Date: Monday, November 9, 2015
Session Type: ACR Poster Session B
Session Time: 9:00AM-11:00AM
Background/Purpose: Inhibitor of DNA-binding 1 (Id1) is a nuclear transcription factor actively transcribed in endothelial progenitor cells and in cells that exhibit hyperproliferative responses such as synovial fibroblasts. Previously, we identified Id1 as an angiogenic factor expressed in rheumatoid arthritis (RA) synovial tissues (STs) and upregulated in RA synovial fluids (SFs). Although it is a relatively small protein of approximately 16 kDA, Id1 contains 10 modifiable arginines. As a variety of citrullinated proteins is known to bind to anti-citrullinated protein antibodies (ACPAs), we investigated citrullinated Id1 (citId1) as a potential autoantigen in RA.
Methods: RA SFs were immunodepleted of Id1 and measured by ELISA using anti-modified citrulline (AMC) antibody for total citrullinated antigens pre and post Id1 depletion. Id1 was also immunoprecipitated from homogenized RA STs and analyzed by Western blot (WB) using anti-human Id1 or AMC antibodies. CitId1 was prepared in vitro from recombinant human (rh) Id1 by incubation with rh peptidyl arginine deiminase 4 (PAD4); noncitrullinated Id1 (noncitId1) was prepared identically without rhPAD4. To confirm the citrullination sites, citId1 and noncitId1 were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). To test the presence of ACPAs to citId1, normal (NL) and RA patient peripheral blood (PB) sera immunodepleted of rheumatoid factor (RF) were analyzed using immunodot blot (IDB). CitId1 and noncitId1 were dotted onto nitrocellulose membranes, blocked, and incubated in the patient sera. Bovine serum albumin (BSA) and citBSA were used as antigen controls; anti-human Id1 antibody and rh IgG were used as sera controls. Epithelial-derived neutrophil-activating peptide-78 (ENA-78/CXCL5) and citENA-78/CXCL5 were similarly tested for autoantigenicity.
Results: ELISA analysis of RA SFs showed that the levels of total citrullinated antigens were significantly reduced upon immunodepletion of Id1. WB analysis of immunoprecipitated Id1 from homogenized RA STs showed that a significant portion of the total Id1 was in the modified form. LC-MS/MS analysis provided 97% sequence coverage for citId1, identifying 9 of the 10 total arginines, and 77% sequence coverage for noncitId1, identifying 7 arginines. Of the 9 identified arginines in citId1, citrullination sites were localized specifically at R33, R44, R52, R75, R87, and R121. Of the 7 identified arginines in noncitId1, R87 was identified to be citrullinated, showing that at least one arginine is natively citrullinated in Id1. IDB analysis of mouse anti-human Id1 antibody (previously used in immunodepletion and immunoprecipitation) showed positive signals from both citId1 and noncitId1, suggesting that this binding epitope was not modified by citrullination. IDB analysis of the patient sera showed robust signals for citId1, and weak but significant signals for citENA-78/CXCL5, from multiple RA patient PB sera, displaying a four-fold increase in average reactivity for citId1 as compared to NL patient PB sera.
Conclusion: We show for the first time the presence of ACPAs with specificity to citId1 in RA patient PB sera, and propose citId1 as a novel autoantigen candidate in RA.
To cite this abstract in AMA style:Ohara RA, Edhayan G, Stinson WA, Campbell PL, Remmer HA, Amin MA, Fox DA, Ruth JH. Evidence for Citrullinated Inhibitor of DNA Binding 1 (Id1) As a Novel Autoantigen Candidate in Rheumatoid Arthritis [abstract]. Arthritis Rheumatol. 2015; 67 (suppl 10). https://acrabstracts.org/abstract/evidence-for-citrullinated-inhibitor-of-dna-binding-1-id1-as-a-novel-autoantigen-candidate-in-rheumatoid-arthritis/. Accessed April 16, 2021.
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