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个组成部分。我们的显微镜交联的交联发现了LUKF和HLG2以随机方式组装，以3：4的亚基构成构造构造复合物，3：4：研究项目，我们试图构建纳米标记的谷胱甘肽 - 谷胱甘肽-S-转移酶（GST）的LUKF和LUKF和HLG2的融合蛋白在电子显微镜下，LUKF和HLG2的替代排列。是其对应物的溶血活性。收集和分析了10 nm宽度和10 nm的pore孔的透射显微镜。

项目成果

期刊论文数量（26）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Protein sequence analysis, cloning and expression of flammutoxin, a pore-forming cytolysin from Flammulina velutipes : Maturation of dimeric precursor to monomeric active form by carboxyl-terminal truncation.

金针菇毒素（一种来自金针菇的成孔溶细胞素）的蛋白质序列分析、克隆和表达：通过羧基末端截断将二聚体前体成熟为单体活性形式。

DOI：
发表时间：
2004
期刊：
J.Biol.Chem. 279
影响因子：
0
作者：
Tomita;T.;Mizumachi;Y;Chong;K;Ogawa.;Konishi;Sugawara Tomita;N.;Dohmae;N;Hashimoto;Y.;Takio;K.
通讯作者：
K.

Cloning expression, and pore-forming properties of mature and precursor forms of pleurotolysin, a sphingomyelin-specific two-component cytolysin from the edible

侧耳溶素的成熟和前体形式的克隆表达和成孔特性，侧耳溶素是一种来自食用植物的鞘磷脂特异性双组分溶细胞素

DOI：
发表时间：
2004
期刊：
Biochim.Biophys.Acta 1679
影响因子：
0
作者：
Sakurai;N.;Kaneko;J.;Kamio;Y.;Tomita;T.
通讯作者：
T.

Cloning, expression, and pore-forming properties of mature and precursor forms of pleurotolysin, a sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus.

侧耳溶素的成熟和前体形式的克隆、表达和成孔特性，侧耳溶素是一种来自食用蘑菇平菇的鞘磷脂特异性双组分溶细胞素。

DOI：
发表时间：
2004
期刊：
Biochim.Biophys.Acta 1679
影响因子：
0
作者：
Sakurai;N.;Kaneko;J.;Kamio.;Y.;Tomita;T.
通讯作者：
T.

Sawada, H., Kawase, T., Inaba, Y., Baba, M., Tomita, T.: "Synthesis of fluoroalkyl end-capped oligomers containing pendant phosphinic acid and phosphonic acid segments"International J.Polymeric Mater.. (in press). (2004)

Sawada, H.、Kawase, T.、Inaba, Y.、Baba, M.、Tomita, T.：“含次膦酸和膦酸链段的氟烷基封端低聚物的合成”International J.Polymeric Mater..（

DOI：
发表时间：
期刊：
影响因子：
0
作者：
通讯作者：

Ito, Y., Tomita, T., Roy, N., Sugawara-Tomita, N., Abe, N., Kamio, Y.: "Cloning, expression, and cell-surface localization of Paenibacillus sp.W-61 xylanase 5, a multidomain xylanase"Appl.Environ.Microbiol.. 69・12. 6969-6978 (2003)

Ito, Y.、Tomita, T.、Roy, N.、Sugarara-Tomita, N.、Abe, N.、Kamio, Y.：“类芽孢杆菌 sp.W-61 木聚糖酶的克隆、表达和细胞表面定位5、多结构域木聚糖酶“Appl.Environ.Microbiol..69・12.6969-6978(2003)