Mutational analysis of the ganglioside‐binding activity of the type II Escherichia coli heat‐labile enterotoxin LT‐IIb

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Abstract
The Escherichia coli type II heat-labile enterotoxin LT-IIb IIb consists of a single A polypeptide and five B polypeptides. The A polypeptide is responsible for the toxic activity, and the B polypeptides function to bind the toxin to gangliosides on the surface of the plasma membrane. Previous studies on the related type II enterotoxin LT-IIa demonstrated the importance of threonine (Thr) residues at positions 13, 14, and 34 in the mature B polypeptide for ganglioside GD1bp-binding activity. In this study, we used sitespecific mutagenesis to investigate ganglioside GD1a-binding activity of the B polypeptide of LT-IIb. We determined that Thr-13 and Thr-14 were involved in binding of ganglioside GD1a by the B polypeptides of LT-IIb but that Thr-34 was not essential. Substitution of serine, but not other amino acids, at position 13 or 14 in the B polypeptide of LT-IIb resulted in retention of ganglioside-binding activity equivalent to that of the wild-type enterotoxin, providing strong evidence that the hydroxyl groups of threonine or serine at positions 13 and 14 are important for the ganglioside-binding activity of LT-IIb. Chimeric genes that expressed hybrids of the B polypeptides of LT-IIb and LT-IIa were also constructed, and analysis of the hybrids showed that the specificity of their ganglioside-binding activity was determined by the N-terminal half of the molecule.