Identification of amino acids in HLA-DPw4b beta and -DR5 beta 1 chains that are involved in antibody binding epitopes using site-directed mutagenesis and DNA-mediated gene transfer.
Based on comparisons of the amino acid sequences of the beta chains of HLA class II molecules that do or do not bind the I-LR1 monoclonal antibody, we predicted that glutamic acid 56 of I-LR1-positive DPw2, DPw3, and DPw4b beta chains and the analogous glutamic acid 58 of I-LR1-positive DR5 beta 1 chains are involved in the I-LR1 epitope. Site-directed mutagenesis of DPw4b beta and DR5 beta 1 cDNAs was used to change the codons for glutamic acid 56 in DPw4b beta and glutamic acid 58 in DR5 beta 1 to the codon for alanine found in I-LR1-negative beta chains. Transfectants expressing wild-type DPw4b beta chains or DR5 beta 1 chains bind the I-LR1 monoclonal antibody, whereas transfectants expressing the mutant DPw4b beta or DR5 beta 1 chains do not bind I-LR1. Therefore, DPw4b beta glutamic acid 56 and DR5 beta 1 glutamic acid 58 are involved in the epitope recognized by the I-LR1 monoclonal antibody. Interestingly, the DR5 beta 1 glutamic acid----alanine 58 substitution also causes the loss of binding of two DR5-specific monoclonal antibodies to DR5 beta 1 molecules. Because the sequences of amino acids 36 to 64 of the DPw4b beta chain and 38 to 66 of the DR5 beta 1 chain are identical, these data raise some interesting issues about the formation of antibody epitopes on class II molecules.
Veterans Administration Medical Center, Iowa City, IA 52246.
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D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D004247:DNA / Q000235:genetics; D000939:Epitopes; D006682:HLA-DP Antigens / Q000235:genetics; D059868:HLA-DP beta-Chains; D015805:HLA-DR5 Antigen / Q000235:genetics; D006801:Humans; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009154:Mutation; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006682:HLA-DP Antigens; D059868:HLA-DP beta-Chains; C056284:HLA-DPw4 antigen; D015805:HLA-DR5 Antigen; D004247:DNA