Pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembles into a transmembrane pore complex
Pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembles into a transmembrane pore complex
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DOI：
10.1074/jbc.m402676200
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发表时间：
2004-06-25
影响因子：
4.8
通讯作者：
Hashimoto, Y
中科院分区：
文献类型：
--
作者：
Tomita, T;Noguchi, K;Hashimoto, Y
自组装、成孔溶细胞素是用于研究跨膜孔的组装和膜插入的说明性分子。本研究从平菇担子果中分离纯化了一种新型的鞘磷脂特异性双组分溶细胞素--侧耳溶细胞素，并对其成孔特性进行了研究。Pleurotolysin由非相关的A（17 kDa）和B（59 kDa）组分组成，它们在纳摩尔浓度下协同引起钾离子从人红细胞中泄漏和细胞肿胀，导致胶体渗透性溶血。溶血试验中存在的聚（乙二醇）s与不同的流体动力学直径表明，pleurotolysin形成膜孔的功能直径为3.8-5 nm。通过向细胞外空间添加鞘磷脂-胆固醇脂质体，特异性抑制了侧耳素诱导的人红细胞溶解。Pleurotolysin A特异性结合鞘磷脂-胆固醇脂质体，并与pleurotolysin B一起引起内部羧基荧光素的泄漏。包括在25 ℃下用2%（w/v）SDS溶解经侧耳溶血素处理的红细胞和SDS-聚丙烯酰胺凝胶电泳/Western免疫印迹的实验表明，侧耳溶血素A和B以该序列结合于人红细胞，并组装成SDS稳定的700-kDa复合物。环形结构的外径和内径分别为14和7 nm，分离从溶解的红细胞膜通过蔗糖梯度离心。Pleurotolysin A和B在鞘磷脂-胆固醇脂质体上也形成相同尺寸的SDS稳定的环状复合物。
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 25degreesC 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.