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Inhibition of Murine Neutrophil Serine Proteinases by Human and Murine Secretory Leukocyte Protease Inhibitor
Biochemical and Biophysical Research Communications 254, 614 – 617 (1999) Article ID bbrc.1998.0108, available online at http://www.idealibrary.com on
Inhibition of Murine Neutrophil Serine Proteinases by Human and Murine Secretory Leukocyte Protease Inhibitor Clifford D. Wright,* ,1 John A. Kennedy,* Ralph J. Zitnik,† and Mohammed A. Kashem* *Amgen, Inc., Boulder, Colorado 80301; and †Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520
Received December 8, 1998
Human secretory leukocyte protease inhibitor (SLPI) is a predominant physiologic inhibitor of elastase and cathepsin G, proinflammatory serine proteases released by activated neutrophils. In order to fully evaluate the potential pharmacologic efficacy of human SLPI in animal models of inflammation, it is critical to know the potency of the inhibitor for corresponding proteases from the species of interest. In this report, we compare the inhibitory activity of human and murine SLPI against elastase and cathepsin G from both species. Human and murine neutrophil elastase and cathepsin G display comparable K m values for their specific peptide substrates. Murine SLPI inhibits murine neutrophil elastase and cathepsin G with K i values of 5 and 0.12 nM, respectively, while human SLPI inhibits the both murine serine proteases with K i’s of 0.02 nM. In contrast, murine SLPI inhibits human neutrophil elastase and cathepsin G with K i values of 1.4 and 90 nM, respectively, while human SLPI inhibits the proteases with K i’s of 0.3 and 10 nM, respectively. These results demonstrate species-specific variations in the protease inhibitory activities of SLPI. Such variations should be considered in the evaluation of the activity of human SLPI in murine pharmacologic models. © 1999 Academic Press
Human secretory leukocyte protease inhibitor (SLPI), also known as mucus proteinase inhibitor, is broad spectrum inhibitor of serine protease. SLPI is produced by mucosal epithelial cells, serous cells, and bronchiolar goblet cells (1), serving to airways and other mucosal surfaces from proteolysis by elastase or cathepsin G released from neutrophils in response to inflammatory stimuli (1–3). SLPI also inhibits chy1 To whom all correspondence should be addressed at Department of Pharmacology, Amgen, Inc., 1 Amgen Center Drive, Mailstop 5-1-D, Thousand Oaks, CA 91320.
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
mase (4) and tryptase (5) secreted by activated mast cells. Human SLPI is an 11.7 kDa non-glycosylated protein composed of 107 amino acids. The protein consists of two domains which each include four disulfide bonds. The N-terminal domain mediates the interaction of SLPI with heparin to increase its association with target serine proteases (6,7). The anti-protease activity is primarily associated with the C-terminal domain, where the amino acid residues surrounding the leucine at position 72 comprise the serine protease binding site (2). Recently, we reported the cloning and characterization of the murine form of SLPI (8). At the amino acid level, murine SLPI exhibits 58% identity to human SLPI with conservation of the cysteine residues. However, a methionine residue replaces leucine as the P1 residue involved in interaction with serine proteases. In order to evaluate the potential efficacy of human SLPI in animal models, it is crucial to know its potency against the corresponding serine proteases of the species of interest. For example, Virca et al. (9) reported similarities between human and rat leukocyte elastase and cathepsin G catalytic activities and their inhibition by the neutral protease inhibitors a 1-antiprotease and eglin C. In addition, the effect of human SLPI on immune complex-induced alveolitis in rats was examined (10) based on structural homologies between rat and human neutrophil serine proteases (11). In this study, we have evaluated the inhibitory activities of human and murine SLPI against elastase and cathepsin G from both species. MATERIALS AND METHODS Materials and reagents. Human neutrophil elastase, human neutrophil cathepsin G, and human plasma a 1-proteinase inhibitor were obtained from Calbiochem-Novabiochem International (San Diego, CA). Chromogenic protease substrates MeO-Suc-Ala-Ala-Pro-Val-pnitroanilide and MeO-Suc-Ala-Ala-Pro-Phe-p-nitroanilide were obtained from Sigma Chemical Company (St. Louis, MO). Aprotinin-
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Characterization of Human and Murine Neutrophil Serine Proteases Protease
Substrate
K m (mM)
Human neutrophil elastase Murine neutrophil elastase Human neutrophil cathepsin G Murine neutrophil cathepsin G
MeO-Suc-Ala-Ala-Pro-Val-pNA MeO-Suc-Ala-Ala-Pro-Val-pNA N-Suc-Ala-Ala-Pro-Phe-pNA N-Suc-Ala-Ala-Pro-Phe-pNA
280 340 350 360
Sepharose 4B was obtained from Elastin Products Co., Inc. (Owensville, MO). Recombinant human and murine SLPIs were expressed and purified as previously described (2,8). Murine neutrophil serine protease preparation. Mice (BALB/c or CD1) were administered 4% thioglycollate medium solution (Sigma) by the intraperitoneal route in Dulbecco’s phosphate-buffered saline (GIBCO, Grand Island, NY). After ten hours, peritoneal exudates composed of 70 – 80% neutrophils were collected. Cell preparations were washed and resuspended in Hanks’ balanced salt solution (GIBCO). Contaminating erythrocytes were removed by hypotonic lysis. Neutrophils were resuspended in 50 mM Tris-HCl, pH 8.0 with 50 mM NaCl at 5 x 10 6 cells/ml. Cells were lysed by 3 freeze/thaw cycles with a 5 second sonication at 4°C after each cycle. Cellular debris was removed by centrifugation at 1400 x g for 15 minutes at 4°C. Murine elastase and cathepsin G were purified from the lysates by affinity chromatography using aprotinin-Sepharose according to the procedure of Baugh and Travis (12).
