Proc. Natl. Acad. Sci. USA Vol. 92, pp. 7267-7271, August 1995 Medical Sciences

Autoantibodies to calpastatin (an endogenous inhibitor for calcium-dependent neutral , ) in systemic rheumatic diseases (rheumatoid arthritis/autoimmune disease/autoantigen/cDNA/molecular cloning) TSUNEYO MIMORI*t, KAZUHIRO SUGANUMA*, YUTAKA TANAMI*, TAKAKI NOJIMA*, MAMi MATSUMURA*, TAKAo FuJII*, ToSHIO YOSHIZAWA*, KOICHI SUZUKIt, AND MASASHI AKIZUKI* *Division of Rheumatology, Department of Internal Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan; and *Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoicho, Bunkyo-ku, Tokyo 113, Japan Communicated by Osamu Hayaishi, Osaka Bioscience Institute, Osaka, Japan, March 27, 1995

ABSTRACT We identified an autoantibody that reacts with tin, may play a significant role in the pathogenesis of rheumatic calpastatin [an inhibitor of the calcium-dependent neu- disorders. tral 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 in HeLa METHODS cell extracts. To identify these autoantigens, we used patient sera Sera Sera from 60 Japanese patients who fulfilled the to clone cDNAs from a Agtll expression library. We isolated revised criteria for RA of the American Rheumatism Associ- clones offour that expressed fusion proteins recognized by ation (15) were used to detect autoantibodies specific for RA. RA sera. The 1.2-kb cDNA insert (termed RA-6) appeared to Of these, 47 patients (78%) had rheumatoid factor. Six sera encode a polypeptide corresponding to the 60-kDa antigen from contained antibodies reactive with the 60-, 45-, or 75-kDa HeLa cells, since antibodies bound to the RA-6 fusion protein proteins that were detected in preliminary immunoblot studies also reacted with a 60-kDa HeLa protein. The deduced amino using HeLa cell extracts (1), and these sera were used as probes acid sequence of the RA-6 cDNA was completely identical with in screening and characterizing cDNA clones that expressed the C-terminal 178 amino acids of human calpastatin except for the autoantigens recognized by RA sera. Sera from 37 patients one amino acid substitution. Patient sera that reacted with the with systemic lupus erythematosus (SLE), 21 with scleroderma RA-6 also bound pig muscle calpastatin, and a monoclonal (systemic sclerosis), 21 with polymyositis/dermatomyositis, antibody to human calpastatin recognized the RA-6 fusion and 14 with overlap syndrome were used as controls. protein, confirming the identity ofRA-6 with calpastatin. More- Screening of cDNA Libraries. A Agtll cDNA library con- over, the purified RA-6 fusion protein inhibited the proteolytic structed from HeLa cell mRNA (Clontech) was screened by activity of calpain, and IgG from a serum containing anti- using a mixture of six RA patient sera (preabsorbed with calpastatin antibodies blocked the calpastatin activity of the Escherichia coli lysates and diluted at 1:100 each) as described RA-6 fusion protein. Immunoblots ofthe RA-6 product detected by Young and Davis (16). Positive clones were purified by autoantibodies to calpastatin in 57% of RA patients; this inci- repeated screening until all progeny plaques were positive. dence was significantly higher than that observed in other Recombinant phages were used to infect E. coli Y1089 to systemic rheumatic diseases, including systemic lupus erythem- develop lysogens. DNA from positive clones was digested by atosus (27%), polymyositis/dermatomyositis (24%), systemic restriction EcoRI, fractionated on 1% agarose gel sclerosis (38%), and overlap syndrome (29%6). Thus, anti- containing ethidium bromide (0.5 ptg/ml), and visualized by calpastatin antibodies are present most frequently in patients ultraviolet light. Insert cDNAs were purified from agarose gel with RA and may participate in pathogenic mechanisms of slices by using a Geneclean kit (Bio 101), labeled with [a-32p]- rheumatic diseases. dCTP, and used as probes for DNA blot analysis (17). Immunoblotting. To characterize cDNA clones and to de- Rheumatoid arthritis (RA) is a systemic disease characterized tect autoantibodies, we performed immunoblot analysis using by chronic polyarthritis and joint destruction. Although RA is bacterial lysates prepared from lysogenic E. coli Y1089 carry- categorized among the systemic autoimmune diseases because ing the recombinant phages in which the fusion protein was of the characteristic presence of rheumatoid factor (an auto- induced by adding 5 mM isopropyl ,-D-thiogalactopyranoside. antibody to the Fc portion of IgG), other autoantibodies Briefly, bacteria expressing fusion proteins were dissolved in specific for RA rarely have been described. In an earlier study, SDS-sample buffer [62.5 mM Tris1HCl/1% SDS/5% (vol/vol) we found autoantibodies that reacted with unidentified HeLa 2-mercaptoethanol/10% (vol/vol) glycerol/0.05% bromophe- proteins in patients with RA (1). In the present study, we nol blue, pH 6.8] and boiled for 5 min. Protein components of molecular of their and dem- lysates were fractionated on an SDS/7.5% polyacrylamide slab performed cloning target antigens§ gel and transferred electrophoretically to a nitrocellulose filter onstrated that one of these autoantigens was calpastatin, an (BA-85; Schleicher & Schull) according to Towbin et al. (18). endogenous protein inhibitor of calpain (EC 3.4.22.17), a calci- After blocking with 2% skim milk solids (Morinaga Nyugyo, um-dependent (2-5). Calpain has been identi- Tokyo) dissolved in Tris-buffered saline (TBS; 10 mM fied in synovial fluid from arthritis patients (6-8) and has been Tris-HCl/150 mM NaCl, pH 7.5), the filter was cut and the implicated as a proteoglycanase in cartilage destruction associ- individual filter was incubated serially with a patient serum ated with joint diseases (8, 9). Other functions include mediation with E. and diluted 1:100 in and of the activation of various inflammatory processes (10-14). (preabsorbed coli lysates TBS) Thus, the production of autoantibodies to its inhibitor, calpasta- Abbreviations: RA, rheumatoid arthritis; SLE, systemic lupus ery- thematosus. The publication costs of this article were defrayed in part by page charge tTo whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" in §The sequence reported in this paper has been deposited in the accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. D50827). 7267 Downloaded by guest on September 29, 2021 7268 Medical Sciences: Mimori et al. Proc. Natl. Acad. Sci. USA 92 (1995)

