Citrullinated calreticulin potentiates rheumatoid arthritis shared epitope signaling

Arthritis Rheum. 2013 Mar;65(3):618-26. doi: 10.1002/art.37814.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Surface / immunology
  • Antigens, Surface / metabolism
  • Arginine / metabolism
  • Arthritis, Rheumatoid / immunology
  • Arthritis, Rheumatoid / metabolism*
  • Binding Sites / immunology
  • Calreticulin / chemistry
  • Calreticulin / genetics
  • Calreticulin / metabolism*
  • Cell Line
  • Citrulline / chemistry
  • Citrulline / metabolism*
  • Epitopes / immunology
  • Epitopes / metabolism*
  • Fibroblasts / cytology
  • Fibroblasts / immunology
  • Fibroblasts / metabolism*
  • Gene-Environment Interaction
  • HLA-DRB1 Chains / immunology
  • HLA-DRB1 Chains / metabolism
  • Humans
  • Mice
  • Mice, Knockout
  • Mutagenesis, Site-Directed
  • Protein Structure, Tertiary
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Recombinant Proteins / metabolism
  • Signal Transduction / immunology*
  • Surface Plasmon Resonance
  • Synovial Membrane / cytology
  • Synovial Membrane / immunology
  • Synovial Membrane / metabolism


  • Antigens, Surface
  • Calreticulin
  • Epitopes
  • HLA-DRB1 Chains
  • Recombinant Proteins
  • Citrulline
  • Arginine