Okamoto K, Baba T, Yamanaka H, Akashi N, Fujii Y
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Japan.
J Bacteriol. 1995 Aug;177(16):4579-86. doi: 10.1128/jb.177.16.4579-4586.1995.
The Escherichia coli heat-stable enterotoxin II (STII) is a typical extracellular toxin consisting of 48 amino acid residues, of which 4 are cysteine. There are two disulfide bonds, one between Cys-10 and Cys-48 and one between Cys-21 and Cys-36. We examined the involvement of DsbA in the formation of the disulfide bonds of STII and the role of each in the secretion of STII. A dsbA mutant was transformed with a plasmid harboring the STII gene, and STII was not detected either in the cells or in the culture supernatant. Reducing the level of STII brought about the dsbA mutation restored by introducing the wild-type dsbA gene into the mutant strain. These results showed that DsbA is involved in forming the disulfide bonds of STII and that STII without these disulfide bonds is degraded during secretion. We substituted these four cysteine residues in vivo by oligonucleotide-directed site-specific mutagenesis. The amino acid sequence of the purified STII (C48S) and pulse-chase studies revealed that two intermolecular disulfide bonds must be formed to be efficiently secreted and that cleavage between amino acid residues 14 and 15 is probably the first step in the proteolytic degradation of STII.
大肠杆菌热稳定肠毒素II（STII）是一种典型的细胞外毒素，由48个氨基酸残基组成，其中4个是半胱氨酸。存在两个二硫键，一个在Cys-10和Cys-48之间，另一个在Cys-21和Cys-36之间。我们研究了二硫键形成蛋白A（DsbA）在STII二硫键形成中的作用以及每个二硫键在STII分泌中的作用。用携带STII基因的质粒转化dsbA突变体，在细胞或培养上清液中均未检测到STII。降低STII水平导致通过将野生型dsbA基因导入突变菌株来恢复dsbA突变。这些结果表明DsbA参与STII二硫键的形成，并且没有这些二硫键的STII在分泌过程中被降解。我们通过寡核苷酸定向位点特异性诱变在体内替换了这四个半胱氨酸残基。纯化的STII（C48S）的氨基酸序列和脉冲追踪研究表明，必须形成两个分子间二硫键才能有效分泌，并且氨基酸残基14和15之间的切割可能是STII蛋白水解降解的第一步。
