Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities

It is widely accepted that neutrophil serine proteases (NSPs) play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases. To investigate NSP pathogenic role(s), various mouse experimental models have been developed that mimic acutely or chronically injured human lungs. We and others are using mouse exposure to cigarette smoke as a model for chronic obstructive pulmonary disease with or without exacerbation. However, the relative contribution of NSPs to lung disease processes as well as their underlying mechanisms remains still poorly understood. And the lack of purified mouse NSPs and their specific substrates have hampered advances in these studies. In this work, we compared mouse and human NSPs and generated three-dimensional models of murine NSPs based on three-dimensional structures of their human homologs. Analyses of these models provided compelling evidence that peptide substrate specificities of human and mouse NSPs are different despite their conserved cleft and close structural resemblance. These studies allowed us to synthesize for the first time novel sensitive fluorescence resonance energy transfer substrates for individual mouse NSPs. Our findings and the newly identified substrates should better our understanding about the role of NSPs in the pathogenesis of cigarette-associated chronic obstructive pulmonary disease as well as other neutrophils-associated inflammatory diseases. It is widely accepted that neutrophil serine proteases (NSPs) play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases. To investigate NSP pathogenic role(s), various mouse experimental models have been developed that mimic acutely or chronically injured human lungs. We and others are using mouse exposure to cigarette smoke as a model for chronic obstructive pulmonary disease with or without exacerbation. However, the relative contribution of NSPs to lung disease processes as well as their underlying mechanisms remains still poorly understood. And the lack of purified mouse NSPs and their specific substrates have hampered advances in these studies. In this work, we compared mouse and human NSPs and generated three-dimensional models of murine NSPs based on three-dimensional structures of their human homologs. Analyses of these models provided compelling evidence that peptide substrate specificities of human and mouse NSPs are different despite their conserved cleft and close structural resemblance. These studies allowed us to synthesize for the first time novel sensitive fluorescence resonance energy transfer substrates for individual mouse NSPs. Our findings and the newly identified substrates should better our understanding about the role of NSPs in the pathogenesis of cigarette-associated chronic obstructive pulmonary disease as well as other neutrophils-associated inflammatory diseases. IntroductionNeutrophil serine proteases (NSPs), 3The abbreviations used are: NSPneutrophil serine proteaseCOPDchronic obstructive pulmonary diseasehNEhuman neutrophil elastasehPr3human proteinase 3hCGhuman cathepsin GmNEmouse neutrophil elastasemPr3mouse proteinase 3mCGmouse cathepsin Gα1-Piα 1-protease inhibitorACTantichymotrypsinFRETfluorescence resonance energy transferHPLChigh-performance liquid chromatography. neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (Pr3), are mainly stored in neutrophil primary granules in readily active forms. NSPs are structurally related and share the conserved charge-relay triad, His57–Asp102–Ser195, where Ser is the active residue (chymotrypsinogen numbering) (1.Bode W. Meyer Jr., E. Powers J.C. Biochemistry. 1989; 28: 1951-1963Crossref PubMed Scopus (381) Google Scholar). NSPs contribute to neutrophil oxygen-independent system-mediated protection of the host against invading pathogens (2.Ganz T. Proc. Assoc. Am. Physicians. 1999; 111: 390-395Crossref PubMed Scopus (22) Google Scholar). Indeed, NSPs serve a physiological role for killing of microbes (3.Belaaouaj A. Microbes Infect. 