Site-directed mutagenesis of an invariant amino acid residue at the variable-diversity segments junction of an antibody.
Structural analysis of 21 murine A/J antibodies specific for the hapten p-azobenzenearsonate (Ars), and bearing the major cross reactive idiotype (IdCRI), has revealed an invariant amino acid residue, serine, encoded by the variable-diversity gene segments junction of the heavy chain. To test whether this serine residue is essential for Ars binding, we changed it either to alanine or to threonine by oligonucleotide-directed mutagenesis of a heavy chain gene. Genes containing the mutations were separately introduced into mouse hybridoma cells producing the homologous light chain, and the resulting proteins were tested for antigen binding and idiotypic expression. Whereas the serine to threonine mutant retains full antigen binding activity, the serine to alanine mutant does not bind either to Ars-bovine serum albumin-Sepharose or to the Ars-tyrosine hapten. Both mutants show the same reactivity as wild type towards a series of anti-idiotypic antibodies. These results suggest that a hydroxyl group at the variable-diversity gene segments junction of A/J anti-Ars antibodies is essential for antigen binding.
D000595:Amino Acid Sequence; D000818:Animals; D000915:Antibody Affinity; D001391:Azo Compounds / Q000276:immunology*; D001666:Binding Sites, Antibody; D003001:Cloning, Molecular; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007130:Immunoglobulin Idiotypes / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D009154:Mutation; D010132:p-Azobenzenearsonate / Q000276:immunology*
D001391:Azo Compounds; D007143:Immunoglobulin Heavy Chains; D007130:Immunoglobulin Idiotypes; D007135:Immunoglobulin Variable Region; D010132:p-Azobenzenearsonate