Identification of novel citrullinated autoantigens of synovium in rheumatoid arthritis using a proteomic approach
© Matsuo et al.; licensee BioMed Central Ltd. 2006
Received: 13 July 2006
Accepted: 27 November 2006
Published: 27 November 2006
Recently, autoantibodies to some citrullinated autoantigens have been reported to be specific for rheumatoid arthritis (RA). However, an entire profile of and autoimmunity of the citrullinated proteins have been poorly understood. To understand the profile, we examined citrullinated autoantigens by a proteomic approach and further investigated the significance of citrullination in antigenicity of one of the autoantigens. Specifically, we detected citrullinated autoantigens in synovial tissue of a patient with RA by two-dimensional electrophoresis and Western blotting by using pooled sera from five patients with RA and anti-citrulline antibodies. After identifying the detected autoantigens by mass spectrometry, we investigated the contribution of citrullination to autoantigenicity by using a recombinant protein with or without citrullination on one of the identified novel citrullinated autoantigens. As a result, we found 51 citrullinated protein spots. Thirty (58.8%) of these spots were autoantigenic. We identified 13 out of the 30 detected citrullinated autoantigenic proteins. They contained three fibrinogen derivatives and several novel citrullinated autoantigens (for example, asporin and F-actin capping protein α-1 subunit [CapZα-1]). We further analyzed the contribution of citrullination to autoantigenicity in one of the detected citrullinated autoantigens, CapZα-1. As a result, frequencies of autoantibodies to non-citrullinated CapZα-1 were 36.7% in the RA group tested, 10.7% in the osteoarthritis (OA) group, and 6.5% in healthy donors. On the other hand, those to citrullinated CapZα-1 were 53.3% in the RA group, 7.1% in the OA group, and 6.5% in the healthy donors. This shows that autoantigenicity of citrullinated or non-citrullinated CapZα-1 is relevant to RA. The antibody titers to the citrullinated CapZα-1 were significantly higher than those to the non-citrullinated CapZα-1 in 36.7% of patients; however, the other patients showed almost equal antibody titers to both citrullinated and non-citrullinated CapZα-1. Therefore, the autoantibodies would target citrulline-related and/or citrulline-unrelated epitope(s) of CapZα-1. In conclusion, we report a profile of citrullinated autoantigens for the first time. Even though citrullination is closely related to autoantigenicity, citrullination would not always produce autoantigenicity in RA. Citrullinated and non-citrullinated autoantigens/autoepitopes would have different pathological roles in RA.
Rheumatoid arthritis (RA) is one of the most prevalent rheumatic disorders and is characterized by chronic inflammation of multiple joints. It affects synovium, articular cartilage, and articular bones, which lead to destruction of the joints. Although the pathogenesis of RA is not fully understood, autoimmune reactions are suggested to play pathological roles in chronic synovitis. So far, a variety of candidate autoantigens such as rheumatoid factor, collagen type II, cartilage intermediate layer protein, YKL-39, and calpastatin have been suggested to induce cellular and/or humoral autoimmune responses in RA [1–5]. Autoantibodies directed to proteins with a non-standard amino acid of citrulline, produced by post-translational modification of arginine, have been found to be RA-specific [6, 7]. Filaggrin is a typical example. In early studies, the autoantibodies to filaggrin, previously called 'anti-perinuclear factor antibodies' or 'anti-keratin antibodies,' were reported to be specific for RA. Later, citrullination was found to be essential for the autoantigenicity of filaggrin . Quite recently, the anti-citrullinated protein antibodies have started to be measured using artificial cyclic citrullinated peptides (CCPs) as a clinical laboratory examination. The anti-CCP antibody was reported to have high predictive value for development of RA as well as high sensitivity and specificity for diagnosis of RA [5, 8]. Since then, several autoantibodies against citrullinated proteins have been identified in RA. They include fibrin/fibrinogen , vimentin , and Epstein-Barr virus nuclear antigen-1 (EBVA-1) . Concurrently, association of functional haplotypes of the gene encoding citrullinating enzyme of peptidylarginine deiminase-4 (PADI4) with susceptibility to RA was reported . It was also reported that PADI4 affected levels of the antibody to citrullinated peptides in sera from patients with RA .
