Purification of the ST_B enterotoxin of Escherichia coli and the role of selected amino acids on its secretion, stability and toxicity

The structure ofEscherichia coliheat-stable enterotoxin b by nuclear magnetic resonance and circular dichroism

“…In contrast, R29S had -10% of the activity of the wild-type peptide, and D30V had -40% of wild-type activity, suggesting that R29 and D30 are among the residues important for toxicity. The mutants R29S and D30V could not compete with native STb, suggesting that these mutant peptides d o not efficiently bind the putative receptor (Dreyfus et al, 1992). Recent evidence suggests that the positive residues K22 and K23 are also very important for toxic activity (Fujii et al, 1994).…”

“…Mutants that lack disulfides are more susceptible to periplasmic proteolysis than is wild-type toxin (Dreyfus et al, 1992). Disulfides are also important for activity; reduction and S-carboxyamidomethylation result in loss of toxicity (Dreyfus et al, 1992).…”

“…The disulfide bridges in STb have been identified by tryptic peptide analysis and Edman degradation to be between Cys 10 and Cys 48 and between Cys 21 and Cys 36 (Dreyfus et al, 1992). Figure 1 shows the primary sequence of STb.…”

“…STb is also synthesized as a 71-amino acid precursor (Lee et al, 1983;Picken et al, 1983). Mature STb is a 48-amino acid peptide, with four Cys residues and no homology to STa (Dreyfus et al, 1992). The inferred N-terminus of pro-STb (residues 1-23) has characteristics of a signal sequence that is cleaved, presumably by a signal peptidase, during export to the periplasm, and the periplasmic STb is then translocated to the extracellular medium (Kupersztoch et al, 1990).…”

“…The contribution of some residues in the loop to the toxicity of STb has been examined by site-directed mutagenesis (Dreyfus et al, 1992). The activity of the mutants was determined by directly measuring the volume of the fluid accumulated in ligated rat intestinal loops following STb injection (Whipp, 1990).…”