Nagahama Masahiro, Mukai Masakazu, Morimitsu Shinsuke, Ochi Sadayuki, Sakurai Jun
Department of Microbiology, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Tokushima 770-8514, Japan.
Microbiol Immunol. 2002;46(10):647-55. doi: 10.1111/j.1348-0421.2002.tb02748.x.
Clostridium perfringens alpha-toxin (370 residues) possesses hemolytic and lethal activities as well as the enzymatic activity of phospholipase C (PLC). In this study we examined the role of the C-domain (251-370 residues; CP251- 370) in biological activities of the toxin. The N-domain (1-250 residues; CP1- 250) of the alpha-toxin as well as the Bacillus cereus phospholipase C (BcPLC) possessed PLC activity, but did not bind to rabbit erythrocytes and lyse them. A hybrid protein (BC-CP251-370) consisting of BcPLC and CP251- 370 bound to the red cells and lysed them. Incubation of CP1-250 with CP251-370 completely complemented hemolytic and PLC activities. CP251-370 also conferred hemolytic activity on BcPLC. CP251-340 (251-340 residues) significantly stimulated PLC activity of CP1-250), but did not confer hemolytic activity on CP1-250. Kinetic analysis suggested that CP251-370 increased affinity toward the substrate of CP1-250. The results suggested that CP251-370 plays an important role in binding to erythrocytes and the hemolytic and enzymatic activities of CP1-250. Acrylodan-labeled CP251-370 variants (S263C and S365C) bound to liposomes and exhibited a marked blue shift, and in addition, an N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1,3-diazolyl)ethylene diamine (NBD)-labeled CP251-370 (S365C) variant also bound to liposomes and the fluorescence intensity significantly increased, suggesting movement of CP251-370 to a hydrophobic environment. These observations suggest that interaction of CP251-370 of alpha-toxin with fatty acyl residues of phosphatidylcholine plays an important role in the biological activities of CP1-250.
产气荚膜梭菌α毒素（370个氨基酸残基）具有溶血和致死活性以及磷脂酶C（PLC）的酶活性。在本研究中，我们研究了C结构域（251 - 370个氨基酸残基；CP251 - 370）在该毒素生物学活性中的作用。α毒素的N结构域（1 - 250个氨基酸残基；CP1 - 250）以及蜡样芽孢杆菌磷脂酶C（BcPLC）具有PLC活性，但不与兔红细胞结合并使其裂解。由BcPLC和CP251 - 370组成的杂合蛋白（BC - CP251 - 370）与红细胞结合并使其裂解。CP1 - 250与CP251 - 370共同孵育完全互补了溶血和PLC活性。CP251 - 370也赋予BcPLC溶血活性。CP251 - 340（251 - 340个氨基酸残基）显著刺激CP1 - 250的PLC活性，但未赋予CP1 - 250溶血活性。动力学分析表明CP251 - 370增加了对CP1 - 250底物的亲和力。结果表明CP251 - 370在与红细胞结合以及CP1 - 250的溶血和酶活性中起重要作用。丙烯罗丹标记的CP251 - 370变体（S263C和S365C）与脂质体结合并表现出明显的蓝移，此外，N，N'-二甲基 - N -（碘乙酰基）- N'-（7 - 硝基苯并 - 2 - 恶唑 - 1,3 - 二唑基）乙二胺（NBD）标记的CP251 - 370（S365C）变体也与脂质体结合且荧光强度显著增加，表明CP251 - 370移动到了疏水环境。这些观察结果表明α毒素的CP251 - 370与磷脂酰胆碱的脂肪酰基残基之间的相互作用在CP1 - 250的生物学活性中起重要作用。
