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<0.0001). PAD2 citrullination also lead to enhanced stability of proteases, with persistently detectable activity after 72 hours in-vitro, and evidence of protection from autoproteolysis in PR3 and NE, which was not observed with PAD4 (Figure 1B). Protection from Trypsin degradation in cit-PR3 was also observed. In the recombinant proteases, supernatant, and NETs, PAD2 citrullination opened new catalytic sites as demonstrated by labeling using an activity-based probe (Figure 1C), suggestive of a conformational change which was confirmed by in-silico modeling. Aggrecan degradation was enhanced by PAD2-citrullination of NE and NETs. Serum cit-PR3 and serum PR3 activity was higher in RA compared to healthy controls (Figure 1D). In synovial fluid, PR3, NE and CTG activity were all increased in RA compared to OA (Figure 1E). Autoantibodies to PAD4/2 cit-proteases were higher in RA and ACPA positive controls compared to ACPA negative controls (Figure 1F). Conclusion PAD enhances neutrophil serine protease activity and stability through conformational changes induced by citrullination, possibly providing a biological advantage for host defense. Enhanced proteolytic activity was apparent in RA biospecimens, leading to degradation of cartilage components and autoantibody formation. PAD isoforms may play functionally distinct roles in the pathogenesis of RA. Best Abstract on Research by Early Career Faculty Award