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

Peter Sahlström, Monika Hansson, Johanna Steen, Khaled Amara, Philip J. Titcombe, Björn Forsström, Ragnhild Stålesen, Lena Israelsson, Luca Piccoli, Karin Lundberg, Lars Klareskog, Daniel L. Mueller, Anca I. Catrina, Karl Skriner, Vivianne Malmström, Caroline Grönwall

Research output: Contribution to journalArticlepeer-review


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 histone-derived 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 citrullinated 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 recognized set of RA autoantigens. So far, KAc reactivity has been detected only in the context of anti–carbamylated and anti–citrullinated peptide autoantibody responses, postulating the existence of hierarchies of autoreactivity in RA. Future investigations of ACPA fine specificities and functionality should take into consideration the presence of consensus Cit/Carb/KAc motifs and the multireactivity of these autoantibodies in patients with RA.

Original languageEnglish (US)
Pages (from-to)1643-1657
Number of pages15
JournalArthritis and Rheumatology
Issue number10
StatePublished - Oct 1 2020

Bibliographical note

Funding Information:
We are grateful to Ute Nonhoff and Zolt?n Konthur (Engine GmbH) for providing the macroarray analysis platform, Ulf Reimer (JPT Peptide Technologies) for the Histone Code array analysis platform, and Linda Mathsson-Alm (ImmunoDiagnostics) for providing the Thermo Fisher Scientific RA autoantigen array. We thank Heidi W?h?maa (Karolinska Institutet) for preparation of the polyclonal anti-CCP2, Lisa Liljefors and Anna Angelopoulou (Karolinska Institutet) for the modified antigens and ELISA assays, Madeline Jenning and Bianka Marklein (Charit?) for the antigen expression and macroarray validation assays, and Eric Meffre (Yale University) for providing positive and negative human IgG clones for the quality control polyreactivity/nonspecificity assays. We would also like to thank Holger Bang (Orgentec Diagnostika GmbH) for generously providing the modified peptide assays, reagents, and protocols, and UCB Pharma for their kind contribution of recombinant IgG.

Publisher Copyright:
© 2020 The Authors. Arthritis & Rheumatology published by Wiley Periodicals LLC on behalf of American College of Rheumatology

PubMed: MeSH publication types

  • Journal Article
  • Research Support, Non-U.S. Gov't


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