2002; 4: 1259-1264Crossref PubMed Scopus (65) Google Scholar). Activated neutrophils are also known to release NSPs in the setting of inflammation. In vitro, NSPs are capable of cleaving a panoply of substrates. These include extracellular matrix proteins, pro-inflammatory mediators, coagulation factors, and immunoglobulins (4.Lee W.L. Downey G.P. Am. J. Respir. Crit. Care Med. 2001; 164: 896-904Crossref PubMed Scopus (266) Google Scholar). NE degrades pro-inflammatory mediators such as tumor necrosis factor-α and interleukin-1β, hence could alter the inflammatory response. The enzyme is capable of inducing the secretion of granulocyte macrophage-colony stimulating factor and interleukin-8, which could amplify the inflammation. Consequently, the release of these potent proteinases in diseased situations could create a proteolytic environment where degradation of different host molecules might occur and result in inappropriate inflammatory response. Because of their large substrate repertoire, NSPs have been implicated in the pathogenesis of various inflammatory and tissue-destructive diseases, including acute lung injury, cystic fibrosis, and COPD (5.Birrer P. Respiration. 1995; 62: 25-28Crossref PubMed Scopus (53) Google Scholar, 6.Janoff A. Am. Rev. Respir. Dis. 1985; 132: 417-433PubMed Google Scholar, 7.Kawabata K. Hagio T. Matsuoka S. Eur. J. Pharmacol. 2002; 451: 1-10Crossref PubMed Scopus (248) Google Scholar).COPD is recognized as a major health problem whose worldwide incidence is increasing dramatically. Cigarette smoke represents the major etiologic factor contributing to the development and progression of COPD. Advances in therapy against COPD have been limited due in part to poor understanding of the pathogenesis of this disease. Various animal models and injurious stimuli mimicking human COPD are now available to help understand the development of this pathology (reviewed in Ref. 8.Mahadeva R. Shapiro S.D. Thorax. 2002; 57: 908-914Crossref PubMed Scopus (138) Google Scholar). For example, several inbred strains of mice spontaneously develop emphysema, the main component of COPD, due to genetic abnormalities (reviewed in Ref. 8.Mahadeva R. Shapiro S.D. Thorax. 2002; 57: 908-914Crossref PubMed Scopus (138) Google Scholar). And mice exposed to cigarette smoke provide a valuable model of human COPD (9.Wright J.L. Cosio M. Churg A. Am. J. Physiol. Lung Cell Mol. Physiol. 2008; 295: L1-L15Crossref PubMed Scopus (309) Google Scholar).The contribution of NSPs to COPD was first evidenced by the observation that patients lacking α1-Pi, the main physiological inhibitor of neutrophil elastase, were at greater risk of developing emphysema (10.Eriksson S. Acta Med. Scand. 1965; 432: 1-85Google Scholar, 11.Sandford A.J. Weir T.D. Paré P.D. Eur. Respir. J. 1997; 10: 1380-1391Crossref PubMed Scopus (181) Google Scholar). Subsequently, various studies reported the induction of emphysema in animal models following instillation of NSPs. Intratracheal instillation of mice with human neutrophil elastase or proteinase 3 leads to tissue destruction and airspace enlargement, characteristic features of emphysema (12.Lafuma C. Frisdal E. Harf A. Robert L. Hornebeck W. Eur. Respir. J. 1991; 4: 1004-1009PubMed Google Scholar). In recent years, it has been shown that mice deficient in NE are considerably protected against cigarette-induced emphysema. Despite these compelling observations, the mechanisms by which NSPs injure the lung are not understood. Part of this concern may be because full characterization of mouse NSPs is still lacking. Although we and others have cloned and characterized murine NSPs genes/cDNAs (13.Belaaouaj A. Moog-Lutz C. Just J. Houzel-Charavel A. Shapiro S.D. Cayre Y. Mamm. Genome. 1999; 10: 210-212Crossref PubMed Scopus (9) Google Scholar, 14.Belaaouaj A. Walsh B.C. Jenkins N.A. Copeland N.G. Shapiro S.D. Mamm. Genome. 1997; 8: 5-8Crossref PubMed Scopus (16) Google Scholar, 15.Hohn P.A. Popescu N.C. Hanson R.D. Salvesen G. Ley T.J. J. Biol. Chem. 1989; 264: 13412-13419Abstract Full Text PDF PubMed Google Scholar), their corresponding proteins have not been purified. More importantly, unlike human NSPs, no specific peptides substrates for mouse NSPs are available to detect and quantify their specific activities especially in disease models.To address these concerns, we purified the three mouse NSPs and carried out a detailed analysis by comparison to human NSPs. Our studies revealed critical differences between mouse and human NSPs, which prompted us to develop new specific FRET peptides substrates for mouse NSPs. Molecular modeling approach in association with kinetic studies enabled