SUBSTITUTIONS OF CYSTEINE RESIDUES OF ESCHERICHIA-COLI HEAT-STABLE ENTEROTOXIN BY OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS
SUBSTITUTIONS OF CYSTEINE RESIDUES OF ESCHERICHIA-COLI HEAT-STABLE ENTEROTOXIN BY OLIGONUCLEOTIDE-DIRECTED MUTAGENESIS
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DOI：
10.1128/iai.55.9.2121-2125.1987
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发表时间：
1987-09-01
影响因子：
3.1
通讯作者：
MIYAMA, A
中科院分区：
文献类型：
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作者：
OKAMOTO, K;OKAMOTO, K;MIYAMA, A
大肠杆菌18-氨基酸热稳定肠毒素STp具有通过三个二硫键在分子内连接的六个半胱氨酸残基。这些二硫键对毒性活性很重要，但每个键的确切作用尚不清楚。我们取代半胱氨酸残基的STp在体内的谷胱甘肽定向位点特异性诱变解离每个二硫键，并检查所产生的突变体的生物活性。cys-6 →Ala和Cys-17 →Ala突变导致毒性活性完全丧失。Cys-5 →Ala，Cys-10 →Ser和Gly-16，Cys-17 →。Cys-16、Gly-17突变导致毒性活性大幅降低。这些结果意味着在固定位置形成的所有三个二硫键是STp的生物活性的完全表达所需的。然而，在三个突变Cys-5 →之后，仍然存在微弱但显著的毒性。Ala，Cys-10 →Ser和Gly-16，Cys-17 →。半胱氨酸16甘氨酸17这表明STp在其构象上具有一定的灵活性以发挥毒性活性，并且每个二硫键在发挥毒性活性中的作用不完全相同。
The Escherichia coli 18-amino-acid, heat-stable enterotoxin STp has six cysteine residues linked intramolecularly by three disulfide bonds. These disulfide bonds are important for toxic activity, but the precise role of each bond is not clear. We substituted cysteine residues of STp in vivo by oligonucleotide-directed site-specific mutangenesis to dissociate each disulfied bond and examined the biological activities of the resulting mutants. The cys-6 .fwdarw. Ala and Cys-17 .fwdarw. Ala mutations caused a complete loss of toxic activity. The Cys-5 .fwdarw. Ala, Cys-10 .fwdarw. Ser, and Gly-16, Cys-17 .fwdarw. Cys-16, Gly-17 mutations caused a large decrease in toxic activity. These results mean that all three disulfied bonds formed at fixed positions are required for full expression of the biological activity of STp. However, a weak but significant toxicity still remained after three mutations, Cys-5 .fwdarw. Ala, Cys-10 .fwdarw. Ser, and Gly-16, Cys-17 .fwdarw. Cys-16, Gly-17. This indicates that STp has some flexibilities in its conformation to exert toxic activity and that the role of each disulfide bond is exerting toxic activity is not quite the same.