k cat/K m (M 21 s 21) 2.1 3 10 4 1.6 3 10 4 8.4 3 10 3 2.4 3 10 4
Inhibition of human and murine elastase and cathepsin G by human and murine SLPI. The inhibitory activities of human and murine SLPIs versus neutrophil elastase and cathepsin G of both species are illustrated in Figs. 1 and 2, respectively and summarized in Table 2. Human SLPI inhibits human elastase and cathepsin G with a 4.7- and 9-fold increase in potency, respectively, compared with murine SLPI. In comparison, human inhibitor is also a more potent inhibitor of the murine elastase and cathepsin G (250- and 6-fold, respectively), than murine SLPI. In contrast, human plasma a 1-proteinase inhibitor was active against human elastase and cathepsin G with K i ’s of 6 and 16 nM,
Enzyme assays. Neutrophil elastase and cathepsin G were assayed using enzyme-specific peptide-p-nitroanilide (pNA) substrates. Human neutrophil elastase was assayed in a reaction mixture containing 7.5 nM enzyme and 0.3 mM MeO-Suc-Ala-Ala-Pro-Val-pNA in 100 mM Tris-HCl, pH 8.3 with 0.96 M NaCl and 1% BSA (13). Murine neutrophil elastase was assayed in the same buffer using 0.9 nM active enzyme and 0.5 mM elastase substrate. Human neutrophil cathepsin G was assayed in a reaction mixture containing 16 nM enzyme and 0.4 mM N-Suc-Ala-Ala-Pro-Phe-pNA in 625 mM TrisHCl, pH 7.5 with 2.5 mM MgCl 2 and 0.125% Brij 35 (14). Murine neutrophil cathepsin G was assayed in the same buffer using 0.7 nM active enzyme and 0.5 mM cathepsin G substrate. The p-nitroaniline product of proteolysis was quantified at 405 nm on a SpectraMAX 340 plate reader (Molecular Devices, Sunnyvale, CA). The K m for the each enzyme with its selective substrate was determined by iterative fitting of the initial velocities of substrate hydrolysis to the Michaelis-Menton equation using Sigma Plot (Jandel Scientific, San Rafael, CA). Data were plotted according to the Lineweaver-Burk transformation from initial velocities of substrate hydrolysis. The enzyme concentrations were determined by active site titration with human SLPI (1). Each protease was incubated with varying concentrations of SLPI or a 1-proteinase inhibitor for 15 minutes at 37°C in specific assay buffer. The residual protease activity was measured following addition of the respective substrate. The inhibition constants (K i’s) for human and murine SLPI versus each protease were determined as previously described (8). Proteases prepared from different mouse strains exhibited similar kinetics and inhibition profiles.
RESULTS Enzymatic characterization of human and murine neutrophil elastase and cathepsin G. The enzymatic characterization of human and murine neutrophil serine proteases is summarized in Table 1. Comparable K m and k cat/K m values were calculated for elastases and cathepsin G’s of both species.
