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

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.

Figure 1

Imaging of autoantibody binding to citrullinated and non-citrullinated protein arrays. Pooled plasma from 15 anti-CCP-positive RA patients or 10 anti-CCP-negative RA patients was diluted 1:200 and added to microarray plates containing 1631 human proteins that had been citrullinated by PAD2 or PAD4 or kept in native form. The binding of IgG antibodies was visualized using Cy3-labelled rabbit anti-human IgG antibodies. (A) Slide with proteins citrullinated by PAD2 and incubated with anti-CCP-positive plasma. (B) Proteins citrullinated by PAD4 incubated with anti-CCP-positive plasma. (C) Native proteins incubated with anti-CCP-positive plasma. (D) Proteins citrullinated by PAD2 incubated with anti-CCP-negative plasma. (E) Proteins citrullinated by PAD4 incubated with anti-CCP negative plasma (F). Native proteins incubated with anti-CCP-negative plasma. (C,F) have previously been published.

Figure 2

Quantitative analysis of arrayed proteins recognized by autoantibodies. Bar chart showing the number of proteins recognized by autoantibodies to a higher degree than native proteins. Plasma from anti-CCP-positive or anti-CCP-negative RA patients was incubated with Immunome protein microarray slides containing native proteins or proteins citrullinated by PAD2 or PAD4. The number above the bars indicates the number of proteins recognized by autoantibodies under the given conditions (defined as more than two-fold differences in fluorescence intensity compared to native proteins, an intraprotein CV < 15, and a P value < 0.05). Specific targets for autoantibodies are shown in Supplementary Dataset 2.

Figure 3

Immunome slide design and protein categories on the protein microarray. (A) The Immunome protein microarray consists of four identical subarrays, each containing 1631 different proteins in addition to 11 control proteins (BCCP, BSA, Cy3BSA, IgA, IgG, IgM, and four different control probes for the BCCP tag acting as negative controls). The control proteins are located between the subarrays. (B) The proteins spotted on the microarray represent different protein groups, such as cancer-associated antigens, transcription factors, kinases, and signaling proteins. The number of proteins in each category is shown. BCCP biotin carboxyl carrier protein, BSA bovine serum albumin.