with alkaline phosphatase-conjugated anti-human IgG (dilut- #4 #8 #18 #21 #42 #55 NHS ed 1:7500 in TBS) (Promega). The filter then was developed by incubating with nitroblue tetrazolium and 5-bromo-4-chloro- M 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 3-indolyl phosphate according to the manufacturer's protocol (Promega). In some experiments, pig calpastatin, purified from pig muscle as described previously (19), was used as a 67-L- substrate (1 ,ug per lane) instead of bacterial lysates. 30- i ..-W Affi'nity Purification ofAntibodies and Immunoblotting. To characterize the isolated cDNA clones, elution blotting was performed. Recombinant phage (104 plaque-forming units) -'tZi .iai$, ''' ...... 9. - ' .: was incubated with E. coli Y1090 for 15 min at 37°C and plated FIG. 1. Antigenicity of fusion proteins characterized by immuno- on an agar plate (90 mm in diameter) with a melted top agar. blotting. Amido black staining of E. coli lysates with recombinant After 3 h of incubation at 42°C, a nitrocellulose filter (BA-85; Agtll phages shows expressed fusion proteins (lanes 1-3, left-most gel; 82 mm in diameter) dipped into 10 mM isopropyl 3-D- lane M, molecular mass markers). On immunoblots with RA patient thiogalactopyranoside was overlaid on the agar for 3 h at 37°C. sera (gels #4, #8, #18, #21, #42, and #55) and a normal human After blocking with 2% skim milk solids, the filter was serum (NHS), only one serum each recognized fusion proteins ex- incubated with 10 ml of a diluted mixture of RA sera. After pressed by the clone RA-2 (#8, lane 1) and RA-3 (#4, lane 2), while three washes with TBS containing 0.05% Nonidet P-40, filter- four independent sera (#4, #8, #18, and #21) reacted with the RA-6 bound antibodies were eluted with 5 ml of 0.2 M glycine HCl fusion protein (lanes 3). at pH 2.8, neutralized immediately by adding Tris base, and The reactivities of fusion proteins expressed from lysogenic used for immunoblotting against HeLa cell extracts that were E. coli bearing recombinant phages were examined by immu- fractionated on a SDS/10% polyacrylamide gel, as described noblotting with patient sera. In Fig. 1, left-most gel, fusion above. proteins (lanes 1-3) were visible in amido black staining of DNA Sequencing. cDNA inserts isolated from Agtll recom- total proteins. Four RA patient sera (gels #4, #8, #18, and binant phages (EcoRI fragments) were digested with each of #21) recognize the 130-kDa fusion protein in the RA-6 lysate several restriction (Alu I, Pvu II, Pst I, and Xba I; (lanes 3). Since these sera all showed antibodies to a 60-kDa Takara Shuzo, Kyoto), and the DNA fragments were ligated protein in HeLa cell protein immunoblots, this clone appears into the polylinker of M13mpl8 or M13mpl9 replicative form to encode the 60-kDa antigen. On the other hand, fusion DNA. Nucleotide sequences were determined by using from the RA-2 and RA-3 Sanger's dideoxynucleotide chain-termination method em- proteins (lanes 1) (lanes 2) lysogenes ploying modified T7 DNA polymerase (Sequenase; United were recognized by only one patient serum each, #8 and #4, States Biochemical) (20). Sequences were aligned by using a respectively. Two patient sera (gels #42 and #55) and a normal computer program for DNA analysis (MicroGenie; Beckman). human serum (gel