Disulfide bond formation and secretion of Escherichia coli heat-stable enterotoxin II.
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.
Okamoto
K
K
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Japan.
Baba
T
T
Yamanaka
H
H
Akashi
N
N
Fujii
Y
Y
D001427:Bacterial Toxins / Q000378:metabolism*; D001483:Base Sequence; D001692:Biological Transport; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism*; D004768:Enterotoxins / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000502:physiology*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D007535:Isomerases / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019704:Protein Disulfide-Isomerases; D011499:Protein Processing, Post-Translational
D001427:Bacterial Toxins; D004220:Disulfides; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C034151:heat stable toxin (E coli); D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D003545:Cysteine