Global Substrate Profiling of Proteases in Human Neutrophil Extracellular Traps Reveals Consensus Motif Predominantly Contributed by Elastase

Abstract

Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a “proteolytic signature” from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.

Cystic Fibrosis Sputum DNA Has NETosis Characteristics and Neutrophil Extracellular Trap Release Is Regulated by Macrophage Migration-Inhibitory Factor

“…22 proteins were unique for the PA581 -stimulated NETs (Figure 2) and 39 proteins were unique for the 2192 NETs (Figure 2). Functional analysis of the proteome showed that the P. aeruginosa -induced NETs were decorated with the “classical” NET members (e.g., histones, elastase, lysosyme, myeloperoxidase, superoxide dismutase) similar to the previously published PMA-induced NETs [26]. The NET proteins could be segregated into different functional categories such as nucleic acid binding, enzymes, structural proteins, and proteins with previously reported antimicrobial activity (Supplementary Table 1).…”

“…Fractions enriched for HMW DNA and NE were pooled and analyzed. While 24 NET-associated proteins were previously reported [24–26], the number of proteins identified in this study ranged from 45 to 80 depending on the nature of the stimuli (Supplementary Table 1). There were 33 common NET-associated proteins when NETosis was triggered by the three different P. aeruginosa strains.…”

TAILS N-terminomics and proteomics reveal complex regulation of proteolytic cleavage by O-glycosylation

“…Together these results indicate that O-glycosylation occurs more frequently than expected around selected MMPs and selected serine protease cleavage sites. protease-specific peptides in this dataset ( figure 3B) (48,49). Comparison of these N-termini to those reported in the TopFIND database indicated that the majority of sites were novel.…”

Distinct Susceptibilities of Corneal Pseudomonas aeruginosa Clinical Isolates to Neutrophil Extracellular Trap-Mediated Immunity

“…To functionally prove that the cytotoxic PA14 strain induced NETosis, separate experiments were carried out with differentiated HL-60 cells (used as a model system for neutrophil responses) exposed to P. aeruginosa strain PA14 (MOI, 10) in the presence or absence of DNase I. Based on prior studies, it was expected that the addition of DNase I will fragment NET DNA and liberate active NE (26,27). Consistent with this expectation, the extracellular proteome during PA14 infection should be significantly altered in the presence or absence of released NE.…”