A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin in cells and planar lipid bilayers.
A conserved tryptophan in pneumolysin is a determinant of the characteristics of channels formed by pneumolysin in cells and planar lipid bilayers.
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肺炎球菌溶血素中的保守色氨酸是细胞和平面脂质双层中肺炎球菌溶血素形成的通道特征的决定因素。
DOI：
10.1042/bj3290571
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
J. Mitchell
中科院分区：
文献类型：
--
作者：
E. Korchev;C. Bashford;Cecilia Pederzolli;A. Pasternak;J. Morgan;W. Andrew;J. Mitchell
肺炎球菌溶血素是硫醇激活的溶细胞毒素家族之一。在这些毒素中，氨基酸序列 Trp-Glu-Trp-Trp 是保守的。在肺炎球菌溶血素的该区域（包括残基433-436）中进行的突变不影响细胞结合或靶细胞膜中毒素寡聚体的形成。然而，这些突变确实影响了溶血、Lettre 细胞中低分子量代谢物的渗漏以及跨平面脂质双层的电导通道的诱导。在检查的八种改良肺炎球菌溶血素中，Trp-433-->Phe 显示出最小量的溶血或渗漏（少于野生型的 5%）。肺炎球菌溶血素诱导的 Lettre 细胞渗漏对二价阳离子的抑制敏感，但抑制程度因修饰而异。突变体 Trp-433-->Phe 的泄漏对阳离子抑制最不敏感。跨平面脂质双层形成的离子传导通道表现出小（小于 30 pS）、中（30 pS-1 nS）和大（大于 1 nS）电导阶跃。中小型通道优先被二价阳离子封闭。与主要形成小通道的野生型毒素相反，修饰毒素Trp-433-->Phe形成对阳离子诱导的闭合不敏感的大通道。分子量大于15 kDa的多糖可抑制野生型毒素引起的溶血，但分子量高达40 kDa的多糖不能防止Trp-433-->Phe引起的溶血。电镜显示Trp-433-->Phe形成寡聚弧和环结构，其尺寸与野生型毒素相同，并且形成的弧与环的比例对于野生型毒素和Trp-433-->Phe变体是相同的。我们得出的结论是，Trp-433-->Phe 的变化会影响与细胞膜结合后的通道形成和寡聚物的形成，并且电子显微镜显示的弧形和环形结构的大小并不反映通道的功能状态。
Pneumolysin is one of the family of thiol-activatable, cytolytic toxins. Within these toxins the amino acid sequence Trp-Glu-Trp-Trp is conserved. Mutations made in this region of pneumolysin, residues 433-436 inclusive, did not affect cell binding or the formation of toxin oligomers in the target cell membrane. However, the mutations did affect haemolysis, leakage of low-molecular-mass metabolites from Lettre cells and the induction of conductance channels across planar lipid bilayers. Of eight modified pneumolysins examined, Trp-433-->Phe showed the smallest amount of haemolysis or leakage (less than 5% of wild type). Pneumolysin-induced leakage from Lettre cells was sensitive to inhibition by bivalent cations but the extent of inhibition varied depending on the modification. Leakage by the mutant Trp-433-->Phe was least sensitive to cation inhibition. The ion-conducting channels formed across planar lipid bilayers exhibit small (less than 30 pS), medium (30 pS-1 nS) and large (more than 1 nS) conductance steps. Small- and medium-sized channels were preferentially closed by bivalent cations. In contrast with wild-type toxin, which formed predominantly small channels, the modified toxin Trp-433-->Phe formed large channels that were insensitive to cation-induced closure. Polysaccharides of molecular mass more than 15 kDa inhibited haemolysis by wild-type toxin, but polysaccharide of up to 40 kDa did not prevent haemolysis by Trp-433-->Phe. Electron microscopy revealed that Trp-433-->Phe formed oligomeric arc and ring structures with dimensions identical with those of wild-type toxin, and that the ratio of arcs to rings formed was the same for wild-type toxin and the Trp-433-->Phe variant. We conclude that the change Trp-433-->Phe affects channel formation at a point subsequent to binding to the cell membrane and the formation of oligomers, and that the size of arc and ring structures revealed by electron microscopy does not reflect the functional state of the channels.