Isolation, primary structure and synthesis of heat-stable enterotoxin produced by Yersinia enterocolitica

Abstract

Five heat-stable enterotoxins were isolated from the culture supernatant of Yersinia enterocolitica and purified to homogeneity by DEAE-Sephacel and high-performance liquid chromatographies. They caused acute fluid accumulation in the intestine of suckling mice. The amino acid sequence of one of the enterotoxins was determined to be Ser-Ser-Asp-Trp-Asp-Cys-Cys-Asp-Val-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys, by Edman degradation of its pyridylethylated derivative and a combination of fast atom bombardment mass spectrometry and carboxypeptidase B digestion. This structure was unambiguously confirmed by chemical synthesis. The other enterotoxins had longer or shorter amino acid sequences at their N termini, but the same sequence at their C termini. The six half-cystine residues formed intramolecular disulfide linkages, as shown by measurement of the molecular masses of the enterotoxins by fast atom bombardment mass spectrometry. The sequence of 13 amino acid residues at the C terminus showed similarity to those of heat-stable enterotoxins isolated from enterotoxigenic Escherichiu c d i [Aimoto, S . et al. (1982) high-performance liquid chromatography (HPLC) and determined the primary structure of one of them [22]. Part of the primary structure of Yersiniu ST determined was similar to the active-site sequence of E. coli ST. These similar sequences were deduced to be responsible for the common biological and immunological properties of E. coli ST and Yersinia ST and to be characteristic of, and conserved in heat-stable enterotoxins produced by, enteric bacteria such as enterotoxigenic E. cwli and Y. mtrrocolitica. Therefore, it seemed important to examine whether the other toxic fractions that we isolated from the culture supernatant of Y . enterocolitica also had a similar primary structure.Here we report purification of toxic fractions from the culture supernatant of Y. enterocolitica by a different chromatographic procedure from that used in previous work [22] and determination of their amino acid sequences. We also describe the chemical synthesis of Yersiniu ST, establishing the amino acid sequence of heat-stable enterotoxin of Y . enterocoliticu. MATERIALS AND METHODS Mutrriuls and upparutusReagent-grade chemicals from several suppliers were used and were purified for analytical experiments before use. Carboxypeptidase B and aminopeptidase M were purchased from Boehringer (Mannheim, FRG) and the Protein Research Foundation (Minoh, Japan) respectively. YMC-ODS ( S -5 ) was obtained from Yamamura Chemical Laboratories Co. Ltd (Kyoto, Japan) and packed into columns (4 x 250 mm, 6 x 150 mm and 8 x 300 mm) in our laboratory for preparative experiments. A microsorb psCls column ( 5 pm,

Mode of disulfide bond formation of a heat‐stable enterotoxin (STh) produced by a human strain of enterotoxigenic Escherichia coli

“…Furthermore, the 1H-NMR spectra of these peptides were identical with that of standard STh (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), as shown in fig.5. Thus, we conclude that the disulfide linkages in STh (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) are formed between Cys-6 and Cys-11, Cys-7 and Cys-15, and Cys-10 and Cys-18, as shown in fig.6. We recently found that STp(5-17), which has Ala in place of Thr in STn (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), has the same disulfide bonds as STh(6--18) (not shown).…”

“…Thus, we conclude that the disulfide linkages in STh (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18) are formed between Cys-6 and Cys-11, Cys-7 and Cys-15, and Cys-10 and Cys-18, as shown in fig.6. We recently found that STp(5-17), which has Ala in place of Thr in STn (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18), has the same disulfide bonds as STh(6--18) (not shown). This indicates that E. coli-STh and STp have the same disulfide bonds as those shown in fig.6.…”

“…The amino acid compositions and molecular masses of purified peptides were examined by amino acid analysis and fast atom bombardment mass spectrometry, respectively, as described [12].…”

“…The full enterotoxigenic activity of STI has been found to be expressed by a segment with 13 amino acid residues, which includes 6 halfcystines [10]. Recently, STI-like enterotoxins from Yersinia enterocolitica [11,12], Vibrio cholerae non-01 [13], and V. mimicus (unpublished) have been isolated and sequenced ( fig.l). These enterotoxins have common regions with 13 amino acid residues including 6 Cys residues, which are located in the same relative positions and linked intramolecularly by three disulfide bonds.…”

Characterization of a highly toxic, large molecular size heat‐stable enterotoxin produced by a clinical isolate of Yersinia enterocolitica

“…Heat-stable enterotoxins (STs) are peptide toxins produced by bacterial enteropathogens such as enterotoxigenic Escherichia coli [1,2], Yersinia enterocolitica [3][4][5] and Vibrio cholerae [6][7][8], and are responsible for acute diarrhea in humans. ST binds to a membrane-bound guanylate cyclase which serve as a specific cell surface receptor for ST, followed by elevation of the intracellular cGMP level [9][10][11].…”