Pathologically, the antibodies to citrullinated proteins are expected to be produced in the synovial compartment  given that the anti-CCP antibodies constituted a higher proportion of immunoglobulin (Ig) G) in synovial fluid (SF) than that in serum of patients with RA [13, 14] and given that B cells producing the anti-CCP antibodies have been isolated from RA synovium . Furthermore, peptidylarginine deiminase (PAD) generates citrulline residues by deimination of arginine residues of proteins. Isoforms 2 and 4 of PAD were expressed in mononuclear cells isolated from SF . These data suggest that presence of citrullinated proteins in the RA synovium causes antigen-driven maturation of B cells at the site of inflammation. However, it is poorly understood what kind of proteins are citrullinated and recognized as targets of autoantibodies in the synovial tissue of patients with RA. To answer these questions, comprehensive analysis of autoantigenic citrullinated proteins in RA would be needed. Based on this background, we performed a screening of autoantigenic citrullinated proteins in synovial tissue proteins from a patient with RA and evaluated the contribution of citrullination to autoantigenicity by using recombinant proteins.
Materials and methods
Patients and synovial tissues
Serum samples were obtained from 30 patients with RA (26 women, 4 men; ages 29 to 78 years, mean 60.1 years) and 28 patients with osteoarthritis (OA) (23 women, 5 men; ages 23 to 84 years, mean 64.4 years). The patients were diagnosed according to the respective classification criteria for each of the two diseases [16, 17]. Serum samples from age- and gender-matched healthy donors were used as a control (27 women, 4 men; ages 32 to 80 years, mean 60.0 years). Non-RA rheumatological control samples were obtained from patients with systemic lupus erythematosus (SLE) (17 women, 2 men; ages 39 to 65 years, mean 49.7) who were diagnosed according to the published classification criteria . Serum samples from age- and gender-matched health donors (21 women, 1 man; ages 32 to 64 years, mean 52.7 years) were used as a control for SLE. Four synovial tissue samples were obtained from three women 53 to 68 years old and a 68-year-old man with RA during their operation of knee joint arthroplasty. All the samples were obtained after the patients gave their informed consent, and this study was approved by the local institutional ethics committee.
Sample preparation, two-dimensional electrophoresis, and subsequent Western blotting
A synovial tissue sample from a 53-year-old-woman with RA was washed in phosphate-buffered saline (PBS) and then homogenized in a deionized lysis buffer (7 M urea, 2 M thiourea, 4% 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate) using HG30 homogenizer (Hitachi Koki Co., Ltd., Tokyo, Japan) on ice. Next, the sample was frozen and thawed five times and then centrifuged at 4°C for 30 minutes. Finally, the supernatant was collected and its protein concentration was determined using the Bradford method. The supernatant was stored at -80°C until use.
The two-dimensional electrophoresis (2DE) was performed as described elsewhere [19, 20]. The first electrophoresis is isoelectric focusing (IEF), in which the extracted proteins were loaded onto 11-cm Imobiline drystrip gels (pH range 4 to 7; GE Healthcare, Buckinghamshire, UK) at 20°C for 12 hours. One hundred fifty micrograms of the extracts was applied to the drystrip gels for detection of antigens and 500 μg for identification of proteins by mass spectrometry (MS). IEF was performed using Ettan IPGphor (GE Healthcare Bio-Sciences Corp.). The second electrophoresis was 12.5% SDS-PAGE. After the electrophoresis, the gels were stained with a fluorescent dye of SYPRO Ruby (Molecular Probes, now part of Invitrogen Corporation, Carlsbad, CA, USA) and then used for protein transfer onto nitrocellulose membranes.
Western blotting after 2DE was performed as described previously . Briefly, the proteins transferred onto membranes were reacted with pooled serum samples from five patients with RA or pooled serum samples from five healthy donors to detect autoantigens. The sera were used at a dilution factor of 1:500 per patient. After washing in PBS, bound antibodies were reacted with horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Zymed Laboratories, Inc., now part of Invitrogen Corporation), and were then visualized with diaminobendzidine. Citrullinated proteins on the membranes were detected by Western blotting by using anti-citrulline (modified) detection kit (Upstate Biotechnology, Lake Placid, NY, USA). Specifically, citrulline residues of the proteins immobilized on the membranes were modified by 2, 3-butanedione monoxime and antipyrine in a strong acid solution in accordance with the manufacturer's instructions. Then, the modified citrulline residues were detected by the rabbit polyclonal anti-modified citrulline (anti-MC) antibodies (Upstate Biotechnology) and goat anti-rabbit IgG-HRP antibody conjugate (Upstate Biotechnology). F-actin capping protein α-1 subunit (CapZα-1) was detected by Western blotting using an anti-CapZα-1 polyclonal antibody (Chemicon International, Temecula, CA, USA) and a goat polyclonal anti chicken IgY (H+L)-HRP antibody (Abcam, Cambridge, UK). Rabbit Ig fraction (Dako Denmark A/S, Glostrup, Denmark) was used as a negative control for the anti-MC antibody, and normal chicken IgY (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) was used as a negative control for anti-CapZα-1 antibody.
