Fu Z , Thorpe M , Akula S , Chahal G , Hellman LT .

                pulmonary emphysema

	Figure 1. Phylogenetic analysis of the hematopoietic serine proteases. A phylogenetic tree of a large number of different vertebrate hematopoietic serine proteases using the MrBayes program. The proteases encoded within the metase locus are enlarged and highlighted and the three proteases analyzed in this communication, hNE, hPR-3, and Xenopus PR-3 are marked by red arrows.
	Figure 2. Analysis of the purified hNE, hPR-3, hCG, Xenopus PR-3, human thrombin (Th) and human granzyme B (GB) used in the chromogenic substrate assay and in the determination of the extended cleavage specificity. The three human neutrophil enzymes were commercial preparations purified from peripheral blood neutrophils. The Xenopus PR-3 was produced in the human cell line HEK293-EBNA. The xPR-3 proenzyme was first purified on Ni-NTA beads (–EK) and then activated by removal of the His6-tag by enterokinase digestion (+EK). The enzymes were analyzed by separation on SDS-PAGE and visualized with Coomassie Brilliant Blue staining.
	Figure 3. Chromogenic substrate assay. (A–K) A panel of different chromogenic substrates was used to determine the primary specificity of hNE, HPR-3, xPR-3, hCG, human granzyme B, and human thrombin. The panel included different chymase, elastase, tryptase and aspase substrates. The amino acid sequences of the substrates are listed on the left side of the figure. Human thrombin, hCG, and human granzyme B were included as reference enzymes for tryptase, chymase, and asp-ase activities, respectively.
	Figure 4. Phage displayed nonamers susceptible to cleavage by hNE, hPR-3 and hCG after five biopannings. After the last selection step, phages released by proteolytic cleavage of the three proteases were isolated and the sequences encoding the nonamers were determined. The general sequence of the T7 phage capsid proteins are PGG(X)9HHHHHH, where (X)9 indicates the randomized nonamers. The protein sequences were aligned into a P5-P4′ consensus, where cleavage occurs between positions P1 and P1′. Sequences occurring more than once are marked by the corresponding number to the right of the sequence. The aa are color coded according to the side chain properties as indicated in the legend.
	Figure 5. Analysis of the cleavage specificity of hNE by the use of recombinant protein substrates. (A) Shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by the different enzymes in Figures 5–10. In these substrates two thioredoxin (trx) molecules are positioned in tandem and the adjacent trx has a His6-tag positioned in its C terminus. The different cleavable sequences are inserted in the linker region between the two trx molecules by the use of two unique restriction sites, one Bam HI and one Sal I site, which are indicated in A. (B) A hypothetical cleavage is shown to highlight possible cleavage patterns. (C–E) Cleavage of recombinant substrates by hNE at different enzyme concentrations (50 or 500 ng). The name and sequence of the different substrates are indicated above the pictures of the gels. The time of cleavage in minutes is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of ~25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa. The cleavage of a panel of substrates with a more strict elastase, as represented by mouse mast cell protease 5, was included in (F).
	Figure 6. Analysis of the cleavage specificity of hNE by the use of recombinant protein substrates. (A–D) Shows the cleavages of a number of substrates by hNE. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in minutes is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of ~25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa.
	Figure 7. Analysis of the cleavage specificity of hPR-3 by the use of recombinant protein substrates. (A–C) Shows the cleavages of a number of substrates by hPR-3. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in minutes is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of ~25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa.
	Figure 8. Analysis of the cleavage specificity of Xenopus PR-3 by the use of recombinant protein substrates. (A–D) Shows the cleavages of a number of substrates by hPR-3. The sequences of the different substrates are indicated above the pictures of the gels. The time of cleavage in minutes is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of ~25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa.
	Figure 9. Analysis of the effect of different concentrations of SDS on the cleavage efficiency by five different enzymes, hPR-3, hNE, hCG, human thrombin and xPR-3. Consensus substrate sequences for the different enzymes were inserted in the 2x Trx substrates as shown in Figure 5. These substrates were then were used to analyze the effect of different SDS concentration on the cleavage efficiency by the five different enzymes. We used the substrate (VLLVSEVL) for hNE, the substrate (VLLFSEVL) for hCG, the substrate (VLLVSEVL) for hPR-3, the substrate (LTPRGVRL) for human thrombin and the substrate (VLLVSEVL) for Xenopus PR-3. Lane C for the different enzymes is a control without SDS. The concentrations of the SDS are then listed above the corresponding lane from 0.1 to 8%. The uncleared substrates have a molecular weight of ≈25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa. Due to the effect of the SDS to open the protein structure of the Trx molecules cleavage also occur within the Trx molecules, which results in additional bands of different molecular weights.
