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J. Biol. Chem., Vol. 279, Issue 26, 26975-26982, June 25, 2004

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Pleurotolysin, a Novel Sphingomyelin-specific Two-component Cytolysin from the Edible Mushroom Pleurotus ostreatus, Assembles into a Transmembrane Pore Complex^*

Toshio Tomita, Kayoko Noguchi¶, Hitomi Mimuro¶, Fumio Ukaji¶, Kiyoshi Ito¶, Noriko Sugawara-Tomita, and Yohichi Hashimoto¶

From the Department of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan and ^¶Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-ohkubo, Sakura-ku, Saitama 388-8570, Japan

Self-assembling, pore-forming cytolysins are illustrative molecules for the study of the assembly and membrane insertion of transmembrane pores. Here we purified pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the basidiocarps of Pleurotus ostreatus and studied the pore-forming properties of the cytolysin. Pleurotolysin consisted of non-associated A (17 kDa) and B (59 kDa) components, which cooperatively caused leakage of potassium ions from human erythrocytes and swelling of the cells at nanomolar concentrations, leading to colloid-osmotic hemolysis. Hemolytic assays in the presence of poly(ethylene glycol)s with different hydrodynamic diameters suggested that pleurotolysin formed membrane pores with a functional diameter of 3.8–5 nm. Pleurotolysin-induced lysis of human erythrocytes was specifically inhibited by the addition of sphingomyelin-cholesterol liposomes to the extracellular space. Pleurotolysin A specifically bound to sphingomyelin-cholesterol liposomes and caused leakage of the internal carboxyfluorescein in concert with pleurotolysin B. Experiments including solubilization of pleurotolysin-treated erythrocytes with 2% (w/v) SDS at 25 °C and SDS-polyacrylamide gel electrophoresis/Western immunoblotting showed that pleurotolysin A and B bound to human erythrocytes in this sequence and assembled into an SDS-stable, 700-kDa complex. Ring-shaped structures with outer and inner diameters of 14 and 7 nm, respectively, were isolated from the solubilized erythrocyte membranes by a sucrose gradient centrifugation. Pleurotolysin A and B formed an SDS-stable, ring-shaped complex of the same dimensions on sphingomyelin-cholesterol liposomes as well.

Received for publication, March 9, 2004 , and in revised form, April 13, 2004.

^* This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Microbial Biotechnology, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai 981-8555, Japan. Tel.: 81-22-717-8781; Fax: 81-22-717-8780; E-mail: tomita{at}biochem.tohoku.ac.jp.

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