Antihomocitrullinated Fibrinogen Antibodies are Specific to Rheumatoid Arthritis and Frequently Bind Citrullinated Proteins/peptides

Abstract

Anticitrullinated protein/peptide antibodies (ACPA) are implicated in rheumatoid arthritis (RA) pathogenesis and linked to the shared epitope (SE). Citrulline modification is very similar to a different modified amino acid, homocitrulline. We investigated antihomocitrullinated protein/ peptide antibody (AHCPA) specificity for RA, whether ACPA were also able to bind homocitrullinated targets, and whether the SE could accommodate homocitrullinated peptide. Homocitrullinated fibrinogen was used to screen sera from patients with RA, psoriatic arthritis, and systemic lupus erythematosus, and healthy subjects for AHCPA using ELISA. Homocitrullination sites on fibrinogen were identified by mass spectrometry. ACPA were affinity-purified using a synthetic citrullinated peptide and tested for binding to homocitrullinated protein/peptide. Inhibition of antihomocitrullinated fibrinogen antibody binding was examined. Homocitrullinated peptide interaction with the SE was studied using computer modeling. IgG antihomocitrullinated fibrinogen antibodies were found specifically in 49% of patients with RA. Enrichment of ACPA by affinity purification from 5 patients with RA also enriched AHCPA. Serum AHCPA was inhibited by citrullinated fibrinogen and more significantly by homocitrullinated fibrinogen. Computer modeling indicated that the SE could accommodate a homocitrullinated peptide without steric hindrance. Mass spectrometry identified that 89/103 lysines of fibrinogen could be homocitrullinated, and 5 peptides that could be both citrullinated and homocitrullinated and are predicted to bind the SE. Antihomocitrullinated fibrinogen antibodies are specific to RA. The presence of AHCPA coincides with ACPA, and AHCPA copurifies with ACPA in affinity purification and is inhibited by citrullinated and homocitrullinated antigens. Thus AHCPA and ACPA are frequently cross-reactive and homocitrullinated proteins/peptides may bind the SE.