title: Citrullinated calreticulin potentiates rheumatoid arthritis shared epitope signaling.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: University of Michigan School of Medicine, Ann Arbor, MI 48109-5680, USA.
authors.forename: Song
authors.initials: S
authors.lastname: Ling
authors.forename: Erika N
authors.initials: EN
authors.lastname: Cline
authors.forename: Timothy S
authors.initials: TS
authors.lastname: Haug
authors.forename: David A
authors.initials: DA
authors.lastname: Fox
authors.forename: Joseph
authors.initials: J
authors.lastname: Holoshitz
mesh_terms: D000818:Animals; D000954:Antigens, Surface / Q000276:immunology / Q000378:metabolism; D001120:Arginine / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000378:metabolism*; D001665:Binding Sites / Q000276:immunology; D037282:Calreticulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D002956:Citrulline / Q000737:chemistry / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology / Q000378:metabolism*; D005347:Fibroblasts / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D059647:Gene-Environment Interaction; D059811:HLA-DRB1 Chains / Q000276:immunology / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology*; D020349:Surface Plasmon Resonance; D013583:Synovial Membrane / Q000166:cytology / Q000276:immunology / Q000378:metabolism
chemical_list: D000954:Antigens, Surface; D037282:Calreticulin; D000939:Epitopes; D059811:HLA-DRB1 Chains; D011994:Recombinant Proteins; D002956:Citrulline; D001120:Arginine