Autocitrullination of human peptidyl arginine deiminase type 4 regulates protein citrullination during cell activation

Arthritis Rheum. 2010 Jun;62(6):1630-40. doi: 10.1002/art.27439.


Objective: To address mechanisms that control the activity of human peptidyl arginine deiminase type 4 (PAD-4).

Methods: PAD-4 autocitrullination was determined by anti-modified citrulline immunoblotting, using purified recombinant and endogenous PAD-4 from activated human primary neutrophils and cell lines expressing PAD-4. The citrullination sites in PAD-4 were determined by mass spectrometry. Mechanisms of autocitrullination-induced inactivation and the functional consequences of autocitrullination in PAD-4 polymorphic variants were addressed using purified components and cell lines expressing PAD-4 wild-type, PAD-4 mutant, and PAD-4 polymorphic variants relevant to rheumatoid arthritis (RA).

Results: PAD-4 is autocitrullinated in vitro and during activation of primary cells and cell lines expressing PAD-4. Interestingly, this modification inactivated the function of the enzyme. The efficiency of inactivation differed among genetically defined PAD-4 variants relevant to RA. PAD-4 was citrullinated at 10 sites, which are clustered into 3 distinct regions, including a cluster of arginines around the active site cleft where Arg-372 and -374 were identified as the potential autocitrullination targets that inactivate the enzyme. Autocitrullination also modified the structure of PAD-4, abrogating its recognition by multiple rabbit antibodies, but augmenting its recognition by human anti-PAD-4 autoantibodies.

Conclusion: Our findings suggest that autocitrullination regulates the production of citrullinated proteins during cell activation, and that this is affected by structural polymorphisms in PAD-4. Autocitrullination also influences PAD-4 structure and immune response.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arthritis, Rheumatoid / immunology
  • Arthritis, Rheumatoid / metabolism
  • Autoantibodies / immunology
  • Autoantibodies / metabolism
  • Blotting, Western
  • Cell Line
  • Cells, Cultured
  • Humans
  • Hydrolases / immunology
  • Hydrolases / metabolism*
  • Immunoprecipitation
  • Mass Spectrometry
  • Neutrophils / immunology
  • Neutrophils / metabolism*
  • Protein-Arginine Deiminase Type 4
  • Protein-Arginine Deiminases


  • Autoantibodies
  • Hydrolases
  • PADI4 protein, human
  • Protein-Arginine Deiminase Type 4
  • Protein-Arginine Deiminases