Novel anti-citrullinated peptide autoantibodies identified by proteomics with in vitro citrullinated proteins in patients with rheumatoid arthritis

抄録

Rheumatoid arthritis (RA) is immunologically characterized by generation of anti-citrullinated peptide antibodies (ACPAs). To promote understanding of ACPAs, we here identified novel ACPA antigens by proteomics using in vitro citrullination and investigated effects of citrullination on one of the ACPA antigens. Specifically, we citrullinated protein extracts from Jurkat cells in vitro and separated the citrullinated protein extracts as well as non-treated ones by 2-dimensional electrophoresis (2DE). By western blotting, we detected protein spots that reacted to RA serum samples only when they were citrullinated and identified them by mass spectrometry. On two of the identified proteins, adenosine deaminase (ADA) 1 and proliferation associated protein 2G4 (PA2G4), we confirmed the citrulline-dependent recognition by ACPAs using enzyme-linked immunosorbent assay (ELISA). Finally, we compared the activity of citrullinated ADA1 and non-treated one. As a result, we detected 8 candidate antigens for ACPAs, and identified 7 of them. In ELISA, 6 (24%) out of the tested 25 RA serum samples reacted to citrullinated ADA1, but none (0%) to non-treated ADA1 (p=0.022). Similarly, 9 (36.0%) out of the same RA samples reacted to citrullinated PA2G4, but only 1 (4.0%) to non-treated PA2G4 (p=0.01). In addition, we found that citrullination enhanced activity of ADA. In conclusion, we demonstrated novel autoantigens for ACPAs including ADA1 and PA2G4 and enhancement of the ADA activity by citrullination. The enhanced ADA activity may decrease anti-inflammatory adenosine and thus may hamper the pharmaceutical increase of adenosine by methotrexate in RA.