Slot-machine mutagenesis of a polymorphic residue on the A kappa alpha-chain.
This study explores the limitations on variability at a polymorphic position of an MHC class II molecule. Using a convenient and rapid method termed "slot-machine mutagenesis," we have converted Glu75 on the A kappa-chain to 15 alternative amino acids. This residue is of interest because it is an immunodominant site on the A kappa alpha chain and because it participates in certain T cell epitopes. The wild-type and mutant A kappa alpha cDNA were transfected into L cells (together with the A kappa beta cDNA and a selection marker), and transfectants displaying high surface levels of the A kappa complex were selected and expanded. We sought to examine three questions: what is the effect of these mutations on the expression and overall conformation of the A alpha: A beta complex? How do these diverse mutations influence mAb epitopes for which Glu75 makes a direct contribution to specificity? Do such substitutions affect T cell recognition of the A kappa alpha:A kappa beta complex? The answers to these three questions are quite different. Position 75 of the A alpha chain can accommodate essentially all chemically divergent amino acids without major consequences for expression and overall A alpha:A beta structure. In contrast, mAb that recognize Glu75-dependent epitopes are extremely particular about the amino acid residing at this position. T cells are less fastidious: those that are affected by the mutations still recognize a number of substitutions. These data emphasize the tolerance of MHC molecules to evolutionary tampering.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D005786:Gene Expression Regulation; D005802:Genes, MHC Class II; D005971:Glutamates / Q000235:genetics / Q000276:immunology; D018698:Glutamic Acid; D000949:Histocompatibility Antigens Class II / Q000235:genetics* / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011110:Polymorphism, Genetic; D011487:Protein Conformation; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D005971:Glutamates; D000949:Histocompatibility Antigens Class II; D018698:Glutamic Acid