Background/Purpose:

Antibodies to citrullinated proteins/peptides (ACPA) are specific for Rheumatoid Arthritis (RA). One of ACPA’s targets is citrullinated fibrinogen (citfib) and it is found in inflamed synovium.  Recently, it has been reported that antibodies to homocitrullinated proteins/peptides (AHPA) also occur in RA. Citrulline and homocitrulline are structurally similar. Both can be generated during inflammation, but by different processes. ACPA from RA patients, including those targeting citfib, are arthritogenic.  However, the role of AHPA in RA is not known.  The purpose of this study was to determine whether:  1) RA patients have antibodies to homocitrullinated fibrinogen (AHFA) and 2) ACPA and AHPA are cross-reactive.

Methods:

Serum was obtained from patients who met ACR criteria for RA, Psoriatic Arthritis or Systemic Lupus Erythematosus and were compared to healthy controls. It was tested against the following antigens: fibrinogen (fib), citfib, homocitrullinated fibrinogen (homocitfib), JED (a proprietary synthetic citrullinated peptide) and homocitrullinated JED (homoJED). Citrullination was done in vitro using peptidyl arginine deiminase enzyme (PAD2) [1]. Homocitrullination was done using potassium isocyanate. Both modifications were confirmed by mass spectrometry using ESI-MSMS and MASCOT server analysis. Shared Epitope (SE) binding was predicted by a computer algorithm [2]. ACPA was purified by affinity chromatography using JED. Antibodies to the above antigens and cyclic citrullinated peptide 2 (CCP2) were detected by ELISA. Inhibition assays using fib and homocitfib were conducted by ELISA.  

Results:

Mass spectrometry of modified fibrinogen revealed 55/79 (70%) of arginines were citrullinated and 89/103 (86%) of lysines were homocitrullinated. Approximately 25% of MHCII-binding peptides were both citrullinated and homocitrullinated. Five of these peptides were predicted to bind to the SE. The majority of RA patients were anti-CCP2 positive (89%) and 50% expressed AHFA. None of the normal controls and <5% of PsA and SLE were AHFA positive. All AHFA positive patients were also anti-CCP2 positive. Reactivity to homocitrullinated sites on fibrinogen was confirmed by inhibition assays. Affinity purified ACPA using a citrullinated peptide (JED) had reactivity to JED, anti-CCP2, as well as the homocitrullinated peptides, homoJED and homocitfib.

Conclusion:

Antibodies to homocitrullinated peptides/proteins are specific for RA. These antibodies bind citrullinated and homocitrullinated antigens, suggesting cross-reactivity and possible pathogenicity. 

[1] Mydel et al. 2010. J Immunol 184(12):6882.

[2] Hammer et al. 1994. J Exp Med 180(6): 2353.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/antibodies-to-citrulline-from-rheumatoid-arthritis-patients-also-bind-homocitrulline/