AbstractObjectiveTo address mechanisms that control the activity of human peptidyl arginine deiminase type 4 (PAD‐4).MethodsPAD‐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).ResultsPAD‐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.ConclusionOur 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.