Peptidylarginine deiminase 2, 3 and 4 have distinct specificities against cellular substrates: novel insights into autoantigen selection in rheumatoid arthritis
- 1Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
- 2Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA
- 3Division of Rheumatology, Columbia University, College of Physicians and Surgeons, New York, New York, USA
- Correspondence to Felipe Andrade, Division of Rheumatology, Department of Medicine, Johns Hopkins University School of Medicine, 5200 Eastern Avenue, Mason F. Lord Bldg, Center Tower, Suite 6000, Room 608, Baltimore MD 21224, USA;
- Accepted 21 July 2011
- Published Online First 21 August 2011
Objective To define the relationship between autoantigen citrullination and different peptidylarginine deiminase (PAD) enzymes in rheumatoid arthritis (RA).
Methods Citrullinated autoantigens were identified by immunoblotting control and ionomycin-activated human primary neutrophil lysate with RA sera. Autoantigen identity and citrullination sites were defined by mass spectrometry. PAD isoenzyme expression in human neutrophils was determined by immunoblotting. PAD substrate specificity was addressed in HL-60 cell lysates co-incubated with human recombinant PAD2, PAD3 and PAD4.
Results Although prominent protein citrullination is observed in ionomycin-activated neutrophils, RA sera only recognised a limited number of these citrullinated molecules. Among these, the authors identified that β and γ-actins are citrullinated on at least 10 arginine residues, generating a novel 47 kDa species that is frequently recognised by RA autoantibodies. Interestingly, the authors showed that the PAD enzymes expressed in human neutrophils (ie, PAD2, PAD3 and PAD4) have unique substrate specificities, independent of their subcellular distribution. Thus, only PAD2 was able to citrullinate native β/γ-actin, while histone H3 was only citrullinated by PAD4.
Conclusion These studies identified β and γ-actins as novel citrullinated autoantigens in RA, allowing enzyme specificity against intracellular substrates to be addressed. The studies provide evidence that PAD enzymes have the intrinsic capacity to select unique protein targets. The authors propose that unique PAD specificity may play a role in autoantigen selection in RA.
Competing interests None.
Patient consent Obtained.
Ethics approval This study was conducted with the approval of the Johns Hopkins Institutional Review Board.
Provenance and peer review Not commissioned; externally peer reviewed.