us to generate/synthesize new sensitive substrates to both detect and measure all three mouse NSPs activities in the subnanomolar range.RESULTS AND DISCUSSIONIn the present work, we purified mouse neutrophil serine proteases and characterized them by comparison to their human counterparts. This allowed us to generate sensitive peptide substrates to individual murine proteases namely mNE, mCG, and mPr3.Purification of Mouse NE, CG, and Pr3As shown in Fig. 1A, intraperitoneal instillation of glycogen resulted in massive recruitment of inflammatory cells. In accordance with our previous work and as judged by morphologic criteria, the cellular infiltrate was predominately neutrophils. Mouse peritonea were lavaged, and collected cells were processed for NSPs purification as outlined under “Experimental Procedures.” To ensure the presence of mouse NSPs, aliquots of cell protein extracts were subjected to Western blotting using antibodies specific to mNE, mCG, and mPr3 (Fig. 1B). Next, mouse NSPs were extracted/purified as outlined in Fig. 1C. Following loading of neutrophil protein extracts on heparin-Sepharose column, we detected peptidase activity in both flow-through and NaCl gradient-eluted fractions using our polyvalent FRET substrate Abz-HPVPVYAFSPQ-EDDnp, a peptide sensitive to both trypsin- and chymotrypsin-like human proteases. The eluted fraction showed an asymmetric peak and was subjected to further fractionation using cation-exchange chromatography. This step allowed us to identify/separate peptidase activities into two fractions. The flow-through fraction of the heparin column was loaded onto phenyl-Sepharose, and peptidase activity was found in the fraction eluted with 50% isopropanol (Fig. 1C). All three fractions were HPLC-purified and processed for N-terminal amino acid sequencing. Peptide sequence analyses found that the phenyl-Sepharose fraction corresponded to mPr3, whereas each of the two fractions with peptidase activity eluted from the cation-exchange column corresponded to mNE or (Fig. of the of mouse and human NSPs showed that the murine NSPs were their human with and mPr3 the and amino acid sequence analyses in to mouse NSPs, a protein This protein is a of the protein also known as cystic It is in and cells K. P. E. PubMed Scopus (22) Google Scholar). presence might with in of this is analysis of the of murine NSPs from their amino acid our purification Next, the kinetic of all three proteases were of mouse and human neutrophil serine in a new of Mouse recent years, we have generated specific FRET substrates us to detect human NSPs in the Because of the of the active and the between human and mouse NSPs, we first kinetic of purified mNE, mCG, and mPr3 using human NSP substrates. Although of FRET peptide with resulted in a fluorescence no in fluorescence were detected with and substrates in the presence of mNE or mPr3 and These that mNE and mPr3 substrate specificities from of their human counterparts. In of these differences in substrate between human and mouse NSPs, the potent human elastase inhibitor not with mNE S. A. P. P. J. Pharmacol. PubMed Scopus Google Scholar). However, the human both mNE and mPr3, and human In this we were to the activities of the three murine proteases using Abz-HPVPVYAFSPQ-EDDnp, against mNE and mPr3, and the substrate against (Fig. The mouse proteases were used to new specific mouse NSPs substrates and to the kinetic of their To this we first identified the structural differences between mouse and human proteases that could for their differences in substrate We models of the three mouse NSPs using the three-dimensional structures of their human as and compared the structures of the critical identified as in substrate for the of FRET elastase in a new for the of proteinase FRET in a new of purified active mNE and mPr3 against human and against human proteases were with increasing of for the with FRET substrates. was for were all in the of were and for mNE, mPr3, and mCG, in accordance with the and of mNE reported in FRET substrates were not by We that the of mNE to such peptides could be due to the presence of at and used for the individual amino acid of a substrate and corresponding of the enzyme is that of and A. PubMed Scopus Google Scholar). In of this the peptide substrate Abz-HPVPVYAFSPQ-EDDnp, which is by both and mNE at