Analysis of structure and function of the B subunit of cholera toxin by the use of site-directed mutagenesis.
Oligonucleotide-directed mutagenesis of ctxB was used to produce mutants of cholera toxin B subunit (CT-B) altered at residues Cys-9, Gly-33, Lys-34, Arg-35, Cys-86 and Trp-88. Mutants were identified phenotypically by radial passive immune haemolysis assays and genotypically by colony hybridization with specific oligonucleotide probes. Mutant CT-B polypeptides were characterized for immunoreactivity, binding to ganglioside GM1, ability to associate with the A subunit, ability to form holotoxin, and biological activity. Amino acid substitutions that caused decreased binding of mutant CT-B to ganglioside GM1 and abolished toxicity included negatively charged or large hydrophobic residues for Gly-33 and negatively or positively charged residues for Trp-88. Substitution of lysine or arginine for Gly-33 did not affect immunoreactivity or GM1-binding activity of CT-B but abolished or reduced toxicity of the mutant holotoxins, respectively. Substitutions of Glu or Asp for Arg-35 interfered with formation of holotoxin, but none of the observed substitutions for Lys-34 or Arg-35 affected binding of CT-B to GM1. The Cys-9, Cys-86 and Trp-88 residues were important for establishing or maintaining the native conformation of CT-B or protecting the CT-B polypeptide from rapid degradation in vivo.
Department of Microbiology, F. Edward Herbert School of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799.
M G
MG
Jobling
R K
RK
Holmes
D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004768:Enterotoxins / Q000737:chemistry* / Q000235:genetics; D005732:Gangliosides / Q000378:metabolism*; D015870:Gene Expression; D006461:Hemolysis / Q000276:immunology; D007118:Immunoassay; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D014176:Protein Biosynthesis; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic
D004768:Enterotoxins; D005732:Gangliosides; C037420:stN protein, Vibrio cholerae