	Figure 10. Analysis of the difference in efficacy in cleavage of a number of consensus substrates identified through phage display and an MS-based method for four human neutrophil proteases. The cleavage of a number of consensus cleavage sites for four different human neutrophil proteases (N-elastase, hCG, hPR3, and NSP4) were studied with the 2x-Trx system. The “A” consensus sites come from phage display analyses performed in our lab and the substrates B and C comes from a proteomics study by O'Donoghue et al. (39). The substrates originating from the two different methods for N-elastase and proteinase 3 both show very good cleavage, whereas for hCG the consensus sites obtained by the proteomics method shows only minor cleavage, indicating a relatively poor site. No phage display has yet been performed on hNSP4, therefore only a proteomics site was studied where minor cleavage after using a relatively high concentration of the enzyme was seen.
	Figure 11. Alignment of a panel of NE, PR-3, and NSP-4 sequences. The sequences of a panel of NSP-4, NE, and PR3 sequences from mammals and amphibians were aligned using the Clustal W algorithm in the DNASTAR program together with a small subfamily of fish proteases that cluster between NSP-4 and NE and PR-3 in the phylogenetic tree shown (from Figure 1). The conserved cysteines are marked with red dots and the three residues of the catalytic triad, His-Asp-Ser, with larger green dots. The three residues with positions 189, 216, and 226 in bovine chymotrypsin are marked with purple arrows. These three residues, are in many trypsin related serine proteases, located in the bottom and the sides of the pocket of the protease where the P1 residue of the substrate is positioned upon cleavage.

Akula, Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution. 2015, Pubmed

Akula, Granule Associated Serine Proteases of Hematopoietic Cells - An Analysis of Their Appearance and Diversification during Vertebrate Evolution. 2015, Pubmed
Andersson, The extended cleavage specificity of the rodent beta-chymases rMCP-1 and mMCP-4 reveal major functional similarities to the human mast cell chymase. 2008, Pubmed
Andersson, Arg143 and Lys192 of the human mast cell chymase mediate the preference for acidic amino acids in position P2' of substrates. 2010, Pubmed
Belaaouaj, Degradation of outer membrane protein A in Escherichia coli killing by neutrophil elastase. 2000, Pubmed
Belaaouaj, Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. 1998, Pubmed
Campanelli, Azurocidin and a homologous serine protease from neutrophils. Differential antimicrobial and proteolytic properties. 1990, Pubmed
Chahal, The Importance of Exosite Interactions for Substrate Cleavage by Human Thrombin. 2015, Pubmed
Cowland, Granulopoiesis and granules of human neutrophils. 2016, Pubmed
Felsenstein, Evolutionary trees from DNA sequences: a maximum likelihood approach. 1981, Pubmed
Fu, Highly Selective Cleavage of Cytokines and Chemokines by the Human Mast Cell Chymase and Neutrophil Cathepsin G. 2017, Pubmed
Fu, rMCP-2, the Major Rat Mucosal Mast Cell Protease, an Analysis of Its Extended Cleavage Specificity and Its Potential Role in Regulating Intestinal Permeability by the Cleavage of Cell Adhesion and Junction Proteins. 2015, Pubmed
Gallwitz, The extended substrate recognition profile of the dog mast cell chymase reveals similarities and differences to the human chymase. 2010, Pubmed
Gallwitz, The extended cleavage specificity of human thrombin. 2012, Pubmed
Guyot, Unopposed cathepsin G, neutrophil elastase, and proteinase 3 cause severe lung damage and emphysema. 2014, Pubmed
Hahn, Cathepsin G and neutrophil elastase play critical and nonredundant roles in lung-protective immunity against Streptococcus pneumoniae in mice. 2011, Pubmed
Heutinck, Serine proteases of the human immune system in health and disease. 2010, Pubmed
Karlson, Rat mast cell protease 4 is a beta-chymase with unusually stringent substrate recognition profile. 2002, Pubmed
Karlson, Extended substrate specificity of rat mast cell protease 5, a rodent alpha-chymase with elastase-like primary specificity. 2003, Pubmed
