Functional Properties of Pro Region of <i>Escherichia coli</i> Heat‐Stable Enterotoxin

Yamanaka, Hidetoshi; Fuke, Yasunori; Hitotsubashi, Shunji; Fujii, Y.; Okamoto, Keinosuke

doi:10.1111/j.1348-0421.1993.tb03200.x

Cited by 18 publications

(24 citation statements)

References 41 publications

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“…E. coli JCB571/ pDS01 was shaken overnight at 37°C in Luria broth containing ampicillin (50 ,ug/ml). The cells were harvested by centrifugation, and the periplasmic fraction was prepared with polymyxin B (34). This fraction was dialyzed against 10 mM phosphate buffer (pH 7.0) at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, we used fusion proteins containing the amino-terminal region of STp (pre and pro regions) and the nuclease A mature region (the entire fusion protein was referred to as pre-pronuclease A). The processed fusion protein is located in periplasm and can be quantified by SDS-PAGE (20,34). Plasmid pKK605 carrying the gene for fusion protein composed of the native amino terminus of STp and nuclease A (20) was introduced into JCB570 (dsbA +) and JCB571 (dsbA mutant).…”

Section: Methodsmentioning

confidence: 99%

“…SDS-PAGE. SDS-PAGE was performed as described by Laemmli (14 regions (20,34). As the intermediates are soon translocated into culture medium, they are difficult to quantify (36).…”

Section: Methodsmentioning

confidence: 99%

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Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation

et al. 1994

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The Escherichia coli heat-stable enterotoxin STp is synthesized as a precursor consisting of pre, pro and mature regions. Mature STp is released into the culture supernatant and is composed of 18-amino-acid residues which contain three intramolecular disulfide bonds. The involvement of DsbA in the formation of the disulfide bonds of STp was examined in this study. A dsbA mutant was transformed with a plasmid harboring the STp gene, and the ST activity was significantly lower than that of the parent strain harboring the same plasmid. Furthermore, purified DsbA induced the conversion of synthetic STp peptide (inactive form) to the active form and increased the ST activity of the culture supernatant derived from the dsbA transformants. These results showed that DsbA directly catalyzes the formation of the disulfide bonds of STp. DsbA is located in periplasmic space, where STp is released as an intermediate form consisting of the pro and mature regions. To examine the effect of the pro region on the action of DsbA, we replaced the cysteine residue at position 39 and tested the effect in vivo. The substitution caused a significant decrease of ST activity in the culture supernatant, the accumulation of inactive ST in periplasmic space, and an alteration in the cleavage site of the intermediate of STp. We conclude that Cys-39 is important for recognition by the processing enzymes required for the maturation of STp.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation

et al. 1994

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“…The levels of DsbA in the periplasmic fraction of these cells were examined by SDS-PAGE. The polymyxin B method was used to prepare the periplasmic fraction of the cells (8,20,28). Figure 2 shows that DsbA was detected in the sample prepared from JCB571/ pDS11 (lane 2) but not in that from JCB571/pDS12 (lane 1).…”

Section: Resultsmentioning

confidence: 99%

Disulfide bond formation and secretion of Escherichia coli heat-stable enterotoxin II

et al. 1995

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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.Enterotoxigenic Escherichia coli strains produce two heatstable enterotoxins (STs), which cause intestinal secretion and diarrhea (5). One is termed STI (also referred to as STa), and the other is termed STII (also referred to as STb). STI is an 18-or 19-amino-acid acidic peptide containing three disulfide bonds and a protease-stable peptide (1,19,25). STI is active in the suckling-mouse assay. On the other hand, STII was reported to be active only in the weaned-pig ligated intestinal loop assay (26). Because of the lack of a convenient assay, STII was not purified for some time after its detection. We and Whipp found that STII is positive in the mouse intestinal loop assay in the presence of a protease inhibitor (9,11,27). We purified STII and confirmed that the mature form released into culture supernatant is composed of 48 amino acid residues that contain disulfide bonds between Cys-10 and Cys-48 and between Cys-21 and Cys-36 (9).The gene encoding STII (estII) has been isolated and the DNA sequence has been determined (15, 21). The sequence indicates that STII is synthesized as a 71-amino-acid-residue peptide. The 48-amino-acid sequence that we determined is identical to that of the 48 carboxy-terminal amino acids of STII predicted from the DNA sequence. This means that STII is synthesized as a precursor consisting of 71 amino acid residues and that the 23 amino-terminal amino acids consist of a conventional leader peptide which is cleaved by signal peptidase after the STII mRNA translation is initiated. This cleavage allows the resulting peptide to translocate through the inner membrane to the periplasm (13). To be converted into active STII, the intramolecular disulfide bonds must be correctly formed. However, the pathway in which the disulfide bond is formed has not been elucidated.A periplasmic protein, termed DsbA, which...

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“…4 and 5). Almost all of the amino acid residues of STIp are hydrophobic (20,27), and therefore mutant STIps in which amino acid residue Phe-3 or Tyr-18 is deleted may interact less efficiently with the secretory machinery via other hydrophobic amino acid residues. Further substitutions may promote our understanding of the amino acid residues of STIp involved in the extracellular secretion.…”

Section: Resultsmentioning

confidence: 99%

Mutation of Aromatic Amino Acid Residues Located at the Amino‐ and Carboxy‐Termini of Escherichia coli Heat‐Stable Enterotoxin Ip Reduces the Efficiency of the Toxin to Cross the Outer Membrane

Yamanaka

Okamoto

2000

Microbiology and Immunology

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Heat-stable enterotoxin Ip (STIp) of Escherichia coli is synthesized as a precursor form consisting of pre-(amino acid residues 1 to 19), pro-(amino acid residues 20 to 54) and mature (amino acid residues 55 to 72) regions. Mature STIp (bioactive STIp) is formed in the periplasmic space after the precursor is proteolytically processed and the mature STIp translocates across the outer membrane through the secretory system including TolC, an outer membrane protein of E. coli. However, it remains unknown how the mature STIp is recognized by this secretory system. In this study, we investigated the amino acid residues of STIp involved in its translocation across the outer membrane. We prepared mutant STIp genes by sitedirected mutagenesis and analyzed translocation of the mutant STIps across the outer membrane. Deletion of the Phe or Tyr residue at position 3 or 18, respectively, decreased the efficiency of translocation of STIp across the outer membrane. To confirm the involvement of these amino acid residues, we further mutated the codons for these amino acid residues to that for Gly. These mutations also decreased the efficiency of extracellular secretion of STIp. In contrast, substitution of Phe-3 and Tyr-18 with Tyr and Phe, respectively, did not affect the efficiency of translocation of the toxin. These results indicated that the aromatic amino acid residues at positions 3 and 18 in the mature region are important for the ability of STIp to cross the outer membrane.

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Functional Properties of Pro Region of Escherichia coli Heat‐Stable Enterotoxin

Cited by 18 publications

References 41 publications

Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation

Maturation pathway of Escherichia coli heat-stable enterotoxin I: requirement of DsbA for disulfide bond formation

Disulfide bond formation and secretion of Escherichia coli heat-stable enterotoxin II

Mutation of Aromatic Amino Acid Residues Located at the Amino‐ and Carboxy‐Termini of Escherichia coli Heat‐Stable Enterotoxin Ip Reduces the Efficiency of the Toxin to Cross the Outer Membrane

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