FIG. 1. Comparison of the inhibition of human and murine elastase by purified recombinant human SLPI and recombinant murine SLPI. The residual protease activity was measured in the presence of varying concentrations of SLPI. The inhibition constants (K i’s) for human and murine SLPI against each protease were determined as previously described (8) assuming a tight-binding model of inhibition. The curves shown are representative of 3 replicate experiments performed with each protease.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
These results are in contrast to the report of Jin, et al. (15) that human SLPI failed to inhibit murine elastase. The demonstrated ability of human SLPI to inhibit murine neutrophil serine proteases supports the use of the inhibitor to assess its potential therapeutic utility in murine animal models. However, in vivo activity should also be evaluated in the context of the increased potency of human SLPI against murine leukocyte proteases as well as its potential breadth of inhibitory activity against additional murine serine proteases. Cellular effects of SLPI which are independent of its antiprotease activity should also be considered (16). While the amino acid sequence of murine SLPI exhibits 58% identity with the human analog, variations in the serine protease binding region may account for differences in their inhibitory activities. Comparison of the P5-P59 region of murine SLPI is shown below: P5 P4 P3 P2 P1 P19 P29 P39 P49 P59 Human SLPI Tyr 68-Gly 69-Gln 70-Cys 71-Leu 72-Met 73Leu 74-Asn 75-Phe 76-Phe 77 Murine SLPI Gln 68-Ala 69-Ala 70-Cys 71-Met 72-Met 73Leu 74-Asn 75-Phe 76-Phe 77
FIG. 2. Comparison of the inhibition of human and murine cathepsin G by purified recombinant human SLPI and recombinant murine SLPI. The residual protease activity was measured in the presence of varying concentrations of SLPI. The inhibition constants (K i’s) for human and murine SLPI against each protease were determined as previously described (8) assuming a tight-binding model of inhibition. The curves shown are representative of 3 replicate experiments performed with each protease.
respectively, while inhibiting murine enzymes with K i’s of 0.6 and 2 nM. Human and murine SLPI are also be distinguished by their relative potencies against serine proteases of the respective species. Human SLPI has a 33-fold selectivity for human elastase versus human cathepsin G, while murine SLPI has a 42-fold selectivity for murine cathepsin G versus murine elastase. These results demonstrate differences in potency and selectivity for human and murine SLPI. DISCUSSION Murine and human forms of SLPI are shown to inhibit neutrophil elastase and cathepsin G from both species.
Residues of the serine protease binding site have been shown to contribute to the selectivity and potency of human SLPI for serine proteases. Mutagenesis studies with human SLPI (2) demonstrated that substitution of leucine at residue 72 with the polar cationic amino acids arginine or lysine results in increased potency and selectivity of the inhibitor for trypsin. In contrast, substitution with the aromatic amino acid phenylalanine increases the inhibitory activity versus trypsin and chymotrypsin while reducing its activity against elastase. Data presented in this report suggests that presence of a methionine residue at position 72 in murine SLPI may contribute to increased activity against chymotryptic enzymes such cathepsin G. The relative contributions of P5-P3 substitutions are not known. Little has been reported regarding the in vivo pharmacologic activity of human SLPI in murine models. We have found that eosinophil and lymphocyte infiltration into airways of mice following chronic allergen challenge is reduced by i.t. administration of human SLPI (17). In rats, human SLPI was effective in preventing immune complex-induced acute alveolitis (10) as well as the proteolytic activation of influenza A and Sendai virus infectivity by the rat serine protease, tryptase Clara (18).
TABLE 2
Inhibition of Neutrophil Serine Proteases by Human and Murine SLPI (K i, nM) Inhibitor
Human neutrophil elastase
Murine neutrophil elastase
Human neutrophil cathepsin G
Murine neutrophil cathepsin G
Human SLPI Murine SLPI
0.3 1.4
0.02 5.0
10 90
0.02 0.12
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Pharmacologic activity of human SLPI in hamsters (19), guinea pigs (20), and sheep (5,21,22) has also been reported. The inhibitory activity of human SLPI against of murine leukocyte serine proteases by human SLPI supports its use in murine models of inflammation. REFERENCES 1. Thompson, R. C. and Ohlsson, K. (1986) Proc. Natl. Acad. Sci. USA 83, 6692– 6696. 2. Eisenberg, S. P., Hale, K. K., Heimdal, P., and Thompson, R. C. (1990) J. Biol. Chem. 265, 7976 –7981. 3. Vogelmeier, C., Hubbard, R. C., Fells, G. A., Schnebli, H-P., Thompson, R. C., Fritz, H., and Crystal, R. G. (1991) J. Clin. Invest. 87, 482– 488. 4. Walter, M., Plotnick, M., and Schechter, N. M. (1996) Arch. Biochem. Biophys. 327, 81– 88. 5. Abraham, W. M., Forteza, R., Ahmed, A., Cortes, A., Kashem, M., and and Wright, C. (1997) Am. Rev. Respir. Crit. Care Med. 155, A880. 6. Mellet, P., Ermolieff, J., and Bieth, J. G. (1995) Biochemistry 34, 2645–2652. 7. Fath, M. A., Wu, X., Hileman, R. E., Linhardt, R. J., Kashem, M. A., Nelson, R. M., Wright, C. D., and Abraham, W. M. (1998) J. Biol. Chem. 273, 13563–13569. 8. Zitnik, R. J., Zhang, J., Kashem, M. A., Kohno, T., Lyons, D. E., Wright, C. D., Rosen, E., Goldberg, I., and Hayday, A. C. (1997) Biochem. Biophys. Res. Commun. 232, 687– 697. 9. Virca, G. D., Metz, G., and Schnebli, H-P. (1984) Eur. J. Biochem. 144, 1–9.
