Extensive citrullination of human serum albumin is physiological and not inherently immunogenic in rheumatoid arthritis.

Autoantibodies against citrullinated proteins are diagnostic of rheumatoid arthritis (RA), a chronic and systemic autoimmune condition that affects synovial joints. Proteomic studies have revealed that human serum albumin is among the proteins that are citrullinated in RA. Anti-citrullinated protein antibodies reacting with albumin have also been reported. Here, we show that albumin is citrullinated at 11 arginine residues in the blood of both RA patients and, surprisingly, in healthy donors and to a very similar stoichiometry, albeit with some subtle differences. Albumin citrullination exhibited slightly different patterns in synovial fluid from RA patients compared to RA serum-derived albumin, although overall stoichiometry was similar. Incubation of albumin with live neutrophils or recombinant protein arginine deiminases 2 or 4 at 37°C resulted in its rapid citrullination at multiple sites. Albumin citrullination reduced thyroxin binding in vitro. IgG antibodies in the serum of RA patients and healthy donors displayed comparable reactivities to physiologically citrullinated albumin. Similarly, citrullinated peptides corresponding to 14 citrullination sites, were not significantly better recognized by IgG in serum from 86 RA patients than from healthy controls, and surprisingly some were even recognized to a lesser degree in RA. The very few RA patient antibodies exceeding the 95^th percentile of the signal in healthy donors may simply represent weak cross-reactivity of antibodies against unrelated citrullinated antigens. Our findings reveal that albumin citrullination is likely physiological and of little interest to the immune system in RA patients, presumably because of persisting immunological tolerance. We discuss potential physiological functions of albumin citrullination. 250 words.