The rheumatoid arthritis citrullinome is enriched in antigenic complement proteins

TL;DR

This study finds that citrullinated complement proteins, especially C9, are more common in rheumatoid arthritis patients and may trigger immune responses.

Contribution

The study identifies citrullinated complement proteins as novel autoantigens in rheumatoid arthritis.

Findings

Citrullinated C9 and CFI levels are higher in RA patients compared to controls.

Autoantibodies to cit-C9 are elevated in RA patients and ACPA+ first-degree relatives.

Structural changes in cit-C9 due to citrullination were observed using in-silico modeling.

Abstract

Citrullination is a post-translational modification that serves as an autoantigen in Rheumatoid Arthritis (RA). It remains unclear whether there is an excess production of specific citrullinated proteins in RA patients that is driving the autoantibody response. The aim of this study was to undertake an unsupervised proteomic analysis of the circulating RA citrullinome, and determine the consequences of citrullination in RA.

Citrullinated serum proteins were isolated and measured by mass spectrometry from 10 anti-citrullinated protein antibody (ACPA) positive RA patients and 10 controls. Cit-proteins and cit-immune complexes (IC) were measured in a larger cohort of unaffected first-degree relatives (FDR, ACPA- n = 31, ACPA+ n = 26) and RA (n = 31). Autoantibodies to citrullinated C9, and linear peptides of citrullinated C9 were measured. Structural changes imparted by citrullination were studied in-silico using Alphafold 3.0.

Differential analysis of the RA citrullinome revealed a higher expression of complement proteins. Validation studies revealed that the levels of cit-C9 (p = 0.02) and cit-CFI (Complement Factor I, p = 0.019) were higher in RA than controls. Cit-C9 IgG, but not cit-CFI, immune complexes were elevated in ACPA+ FDR (p < 0.0001) and RA (p < 0.0001) compared to ACPA- FDR. Autoantibodies to cit-C9 were also higher in ACPA+ FDR (p = 0.01) and RA (p = 0.002). Reactivity to linear peptides of cit/homocit C9 were enriched in RA patients compared to controls. In-silico modelling revealed structural changes to the monomeric and multimeric structure of cit-C9.

This analysis of the circulating citrullinome in RA reveals the generation of citrullinated complement proteins that serve as autoantigens.

The online version contains supplementary material available at 10.1186/s13075-026-03750-9.

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