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Studies of the Tertiary Structure and Structural Requirements for Toxic Expression of Heat-stable Enterotoxins Produced by Enterotoxigenic Escherichia coli

产肠毒素大肠杆菌产生的耐热肠毒素的三级结构和毒性表达的结构要求的研究

基本信息

批准号：
62470022
负责人：
SHIMONISHI Yasutsugu
金额：
$ 3.52万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1987
资助国家：
日本
起止时间：
1987 至 1988
项目状态：
已结题

项目摘要

Heat-stable enterotoxin (ST) produced by enterotoxigenic Escherichia coli causes acute diarrhea in human. Previously, we and others determined the amino acid sequences of the STs of enterotoxigenic E. coli, which consist of 19 and 18 residues. The present work was carried out with the aim to elucidate the tertiary structure of ST and the structural requirement for expression of its toxicity. The following tesults were obtained: 1) The positions of three intramolecular disulfide linkages not only in two STs of enterotoxigenic E. coli but also in sTs of yersinia entero colitica and vibrio cholerae non-01 were determined. The mode of disulfide linkages were same in all of the STs examined. 2) Analogs of the ST of enterotoxigenic E. coli with one or two among three disulfide linkages were synthesized and their activities were examined, demonstrating tha the special one of three disulfide linkages was essential for expression of the toxicity of the ST. 3) Analogs of STp (ST produced by a porcine stratin of enterotoxigenic E. coli) replaced by D-amino acid residues at a single position were synthesized. The analog of STp with D-Cys at position 5 had the same activity as that of STp and moreover the toxicity of the analog lasted after its administration into suckling mouse more than 24 hours, while the toxicity of STp decreased after 6-10 hours.

产肠毒素大肠杆菌产生的热稳定肠毒素（ST）可引起人类急性腹泻。在此之前，我们等人确定了产肠毒素大肠杆菌STs的氨基酸序列，其中包括19和18个残基。本研究旨在阐明ST的三级结构及其表达毒性的结构要求。结果如下：1)确定了两个产肠毒素大肠杆菌STs中三个分子内二硫键的位置，也确定了大肠耶尔森菌和非01型霍乱弧菌STs中的三个分子内二硫键的位置。二硫键的模式在所有STs中都是相同的。2)合成了产肠毒素大肠杆菌中具有三种二硫键中的一种或两种的ST类似物，并对其活性进行了检测，证明了三种二硫键中特殊的一种对于表达ST的毒性至关重要。3)合成了由产肠毒素大肠杆菌的猪层蛋白产生的ST类似物（由d -氨基酸残基在单个位置取代）。含有5位D-Cys的STp类似物与STp具有相同的活性，并且其毒性在给药后持续24小时以上，而STp的毒性在6-10小时后下降。