Molecular genetic analysis of ganglioside GD1b-binding activity of Escherichia coli type IIa heat-labile enterotoxin by use of random and site-directed mutagenesis.
Mutagenesis of the B-subunit gene of Escherichia coli heat-labile enterotoxin LT-IIa was performed in vitro with sodium bisulfite. Mutants were screened initially by radial passive immune hemolysis assays for loss of binding to erythrocytes. Mutant B polypeptides were characterized for immunoreactivity; for binding to gangliosides GD1b, GD1a, and GM1; for formation of holotoxin; and for biological activity. Mutant alleles that determined altered binding specificities were sequenced. Three such mutant alleles encoded Thr-to-Ile substitutions at residues 13, 14, and 34 in the mature B polypeptide of LT-IIa. Each mutant protein failed to bind to ganglioside GD1b, although the Ile-14 mutant retained the ability to bind to ganglioside GM1. Site-specific mutagenesis was used to construct mutants with various amino acid substitutions at residue 13, 14, or 34. Only those mutant proteins with Ser substituted for Thr at position 13, 14, or 34 retained the ability to bind to ganglioside GD1b, thereby suggesting a role for the hydroxyl group of Thr or Ser in ganglioside GD1b binding.
Connell
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Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
Holmes
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D000595:Amino Acid Sequence; D000818:Animals; D001427:Bacterial Toxins / Q000378:metabolism*; D001681:Biological Assay; D004768:Enterotoxins / Q000502:physiology*; D004926:Escherichia coli / Q000472:pathogenicity*; D029968:Escherichia coli Proteins; D005677:G(M1) Ganglioside / Q000378:metabolism*; D005732:Gangliosides / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D006461:Hemolysis; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000187:drug effects; D015345:Oligonucleotide Probes / Q000235:genetics; D010957:Plasmids; D011863:Radioimmunoassay; D013447:Sulfites; D014315:Triterpenes / Q000378:metabolism
D001427:Bacterial Toxins; D004768:Enterotoxins; D029968:Escherichia coli Proteins; D005732:Gangliosides; D006027:Glycosides; D015345:Oligonucleotide Probes; D013447:Sulfites; D014315:Triterpenes; C021298:ganglioside, GD1a; C033851:ganglioside, GD1b; C001883:stichoposide; D005677:G(M1) Ganglioside; C027538:heat-labile enterotoxin, E coli; C042345:hydrogen sulfite