Mapping epitopic regions of cholera toxin B-subunit protein.
Continuous overlapping synthetic hexapeptides representing the entire 103 amino acid sequence of the immunodominant B-subunit protein of cholera enterotoxin were used to examine reactivities of a variety of antisera in attempts to detect and define sequence-related (continuous) antigenic regions. The validity of the methods was established by the reactions of polyclonal antisera raised against longer synthetic peptides with appropriate synthetic hexapeptides. An unexpected cross-reaction is attributed to the presence of three identical amino acids (Gln16-Ile17-His18)--although in different order (Gln56-His57-Ile58)--in two parts of the B-subunit chain. Adsorption studies using polyclonal rabbit antisera revealed that, in many instances, denatured B-subunit protein more effectively removed reactivity with hexapeptides than did the native protein. Native holotoxin was more effective than native B-subunit. Sera from human cholera convalescents gave diffuse patterns of reactivity with synthetic hexapeptides--primarily against regions of reactive hexapeptides rather than with clearly defined continuous epitopes. Among many epitopic regions encountered, a strongly reactive tetramer, Ser-Gln-His-Ile (SQHI), was discovered in a highly conserved region, residues 55-58, of the B-subunit amino acid sequence. Adsorption studies revealed that this epitope is apparently exposed on the surface of the native protein. Amino acid substitution revealed the essentiality of Gln and His residues to this epitope. Gly54 was not part of the epitope but substitution of acidic residues Glu and Asp for Gly eliminated reactivity with antibody. The results suggest that continuous epitopes may contribute to the antigenicity of the native toxin protein and may be potentially useful for development of a peptide vaccine.
Kazemi
M
M
Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri-Columbia 65212.
Finkelstein
R A
RA
D000595:Amino Acid Sequence; D000818:Animals; D002772:Cholera Toxin / Q000235:genetics* / Q000276:immunology; D000939:Epitopes / Q000235:genetics* / Q000276:immunology; D007106:Immune Sera / Q000276:immunology; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics* / Q000276:immunology; D051381:Rats
D000939:Epitopes; D007106:Immune Sera; D010446:Peptide Fragments; D002772:Cholera Toxin