medicine, Fujita Health University, Toyoake, Aichi, Japan
Department of Chemistry of Functional Molecule, Faculty of Pharmaceutical Science, Tokushima Bunri University, Tokushima, Japan

The online version of this article (available at http://www.jlr.org) contains supplementary data in the form of three figures.

Published, JLR Papers in Press, February 8, 2008.

¹ To whom correspondence should be addressed. e-mail: sakurai{at}ph.bunri-u.ac.jp

Clostridium perfringens -toxin induces the hemolysis of sheep erythrocytes by activating the metabolism of sphingomyelin (SM) via a GTP binding protein in membranes. -Toxin stimulated the formation of 15-N-nervonoyl sphingosine (C_24:1-ceramide), which was identified by positive ion fast atom bombardment-MS and ¹H-NMR spectroscopy. C_24:1-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 C_24:1-ceramide were significantly attenuated by treatment with dihydrosphingosine, a sphingosine kinase inhibitor. TLC showed that the level of C_24:1-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 (C_24:1-SM) in DRMs. Treatment of the cells with pertussis toxin (PT) inhibited the -toxin-induced formation of C_24:1-ceramide from C_24:1-SM in DRMs and hemolysis, indicating that endogenous sphingomyelinase, which hydrolyzes C_24:1-SM to C_24:1-ceramide, is controlled by PT-sensitive GTP binding protein in membranes. These results show that the toxin-induced metabolism of C_24:1-SM to S1P in DRMs plays an important role in the toxin-induced hemolysis of sheep erythrocytes.

Supplementary key words C_24:1-sphingomyelin • LC-MS/MS • detergent-soluble fractions

Abbreviations: C_16:0-ceramide, N-palmitoyl sphingosine (d18:1/16:0); C_24:0-ceramide, N-lignoceroyl sphingosine (d18:1/24:0); C_24:1-ceramide, 15-N-nervonoyl sphingosine (d18:1/24:1); C_24:2-ceramide, 15,18-N-tetracosadienoyl sphingosine (d18:1/24:2); C_24:1-SM, N-nervonoic sphingomyelin; DGK, 1,2-diacylglycerol kinase; DHS, dihydrosphingosine; DRM, detergent-resistant membrane; FAB, fast atom bombardment; MβCD, methyl-β-cyclodextrin; N-OE, N-oleoylethanolamine; PT, pertussis toxin; S1P, sphingosine 1-phosphate; SM, sphingomyelin; SMase, sphingomyelinase

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