Protein spots on the gel stained with SYPRO Ruby which corresponded to the positive spots by Western blotting were recovered and then subjected to in-gel digestion with trypsin. Next, the mass of the digested proteins, extracted by C18 beads, was measured using matrix-assisted laser disorption/ionization-time of flight (MALDI-TOF) MS as described previously [21, 22]. Mass spectra of positively charged ions were recorded on a Bruker Ultraflex TOF/TOF instrument (Bruker Daltonik GmbH, Bremen, Germany) operated in the reflector mode. The flexControl, flexAnalysis, and Biotools software packages provided by manufacturer were used for data processing. A list of determined peptide masses were subjected to mass fingerprinting by using the Mascot Search software program (Matrix Science Ltd., London, UK), in which the National Center for Biotechnology Information (NCBI) (Bethesda, MD, USA) protein databases were searched.
Preparation of recombinant proteins
In accordance with the nucleotide sequence of the human CapZα-1, two DNA primers of 5'-tttccatggccgacttcgatgatcg and 3'-tttctcgagagcattctgcatttctttgccaatc were prepared. A DNA fragment for the entire protein-coding region of CapZα-1 was amplified from cDNA prepared from human synoviocytes by using reverse transcription-polymerase chain reaction. The amplified DNA fragment for CapZα-1 was cloned into a plasmid expression vector of pETBlue-2 (Novagen; Merck KGaA, Darmstadt, Germany) by using restriction endonucleases of Nco I and Xho I. Thereby, recombinant CapZα-1 with a tag of six histidines in its C-terminal was produced in Escherichia coli (DE3). The recombinant protein was purified using histidine-Ni+ affinity (His Trap; GE Healthcare Bio-Sciences Corp.) as described previously .
Citrullination of CapZα-1
The recombinant CapZα-1 was citrullinated in several concentrations of rabbit muscle PAD (Sigma-Aldrich, St. Louis, MO, USA). One milligram of the recombinant CapZα-1 was loaded into a Ni+-bound column (His Trap). After washing, the column-bound CapZα-1 was reacted with 20 U/mg of PAD in a buffer containing 0.1 M Tris-HCl (pH 7.6), 10 mM CaCl2, and 5 mM dithioerythritol and incubated for 2 hours at 37°C. After the second washing, the citrullinated CapZα-1 was eluted by addition of an elution buffer containing 500 mM imidazole. Citrullination of the recombinant CapZα-1 was estimated by Western blotting using an anti-citrulline (modified) detection kit (Upstate Biotechnology).
Enzyme-linked immunosorbent assay
Enzyme-linked immunosorbent assay (ELISA) was performed as described previously . Briefly, each well of a multititer plate (Immulon; Thermo Labsystems, Franklin, MA, USA) was coated with 2.5 μg/ml CapZα-1 or citrullinated CapZα-1 in a carbonate buffer (50 mM sodium carbonate, pH 9.6). The serum samples diluted at 1:400 were reacted with the immobilized CapZα-1. Then, the bound antibodies were reacted with HRP-conjugated goat anti-human IgG (Invitrogen Corporation). The reactivity of the serum samples in response to the citrullinated or non-citrullinated recombinant CapZα-1 was expressed as optical density (OD) or as arbitrary binding units according to the following formula: sample (binding units) = (OD sample/[mean OD normal sera + 2 standard deviations of normal sera] × 100). According to this formula, 100 binding units was defined as the cutoff point for reactivity.
