Clinical and pathological significance of autoantibodies to calpastatin in rheumatoid arthritis

我们在类风湿性关节炎（RA）患者中发现了针对钙依赖性中性蛋白酶（calpain）的天然抑制剂钙pastatin的新型自身抗体。由于钙蛋白酶被认为是一种可能参与关节破坏和炎症过程的中性蛋白酶，其抑制剂钙pastatin的自身抗体的产生可能与RA的致病机制有关。在本研究中，我们打算阐明1)calpastatin自身抗体的临床意义，2)患者自身抗体识别的自身抗原表位分析，以及3)抗clpastatin抗体的致病作用。利用大肠杆菌表达的含有编码人calpastatin c -末端178个氨基酸的部分cDNA （RA-6）的融合蛋白，通过免疫印迹法筛选各种风湿性疾病的calpastatin自身抗体。抗calpastatin抗体在57%的RA患者中检测到，明显高于SLE（27%）、硬皮病（38%）和肌炎（24%）。用限制性内切酶酶切的RA-6 cDNA构建表位文库，亚克隆到表达载体上，分析抗原表位。患者血清中的自身抗体识别c端区（C1; aa）。152-178)和n端区(C2；1-76)，而小鼠单克隆抗体（CSF3-3）识别了包含calpain结合位点（B2; aa）的完全不同的区域。（77-130）与Cl表位反应的RA患者的血清比那些没有Cl反应的RA患者的关节炎进展更严重。抗calpastatin抗体也在RA患者血清中抗calpastatin的滑液中检测到。血清中含有抗calpastatin抗体的IgG组分选择性阻断calpastatin的功能，并以剂量依赖的方式恢复calpain的蛋白水解活性。相比之下，正常血清或不含抗calpastatin抗体的患者血清中的IgG不能恢复calpain的活性。这些结果强烈提示，类风湿性关节炎患者钙帕斯汀自身抗体的产生不仅仅是一种附带现象，而可能通过抑制钙帕斯汀的功能和增加滑膜组织钙蛋白酶活性参与致病机制。少

We found novel autoantibodies to calpastatin, natural inhibitor for calcium-dependent neutral proteinase (calpain), in patients with rheumatoid arthritis (RA). Since calpain is thought to be one of neutral proteinases that may be involved in joint destruction and inflammation process, the production of autoantibodies to its inhibitor protein, calpastatin, might be associated with pathogenic mechanisms of RA.In this study, we intended to elucidate 1) the clinical significance of autoantibodies ot calpastatin, 2) analysis of autoantigenic epitopes recognized by patient autoantibodies, and 3) the pathogenic role of anti-clpastatin antibodies.Autoantibodies to calpastatin in various rheumatic diseases were screened by immunoblotting using the fusion protein expressed from E.coli with a partial cDNA (RA-6) encoding the C-terminal 178 amino acids of human calpastatin. Anti-calpastatin antibodies were detected in 57% of patients with RA and were significantly more frequent than in SLE (27%), … More scleroderma (38%) and myositis (24%).Antigenic epitopes were analyzed by using an epitope library constructed by restriction enzyme-digested RA-6 cDNA that was subcloned into an expression vector. Autoantibodies from patient sera recognized a C-terminal region (C1 ; aa. 152-178) and a N-terminal region (C2 ; aa. 1-76), whereas a mouse monoclonal antibody (CSF3-3) recognized an entirely different region containing the calpain-binding site (B2 ; aa. 77-130) RA patients whose sera reacted with the Cl epitope represented a more progressed and severe state of arthritis than those not reacting with Cl.Anti-calpastatin antibodies were also detected in synovial fluids from RA patients who had anti-calpastatin in sera. IgG fractions from sera containing anti-calpastatin antibodies selectively blocked the function of calpastatin and recovered the proteolytic activity of calpain in dose-dependent manner. In contrast, IgG from normal sera or patient sera without anti-calpastatin antibodies did not recover the calpain activity.These results strongly suggest that the production of autoantibodies to calpastatin in patients with RA is not merely epiphenomenon but may be involved in pathogenic mechanisms by inhibiting the function of clpastatin and increasing the calpain activity in synovial tissue. Less