Katoh, MAFFT multiple sequence alignment software version 7: improvements in performance and usability. 2013, Pubmed
Kawabata, The role of neutrophil elastase in acute lung injury. 2002, Pubmed
Kobayashi, Neutrophils in the innate immune response. 2005, Pubmed
Korkmaz, Neutrophil elastase, proteinase 3, and cathepsin G as therapeutic targets in human diseases. 2010, Pubmed
Korkmaz, Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions. 2008, Pubmed
Kurnikova, Four novel ELANE mutations in patients with congenital neutropenia. 2011, Pubmed
Laurell, Is emphysema in alpha 1 -antitrypsin deficiency a result of autodigestion? 1971, Pubmed
López-Boado, Neutrophil serine proteinases cleave bacterial flagellin, abrogating its host response-inducing activity. 2004, Pubmed
Massberg, Reciprocal coupling of coagulation and innate immunity via neutrophil serine proteases. 2010, Pubmed
Moraes, Proteases and lung injury. 2003, Pubmed
Nanua, Activation of the unfolded protein response is associated with impaired granulopoiesis in transgenic mice expressing mutant Elane. 2011, Pubmed
Neuhoff, Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. 1988, Pubmed
O'Donoghue, Global substrate profiling of proteases in human neutrophil extracellular traps reveals consensus motif predominantly contributed by elastase. 2013, Pubmed
Papayannopoulos, NETs: a new strategy for using old weapons. 2009, Pubmed
Penn, GUIDANCE: a web server for assessing alignment confidence scores. 2010, Pubmed
Pereira, CAP 37, a 37 kD human neutrophil granule cationic protein shares homology with inflammatory proteinases. 1990, Pubmed
Perera, NSP4, an elastase-related protease in human neutrophils with arginine specificity. 2012, Pubmed
Perera, Perspectives and potential roles for the newly discovered NSP4 in the immune system. 2012, Pubmed
Pham, Neutrophil serine proteases: specific regulators of inflammation. 2006, Pubmed
Polanowska, Specificity of human cathepsin G. 1998, Pubmed
Raymond, How immune peptidases change specificity: cathepsin G gained tryptic function but lost efficiency during primate evolution. 2010, Pubmed
Reeves, Killing activity of neutrophils is mediated through activation of proteases by K+ flux. 2002, Pubmed
Relle, Genetics and pathophysiology of granulomatosis with polyangiitis (GPA) and its main autoantigen proteinase 3. 2016, Pubmed
Ronquist, MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. 2012, Pubmed
Schechter, On the size of the active site in proteases. I. Papain. 1967, Pubmed
Standish, Human neutrophils kill Streptococcus pneumoniae via serine proteases. 2009, Pubmed
Steinwede, Cathepsin G and neutrophil elastase contribute to lung-protective immunity against mycobacterial infections in mice. 2012, Pubmed
Tanaka, Human leukocyte cathepsin G. Subsite mapping with 4-nitroanilides, chemical modification, and effect of possible cofactors. 1985, Pubmed
Thorpe, Extended cleavage specificity of the mast cell chymase from the crab-eating macaque (Macaca fascicularis): an interesting animal model for the analysis of the function of the human mast cell chymase. 2012, Pubmed
Thorpe, Channel catfish granzyme-like I is a highly specific serine protease with metase activity that is expressed by fish NK-like cells. 2016, Pubmed
Thorpe, Extended cleavage specificity of human neutrophil cathepsin G: A low activity protease with dual chymase and tryptase-type specificities. 2018, Pubmed
Tkalcevic, Impaired immunity and enhanced resistance to endotoxin in the absence of neutrophil elastase and cathepsin G. 2000, Pubmed
Walter, Cathepsin G in Experimental Tuberculosis: Relevance for Antibacterial Protection and Potential for Immunotherapy. 2015, Pubmed
Wiesner, Antimicrobial peptides: the ancient arm of the human immune system. 2010, Pubmed
Yang, New Insights into Neutrophil Extracellular Traps: Mechanisms of Formation and Role in Inflammation. 2016, Pubmed
van de Vosse, Severe congenital neutropenia in a multigenerational family with a novel neutrophil elastase (ELANE) mutation. 2011, Pubmed