Citrullination enhances the activity, stability and autoantigenicity of serine proteases in Rheumatoid Arthritis

Abstract Protein citrullination, catalysed by peptidyl arginine deiminase (PAD) enzymes, is the primary target of autoantibodies in rheumatoid arthritis (RA), yet the functional consequences of citrullination have remained unexplored. Using the serine protease trypsin as a model system, we show that citrullination increases maximum velocity and catalytic rate, stability, while preserving substrate affinity. These findings extend to neutrophil serine proteases (NSP) proteinase-3, neutrophil elastase and cathepsin G, where PAD-mediated citrullination enhances activity and stability across both purified and neutrophil-derived preparations. We also demonstrate that citrullinated neutrophil serine proteases are recognised as autoantigens in RA, forming immune complexes with serum from RA patients. Further elevated NSP activity at mucosal sites is associated with autoantibody production in individuals at-risk to develop future RA. In the joint, citrullinated neutrophil elastase drives enhanced aggrecan degradation, while citrullinated proteinase-3 activates fibroblast-like synoviocytes via protease activated receptor-2 signalling. PAD inhibition significantly attenuates citrullination-mediated NSP activation in recombinant enzymes and human RA synovial fluid, suggesting a potential role for pharmacological targeting of PAD to treat RA. Overall, we demonstrate that PAD-mediated citrullination of serine proteases leads to a gain-of-function phenotype, enhancing catalytic activity and conferring resistance to degradation.