Citrullination of Neutrophil Serine Proteases Enhances Proteolytic Activity, Stability and Autoantigenicity in Rheumatoid Arthritis

Objectives In Rheumatoid Arthritis (RA), the generation of a post-translational modification called citrulline leads the formation of autoantibodies (ACPA, anticitrullinated proteins antibodies). Although citrullination is pathogenic in RA, the underlying triggers and mechanisms driving this modification remains poorly understood. Neutrophils contain 2 major isoforms of PAD (2/4) and readily form citrullinated proteins during neutrophil extracellular trap (NETs) formation. We hypothesized that citrullination influences the proteolytic activity of neutrophil serine proteases, namely Neutrophil Elastase (NE), Proteinase-3 (PR3) and Cathepsin G (CTG). Methods The activity of proteases (NE, PR3, CTG) and various biospecimens were analyzed using fluorogenic substrate after citrullination with PAD2/4 (confirmed by a cit-specific probe). Active protease sites were labeled and visualized using a probe (TAMRA-FP). Degradation of Aggrecan, a proteoglycan found in cartilage, was determined using various biospecimens exposed to PAD isoforms. ACPA targeting cit-proteases was measured in serum from RA patients, ACPA positive and ACPA negative controls with ELISA. Results Using both recombinant proteases and neutrophil supernatant, proteolytic activity of NE, PR3 and CTG increased markedly after citrullination with PAD2, and to a lesser extent, PAD4 (Figure 1A). For example, the activity of cit-PR3 increased by 13-fold compared to native PR3. Citrullination level with increasing concentrations of PAD2 correlated strongly with proteolytic activity (R=0.95, p