NHS) did not react with any of the fusion Assay of Calpain and Calpastatin. m-Calpain was purified proteins. The RA-13 clone did not express a fusion protein from rabbit liver as described previously (21). RA-6 fusion recognized by any patient sera (data not shown). protein was prepared from bacterial lysates as described by To confirm the identity of the clones as the true autoanti- Rothfield et al. (22) and then further purified by using the gens, affinity-purified antibodies were eluted from membrane- electro-eluter (model 422, Bio-Rad) after fractionation by bound fusion proteins and used for immunoblots against the SDS/polyacrylamide gel electrophoresis. Proteolytic activity HeLa cell extracts (Fig. 2). The unpurified mixed RA serum of calpain was assayed with casein (Merck) as substrate (23). reacted with a 60-kDa HeLa cell protein (lane 1). The same Reaction mixture containing 1 jig of m-calpain in 250 ,ul of a protein was recognized by antibodies eluted after binding the reaction buffer (3 mg of casein per mi/5 mM CaCl2/10 mM RA-6 fusion protein (lane 2), but not by antibodies eluted from 2-mercaptoethanol/100 mM Tris HCl, pH 7.5) was incubated an indifferent clone (wild-type Agtll) (lane 3). Antibodies for 20 min at 30°C. Then 250 ,ul of ice-cold 10% (wt/vol) trichloroacetic acid was added to stop the reaction. After 2 4M centrifugation, absorbance at 280 nm (A2N) of acid-soluble :i supernatant was measured. For the assay of calpastatin, the kDa purified RA-6 fusion protein was added into the reaction :P. mixture, and inhibition of calpain activity on casein was I -94 determined as described above. In an experiment to determine the effect of anti-calpastatin antibodies on inhibitory activity | 1. -67 of calpastatin, 0.5 ,ug of RA-6 fusion protein was incubated with various amounts of IgG purified from a patient's serum or a normal control serum for 1 h at room temperature before 43 the reaction mixture was added. RESULTS -30 Isolation of cDNA by Using RA Patient Sera. To begin to characterize autoantigens recognized by RA sera in an earlier immunoblot study, we isolated cDNA clones encoding these autoantigens. Using mixed RA patient sera as a probe, we screened 1 x 106 clones of Agtll human cDNA library and FIG. 2. Antigenic specificities of the RA-6 clone as determined by isolated 22 positive clones, designated RA-1 to RA-22. When elution blotting. Antibodies purified from the membrane-bound fusion these cDNAs were fell into four protein that had reacted with mixed RA sera were used as primary cross-hybridized, they groups, antibodies on immunoblots of whole HeLa cell extracts. Both the apparently encoding different proteins. Restriction enzyme original mixed sera (lane 1) and antibodies eluted from the RA-6 EcoRI digestion of representative recombinant phage DNA, fusion protein (lane 2) bound specifically to a 60-kDa HeLa protein termed RA-2, RA-3, RA-6, and RA413, demonstrated 1.5-, (arrowhead), while antibodies eluted from a wild-type Agtll (,B- 1.9-, 1.2-, and 2.0-kb cDNA inserts, respectively (data not galactosidase) did not react with that protein (lane 3). Lane 4 is amido shown). black stain of whole HeLa cell extracts. Downloaded by guest on September 29, 2021 Medical Sciences: Mimori et al. ~~Proc. Nati. Acad. Sci. USA 92 (1995) 7269