Purification of autoantibodies to citrullinated CapZα-1 and subsequent Western blotting
A citrullinated CapZα-1-bound column was created by coupling the citrullinated recombinant CapZα-1 into an N-hydroxysuccinimide (NHS)-activated sepharose column (HiTrap NHS-activated HP; GE Healthcare Bio-Sciences Corp.) in accordance with the manufacturer's instructions. Next, a mixture of serum samples from four patients with RA was loaded into the citrullinated CapZα-1-bound column. After washing, autoantibodies against citrullinated CapZα-1 in RA sera were eluted. The concentration of purified antibodies was measured using a human IgG quantitation kit (Bethyl Laboratories, Inc., Montgomery, TX, USA). The purified antibodies and control human IgG (Invitrogen Corporation) diluted to the concentration of 1 μg/ml and the RA serum mixture diluted at 1:500 were reacted with proteins from three synovial samples separated by SDS-PAGE.
Differences of prevalence of the anti-non-citrullinated and citrullinated CapZα-1 antibodies among disease categories were compared using the χ2 test. The differences in mean binding units between the disease categories and in mean clinical parameters between groups were compared by Student's t test. The differences in the mean clinical parameter among four groups separated by the patterns of autoantibodies to non-citrullinated and citrullinated CapZα-1 were compared by one-factor analysis of variance or Kruskal-Wallis test. Spearman's correlation coefficient by rank test was used to measure the correlation between titers of antibody and clinical parameters.
Detection of citrullinated autoantigens in synovial tissue of a patient with RA
Numbers of citrullinated and/or autoreactive synovial protein spots
Rheumatoid arthritis sera
Identification of the citrullinated autoantigens by mass spectroscopy
Citrullinated autoantigens identified using mass spectrometry
Number of peptides
Cathepsin D, chain B
F-actin capping protein α-1 subunit (CapZα-1)
Periodontal ligament associated protein-1
Chain B, crystal structure of fibrinogen fragment D
48-kDa histamine receptor subunit peptide-4 (internal fragment)
Protein disulfide-isomerase (EC 5. 3. 4. 1.)
ER60 precursor Glucose-related protein 58 kDa
Chain A, human mitochondrial aldehyde dehydrogenase complexed with NAD+ and Mn2+
Human serum albumin in a complex with myristic acid and tri-iodobenzoic acid
Chain A, crystal structure of human serum albumin
Recombinant human gamma-fibrinogen carboxyl terminal fragment
Autoantigenicity of citrullinated and non-citrullinated CapZα-1
Autoantibodies to citrulline-related epitope(s) and non-related epitope(s) on CapZα-1
Cross-reactivity of the autoantibodies to citrullinated CapZα-1
Comparison of clinical parameters among the different reactivity groups
Comparison of clinical data among groups A-D, between antibodies to citrulline-related epitope (positive and negative), and between antibodies non-related epitope (positive and negative)
Number of patients
Age in years
56.1 ± 10.8a
12.2 ± 9.2
89.6 ± 116.7
63.3 ± 66.9c
1.55 ± 1.86
33.2 ± 16.8
66.6 ± 2.1a
11.4 ± 5.4
422.6 ± 574.5
385.6 ± 396.1
0.88 ± 0.79
41.8 ± 23.4
65.7 ± 7.8
17.2 ± 12.7
139.3 ± 146.2
63.8 ± 55.5d
0.97 ± 0.95
38.3 ± 27.9
61.2 ± 21.0
6.3 ± 5.8
47.5 ± 47.0
164.0 ± 84.9c, d
1.18 ± 1.48
31.0 ± 19.1
64.6 ± 12.0
11.6 ± 9.2
199.2 ± 348.5
195.7 ± 253.2
1.01 ± 1.03
37.1 ± 22.9
56.1 ± 10.8
12.2 ± 9.2
89.6 ± 116.7
63.3 ± 66.9
1.55 ± 1.86
33.2 ± 16.8
63.6 ± 14.6
11.7 ± 10.9
97.6 ± 117.8
109.3 ± 84.7
1.06 ± 1.16
35.0 ± 23.4
58.8 ± 10.4
12.0 ± 8.2
177.3 ± 325.4
148.1 ± 243.7
1.37 ± 1.65
35.5 ± 18.5
66.1 ± 5.7b
14.3 ± 9.7
268.1 ± 405.7
210.1 ± 304.2
0.93 ± 0.84
39.9 ± 24.7
57.4 ± 13.7b
10.7 ± 8.7
78.6 ± 103.4
89.8 ± 83.2
1.45 ± 1.74
32.6 ± 16.9
Here, we examined autoantigenic citrullinated proteins in a synovial tissue sample from a patient with RA by using the proteomic approach and then investigated the contribution of citrullination to autoantigenicity on one of the identified autoantigens, CapZα-1. Our findings are as follows: (a) Out of the 990 synovial tissue protein spots detected by 2DE, 51 protein spots were citrullinated and 94 protein spots were autoantigenic in RA. Thirty protein spots were both citrullinated and autoantigenic. (b) Among the 30 citrullinated and autoantigenic protein spots, 13 protein spots were identified in which derivative peptides of fibrinogen accounted for 3. (c) New identified citrullinated autoantigens were asporin, cathepsin D, β-actin, CapZα-1, albumin, histamine receptor, PDI (EC 5. 3. 4. 1.) ER60 precursor, and ALDH2. (d) In the investigation of autoantigenicity of CapZα-1, citrullination of CapZα-1 was confirmed in all four of the synovial tissue samples from patients with RA. Some patients with RA carried autoantibodies only to citrulline-unrelated epitope(s) on CapZα-1, some carried autoantibodies only to citrulline-related epitope(s) on CapZα-1, and others carried both autoantibodies. (e) Clinically, the patients who had autoantibodies to neither citrullinated CapZα-1 nor non-citrullinated CapZα-1 appeared to be younger than the others. Also, the groups that do not have antibodies to the citrulline-related epitope(s) were significantly younger than the other groups. The anti-CCP antibody titers were not correlated with the titers to CapZα-1.