the no at these as well we compared the of mNE and we found that the of mNE active was that of (Fig. which may mNE not the at in substrates the the two amino acid at and in mNE also create a environment close to and (Fig. This may substrates with at are not by in substrates of or the two at and by or not by mNE Because is by all three mouse NSPs, we to a FRET substrate from a sequence in a NE which has at and a for cathepsin G. We identified such a sequence in both mouse and human protein Ley T.J. Full Text Full Text PDF PubMed Scopus Google Scholar). The substrate was by mNE with a in the Although this peptide was to by mCG, it was at the by we to by a residue at the the substrate was no by mNE due to the of mNE Our to a at as well that the substrate was not by mNE because of of murine elastase compared with that of human the sequence of murine neutrophil elastase was with human using are shown against a based on the sequence are the the of the amino of the and acid were from and for mouse and human elastase, the of each is to and with The of each which to the active has been to the of to as from the of the of inhibitor to In the the active serine is critical that in the of the active are The main in the of both proteases where the in is by a residue in The of for at in mNE may also have an on the The of are and of murine elastase was as under “Experimental and the for human NE were from of an mPr3 following in have identified critical in the of human that contribute to substrate These are at the at both and and at the E. T. M. T. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). and conserved was with in mPr3 E. PubMed Scopus Google (Fig. of substrates are characterized by a residue at that is the We the residue by an residue in the human of the new resulted in a of mPr3 by as judged by comparison of the time of the peptide with that of E. T. M. T. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). mNE To the of this substrate to mPr3 we the sequence with the sequence which of substrates E. T. M. T. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google The for both mPr3 and that the are for between and Mouse not the substrate from the sequence that also an residue at E. T. M. T. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google further the of the for between human and mouse In of these observations, analysis of the three-dimensional model of mPr3 that the main between the mouse and human in the (Fig. the and generated an environment the in This is also by the that mouse a human their the of the of the mouse and human to be (Fig. of murine and human proteinase 3 active of murine and human proteinase 3 and between both acid to of the the of the amino of the and of as in Fig. of murine and human proteinase 3 were onto the of each and of the are and in substrate are in the of the active the and of the substrate in are by the and in mPr3, The structural model of mPr3 was from by modeling as under “Experimental Procedures.” of the to was as in Fig. The serine is in to mNE and mPr3, substrates the FRET peptide with a better This peptide is from protein the S. L. E. C. M. S. P. J. 2002; PubMed Google Scholar). at the was conserved in The other sensitive a peptide from the of the human was also by at the human not also for the of cathepsin FRET in a new To better our understanding of peptide substrate modeling studies revealed that differences in the of between and in the of active (Fig. that and might substrate namely the at in the of the that is with in (Fig. not and from a residue at as using the However, a residue was present at it could with not to a To this we the residue in the polyvalent substrate at the by by an of substrate found that the substrate was by and by that the in cathepsin G is for substrate and that residue at is in the of cathepsin G between and G is the of at the of the in (Fig. This residue could both and are in the P. R. E. J. J. Powers J.C. W. J. PubMed Scopus Google Scholar). Consequently, unlike human should not This was by of two FRET substrates that by or at by and experimental at a whereas both and the peptide was structures of the active of murine and human cathepsin G. sequence for murine and human cathepsin G. the of the amino of the and The critical (chymotrypsinogen numbering) of the of is by an residue in are shown against a