eluted from the RA-2 and RA-3 fusion proteins did not react RA Sera with any significant protein component in HeLa cells (data not HRS OT NH KH MoAb NHS shown). 1 2 1 2 12 12 12 12 12 12 A Polypeptide Encoded by the RA-6 cDNA Is Identical to kDa Human Calpastatin The nucleotide sequence of the 1.2-kb cDNA encoding the RA-6 clone was determined. The primary nucleotide sequence and its deduced amino acid sequence (Fig. 3) suggested that the RA-6 cDNA was partial, since a potential ribosomal was not present in the se- quence. Its longest reading frame encodes 178 amino acids, and the predicted molecular weight for the encoded polypep- tide is 19,229. A search of the protein data bank (Swiss-Prot Version 26) with the amino acid of the RA-6 cDNA predicted sequence 30 revealed a high homology with domain IV of human, pig, rabbit, and rat calpastatins (2-5). Its only difference from the published human sequence was a Glu residue at position 61 of Amido Immunoblotting Black the RA-6 instead of a Gly residue at position 557 of human calpastatin (Fig. 3) (4). Therefore, we concluded that the RA-6 FIG. 4. Reactivity of patient sera with human and pig calpastatin. cDNA encoded a of human pailtial sequence calpastatin. Bacterial lysates expressing the RA-6 fusion protein (lanes 1) and pig of Patient Sera and Monoclonal Antibodies with React'ivi'ty calpastatin (lanes 2) were fractionated by SDS/7.5% PAGE, trans- the Fusion Protein and Purified Pig Calpastatin. When ferred to nitrocellulose membrane, and allowed to react with patient purified pig muscle calpastatin was used for immunoblot sera or a monoclonal anti-calpastatin antibody (CSF3-3, domain II- is amido detection, all five sera that reacted with the RA-6 fusion and IV-specific; Takara Shuzo, Kyoto). The left-most gel protein (130 kDa; Fig. 4, lane 1) also recognized pig calpastatin black staining of bacterial lysate and pig calpastatin. All patient sera (HS, DT, NH, KH, and SY) recognize pig calpastatin as well as the (68 kDa; lane 2), indicating that these sera contain antibodies fusion protein. The monoclonal antibody-(MoAb) recognizes only the to calpastatin. However, except in serum SY, the reactivity to RA-6 fusion protein. Normal human serum (NHS) recognizes neither pig calpastatin was weaker than that against the fusion protein; the fusion protein nor pig calpastatin. this was probably because autoantibodies in patient sera recognized epitopes that are specific to human calpastatin. Calpastatin Activity of Fusion Protein and Blocking Act'iv'ity Antibodies eluted from the RA-6 fusion also bound protein pig of Patient IgG on Calpastatin. The proteolytic activity of A to hu- calpastatin (data not shown). monoclonal antibody calpain was inhibited by the purified RA-6 fusion protein man calpastatin (CSF3-3, domain II- and IV-specific; pur- dose-dependently (93% inhibition at 1 tLg/ml) but not by chased from Takara Shuzo, Kyoto) bound to the RA-6 fusion f3-galactosidase (Fig. 5A). IgG from an RA patient serum protein (MoAb, Fig. 4), while another monoclonal anti- containing anti-calpastatin antibodies inhibited the calpastatin calpastatin (CSF1-2, domain I-specific) did not bind (data not activity of RA-6 fusion protein. Fig. SB shows a typical study shown). Neither monoclonal antibody reacted with pig cal- in which the proteolytic activity of calpain was recovered, up pastatin, as they were specific to human. Neither normal serum to 75% of original activity, by prei'ncubating the RA-6 fusion (NHS; Fig. 4) nor negative patient sera (data not shown) protein with 400 ptg of patient IgG but not with the same reacted with either the fusion protein or pig calpastatin. amount of normal IgG.

