Disparate folding and stability of the ankylosing spondylitis–associated HLA–B1403 and B2705 proteins

• Published in: Arthritis and rheumatism 2008-12, Vol.58 (12), p.3693-3704
• Main Authors: Merino, Elena, Galocha, Begoña, Vázquez, Miriam N., López De Castro, José A.
• Format: Article
• Language: English
• Subjects: Alleles; Antigens, Surface - chemistry; Antigens, Surface - metabolism; Biological and medical sciences; Cell Line; Dimerization; Diseases of the osteoarticular system; Diseases of the spine; Endoplasmic Reticulum - metabolism; HLA-B Antigens - chemistry; HLA-B Antigens - genetics; HLA-B Antigens - metabolism; HLA-B14 Antigen; HLA-B27 Antigen - chemistry; HLA-B27 Antigen - genetics; HLA-B27 Antigen - metabolism; Hot Temperature; Humans; Immunoprecipitation; Inflammatory joint diseases; Lymphocytes - cytology; Medical sciences; Membrane Transport Proteins - metabolism; Protein Folding; Protein Transport - immunology; Spondylitis, Ankylosing - immunology; Spondylitis, Ankylosing - metabolism; Transfection
• ISSN: 0004-3591; 1529-0131
• DOI: 10.1002/art.24045

Objective To investigate the folding, assembly, maturation, and stability of HLA–B*1402 and B*1403, which differ by 1 amino acid change and are differentially associated with ankylosing spondylitis (AS), and to compare these features with those of B*2705. Methods Stable transfectants expressing B*1402, B*1403, and B*2705 were used. Folding rates were estimated from the ratio of unfolded heavy chains to folded heavy chains that had been immunoprecipitated with specific antibodies in pulse–chase experiments. Heavy chain misfolding was measured as the half‐life of endoglycosidase H (Endo H)–sensitive β2‐microglobulin–free heavy chains. Maturation/export rates were measured by acquisition of Endo H resistance. Association with calnexin or tapasin was analyzed by coprecipitation with chaperone‐specific antibodies, and surface expression was estimated by flow cytometry. Thermostability of HLA–peptide complexes was assessed by immunoprecipitation after incubation at various temperatures. Heavy chain expression was quantified by Western blotting. Results The folding rates of B*1402 and B*1403 were similar, and both were faster and more efficient than B*2705, but some unfolded heavy chains from both B14 subtypes remained in the endoplasmic reticulum (ER) with a long half‐life. The export rates of B*1402 and B*1403 were slow, and the heterodimers partially dissociated after exiting the ER, as revealed by significant amounts of Endo H–resistant and surface‐expressed free heavy chains. Both interaction with tapasin and thermostability were higher for B*2705 than for B*1402 and higher for B*1402 than for B*1403, suggesting that the repertoires of the B*1402‐bound peptide and especially the B*1403‐bound peptide were less optimized than that of B*2705. Conclusion Our results indicate that the folding, maturation, and stability of B*1403 differ more from B*2705 than from B*1402. Thus, these features cannot account for the fact that only the 2 former allotypes are associated with AS.