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 HLA-DRB1 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: CRT-SE 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 Arg(205), which is spatially adjacent to the SE binding site in the CRT P-domain, as a dominant inhibitor of SE-CRT interaction and signaling, while a more remote arginine residue, Arg(261), 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 gene-environment interaction in RA.