1 30 60 90 120

RA-6I E DA K LA A A IS3EV V STP A ST TQ0AOA P P RDT S0SD KD L DD A 40 HCS 496------535

150 180 210 240

RA-6 41 L DK LSDS3LOQR0P DP D E NK PM ED K VKE KA K AE H R DK LOER HCS 536------G------0------575

270 300 330 360

RM-6 81 D T P P E Y R H L L D D N 0 0 D K P V K PP T K K S E D S K K P AD D 0Q 0 P 120 HCS 576------615 390 420 450 480

-6 121 -I D ALSO D LOD3C PSTT E T S0NT A KD K CK K AA S SSK A PK N 160 HCS 616------655- -

s1n i.PT%o540l I..# I v %1%1- AAAGCOAAGGATTCAOCAAAGACAACAC%AGGWCTTCCAAGCCAAAAGATGACTAAAGAAATACAAGTTAAGOTATCTGOTATCTGCATOTAAAATCTTCAOCTOOTOGATOOTGACTT;.o I v 178 HCS 656 ------673 630 660 690 720 4-1 750 780 810 840 870 900 930 960 990 1020 1050 1080

1140 1156 CATTGGGCACATATCTCCTCTTGGGCfTCT'AAATAAIATAATAATCAGOTAACCTGGACAAACCAGGAAGCATT (A)30 FIG. 3. Nucleotide and deduced amino acid sequences of the RA-6 cDNA and homology between the RA-6 polypeptide and calpastatins. The nucleotide sequence of the RA-6 cDNA and its deduced amino acid sequence in what appears to be the open reading frame are illustrated. The asterisk indicates a presumptive stop codon. Underline indicates a poly(A) addition signal (AATAAA, nucleotides 1115-1 120). Arrow (nucleotide 734) indicates the 3'-end nucleotide of the previously described human calpastatin (4). The sequence of the 422 bases from nucleotide 735 is newly reported here. Amino acid sequences of the RA-6 clone and human calpastatin (4) were compared. Identical amino acids in human calpastatin (HCS) are indicated by hyphens. The RA-6 was completely identical with human calpastatin except for a single amino acid substitution: E is at position 62 of the RA-6, whereas G is at position 557 of human calpastatin. Downloaded by guest on September 29, 2021 7270 Medical Sciences: Mimori et al. Proc. Natl. Acad. Sci. USA 92 (1995)

A B We isolated cDNA clones from a Agtll HeLa cell cDNA library by using patient sera as a probe, and we identified 100 )o , 1C several cDNAs that expressed fusion proteins recognized by 24 80 10 RA patient sera. One of these cDNAs, RA-6, appeared to

