In vitro mutagenesis of HLA-B27. Substitution of an unpaired cysteine residue in the alpha 1 domain causes loss of antibody-defined epitopes.

Abstract

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 a, 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 a, 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.

The Solvent-Inaccessible Cys67 Residue of HLA-B27 Contributes to T Cell Recognition of HLA-B27/Peptide Complexes

“…After preincubation at 37°C the binding of the HLA-B27.Ser 67 aggrecan peptide tetramer was significantly reduced after 1 h suggesting that the staining of ILT-2-transfected cells strongly depends on the stability of the HLA-B27 complex. These findings are in line with the hypothesis that the stability of the molecule might have been altered by the mutation at position 67 of the HLA-B27 H chain (25), which might also be the reason for an abrogation of mAb recognition of HLA-B27 molecules after site-directed mutagenesis of residue 67 Cys to tyrosine (45,46).…”

“…6). Its hydrophobic part forms van der Waals contacts to Tyr 7 and Ile 66 , while the charged guanidyl moiety is stabilized by a bifurcated salt bridge to Glu 45 and a hydrogen bond to Thr 24 . The side chain of Cys 67 (Fig.…”

“…A cysteine is present at position 67 in all subtypes of HLA-B27 (B*2701-2725), with the exception of the rare subtypes B*2718 (67-Ser) and B*2723 (67-Phe) (19,20). The crystal structure of HLA-B27 suggests that the Cys 67 contributes to the specificity of the critical B pocket of the binding site together with the highly conserved Glu 45 and Thr 24 residues. Additionally, Cys 67 allows the formation of homodimeric and oligomeric HLA-B27 H chain complexes (21)(22)(23).…”

How does HLA-B27 confer susceptibility to inflammatory arthritis?

“…(Accepted for publication 25 July 1990) The association of ankylosing spondylitis with HLA-B27 was first described 17 years ago (Brewerton et al, 1973a;Schlosstein et al, 1973); the realization that other seronegative spondyloarthropathies, notably reactive arthritis, had the same association followed rapidly (Brewerton et al, 1973b), There has been an enormous increase in our understanding ofthe structure and function of HLA molecules in the intervening years: the amino acid sequence of several B27 variants is known (Choo et al, 1986) and there is a crystallographic picture of its likely threedimensional structure (Bjorkman et al, 1987); the B27 gene has been cloned (Szots et al, 1986), subjected to mutational analysis (Taurog & El-Zaatari, 1988) and expressed in transgenic mice Nickerson, Luthra & David, 1990), Furthermore, the function of class I MHC antigens in presenting antigenic peptides to T cells has been elegantly demonstrated (Townsend et al, 1986), It is rather frustrating, therefore, that despite this wealth of information, the involvement of B27 in the pathogenesis of these arthropathies remains shrouded in mystery.…”

Autoantibodies to the HLA-B27 sequence cross-react with the hypothetical peptide from the arthritis-associated Shigella plasmid.

“…Crystallographic studies have suggested that the amino acid sequence of the HLA-B27 antigen studied in this report corresponds to a part of the presumed antigen-peptide binding cleft (34, 35). Also, the possible role of the unpaired cysteine residue at position 67 in interacting with ligands has been suggested (20). All ofthese possibilities are not mutually exclusive.…”