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 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.
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Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, USA.
Holmes
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001427:Bacterial Toxins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004305:Dose-Response Relationship, Drug; D004768:Enterotoxins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005732:Gangliosides / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011863:Radioimmunoassay; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D012694:Serine / Q000378:metabolism; D013912:Threonine / Q000378:metabolism*; D014169:Transformation, Bacterial
D000911:Antibodies, Monoclonal; D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D005732:Gangliosides; D011993:Recombinant Fusion Proteins; D013912:Threonine; D012694:Serine; C027538:heat-labile enterotoxin, E coli