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

The Escherichia coli type II heat‐labile enterotoxin LT‐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 GD1b‐binding activity. In this study, we used site‐specific 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 GD1 a 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.