Product(s) used in this publication: Histone Code Tools
Objective: Anti-citrullinated protein antibodies (ACPA) are a hallmark of seropositive rheumatoid arthritis (RA). Yet, the precise disease-relevant autoantigen(s) targeted remains debated. By utilizing patient-derived monoclonal ACPA, rather than serum-analysis, we could characterize the multi-reactivity to different protein modifications and reveal autoantibody subsets.
Methods: A head-to-head comparison of 12 CCP2-positive human monoclonal ACPA generated from six RA patients was performed. We used a cDNA-based protein array (Engine) and three peptide screening platforms with RA autoantigens (Thermo Fisher Scientific), citrullinated (Cit) and carbamylated (Carb) peptides (NimbleGen/Roche), or histone-derived peptides with different post-translational modifications (JPT Histone Code), covering >207000 peptides (>7800 gene-products).
Result: The fine-specificity profiles of the investigated ACPA varied, but all mAbs displayed multi-reactivities to large numbers of citrullinated peptides/proteins without unspecific binding. ACPA subsets could be defined by clone-distinct consensus binding-motifs (e.g. Cit-Gly; Gly-Cit; Arg-Cit-Asp) with the most common mAb-recognition being a glycine in the +1 flanking position, but with additional amino acid preferences. For protein recognition, we observed a preference for citrullinated RNA-binding proteins with high arginine/glycine content. Six of 12 ACPA clones also bound acetylated lysine (KAc) or homocitrulline peptide-motifs with a similar or higher apparent affinity than for Cit-peptides.
Conclusion: ACPA and anti-modified protein autoantibodies (AMPA) represent overlapping facets of RA autoimmunity and bind to a wide variety of modified proteins, well beyond "historical" RA autoantigens. So far, KAc-reactivity was only detected in context of anti-Carb and anti-Cit, postulating autoreactivity hierarchies. Future investigations of ACPA fine-specificities and functionality should take the consensus Cit/Carb/KAc-motifs and multi-reactivity into consideration.