High-Titer Rheumatoid Arthritis Antibodies Preferentially Bind Fibrinogen Citrullinated by Peptidylarginine Deiminase 4

Objective. Most patients with rheumatoid arthritis (RA) harbor antibodies to citrullinated autoantigens such as citrullinated fibrinogen. Two isoforms of peptidylarginine deiminase (PAD), PAD type 2 (PAD2) and PAD4, which catalyze citrullination with different substrate specificities, can be detected in the synovium of RA patients. This study was undertaken to determine whether RA antibodies preferentially bind PAD2-or PAD4-citrullinated fibrinogen. Methods. RA patient and normal donor plasma specimens were tested for binding to PAD2-or PAD4-citrullinated fibrinogen, native fibrinogen, or citrullinated fibrinogen peptides in various dilutions by enzyme-linked immunosorbent assay (ELISA) and Western blotting. Bands corresponding to masses demonstrating RA antibody reactivity by Western blotting were excised and analyzed bymass spectrometry. Results. At low antibody titers (1: 40 and 1: 100), there was no significant difference between RA antibody reactivity to PAD2-and PAD4-citrullinated fibrinogen. When plasma was further diluted to 1: 250 and 1: 1,000, RA patient plasma bound PAD4-citrullinated fibrinogen significantly more than PAD2-citrullinated fibrinogen, as measured by ELISA and Western blotting. An increased antibody titer was associated with increased avidity for both PAD2-and PAD4-citrullinated fibrinogen. Both enzymes hypercitrullinated fibrinogen, but PAD4 citrullinated arginines more intermittently, generating a mix of citrullinated and noncitrullinated arginines. Peptide ELISA and preadsorption assays confirmed that the region of intermittent citrullination accounts for the majority of RA antibody binding to the beta-chain of citrullinated fibrinogen. Conclusion. At high titers, RA antibodies preferentially bind fibrinogen modified by PAD4, because intermittent citrullination offers a more diverse assortment of citrullinated epitopes.