The first finding indicates that citrullination would be one of the major factors for self-proteins to get autoantigenicity in RA, given that a considerable proportion (30/51, 58.8%) of the citrullinated protein spots were autoreactive. On the other hand, approximately 70% of the autoantigenic protein spots detected were found to be non-citrullinated. Citrullination would not be the only way to get autoantigenicity in RA. The degree of the relation between citrullination and autoantigenicity would differ among different patients with RA. Given that only one synovial sample was available in this screening, larger numbers of RA synovial samples should be investigated in the future to evaluate the relation more precisely.
The second point that 3 out of the 13 identified protein spots were derivative peptides of fibrinogen confirmed the significance of fibrinogen as a major citrullinated autoantigen in RA as reported previously . Our screening revealed that two out of the three fibrinogen peptides were assigned to the fibrinogen gamma chain, not alpha or beta chains (Table 2). The fibrinogen gamma chain may be a major citrullinated autoantigen as well as alpha or beta chains in RA, although the previous study reported that fibrinogen gamma chain was not targeted as frequently as alpha or beta chains were . That could be due to a poor efficiency of citrullination by PAD enzyme in vitro as recently described .
Besides the fibrinogen chains, we identified several new citrullinated autoantigens successfully. The first is asporin, one of the extracellular matrix components expressed abundantly in the articular cartilage of patients with OA . Asporin is reported to suppress transforming growth factor (TGF)-β-mediated gene expression of aggrecan and type II collagen by inhibiting TGF-β function, possibly through a direct physical interaction with TGF-β . A genetic association with OA of two functional alleles of the asporin gene was reported recently . Thus, citrullination of asporin and binding of the autoantibodies to asporin may alter chondrocyte metabolism in RA. The second is cathepsin D, a lysosomal aspartic peptidase, which is reported to play roles in destroying synovial tissue and cartilage matrix [30, 31]. It is interesting whether citrullination affects the functions of cathepsin D. The third is histamine, a classic mediator of inflammation, which was reported to enhance production of matrix metalloprotease-1 by rheumatoid synovial fibroblasts via H1 receptors and to enhance interleukin (IL)-1-α-induced IL-6 and IL-1-β synthesis by peripheral blood mononuclear cells via H2 receptors [32–34]. It is also interesting whether citrullination of histamine receptors and/or binding of autoantibodies to histamine receptors affects the inflammatory conditions in RA.
In addition to the three molecules, we identified β-actin, albumin, and PDI ER60 precursor. PDI ER60 precursor was thought to be a thiol protease. Autoantibodies to β-actin were reported in patients with autoimmune inner ear disease [35, 36]. Autoantibodies to N-homocysteinylated albumin have been reported as a marker for coronary artery disease . Autoantibodies to PDI were detected in a spontaneous rat hepatitis model and in patients with SLE, infertility, or allergic rhinitis [38–41]. However, function of the autoantibodies remains to be solved in these diseases as well as in RA. Similarly, effects of citrullination on the functions of these molecules should be investigated. Our screening did not identify some of the major known citrullinated autoantigens such as EBVA-1 and vimentin. A single screening is not ideal for identifying all the citrullinated proteins. Repeated screening would elucidate greater numbers of citrullinated proteins.
