In vitro mutagenesis of HLA-B27. Substitution of an unpaired cysteine residue in the alpha 1 domain causes loss of antibody-defined epitopes.
The HLA class I molecules identified serologically as HLA-B27 are highly associated with ankylosing spondylitis and related human disorders. All known HLA-B27 amino acid sequences contain a cysteine residue at position 67; no other published HLA class I sequence contains a cysteine within the hypervariable region of the alpha 1 domain, which extends from amino acid residues 63-84. To investigate the role of this cysteine residue in the antigenic structure of HLA-B27, we isolated a genomic clone encoding a molecule of the HLA-B27.1 subtype and performed oligonucleotide-directed mutagenesis to convert the cysteine at position 67 to a tyrosine. When transfected into mouse L cells, both the wild-type and Cys67----Tyr67 mutant B27 genes directed the synthesis and surface expression of molecules reactive with the monomorphic anti-HLA class I antibody W6/32. However, only the L cells transfected with the wild-type B27 gene reacted with the anti-B27 antibody ME1; L cells transfected with the mutant B27 were completely unreactive with this antibody. Experiments with hybrid exons created from the HLA-B27 and HLA-A2 genes yielded results consistent with the mapping of the ME1 epitope to the B27 alpha 1 domain. A second anti-B27 antibody, GS145.2, also showed markedly reduced binding to the Cys67----Tyr67 mutant. These studies document the importance of the unique Cys67 residue in the antigenic structure of HLA-B27.
Taurog
J D
JD
Harold C. Simmons Arthritis Research Center, University of Texas, Dallas 75235.
el-Zaatari
F A
FA
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000937:Antigen-Antibody Reactions; D001483:Base Sequence; D003545:Cysteine / Q000235:genetics*; D000939:Epitopes / Q000235:genetics / Q000276:immunology*; D005434:Flow Cytometry; D006680:HLA Antigens / Q000235:genetics*; D015796:HLA-B27 Antigen; D006801:Humans; D007739:L Cells; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D010449:Peptide Mapping; D014162:Transfection
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006680:HLA Antigens; D015796:HLA-B27 Antigen; D003545:Cysteine