the of and has been to of with The in active is by the and residue in in the of each active this leads to major differences in the and of the model was by modeling “Experimental based on the of to in each active was as in Fig. of the active of murine and human in which of a in the of is by an residue in were with W. L. specificities of mouse and human cathepsin in a new in of Our is widely accepted that NSPs play a critical role in neutrophil-associated lung inflammatory and tissue-destructive diseases. To investigate NSP pathogenic role(s), various mouse experimental models have been developed that mimic acutely or chronically injured human lungs. to the relative contribution of NSPs to lung disease processes as well as their underlying mechanisms still poorly understood. And the lack of purified mouse NSPs and their specific substrates have hampered advances in these studies. In this work, we provided evidence that peptide substrate specificities of human and mouse NSPs could be different despite their conserved cleft and close structural resemblance. We on to in the of the active of human and mouse NSPs, which for these It be that the of may not the substrate in both of mouse and human NSPs may well generate different peptide from a For example, and mNE generate peptides that structurally not P.A. Shapiro S.D. J. PubMed Scopus Google Scholar). Because of different mPr3 not the substrate from the sequence of whose and cell J.C. J. Am. 2002; PubMed Scopus Google Scholar). Because mouse has the sequence as human this the of the role of mPr3 as a that the cell inflammation. We also showed that not unlike because it to lack a residue at the of the In poorly in This might the physiological of the mouse which may the of protein substrates. enzyme may different mouse and human NSPs. In of mPr3 not and inhibitor is a poor inhibitor of mNE R.D. PubMed Scopus Google Scholar). is the This as a for lung inflammatory not mNE S. A. P. P. J. Pharmacol. PubMed Scopus Google Scholar, C. S. C. A. M. Harf A. C. Am. J. Respir. Cell Mol. Biol. 2002; PubMed Scopus Google Scholar). It be that structures of mouse neutrophil serine proteases to be to our findings and on the structural differences with their human counterparts. In and in of our generated from mouse models disease pathogenesis or NSP should be to human of human NSPs using mice as experimental models should into the differences in substrate specificities between mouse and human the of mice deficient in NSPs should to help the relative contribution of these proteases in neutrophil-associated lung diseases. The identified structural differences between human and mouse NSPs and the now available peptide substrates to mNE, mCG, mPr3 should better our understanding of NSP in disease IntroductionNeutrophil serine proteases (NSPs), 3The abbreviations used are: NSPneutrophil serine proteaseCOPDchronic obstructive pulmonary diseasehNEhuman neutrophil elastasehPr3human proteinase 3hCGhuman cathepsin GmNEmouse neutrophil elastasemPr3mouse proteinase 3mCGmouse cathepsin Gα1-Piα 1-protease inhibitorACTantichymotrypsinFRETfluorescence resonance energy transferHPLChigh-performance liquid chromatography. neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (Pr3), are mainly stored in neutrophil primary granules in readily active forms. NSPs are structurally related and share the conserved charge-relay triad, His57–Asp102–Ser195, where Ser is the active residue (chymotrypsinogen numbering) (1.Bode W. Meyer Jr., E. Powers J.C. Biochemistry. 1989; 28: 1951-1963Crossref PubMed Scopus (381) Google Scholar). NSPs contribute to neutrophil oxygen-independent system-mediated protection of the host against invading pathogens (2.Ganz T. Proc. Assoc. Am. Physicians. 1999; 111: 390-395Crossref PubMed Scopus (22) Google Scholar). Indeed, NSPs serve a physiological role for killing of microbes (3.Belaaouaj A. Microbes Infect. 