In the investigation of autoantigenicity of CapZα-1, citrullination of CapZα-1 was confirmed in all four of the tested synovial tissue samples from the patients with RA. The frequency of the autoantibodies to non-citrullinated CapZα-1 was significantly higher in the RA group than in the OA, SLE, and healthy groups. Further, the frequency and titers of the autoantibodies to CapZα-1 increased when citrullinated CapZα-1 was used as an antigen only in the RA group. Therefore, both of the autoantibodies to citrulline-related and -unrelated epitope(s) on CapZα-1 would be closely related to RA. However, it appears to depend on individual patients whether they carry autoantibodies to the citrulline-related epitope(s), to the citrulline-unrelated epitope(s), or to both epitope(s). The autoantibodies purified by the citrullinated CapZα-1 showed cross-reactivity to other proteins, and we detected five protein bands reacted with the antibodies. Two of the five protein bands reacted with the polyclonal anti-CapZα-1 antibodies, indicating existence of similar epitope(s) to non-citrullinated CapZα-1. The remaining three would have other epitope(s) similar to citrullinated parts of CapZα-1. The titers of the anti-CCP antibodies were significantly higher in the patients with antibodies to citrulline-unrelated epitope(s) (group D) than those in the patients with only antibodies to citrulline-related epitope(s) (group C). None of the sera of patients with OA or of healthy donors with reactivity to citrullinated CapZα-1 reacted with CCP. Only one patient each in the OA group and the healthy group had anti-CCP antibodies, but they did not have anti-citrullinated CapZα-1 antibodies (data not shown). These results indicate that the titers of the anti-CCP antibodies do not always represent total antibodies to citrullinated epitopes.
Clinically, the mean age of the groups that carry autoantibodies to citrulline-related epitope(s) (groups B and C) was significantly higher than that of the other groups (groups A and D). Further, in the groups not having antibodies to non-citrullinated CapZα-1, the mean age of the group that carries autoantibodies to citrulline-related epitope(s) (group B) is significantly higher than that of the group that carries antibodies neither to citrulline-related nor-unrelated epitope(s) (group A). The differences of the means of duration were not significant among them. Because the number of patients tested is rather small, we need further investigation with larger numbers of samples to clarify the association with clinical data.
F-actin capping protein (CapZ), a heterodimeric protein consisting of subunits of α and β that bind selectively and with high affinity to the barbed ends of actin filaments , is present in a wide variety of tissues and organisms. Vertebrates, including chicken, mice, and humans, contain two α-subunit isoforms and two β-subunit isoforms [43, 44]. The α1 and α2 isoforms share 85% amino acid identity [45, 46]. CapZ regulates the growth of actin filament by capping the barbed ends of actin filaments . Previous studies suggest that CapZ is essential to the assembly and organization of the actin cytoskeleton in vivo [47–50]. Therefore, citrullination of CapZα-1 may inhibit the activity of capping protein and can lead to degeneration of various cells such as synoviocytes and chondrocytes, resulting in the release of antigens to inflammatory cells. Further studies are needed to elucidate whether the pathway is possible.
In summary, we demonstrated a profile of citrullinated autoantigens in RA synovium. Autoantigenicity and citrullination are closely related, but citrullination does not entirely explain autoantigenicity in RA. Our data will help in understanding the roles of citrullination in RA.
= mitochondrial aldehyde dehydrogenase
= anti-modified citrulline
= F-actin capping protein α-1 subunit
= cyclic citrullinated peptide
= Epstein-Barr virus nuclear antigen 1
= enzyme commission number
= enzyme-linked immunosorbent assay
= horseradish peroxidase
= isoelectric focusing
= matrix-assisted laser disorption/ionization-time of flight
= mass spectrometry
= National Center for Biotechnology Information
= optical density
= peptidylarginine deiminase
= peptidylarginine deiminase-4
= phosphate-buffered saline
= protein disulfide-isomerase
= rheumatoid arthritis
= synovial fluid
= systemic lupus erythematosus
= transforming growth factor
= two-dimensional electrophoresis.
The authors thank Ms. M. Kanke and Ms. M. Tamaki for their excellent technical assistance and Prof. M. Beppu and doctors of the Department of Orthopaedic Surgery, St. Marianna University School of Medicine, for the provision of the synovial tissue samples. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan; the Ministry of Health, Labor and Welfare of Japan; and the Japan Rheumatism Foundation.
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