Okamoto K, Okamoto K, Yukitake J, Miyama A
Department of Microbiology, School of Medicine, Fujita-Gakuen Health University, Aichi-ken, Japan.
Infect Immun. 1988 Aug;56(8):2144-8. doi: 10.1128/iai.56.8.2144-2148.1988.
The Escherichia coli heat-stable enterotoxins (STs) are small peptide toxins consisting of 18 (STp) or 19 (STh) amino acids. STp and STh share biologically active sequences which reside in the C-terminal 13 amino acid residues, but the role of each amino acid in the active sequences is not clear. We substituted in vivo Asp, Tyr, His, Gln, Lys, and Arg for the Asn residue at position 11 of STp by oligonucleotide-directed site-specific mutagenesis and examined the biological activities of the resulting mutants. All mutant STs reacted with both monoclonal and polyclonal antibodies, demonstrating that the amino acid substitutions at position 11 did not cause a significant change in the conformation of STp. However, the substitutions invariably caused a significant decrease in enterotoxic activities. The most remarkable decrease was observed with Asn-11----Lys-11 and Asn-11----Arg-11 mutations; that is, enterotoxic activity could not be detected in the culture supernatant of either of these mutant strains. These results indicate that Asn-11 of STp plays an essential role in the enterotoxic activity. The amide group and the length of side chain of Asn-11 seem to be especially important for enterotoxic activity.
大肠杆菌热稳定肠毒素（STs）是由18个氨基酸（STp）或19个氨基酸（STh）组成的小肽毒素。STp和STh具有位于C端13个氨基酸残基中的生物活性序列，但活性序列中每个氨基酸的作用尚不清楚。我们通过寡核苷酸定向位点特异性诱变，将STp第11位的天冬酰胺残基在体内分别替换为天冬氨酸、酪氨酸、组氨酸、谷氨酰胺、赖氨酸和精氨酸，并检测了所得突变体的生物活性。所有突变型STs均能与单克隆抗体和多克隆抗体发生反应，表明第11位的氨基酸替换并未导致STp的构象发生显著变化。然而，这些替换无一例外地导致肠毒素活性显著降低。Asn-11→Lys-11和Asn-11→Arg-11突变导致的活性降低最为显著；也就是说，在这两种突变菌株的培养上清液中均未检测到肠毒素活性。这些结果表明，STp的Asn-11在肠毒素活性中起关键作用。Asn-11的酰胺基团和侧链长度似乎对肠毒素活性尤为重要。
