In vitro mutagenesis of HLA-B27. Amino acid substitutions at position 67 disrupt anti-B27 monoclonal antibody binding in direct relation to the size of the substituted side chain.
The class I MHC molecule HLA-B27 bears an unpaired Cys residue at position 67, which is predicted to face the Ag binding pocket, based on the x-ray crystallographic model of HLA-A2. To investigate the potential of this residue in the antigenic structure of HLA-B27, a panel of 11 mutant HLA-B27 genes has been created, each bearing a separate amino acid substitution at position 67. The genes were transfected into mouse L cells and the resulting cells analyzed by cytofluorography with a panel of antibodies reactive with the wild-type B27 molecule. Although previous studies had indicated that all mAb that bound the B27 molecule on human lymphocytes bound comparably to L cells transfected with the wild-type B27 gene in the absence of h beta 2-m (human beta 2-microglobulin), the first of the mutant B27 genes was found to express several mAb epitopes in the presence but not in the absence of a h beta 2-m gene. Therefore, subsequent analysis of the B27 mutant panel was conducted in L cells coexpressing the h beta 2-m gene. Under these circumstances, all of the mutants bound the monomorphic anti-class I HLA mAb W6/32 and B.9.12.1, as well as the broadly polymorphic mAb B.1.23.2. Binding to the mutant transfectants of three anti-B27 mAb that cross-react with HLA-B7, ME1, GS145.2, and GSP5.3, was directly proportional to the size of the substituted amino acid side chain. The binding of another anti-B27 mAb, B27M2, that recognizes a B27 determinant that includes the region of amino acids 77-81, was not affected by the Cys67- greater than Tyr67 substitution. Rabbit antibodies to a synthetic peptide composed of B27 amino acids 61-84 bound to both the wild-type B27 and to the Tyr67 mutant. This binding, but not the binding of ME1 or B27M2, was inhibited by the synthetic peptide. These data are interpreted as suggesting that the large amino acid substitutions at position 67 induce a limited conformational change that disrupts the epitopes of the three anti-B27, B7 mAb, that are themselves at least partially conformational. The potential implications of these findings for the role of HLA-B27 in disease pathogenesis are discussed.
Harold C. Simmons Arthritis Research Center, University of Texas Southwestern Medical Center, Dallas 75235.
F A
FA
el-Zaatari
K C
KC
Sams
J D
JD
Taurog
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D000954:Antigens, Surface / Q000378:metabolism; D001483:Base Sequence; D001667:Binding, Competitive; D000939:Epitopes; D015796:HLA-B27 Antigen / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D013329:Structure-Activity Relationship; D001613:beta 2-Microglobulin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D000939:Epitopes; D015796:HLA-B27 Antigen; D010455:Peptides; D001613:beta 2-Microglobulin