Citrullinated calreticulin potentiates rheumatoid arthritis shared epitope signaling

Authors

  • Song Ling,

    1. University of Michigan School of Medicine, Ann Arbor
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    • Drs. Ling and Holoshitz are named inventors on six patents and two pending patent applications for devices/substances/procedures related to rheumatoid arthritis. The patents are owned by the University of Michigan, and Drs. Ling and Holoshitz receive no licensing fees.

  • Erika N. Cline,

    1. University of Michigan School of Medicine, Ann Arbor
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  • Timothy S. Haug,

    1. University of Michigan School of Medicine, Ann Arbor
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  • David A. Fox,

    1. University of Michigan School of Medicine, Ann Arbor
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  • Joseph Holoshitz

    Corresponding author
    1. University of Michigan School of Medicine, Ann Arbor
    • University of Michigan, 5520D MSRB1, Box 0680, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5680
    Search for more papers by this author
    • Drs. Ling and Holoshitz are named inventors on six patents and two pending patent applications for devices/substances/procedures related to rheumatoid arthritis. The patents are owned by the University of Michigan, and Drs. Ling and Holoshitz receive no licensing fees.


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 Arg205, 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, 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 gene–environment interaction in RA.

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