Merino Elena, Galocha Begoña, Vázquez Miriam N, López de Castro José A
Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid), Universidad Autónoma, Madrid, Spain.
Arthritis Rheum. 2008 Dec;58(12):3693-704. doi: 10.1002/art.24045.
To investigate the folding, assembly, maturation, and stability of HLA-B1402 and B1403, 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.
Stable transfectants expressing B1402, B1403, 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 beta2-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.
The folding rates of B1402 and B1403 were similar, and both were faster and more efficient than B2705, but some unfolded heavy chains from both B14 subtypes remained in the endoplasmic reticulum (ER) with a long half-life. The export rates of B1402 and B1403 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 B2705 than for B1402 and higher for B1402 than for B1403, suggesting that the repertoires of the B1402-bound peptide and especially the B1403-bound peptide were less optimized than that of B2705.
Our results indicate that the folding, maturation, and stability of B1403 differ more from B2705 than from B*1402. Thus, these features cannot account for the fact that only the 2 former allotypes are associated with AS.
研究HLA - B1402和B1403的折叠、组装、成熟及稳定性，这两种亚型仅有1个氨基酸的差异且与强直性脊柱炎（AS）的关联有所不同，并将这些特征与B*2705的特征进行比较。
使用稳定转染表达B1402、B1403和B*2705的细胞。在脉冲追踪实验中，根据未折叠重链与经特异性抗体免疫沉淀的折叠重链的比例估算折叠速率。重链错误折叠以对内切糖苷酶H（Endo H）敏感的无β2微球蛋白重链的半衰期来衡量。成熟/输出速率通过获得Endo H抗性来测定。通过与伴侣特异性抗体共沉淀分析与钙连蛋白或塔帕辛的结合情况，并通过流式细胞术估算表面表达。在不同温度孵育后，通过免疫沉淀评估HLA - 肽复合物的热稳定性。通过蛋白质印迹法定量重链表达。
B1402和B1403的折叠速率相似，且两者均比B2705更快且更高效，但两种B14亚型的一些未折叠重链在内质网（ER）中保留了较长的半衰期。B1402和B1403的输出速率较慢，并且如大量Endo H抗性和表面表达的游离重链所示，异二聚体在离开内质网后部分解离。B2705与塔帕辛的相互作用和热稳定性均高于B1402，而B1402高于B1403，这表明B1402结合的肽尤其是B1403结合的肽的库比B2705的库优化程度更低。
我们的结果表明，B1403与B2705相比，在折叠、成熟和稳定性方面的差异比与B*1402的差异更大。因此，这些特征无法解释只有前两种同种异型与AS相关这一事实。
