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

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1630-1640
Number of pages11
JournalArthritis and rheumatism
Volume62
Issue number6
DOIs
StatePublished - Jun 2010

ASJC Scopus subject areas

  • Immunology and Allergy
  • Rheumatology
  • Immunology
  • Pharmacology (medical)

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