OBJECTIVE

Association of position 97 (P97) residue polymorphisms in human leucocyte antigen (HLA)-B, including HLA-B27, with ankylosing spondylitis (AS) has recently been reported. We studied the effect of P97 variations on cell surface expression of the AS-associated HLA-B27 and HLA-B51, and the AS-protective HLA-B7.

METHODS

Flow cytometry was used to measure surface expression of HLA-B27 in C1R/HeLa cells expressing HLA-B27 (N97) and six mutants at P97 (N97T, N97S, N97V, N97R, N97W and N97D). Transporter associated with antigen processing-deficient T2, tapasin-deficient 220, β2m-deficient HCT15 and endoplasmic reticulum aminopeptidase 1 or β2m-clustered regularly interspaced short palindromic repeats/Cas9-knockout HeLa cells were used to provide evidence for specific protein interactions. Surface expression of HLA-B7/HLA-B51 P97 mutants was also studied.

RESULTS

Mutation of HLA-B27 P97 to the AS risk residue threonine increased cell surface free heavy chain (FHC) expression. Protective residues (serine or valine) and non-AS-associated residues (arginine or tryptophan) did not alter FHC expression. The N97D mutation reduced expression of conventional and FHC forms of HLA-B27. Differences in FHC expression levels between HLA-B27, HLA-B27-N97T and HLA-B27-N97D were dependent on the presence of functional β2m. HLA-B7, which has an AS-protective serine at P97, expressed lower levels of FHC than HLA-B27 or HLA-B51. Introduction of asparagine at P97 of both HLA-B7 and HLA-B51 increased FHC expression.

CONCLUSIONS

The nature of P97 residue affects surface expression of HLA-B27, B7 and B51, with AS-associated residues giving rise to higher FHC expression levels. The association of P97 amino acid polymorphisms with AS could be, at least in part, explained by its effect on HLA-B27 FHC cell surface expression.