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Updated: Jun 15, 2026

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog
Published on: November 5, 2014
Autocitrullination of human peptidyl arginine deiminase type 4 regulates protein citrullination during cell
Felipe Andrade1, Erika Darrah, Marjan Gucek
1Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA. andrade@jhmi.edu
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.
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