._ encode a protein corresponding to the 60-kDa protein in HeLa 60 7 cells, since all those reference sera that reacted with a 60-kDa C2ct5 404o 0. HeLa protein also recognized the RA-6 fusion protein. More- 0. 4 over, antibodies that were bound to and eluted from this fusion oc 20 protein selectively recognized a 60-kDa HeLa protein. Al- though a 60-kDa protein was recognized in 36% of RA and a 0 - 0.1 0.2 0.5 1.1 50100 200 400 few patients with other rheumatic diseases in early immunoblot Fusion protein, gg IgG, g studies using HeLa cells (1), antibodies were found more frequently in these disorders when the RA-6 fusion protein was FIG. 5. Calpastatin activity of RA-6 fusion protein and its inhibi- used. This superior sensitivity was probably due to the low level tion by IgG fraction from a patient serum containing anti-calpastatin of expression of the antigen in HeLa cells compared with its antibodies. (A) Various amounts of the RA-6 fusion protein (-) or strong expression as a fusion protein by bacteria infected with 13-galactosidase (0) were added to 250 pl of a reaction mixture a cDNA-bearing phage. Alternatively, the 60-kDa HeLa pro- containing 1 ,ig of rabbit m-calpain and casein as substrate. After a tein might delete domain IV of calpastatin, which appears to 20-min incubation at 30°C, 250 ,ul of 10% trichloroacetic acid was be a main target of autoantibodies in patient sera (see below). added andA28o of acid-soluble supernatants was measured. The RA-6 fusion protein inhibited the proteolytic activity of calpain dose- Computer searches of data banks demonstrated that dependently. (B) Various amounts of patient (A) or normal (/A) IgG the deduced amino acid sequence encoded by the RA-6 cDNA were preincubated with 0.5 Zig of the RA-6 fusion protein for 1 h at was completely identical to the C-terminal region of human room temperature before addition to the reaction mixture, and the calpastatin, except for one amino acid substitution (4). Cal- proteolytic activity of calpain was measured as described above. The pastatin is a specific endogenous inhibitor of the calcium- calpain activity was recovered up to 75% when patient IgG was added. dependent cysteine protease calpain (24, 25). The calpastatin molecule consists of an N-terminal domain (domain L) and Detection of Autoantibodies to Calpastatin in Rheumatic four repetitive domains (domains I-IV) (2-4). The RA-6 Diseases. Since it was demonstrated that the RA-6 cDNA cDNA encodes a part of domain III and entire domain IV. encoded a human calpastatin polypeptide, we used the RA-6 Human calpastatin contains 673 amino acid residues, and its fusion protein to screen for autoantibodies to calpastatin in estimated molecular weight is 72,605 (4). However, it has been sera from patients with a variety of rheumatic diseases. On reported to migrate aberrantly, and if its mass were 110 kDa, immunoblots, anti-calpastatin antibodies were detected in 34 in SDS/polyacrylamide gels because of its unique amino acid of 60 sera (57%) from patients with RA (Table 1). Alterna- content (2). When we used HeLa cell extracts as antigens in tively, antibodies were detected in only 10 or 37 sera (27%) immunoblotting, the 60-kDa protein was recognized by patient from patients with SLE (P < 0.005 compared with RA), 5 of sera with anti-calpastatin as well as antibodies affinity-purified 21 (24%) of polymyositis/dermatomyositis (P < 0.01), 8 of 21 from the RA-6 fusion protein. We suspect that