2002; 4: 1259-1264Crossref PubMed Scopus (65) Google Scholar). Activated neutrophils are also known to release NSPs in the setting of inflammation. In vitro, NSPs are capable of cleaving a panoply of substrates. These include extracellular matrix proteins, pro-inflammatory mediators, coagulation factors, and immunoglobulins (4.Lee W.L. Downey G.P. Am. J. Respir. Crit. Care Med. 2001; 164: 896-904Crossref PubMed Scopus (266) Google Scholar). NE degrades pro-inflammatory mediators such as tumor necrosis factor-α and interleukin-1β, hence could alter the inflammatory response. The enzyme is capable of inducing the secretion of granulocyte macrophage-colony stimulating factor and interleukin-8, which could amplify the inflammation. Consequently, the release of these potent proteinases in diseased situations could create a proteolytic environment where degradation of different host molecules might occur and result in inappropriate inflammatory response. Because of their large substrate repertoire, NSPs have been implicated in the pathogenesis of various inflammatory and tissue-destructive diseases, including acute lung injury, cystic fibrosis, and COPD (5.Birrer P. Respiration. 1995; 62: 25-28Crossref PubMed Scopus (53) Google Scholar, 6.Janoff A. Am. Rev. Respir. Dis. 1985; 132: 417-433PubMed Google Scholar, 7.Kawabata K. Hagio T. Matsuoka S. Eur. J. Pharmacol. 2002; 451: 1-10Crossref PubMed Scopus (248) Google Scholar).COPD is recognized as a major health problem whose worldwide incidence is increasing dramatically. Cigarette smoke represents the major etiologic factor contributing to the development and progression of COPD. Advances in therapy against COPD have been limited due in part to poor understanding of the pathogenesis of this disease. Various animal models and injurious stimuli mimicking human COPD are now available to help understand the development of this pathology (reviewed in Ref. 8.Mahadeva R. Shapiro S.D. Thorax. 2002; 57: 908-914Crossref PubMed Scopus (138) Google Scholar). For example, several inbred strains of mice spontaneously develop emphysema, the main component of COPD, due to genetic abnormalities (reviewed in Ref. 8.Mahadeva R. Shapiro S.D. Thorax. 2002; 57: 908-914Crossref PubMed Scopus (138) Google Scholar). And mice exposed to cigarette smoke provide a valuable model of human COPD (9.Wright J.L. Cosio M. Churg A. Am. J. Physiol. Lung Cell Mol. Physiol. 2008; 295: L1-L15Crossref PubMed Scopus (309) Google Scholar).The contribution of NSPs to COPD was first evidenced by the observation that patients lacking α1-Pi, the main physiological inhibitor of neutrophil elastase, were at greater risk of developing emphysema (10.Eriksson S. Acta Med. Scand. 1965; 432: 1-85Google Scholar, 11.Sandford A.J. Weir T.D. Paré P.D. Eur. Respir. J. 1997; 10: 1380-1391Crossref PubMed Scopus (181) Google Scholar). Subsequently, various studies reported the induction of emphysema in animal models following instillation of NSPs. Intratracheal instillation of mice with human neutrophil elastase or proteinase 3 leads to tissue destruction and airspace enlargement, characteristic features of emphysema (12.Lafuma C. Frisdal E. Harf A. Robert L. Hornebeck W. Eur. Respir. J. 1991; 4: 1004-1009PubMed Google Scholar). In recent years, it has been shown that mice deficient in NE are considerably protected against cigarette-induced emphysema. Despite these compelling observations, the mechanisms by which NSPs injure the lung are not understood. Part of this concern may be because full characterization of mouse NSPs is still lacking. Although we and others have cloned and characterized murine NSPs genes/cDNAs (13.Belaaouaj A. Moog-Lutz C. Just J. Houzel-Charavel A. Shapiro S.D. Cayre Y. Mamm. Genome. 1999; 10: 210-212Crossref PubMed Scopus (9) Google Scholar, 14.Belaaouaj A. Walsh B.C. Jenkins N.A. Copeland N.G. Shapiro S.D. Mamm. Genome. 1997; 8: 5-8Crossref PubMed Scopus (16) Google Scholar, 15.Hohn P.A. Popescu N.C. Hanson R.D. Salvesen G. Ley T.J. J. Biol. Chem. 1989; 264: 13412-13419Abstract Full Text PDF PubMed Google Scholar), their corresponding proteins have not been purified. More importantly, unlike human NSPs, no specific peptides substrates for mouse NSPs are available to detect and quantify their specific activities especially in disease models.To address these concerns, we purified the three mouse NSPs and carried out a detailed analysis by comparison to human NSPs. Our studies revealed critical differences between mouse and human NSPs, which prompted us to develop new specific FRET peptides substrates for mouse NSPs. Molecular modeling approach in association with kinetic studies enabled us to generate/synthesize new sensitive substrates to both detect and measure all three mouse NSPs activities in the subnanomolar