Background/Purpose:  Inhibitor of DNA binding-1 (Id1) is a nuclear transcription factor that regulates cell growth and differentiation via selective binding and sequestering of other transcription factors. We have shown Id1 is primarily fibroblast derived with elevated expression in rheumatoid arthritis (RA) synovial fluids (SFs) and functions as a potent angiogenic factor. We have found that in vitro citrullination of Id1 exhibits heterogeneity in the number of modified arginines observed, leading to remarkable differences in autoantigenicity and autoantibody formation in vivo. In this study, we investigated how targeted citrullination affects the potential autoantigenicity of citrullinated Id1 and autoantibody formation in RA.

Methods: RA SFs were immunodepleted of Id1 and measured by ELISA using anti-modified citrulline (AMC) antibody for total citrullinated antigens. RA synovial tissues (STs) were homogenized and immunoprecipitated of Id1 for Western blot analysis using anti-Id1 or AMC antibodies. In vitro citrullination was performed by incubating recombinant human (rh) PAD4 or rabbit PAD with the target protein. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to localize the sites of citrullination. To test the presence of anti-citrullinated protein antibodies (ACPAs) to citrullinated Id1, normal (NL) and RA patient peripheral blood (PB) sera as well as SFs, immunodepleted of rheumatoid factor, were analyzed using immunodot blot (IDB).

Results:  Immunodepletion of Id1 from RA SFs significantly reduced the levels of total citrullinated antigens in RA SFs by as much as 64%, with an average reduction of 31%, as measured by ELISA. Similarly, immunoprecipitated Id1 from homogenized RA STs was also significantly citrullinated as shown by Western blots. IDB analyses revealed antibodies to citrullinated Id1, but not native Id1, which were detected in RA sera and SFs, but not in NL sera. By a series of LC/MS-MS and immunoassays, we determined the citrullination sites of modified Id1. Of the 10 available arginines (R) in Id1, the critical arginines for autoantigenicity were located at positions R33, R52 and R121. Citrullinated Id1 protein lacking modified arginines at those residues did not bind to AMC antibody, did not migrate as citId1 on acrylamide gels due to charge differences, and were not recognized by ACPAs to citId1 in IDBs. In contrast, we observed a robust change in autoantigenicity of epithelial-derived neutrophil-activating peptide-78 (ENA-78)/CXCL5 modified with rabbit PAD, showing that a single modification at R48 converts it so that it is recognized by ACPAs in patient sera and SFs.

Conclusion:  We identify key arginines in Id1 that may be an autoantigenic target for ACPA development in RA. Our findings also show that targeting select arginines for citrullination conversion can produce dramatic changes in autoantigenicity. The degree of citrullination of Id1 may alter the natural folding pattern and immune properties of Id1, leading not only to autoantigenicity, but potentially also to functional changes in this molecule. These findings could explain putative disease-associated properties of Id1: increased severity of certain cancers and perpetuation of inflammatory disease.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/citrullination-of-inhibitor-of-dna-binding-1-at-specific-locations-leads-to-autoantigenicity-in-rheumatoid-arthritis/