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

产肠毒素大肠埃希菌产生的耐热肠毒素(ST)可引起人的急性腹泻。此前，我们和其他人测定了产肠毒素大肠杆菌STS的氨基酸序列，由19个和18个残基组成。本工作旨在阐明ST的三级结构及其毒性表达的结构要求。1)确定了三个分子内二硫键不仅存在于产肠毒素大肠杆菌的两个STS中，而且还存在于肠结肠耶尔森菌和非01霍乱弧菌的STS中。在所考察的所有STS中，二硫键的模式是相同的。2)合成了具有三个二硫键中的一个或两个的产肠毒素大肠杆菌的ST类似物，并对其活性进行了检测，证明三个二硫键中的一个是表达ST的毒性所必需的。3)合成了由D-氨基酸残基取代的STP类似物(由产肠毒素的猪链霉菌产生的ST)。5位D-半胱氨酸的STP类似物具有与STP相同的活性，且其对乳鼠的毒性持续时间超过24小时，而STP的毒性在6-10小时后降低。

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.