Identification of potential autoantigens in anti-CCP-positive and anti-CCP-negative rheumatoid arthritis using citrulline-specific protein arrays

Thomas B G Poulsen,Dres Damgaard,Ladislav Senolt,Jonathan M Blackburn,Malene M Jørgensen,Allan Stensballe,Claus H Nielsen

doi:10.1038/s41598-021-96675-z

Abstract

The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis. The heterogeneous responses of RA patients to various biologics, even among ACPA-positive patients, emphasize the need for further stratification of the patients. We used high-density protein array technology for fingerprinting of ACPA reactivity. Identification of the proteome recognized by ACPAs may be a step to stratify RA patients according to immune reactivity. Pooled plasma samples from 10 anti-CCP-negative and 15 anti-CCP-positive RA patients were assessed for ACPA content using a modified protein microarray containing 1631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PADs) 2 and PAD4. IgG antibodies from anti-CCP-positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Curiously, the binding of IgG antibodies in anti-CCP-negative plasma was also enhanced by PAD2- and PAD4-mediated citrullination of 29 and 26 proteins, respectively. For only four proteins, significantly more ACPA binding occurred after citrullination with PAD2 compared to citrullination with PAD4, while the opposite was true for one protein. We demonstrate that PAD2 and PAD4 are equally efficient in generating citrullinated autoantigens recognized by ACPAs. Patterns of proteins recognized by ACPAs may serve as a future diagnostic tool for further subtyping of RA patients.

Highlights

The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis
We examined the binding of IgG antibodies in pools of plasma from anti-CCP-positive and anti-CCP-negative RA patients to microarrays containing 1631 human proteins in native form or citrullinated on-slide using PAD2 or PAD4 as catalysts
After incubation with a pool of anti-CCP-positive plasma, 632 proteins showed more than twofold higher binding of IgG after citrullination with PAD2 than in their native form, and the corresponding number was 629 proteins after citrullination with PAD4, suggesting that these proteins were recognized by anti-citrullinated protein antibodies (ACPAs) (Fig. 2, Supplementary Dataset 2)

Summary

Introduction

The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis. Pooled plasma samples from 10 anti-CCP-negative and 15 anti-CCP-positive RA patients were assessed for ACPA content using a modified protein microarray containing 1631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PADs) 2 and PAD4. IgG antibodies from anti-CCP-positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Two-thirds of RA patients produce anti-citrullinated protein antibodies (ACPAs)[2,3] These autoantibodies may be present years before the onset of clinical symptoms, underlining their possible involvement in the pathogenesis of early RA and may serve as early biomarkers[4,5]. Utilizing the high-throughput capacity of protein microarrays, we performed an exploratory study quantifying the binding of autoantibodies in plasma pools from well-characterized RA patients to more than 1600 different human proteins in citrullinated and non-citrullinated forms

Methods

Results

Discussion

Conclusion