Structural analysis of heteroheptamer transmembrane pore supramolecule of staphylococcal gamma-hemolysin

葡萄球菌γ-溶血素异七聚体跨膜孔超分子的结构分析

基本信息

批准号：
15590380
负责人：
TOMITA Toshio
金额：
$ 2.37万
依托单位：
Tohoku University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2004
项目状态：
已结题

项目摘要

Staphylococcal γ-hemolysin (Hlg) is a two-component cytolysin secreated by Staphylococcus aureus. We have previously shown that the two-component γ-hemolysin assembles from LukF (or Hlg1, 34 kDa) and Hlg2 (32 kDa) to form ring-shaped transmembrane pores of approximately 200 kDa on human erythrocytes. Our recent study involving electron microscopy and chemical cross-linking suggested that LukF and Hlg2 assemble in a stochastic manner to form alternate complexes with subunit stoichiometries of 3:4 and 4:3. In this research project, we attempted to construct nanogold-labeled glutathione-S-transferase (GST) fusion proteins of LukF and Hlg2 to visualize the alternate arrangements of LukF and Hlg2 under electron microscope. For nanogold labeling of the GST fusion proteins, three Cys residues (except for Cys138) of GST were successfully replaced with Ala by using primer extension procedure of polymerase chain reactions. Recombinant GST fusion proteins of LukF and Hlg2 thus obtained were hemolytically active in the presence of their counterpart. We now attempt to separate nanogold-labeled GST fusion proteins from unlabeled fusion proteins and nanogold particles. In this research project, we also analyzed fine structure of the heteroheptamers of Hlg by electron microscopy. The pore complexes isolated from human erythrocytes were negatively stained with phosphotungstic acid and observed under a transmission electron microscopy. High resolution images of the pore complexes from different angles were collected and analyzed. As a result, the pore complex of Hlg has a barrel morphology with 10-nm width and 10-nm height.

葡萄球菌γ-蛋白酶（HLG）是金黄色葡萄球菌分泌的两组细胞蛋白酶。我们先前已经表明，从LUKF（或HLG1、34 kDa）和HLG2（32 kDa）组装的两组分组γ-蛋白酶在人类红细胞细胞上形成大约200 kDa的环形跨膜孔。我们最近涉及电子显微镜和化学交联的研究表明，LUKF和HLG2以随机方式组装，以3：4和4：4：4和4：3形成替代复合物。在该研究项目中，我们试图构建LUKF和HLG2的纳米标记的谷胱甘肽-S-转移酶（GST）融合蛋白，以可视化电子显微镜下LUKF和HLG2的替代排列。对于GST融合蛋白的纳米质量标记，通过使用聚合酶链反应的质子扩展程序，成功地将三个CYS保留（除Cys138）被ALA成功替换。因此，在其对应物的存在下，LUKF和HLG2的重组GST融合蛋白具有溶血活性。现在，我们试图将纳米标记的GST融合蛋白与未标记的融合蛋白和纳米颗粒分开。在该研究项目中，我们还通过电子显微镜分析了HLG异质膜的精细结构。从人的红细胞中分离出的孔复合物用磷酸烟酸对造成负染色，并在透射电子显微镜下观察到。收集并分析了来自不同角度的孔复合物的高分辨率图像。结果，HLG的孔复合物具有宽度为10 nm的枪管形态，高度为10 nm。