the 60-kDa (38%) of systemic sclerosis (scleroderma) (not significantly HeLa protein is a fragment of calpastatin that was degraded different from RA), and 4 of 14 (29%) of overlap syndrome (P during sample preparation, since calpastatin is known to be < 0.05). Thus, autoantibodies to calpastatin appeared to be fragile and easily digested by endogenous or calpain present in RA patients at the highest frequency, although they itself (26). This is reasonable, since past efforts to purify were also detected at substantial frequencies in other systemic calpastatin suggest that the degradation of calpastatins often produces 60- to 70-kDa fragments, as determined by SDS gel rheumatic diseases. electrophoresis (27). Autoantibodies to calpastatin were detected in patients with DISCUSSION RA at the highest frequency. In immunoblots using the RA-6 fusion protein, anti-calpastatin was detected in 57% of RA We identified three autoantibodies that reacted with uniden- patients, but in only 24-38% of patients with other connective tified 75-, 60-, and 45-kDa proteins in HeLa cells by prelim- tissue diseases. Although RA is classified in the systemic inary immunoblot studies. Among 42 sera from RA patients, autoinmune diseases, RA-specific autoantibodies have only 15 (36%) reacted with a 60-kDa protein, 10 (24%) with a rarely been described. In these limited numbers of reports, 45-kDa protein, and 8 (19%) with a 75-kDa protein (1). Since anti-Sa antibody described by Despres et al. (28) appears to these proteins were not recognized in other patient sera have characteristics similar to those of anti-calpastatin, since (except in one patient with SLE and one patient with poly- anti-Sa recognizes a 50-kDa polypeptide of human placenta myositis whose serum reacted with the 60- and 75-kDa pro- and is found predominantly in RA patients (42.7%). However, teins, respectively), these autoantibodies appeared to be spe- the relationship between calpastatin and Sa antigen should be cific to RA patients. There was no relationship of seroposi- clarified by the exchange of standard sera. tivity and the presence of these antibodies. Calpastatin as well as calpain is distributed widely in the cytoplasmic fraction of almost all mammalian cells (29). While Table 1. Frequency of anti-calpastatin antibodies in systemic calpain has been known to degrade a variety of substrates, such rheumatic disorders as intracellular enzymes, hormone receptors, and various No. No. Frequency, membrane and cytoskeletal proteins (30), the physiologic and Disease tested positive % pathophysiologic roles of calpain and calpastatin are not well understood. It remains unclear how autoantibodies to cal- RA 60 34 57 pastatin can be associated with etiologic and pathogenic SLE 37 10 27* mechanisms of rheumatic disorders. However, since the IgG Polymyositis/dermatomyositis 21 5 24t fraction of RA serum containing anti-calpastatin antibodies Systemic sclerosis 21 8 38 blocked the calpastatin-mediated inhibition of the protease Overlap syndrome 14 4 29* activity of calpain, the presence of autoantibodies would *, P < 0.005 compared with RA (X2 test); t, P < 0.01 compared with increase the relative activity of calpain and might involve in RA (X2 test); *, P < 0.05 compared with RA (Fisher's exact test). tissue injury or inflammation in patients. In fact, there have Downloaded by guest on September 29, 2021 Medical Sciences: Mimori et aL Proc. Natl. Acad. Sci. USA 92 (1995) 7271

