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.

Figure 1. Monitoring of proteolytic activity in neutrophil extracellular traps.

A. Identification of an internally quenched fluorescent substrate that is hydrolyzed by NE, CG and PR3. B. Extracellular proteolytic activity was analyzed from 3 donor neutrophils (Donor 1, dark grey; Donor 2, black; Donor 3, light grey) following treatment with PMA, MNase or a combination of both. Proteolytic activity was measured using (K-Amc) PLGKQVEY(K-Dnp).

Figure 2. Determination of the proteolytic signatures in NETs.

A-C. IceLogos representing the P4 to P4′ sites for NETs isolated from three donor samples. Amino acids that are most frequently observed (above axis) and least frequently observed (below axis) are illustrated. The number of cleavage sites used to make each iceLogo are listed in the bottom right-hand corner. Residues that are highlighted in black text are significantly (p = 0.05) enriched relative to the frequency that these same amino acids are found in the peptide library (5.2 +/- 0.5%). The amino acid ‘n’ corresponds to norleucine. D. Determination of the number of cleavage sites that are common and unique to each donor sample. E. Positional frequency of all donor-derived cleavage sites within the tetradecapeptides (n=85).

Figure 3. Substrate specificity profiling of human Neutrophil Elastase A.

Positional scanning of the P4 to P1 subsites of NE using the PS-SCL assay. B An iceLogo illustrating amino acids that are most frequently (above axis) and least frequently (below axis) observed in the P4 to P4′ sites of NE. Residues that are highlighted in black are significantly (p = 0.05) enriched or de-enriched in the subsites relative to the frequency that these same amino acids are found in the peptide library (5.2 +/- 0.5%). C. A representative “donor signature” consisting of 40 cleavage sites that are common to the three donors. D. A pie chart representing the 40 cleavage sites that are common to the donor samples. 33 of these sites are also hydrolyzed by NE.

Figure 4. Determination of proteolytic signature of PR3, CG and NSP4.

A-C. IceLogos representing the P4 to P4′ sites for PR3, CG and NSP4. Amino acids that are most frequently observed (above axis) and least frequently observed (below axis) are illustrated. The number of cleavage sites used to make each iceLogo are listed in the bottom right-hand corner. Residues that are highlighted in black text are significantly (p ≤ 0.05) enriched relative to the frequency that these same amino acids are found in the peptide library (5.2 +/- 0.5%). The amino acid ‘n’ corresponds to norleucine. D. A 4-way venn diagram illustrating the number of unique and overlapping peptide bonds that are cleaved by each neutrophil serine protease. E. A pie chart representing the 40 cleavage sites that are common to the donor samples. ‘Shared’ corresponds to cleavage sites that are derived from more than one neutrophil serine protease.

Figure 5. Determination of the proteolytic signatures in NE-depleted NETs.

A. After immunodepletion of NE from donor NET samples, the MSP-MS assay was performed at 15-fold higher total protein concentration. A total of 153 cleaved bonds were identified in the 3 donor samples and 76 cleaved bonds were common to each donor. B. An iceLogo illustrating the proteolytic signature of cleavage sites that are common to each NE-depleted donor. C. A pie chart representing the 76 cleavage sites that are common to the donor samples. ‘Shared’ corresponds to cleavage sites that are derived from more than one neutrophil serine protease.