Kashimoto Takashige, Akita Tomoe, Kado Takehiro, Yamazaki Kohei, Ueno Shunji
Laboratory of Veterinary Public Health, School of Veterinary Medicine, Kitasato University, Higashi 23-35-1, Towada, Aomori, Japan.
Laboratory of Veterinary Public Health, School of Veterinary Medicine, Kitasato University, Higashi 23-35-1, Towada, Aomori, Japan.
Microb Pathog. 2017 Aug;109:71-77. doi: 10.1016/j.micpath.2017.05.029. Epub 2017 May 22.
Vibrio vulnificus secretes a hemolysin/cytolysin (VVH) that induces cytolysis against a variety of mammalian cells by forming pores on the cellular membrane. VVH is known to bind to the cellular membrane as a monomer, and then convert to a pore-forming oligomer. However, the structural basis for binding of this toxin to target cells remains unknown. We show here that the polarity and indole ring on the side chain of Trp 246 (W246) of VVH, which sits on a bottom loop, participates in binding to cellular membrane. To clarify the binding mechanisms of VVH, we generated a series of W246 point mutants that were substituted with Arg (W246R), Ala (W246A), or Tyr (W246Y), and tested their binding and cytotoxicity on Chinese hamster ovary (CHO) cells. At a final concentration of 1 μg/ml of VVH, wild type (Wt), W246A and W246Y could bind and induce cytotoxicity to CHO cells, whereas W246R could not. The cytotoxic activity of W246A was significantly lower than that of Wt. These findings indicate that both the polarity and indole ring on the side chain of W246 were involved in the binding of this toxin to the target cellular membrane. The indole ring plays a particularly important role in toxin binding.
创伤弧菌分泌一种溶血素/细胞毒素（VVH），该毒素通过在细胞膜上形成孔道来诱导对多种哺乳动物细胞的细胞溶解。已知VVH以单体形式结合到细胞膜上，然后转变为形成孔道的寡聚体。然而，这种毒素与靶细胞结合的结构基础仍然未知。我们在此表明，位于底部环上的VVH的Trp 246（W246）侧链上的极性和吲哚环参与与细胞膜的结合。为了阐明VVH的结合机制，我们生成了一系列用Arg（W246R）、Ala（W246A）或Tyr（W246Y）取代的W246点突变体，并测试了它们对中国仓鼠卵巢（CHO）细胞的结合和细胞毒性。在VVH终浓度为1μg/ml时，野生型（Wt）、W246A和W246Y能够结合并诱导对CHO细胞的细胞毒性，而W246R则不能。W246A的细胞毒性活性明显低于Wt。这些发现表明，W246侧链上的极性和吲哚环都参与了这种毒素与靶细胞膜的结合。吲哚环在毒素结合中起着特别重要的作用。