been several reports that calpain level is increased in synovial 5. Ishida, S., Emori, Y. & Suzuki, K. (1991) Biochim. Biophys. Acta cells, along with its secretion into the synovial fluid of patients 1088, 436-438. with arthritides, including RA and osteoarthritis (6-8). Thus, 6. Fukui, I., Tanaka, K. & Murachi, T. (1989)Biochem. Biophys. Res. the total activity of calpain exceeded calpastatin in such Commun. 162, 559-566. patients (8). Moreover, at least in vitro, calpain degrades 7. Suzuki, K, Shimizu, K, Hamamoto, T., Nakagawa, Y., Ha- cartilage proteoglycan (9). These findings suggest that in- makubo, T. & Yamamuro, T. (1990) Arthritis Rheum. 33, 728- creased calpain in synovial fluid may act as a matrix proteo- 732. in 8. Yamamoto, S., Shimizu, K., Suzuki, K., Nakagawa, Y. & glycanase and may facilitate articular cartilage degradation Yamamuro, T. (1992) Arthritis Rheum. 35, 1309-1317. arthritis patients (8). 9. Suzuki, K., Shimizu, K., Hatamoto, T., Nakagawa, Y., Murachi, Several reports suggest that calpain may participate in T. & Yamamuro, T. (1992) Biochem. J. 285, 857-862. various inflammatory processes. Calpain irreversibly activates 10. Inoue, M., Kishimoto, A., Takai, Y. & Nishizuka, Y. (1977) J. protein kinase C, a key enzyme of signal transduction, by Biol. Chem. 252, 7610-7616. digesting the membrane-bound form of the protein kinase C 11. Kishimoto, A., Mikawa, K., Hashimoto, K., Yasuda, I., Tanaka, molecule (10, 11). Calpain promotes exocytosis ofgranules and S., Tominaga, M., Kuroda, T. & Nishizuka, Y. (1989) J. Biol. superoxide production in neutrophils (12). Calpain partici- Chem. 264, 4088-4092. pates in the secretion and activation of interleukin-laL, an 12. Pontremoli, S., Melloni, E., Damiani, G., Salamino, F., Spara- inflammatory cytokine, by processing its precursor (13). Au- tore, B., Michetti, M. & Horecker, B. L. (1988)J. Biol. Chem. 263, todigestion of calpain generates an oligopeptide which acts as 1915-1919. a chemotactic factor for neutrophils (14). Taken together, 13. Kobayashi, Y., Yamamoto, K, Saido, T., Kawasaki, H., Oppen- these findings indicate that the presence of anti-calpastatin heim, J. J. & Matsushita, K. (1990) Proc. Natl. Acad. Sci. USA 87, antibodies in patients may increase the enzymatic activity of 5548-5552. calpain that mediates inflammation. Our result that IgG from 14. Kunimatsu, M., Higashiyama, S., Sato, K, Ohkubo, I. & Sasaki, a serum with anti-calpastatin inhibits the function of calpasta- M. (1989) Biochem. Biophys. Res. Commun. 164, 875-882. tin supports this hypothesis. In a preliminary examination, the 15. Arnett, F. C., Edworthy, S., Block, D. A., Moshane, D. J., Fries, of in RA not appear to J. F., Cooper, N. S., Healy, L. K, Kaplan, S. R., Liang, M. H., presence anti-calpastatin patients does Luthra, H. S., Medsger, T. A., Jr., Mitchell, D. M., Neustadt, correlate with titer of rheumatoid factor or activity or severity D. H., Pinals, R. S., Schaller, J. G., Sharp, J. T., Wilder, R. L. & of the disease (data not shown). However, these findings do not Hunder, G. G. (1988) Arthritis Rheum. 31, 315-324. rule out the possibility that anti-calpastatin antibodies might 16. Young, R. A. & Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA be involved in pathogenic processes of RA, since the blocking 80, 1194-1198. activity and autoantibody titer may be more important in the 17. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular association with such clinical manifestation. Quantitative mea- Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, surement and more intensive clinical survey may be necessary Plainview, NY), 2nd Ed., pp. 9.31-9.58. to elucidate the pathogenic roles of anti-calpastatin antibodies. 18. Towbin, H., Staehelin, T. & Gordon, J. (1979) Proc. Natl. Acad. It should be noted also that calpastatin and m-calpain are Sci. US4 76, 4350-4354. increased in cells infected by human T-lymphotropic virus 1 19. Takano, E., Kitahara, A., Sasaki, T., Kannagi, R. & Murachi, T. (31, 32), which is a retrovirus that causes Ra-like arthropathy (1986) Biochem. J. 235, 97-102. (33). This suggests a possibility that a viral infection might 20. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. promote an autoimmune arthropathy by stimulating calpasta- Sci. USA 74, 5463-5467. tin production, with the increased level of this autoantigen 21. Inomata, M., Nomoto, M., Hayashi, M., Nakamura, M., Imahori, driving the production of autoantibodies. Alternatively, a K. & Kawashima, S. (1984) J. Biochem. 95, 1661-1670. molecular mimicry by viral proteins of the autoantigens might 22. Rothfield, N., Whitaker, D., Bordwell, B., Weiner, E., Senecal, J.-L. & Earmshaw, W. (1987) Arthritis Rheum. 30, 1416-1419. be another mechanism of autoantibody generation. Thus, the 23. Imajoh, S., Kawasaki, H. & Suzuki, K. (1986) J. Biochem. 100, identification of anti-calpastatin autoantibodies might be use- 633-642. ful in understanding the etiologic and pathogenic mechanisms 24. Nishiura, I., Tanaka, K., Yamato, S. & Murachi, T. (1978) J. of rheumatic diseases. Biochem. 84, 1657-1659. 25. Murachi, T., Tanaka, K., Hatanaka, M. & Murakami, T. (1981) This work was supported by a Grant-in-Aid for Scientific Research Adv. Enzyme Regul. 19, 407-424. (07670540) from the Ministry of Education, Science and Culture in 26. Shigeta, K., Yumoto, N. & Murachi, T. (1984) Biochem. Int. 9, Japan, a Grant for Rheumatoid Arthritis Research from the Ministry 327-333. of Health and Welfare in Japan, a grant from the Kato Memorial 27. Parker, C. (1986) in Protease Inhibitors, eds. Barrett, A. J. & Foundation, a grant from the Inamori Foundation, and a grant from Salvensen, G. (Elsevier, Amsterdam), pp. 571-587. Keio University. 28. Despres, N., Boire, G., Lopez-Longo, F. 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