|Budget Amount *help
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1996: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1995: ¥1,100,000 (Direct Cost: ¥1,100,000)
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%),
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