Citrullinated calreticulin potentiates rheumatoid arthritis shared epitope signaling

Objective Citrullinated proteins are immunogenic in rheumatoid arthritis (RA), particularly in patients who carry shared epitope (SE)coding HLADRB1 alleles. The mechanism underlying this association is unknown. We have previously identified the SE as a ligand that interacts with cell surface calreticulin (CRT) and activates immune dysregulation. This study was undertaken to determine the effect of CRT citrullination on SE signaling. Methods CRTSE binding affinity was measured by surface plasmon resonance. The role of individual CRT arginine residues was determined by site-directed mutagenesis, and nitric oxide levels were measured using a fluorochrome-based assay. CRT citrullination in synovial tissue samples and cell cultures was determined by 2-dimensional gel electrophoresis, immunoblotting, and mass spectrometry techniques. Results Synovial tissue and fibroblast-like synoviocytes from RA patients were found to express a higher abundance of citrullinated CRT than samples from osteoarthritis patients. Citrullinated CRT showed more robust interaction with the SE ligand, and transduced SE signaling at a 10,000-fold higher potency, compared to noncitrullinated CRT. Site-directed mutation analysis identified Arg205, which is spatially adjacent to the SE binding site in the CRT P-domain, as a dominant inhibitor of SECRT interaction and signaling, while a more remote arginine residue, Arg261, was found to enhance these SE functions. Conclusion Our findings indicate that citrullinated CRT is overabundant in the RA synovium and potentiates SE-activated signaling in vitro. These findings could introduce a new mechanistic model of geneenvironment interaction in RA.