Haag, S.S.HaagSchneider, N.N.SchneiderMason, D.E.D.E.MasonTuncel, J.J.TuncelAndersson, I.E.I.E.AnderssonPeters, E.C.E.C.PetersBurkhardt, H.H.BurkhardtHolmdahl, R.R.Holmdahl2022-03-042022-03-042014https://publica.fraunhofer.de/handle/publica/23884010.1002/art.38383Objective To investigate type II collagen (CII) as a joint-specific target of the anti-citrullinated protein antibody (ACPA) response in rheumatoid arthritis (RA). Methods Potential citrullinated neoepitopes were identified by high-resolution tandem mass spectrometry (MS/MS) of in vitro peptidylarginine deiminase 2 (PAD-2)-treated CII, and the relationship between citrullination and CII conformation was investigated by circular dichroism and conformation-dependent antibodies. Based on the MS analyses, synthetic peptides were designed and analyzed for serum IgG reactivity in the Epidemiological Investigation of RA (EIRA) case-control cohort of 1,949 RA patients and 278 healthy controls. Peptide-specific antibodies were purified from RA patient serum and used to stain RA cartilage specimens. Results We described the conformation-dependent citrullination pattern of CII after PAD-2 treatment at room temperature and 37°C and showed that CII could be citrullinated in its native triple-helical conformation. Screening of Arg and Cit pairs of synthetic peptides revealed new citrullinated B cell epitopes on CII. Antibodies directed to 2 proximal epitopes close to the C-terminus of the CII triple helix were recognized by autoantibodies in 21% and 17% of RA patients, respectively. Affinity-purified antibodies from RA sera directed to these 2 epitopes, but not antibodies directed to citrullinated a-enolase peptide 1, bound to RA cartilage. Conclusion These findings suggest that cartilage-directed anticitrulline immunity contributes to the induction of joint inflammation in RA.en540571572journal article