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


CONCLUSIONS
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.
Andrade
Felipe
F
Division of Rheumatology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21224, USA. andrade@jhmi.edu
Darrah
Erika
E
Gucek
Marjan
M
Cole
Robert N
RN
Rosen
Antony
A
Zhu
Xiaoming
X
D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000378:metabolism; D001323:Autoantibodies / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D006801:Humans; D006867:Hydrolases / Q000276:immunology / Q000378:metabolism*; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism*; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases
D001323:Autoantibodies; D006867:Hydrolases; C000629235:PADI4 protein, human; D000080002:Protein-Arginine Deiminase Type 4; D000076342:Protein-Arginine Deiminases