Autoantibodies to calpastatin (an endogenous inhibitor for calcium-dependent neutral protease, calpain) in systemic rheumatic diseases.
We identified an autoantibody that reacts with calpastatin [an inhibitor protein of the calcium-dependent neutral protease calpain (EC 3.4.22.17)]. In early immunoblot studies, sera from patients with rheumatoid arthritis (RA) recognized unidentified 60-, 45-, and 75-kDa proteins in HeLa cell extracts. To identify these autoantigens, we used patient sera to clone cDNAs from a lambda gt11 expression library. We isolated clones of four genes that expressed fusion proteins recognized by RA sera. The 1.2-kb cDNA insert (termed RA-6) appeared to encode a polypeptide corresponding to the 60-kDa antigen from HeLa cells, since antibodies bound to the RA-6 fusion protein also reacted with a 60-kDa HeLa protein. The deduced amino acid sequence of the RA-6 cDNA was completely identical with the C-terminal 178 amino acids of human calpastatin except for one amino acid substitution. Patient sera that reacted with the RA-6 also bound pig muscle calpastatin, and a monoclonal antibody to human calpastatin recognized the RA-6 fusion protein, confirming the identity of RA-6 with calpastatin. Moreover, the purified RA-6 fusion protein inhibited the proteolytic activity of calpain, and IgG from a serum containing anti-calpastatin antibodies blocked the calpastatin activity of the RA-6 fusion protein. Immunoblots of the RA-6 product detected autoantibodies to calpastatin in 57% of RA patients; this incidence was significantly higher than that observed in other systemic rheumatic diseases, including systemic lupus erythematosus (27%), polymyositis/dermatomyositis (24%), systemic sclerosis (38%), and overlap syndrome (29%). Thus, anti-calpastatin antibodies are present most frequently in patients with RA and may participate in pathogenic mechanisms of rheumatic diseases.
Mimori
T
T
Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan.
Suganuma
K
K
Tanami
Y
Y
Nojima
T
T
Matsumura
M
M
Fujii
T
T
Yoshizawa
T
T
Suzuki
K
K
Akizuki
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001172:Arthritis, Rheumatoid / Q000276:immunology*; D001323:Autoantibodies / Q000097:blood*; D001483:Base Sequence; D001667:Binding, Competitive; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000276:immunology*; D002154:Calpain / Q000037:antagonists & inhibitors*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D006367:HeLa Cells; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D008969:Molecular Sequence Data; D009132:Muscles / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D012216:Rheumatic Diseases / Q000276:immunology; D013045:Species Specificity; D013552:Swine
D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; C033668:calpastatin; D002154:Calpain