0078: Citrullinated Serum Albumin Is Not an Autoantigen in Rheumatoid Arthritis

Background/Purpose: Autoantibodies against citrullinated proteins (ACPAs) are diagnostic of rheumatoid arthritis (RA), an autoimmune disease primarily affecting synovial joints. Proteomic analyses have identified human serum albumin as a target of citrullination in RA, and anti-citrullinated albumin antibodies have been reported. This study aimed to characterize the extent and immunological relevance of albumin citrullination in RA.

Methods: Sera from individuals with RA (n = 87), SLE (n = 24), and healthy controls (n = 33) were obtained from the UW Rheumatology Biorepository, and neutrophils were isolated from freshly collected blood samples of RA patients. Anti-CCP status in RA sera was confirmed by ELISA. Neutrophils from RA blood or recombinant PAD2/PAD4 enzymes were used to citrullinate human albumin in vitro. Citrullination was assessed by AMC blotting and LC-MS/MS after in-gel tryptic digestion. Citrullinated and unmodified peptide pairs (14 sites) were tested by ELISA for IgG binding; responses above the 95^th percentile of controls were considered positive. Thyroxine binding to native versus citrullinated albumin was measured using competitive ELISA. All protocols were IRB-approved. Mann-Whitney U tests were used for statistical analyses, with p< 0.05 considered significant.

Results: This study analyzed serum from 87 RA patients, confirming CCP positivity in 59.5%, and identified 17 citrullinated arginine residues in albumin from both RA patients and healthy donors using LC-MS/MS. Surprisingly, the stoichiometry of citrullination was similar in both groups, suggesting it is a physiological process. Increased citrullination was observed only in RA synovial fluid, albeit at the same sites. The citrullinated residues were surface-exposed and located near ligand-binding pockets, including R81 and R521 near the FcRn binding interface, indicating potential structural or functional consequences. In vitro, PAD2, PAD4, and live RA neutrophils rapidly citrullinated albumin at multiple sites, with PAD4 inducing the most extensive changes. Citrullination significantly reduced thyroxin binding by 64%, suggesting a regulatory role of citrullination in ligand binding. However, ELISA assays showed minimal IgG ACPA reactivity to either physiologically citrullinated albumin or synthetic citrullinated peptides, with only a few RA sera slightly exceeding control thresholds, likely due to cross-reactivity of ACPA against other citrullinated proteins. These findings suggest that albumin citrullination is a physiological process and not a major immune target in RA.

Conclusion: Albumin citrullination appears to be a physiological process and is not immunogenic in RA, with similar stoichiometry to healthy controls and minimal ACPA recognition. We conclude that citrullinated albumin is unlikely to contribute significantly to ACPA responses in RA and that immunological tolerance against citrullinated albumin is maintained in RA.