Different Hierarchies of Anti-Modified Protein Autoantibody Reactivities in Rheumatoid Arthritis

Objective. Anti-citrullinated protein antibodies (ACPAs) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease-relevant autoantigens that are targeted by ACPAs remains a matter of debate. This study utilized patient-derived monoclonal ACPAs, rather than serum autoantibody analysis, to characterize the multireactivity to different protein modifications and to reveal autoantibody subsets in patients with RA. Methods. Twelve human monoclonal ACPAs (positive by the second-generation cyclic citrullinated peptide test) were generated from 6 RA patients, and a head-to-head comparison of their reactivities was performed. For profiling, we used a complementary DNA-based protein array (Engine GmbH) and 3 peptide-screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated and carbamylated peptides (NimbleGen/Roche), or histonederived peptides with different posttranslational modifications (JPT Histone Code), covering >207,000 peptides (>7,800 gene products). Results. The fine-specificity profiles of the investigated ACPAs varied, but all of the monoclonal ACPAs displayed multireactivity to a large number of citrullinated peptides/proteins, each characterized by specific binding properties. ACPA subsets could be defined by clone-distinct consensus binding motifs (e.g., Cit-Gly, Gly-Cit, or Arg-Cit-Asp), with the most common ACPA recognition being that of a Gly in the +1 flanking position, but with additional amino acid preferences. For ACPA protein recognition, we observed a preference for citrullinated RNA-binding proteins with high Arg/Gly content. Six of the 12 ACPA clones also bound acetylated-lysine (KAc) or homocitrulline peptide motifs, displaying a similar affinity or higher apparent affinity than that for Cit peptides. Conclusion. ACPAs and anti-modified protein autoantibodies represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, extending well beyond the historically r
QC 20201201