title: Position 97 of HLA-B, a residue implicated in pathogenesis of ankylosing spondylitis, plays a key role in cell surface free heavy chain expression.
abstract: OBJECTIVE
  Association of position 97 (P97) residue polymorphisms in human leucocyte antigen (HLA)-B, including HLA-B*27, with ankylosing spondylitis (AS) has recently been reported. We studied the effect of P97 variations on cell surface expression of the AS-associated HLA-B*27 and HLA-B*51, and the AS-protective HLA-B*7.
  
  
  METHODS
  Flow cytometry was used to measure surface expression of HLA-B*27 in C1R/HeLa cells expressing HLA-B*27 (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-B*7/HLA-B*51 P97 mutants was also studied.
  
  
  RESULTS
  Mutation of HLA-B*27 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-B*27. Differences in FHC expression levels between HLA-B*27, HLA-B*27-N97T and HLA-B*27-N97D were dependent on the presence of functional β2m. HLA-B*7, which has an AS-protective serine at P97, expressed lower levels of FHC than HLA-B*27 or HLA-B*51. Introduction of asparagine at P97 of both HLA-B*7 and HLA-B*51 increased FHC expression.
  
  
  CONCLUSIONS
  The nature of P97 residue affects surface expression of HLA-B*27, B*7 and B*51, 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-B*27 FHC cell surface expression.
authors: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK.
authors: Liye
authors: L
authors: Chen
authors: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK.
authors: Hui
authors: H
authors: Shi
authors: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK.
authors: Jack
authors: J
authors: Yuan
authors: Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, Oxford, UK.
authors: Paul
authors: P
authors: Bowness
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000172:deficiency / Q000235:genetics; D000626:Aminopeptidases / Q000172:deficiency / Q000235:genetics; D000954:Antigens, Surface / Q000235:genetics* / Q000378:metabolism; D001216:Asparagine / Q000235:genetics; D005434:Flow Cytometry; D015796:HLA-B27 Antigen / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D059949:HLA-B51 Antigen / Q000235:genetics / Q000378:metabolism; D015793:HLA-B7 Antigen / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D026901:Membrane Transport Proteins / Q000172:deficiency / Q000235:genetics; D015778:Minor Histocompatibility Antigens / Q000235:genetics; D009154:Mutation; D012694:Serine / Q000235:genetics; D013167:Spondylitis, Ankylosing / Q000235:genetics* / Q000276:immunology; D013912:Threonine / Q000235:genetics; D014162:Transfection; D014633:Valine / Q000235:genetics; D001613:beta 2-Microglobulin / Q000235:genetics / Q000378:metabolism*
chemical_list: D018528:ATP-Binding Cassette Transporters; D000954:Antigens, Surface; C464936:HLA-B*27:05 antigen; D015796:HLA-B27 Antigen; D059949:HLA-B51 Antigen; D015793:HLA-B7 Antigen; D026901:Membrane Transport Proteins; D015778:Minor Histocompatibility Antigens; D001613:beta 2-Microglobulin; C107762:tapasin; C477327:transporter associated with antigen processing (TAP); D013912:Threonine; D012694:Serine; D001216:Asparagine; D000626:Aminopeptidases; C464841:ERAP1 protein, human; D014633:Valine
keywords: Ankylosing Spondylitis; Autoimmunity; Gene Polymorphism