Background/Purpose: Anti-citrullinated protein antibodies (ACPAs) and rheumatoid factor (RF) are the main diagnostic and pathologic autoantibodies in rheumatoid arthritis. However, autoantibodies develop against proteins with other post-translational modifications, such as homocitrullination, as well as against various native proteins without obvious unifying characteristics of the antigens. Moreover, some rheumatoid arthritis patients generate only RF or only ACPAs (as detected by the anti-cyclic citrullinated peptide (CCP) test) and some produce neither. We have a poor understanding of the autoantibody repertoire in these patients. Our objective was to broadly evaluate autoantibody binding in seronegative and seropositive rheumatoid arthritis to identify general and novel features of autoantibody reactivity.

Methods: An array was created with 172,828 overlapping peptides derived from 122 proteins citrullinated in the rheumatoid joint including variants with arginines or lysines replaced with citrullines or homocitrullines, respectively. IgG and IgM binding to peptides was compared for CCP+RF+, CCP+RF-, CCP-RF+, and CCP-RF- rheumatoid arthritis versus controls. Highly bound peptides were analyzed for amino acid patterns and predictors of structural disorder. Published synovial fluid citrullinomes were also analyzed for amino acid patterns.

Results: Broadly, CCP+RF+ subjects had very high and CCP+RF- and CCP-RF+ subjects had modest citrulline-specific IgG binding. All rheumatoid arthritis groups had low homocitrulline-specific IgG binding and CCP+RF+ subjects had high IgG binding to native peptides. CCP-RF- subjects had very little antibody binding in general. By far, the highest IgG binding in rheumatoid arthritis was to citrulline-containing peptides, irrespective of protein identity, especially if the citrulline was adjacent to glycine or serine. Citrullinated arginines in the rheumatoid joint were also frequently next to glycine and serine. The most highly bound citrulline-containing and native peptides had multiple features predictive of conformational disorder.

Conclusion: In general, we observed a continuum of autoantibody binding in rheumatoid arthritis with a very high amount of binding in CCP+RF+ subjects, modest binding in CCP+RF- and CCP-RF+ subjects, and very little binding in CCP-RF- subjects. Further, citrulline, particularly next to serine or glycine, is a major driver of autoantibody reactivity in rheumatoid arthritis, likely due in part to frequent in vivo citrullination of arginine next to serine or glycine. Finally, structural disorder may be a newly discovered feature that drives rheumatoid arthritis autoantibodies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/autoantibodies-in-rheumatoid-arthritis-target-citrulline-containing-and-native-epitopes-from-conformationally-disordered-regions-of-proteins/