Identification of new citrulline-specific autoantibodies, which bind to human arthritic cartilage, by mass spectrometric analysis of citrullinated type II collagen.
OBJECTIVE
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 α-enolase peptide 1, bound to RA cartilage.


CONCLUSIONS
These findings suggest that cartilage-directed anticitrulline immunity contributes to the induction of joint inflammation in RA.
Karolinska Institutet, Stockholm, Sweden.
Sabrina
S
Haag
Nadine
N
Schneider
Daniel E
DE
Mason
Jonatan
J
Tuncel
Ida E
IE
Andersson
Eric C
EC
Peters
Harald
H
Burkhardt
Rikard
R
Holmdahl
D000818:Animals; D001172:Arthritis, Rheumatoid / Q000378:metabolism*; D001323:Autoantibodies / Q000378:metabolism*; D002358:Cartilage, Articular / Q000378:metabolism*; D016022:Case-Control Studies; D002956:Citrulline / Q000276:immunology*; D024043:Collagen Type II / Q000187:drug effects / Q000378:metabolism*; D000939:Epitopes / Q000276:immunology; D006801:Humans; D006867:Hydrolases / Q000494:pharmacology; D007074:Immunoglobulin G / Q000378:metabolism; D066298:In Vitro Techniques; D000076342:Protein-Arginine Deiminases; D051381:Rats; D053719:Tandem Mass Spectrometry
D001323:Autoantibodies; D024043:Collagen Type II; D000939:Epitopes; D007074:Immunoglobulin G; D002956:Citrulline; D006867:Hydrolases; D000076342:Protein-Arginine Deiminases