Chemical synthesis of an octadecapeptide with the biological and immunological properties of human heat-stable Escherichia coli enterotoxin

Abstract

An eighteen-amino-acid peptide having the linear amino acid sequence of human heat-stable enterotoxin (ST) has been synthesized by solid phase peptide synthesis. The purified peptide could be obtained in yields approaching 25% after purification by size, charge, and high-performance ligand chromatography. This material was pure and identical to native ST by analytical high-performance ligand chromatography, amino acid analysis, paper electrophoresis and thin-layer chromatography. The formation of the disulfide bonds was critical for biological and immunological activity and were tentatively determined to be between cysteines 5 and 14, 6 and 10, and 9 and 17. This synthetic peptide had full immunological and biological activity when compared to native ST by enzyme-linked immunosorbent assay and the suckling mouse assay respectively.The toxins produced by enterotoxigenic Escherichia coli are known to cause a variety of diarrheal symptoms in man and animals. These range from the inconvenience of travelers' diarrhea to the life-threatening affliction in human infants and high death rate in newborn pigs and cattle [I -51. The two forms of toxin commonly found are termed heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST). The heatlabile form [6] is a protein composed of a single 25-kDa subunit (LT-A) in combination with five identical 13-kDa subunits (LT-B). The smaller subunit of LT can be obtained by known methods [6, 71. The heat-stable form of enterotoxin is composed of 18 -20 amino acids, six of which are cysteines [8 -121. Of the sequences known thus far for ST, three were determined by sequencing of the peptide isolated from E. coli strains [8-101 and two were determined from nucleotide sequences [ll, 121. These differ primarily in the N-terminal four residues and the tyrosine and asparagine residues at positions 11 and 18. Natural ST obtained from E. coli and covalently linked to LT-B has been clearly shown in animal studies to be an effective combination for vaccination against the actions of both ST and LT [13]. The difficulty in obtaining sufficient quantities of ST from natural sources for use in conjunction with LT-B, and the recent successful use of synthetic peptides as vaccine agents [14, 151, prompted the synthesis of ST (Fig. 1) for use in the above vaccine. In this paper we report the successful synthesis by the solid phase method [I 61 of ST [9] having full biological and immunological activity. ST prepared in this manner has been successfully used in combination with the B-subunits of LT to immunize against diarrheal symptoms caused by enterotoxigenic E. coli [17 -211. A preliminary report of this synthesis was presented earlier [20].

Disulfide structures of highly bridged peptides: A new strategy for analysis

“…With the relatively stable conotoxin GI, partial reduction by NaBH, in the presence of iodoacetic acid gave unambiguous results (Gray et al, 1984), although some exchange was detected. On the other hand, alkylation of partially oxidized enterotoxin led to incorrect assignments (Houghten et al, 1984). The present work describes a way to circumvent the exchange problem, opening a new route to disulfide assignment.…”

Heat-Labile- and Heat-Stable-Toxoid Fusions (LT R192G -STa P13F ) of Human Enterotoxigenic Escherichia coli Elicit Neutralizing Antitoxin Antibodies

“…Although it has been suggested that STa needs to retain some toxicity in order to become immunogenic when carried by a carrier protein (3,35,36), other studies clearly indicated that nontoxic STa antigens in fusion formats elicited neutralizing antibodies (18,19,20,28,46). It was demonstrated that synthetic mutated pSTa (pSTa N11P , pSTa Y18N ), when chemically cross-linked to an LT B subunit or porcine IgG molecules, elicited anti-STa antibodies.…”

“…It was demonstrated that synthetic mutated pSTa (pSTa N11P , pSTa Y18N ), when chemically cross-linked to an LT B subunit or porcine IgG molecules, elicited anti-STa antibodies. Moreover, these anti-STa antibodies provided protection against ETEC infection (18,19,20). Data from the present study further confirm that the STa 13 toxoid, when fused to a carrier protein, elicits neutralizing anti-STa antibodies.…”

Heat-Stable Enterotoxin of EnterotoxigenicEscherichia colias a Vaccine Target

“…It is worth noting that the synthetic ST toxin used by Klipstein and colleagues was based on the STp sequence published by Chan and Giannella (5), which was later discovered to have two interchanged residues (STp, Asn11Tyr and Tyr18Asn) (54). However, this mutant ST peptide apparently had the same biological and immunological properties as native ST (16,27). In spite of the apparent successful generation of both a chemical conjugate and a synthetic ST vaccine candidate, these promising studies have not been further pursued.…”