10. Mulligan, M. S., Desrochers, P. E., Chinnaiyan, A. M., Gibbs, D. F., Varani, J., Johnson, K. J., and Weiss, S. J. (1993) Proc. Natl. Acad. Sci. USA 90, 11523–11527. 11. Bjork, P. and Ohlsson, K. (1990) Biol. Chem. Hoppe-Seyler 371, 595– 601. 12. Baugh, R. J. and Travis, J. (1976) Biochemistry 15, 836 – 841. 13. Nakajima, K., Powers, J. C., Ashe, B., and Zimmerman, M. (1979) J. Biol. Chem. 254, 4027– 4032. 14. Lottenberg, R., Christensen, U., Jackson, C. M., and Coleman, P. L. (1981) Meth. Enzymol. 80, 341–361. 15. Jin, F-U., Nathan, C., Radzioch, D., and Ding, A. (1997) Cell 88, 417– 426. 16. Zhang, Y., DeWitt, D. L., McNeely, T. B., Wahl, S. M., and Wahl, L. M. (1997) J. Clin. Invest. 99, 894 –900. 17. Wright, C. D., Havill, A. M., Lee, P. A., O’Riordan, T. G., Bevilacqua, M. P., and Abraham, W. M. (1998) 22 nd Symposium of the Collegium Internationale Allergologicum, Abstract 144. 18. Beppu, Y., Imamura, Y., Tashiro, M., Towatari, T., Ariga, H., and Kido, H. (1997) J. Biochem. 121, 309 –316. 19. Rudolphus, A., Stolk, J., Dijkman, J. H., and Kramps, J. A. (1993) Am. Rev. Respir. Dis. 147, 442– 447. 20. Havill, A. M., Middleton, S., Lyons, D., and Wright, C. (1997) Am. Rev. Respir. Crit. Care Med. 155, A654. 21. Gossage, J. R., Perkett, E. A., Davidson, J. M., Starcher, B. C., Carmichael, D., Brigham, K. L., and Meyrick, B. (1995) J. Appl. Physiol. 79, 1163–1172. 22. O’Riordan, T. G., Otero, R. A., Mao, Y., Lopez, J., Wright, C, and Abraham, W. M.. (1997) Am. Rev. Respir. Crit. Care Med. 155, A878.
617
Inhibition of Murine Neutrophil Serine Proteinases by Human and Murine Secretory Leukocyte Protease Inhibitor Clifford D. Wright,* ,1 John A. Kennedy,* Ralph J. Zitnik,† and Mohammed A. Kashem* *Amgen, Inc., Boulder, Colorado 80301; and †Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06520
Received December 8, 1998
Human secretory leukocyte protease inhibitor (SLPI) is a predominant physiologic inhibitor of elastase and cathepsin G, proinflammatory serine proteases released by activated neutrophils. In order to fully evaluate the potential pharmacologic efficacy of human SLPI in animal models of inflammation, it is critical to know the potency of the inhibitor for corresponding proteases from the species of interest. In this report, we compare the inhibitory activity of human and murine SLPI against elastase and cathepsin G from both species. Human and murine neutrophil elastase and cathepsin G display comparable K m values for their specific peptide substrates. Murine SLPI inhibits murine neutrophil elastase and cathepsin G with K i values of 5 and 0.12 nM, respectively, while human SLPI inhibits the both murine serine proteases with K i’s of 0.02 nM. In contrast, murine SLPI inhibits human neutrophil elastase and cathepsin G with K i values of 1.4 and 90 nM, respectively, while human SLPI inhibits the proteases with K i’s of 0.3 and 10 nM, respectively. These results demonstrate species-specific variations in the protease inhibitory activities of SLPI. Such variations should be considered in the evaluation of the activity of human SLPI in murine pharmacologic models. © 1999 Academic Press
Human secretory leukocyte protease inhibitor (SLPI), also known as mucus proteinase inhibitor, is broad spectrum inhibitor of serine protease. SLPI is produced by mucosal epithelial cells, serous cells, and bronchiolar goblet cells (1), serving to airways and other mucosal surfaces from proteolysis by elastase or cathepsin G released from neutrophils in response to inflammatory stimuli (1–3). SLPI also inhibits chy1 To whom all correspondence should be addressed at Department of Pharmacology, Amgen, Inc., 1 Amgen Center Drive, Mailstop 5-1-D, Thousand Oaks, CA 91320.
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
mase (4) and tryptase (5) secreted by activated mast cells. Human SLPI is an 11.7 kDa non-glycosylated protein composed of 107 amino acids. The protein consists of two domains which each include four disulfide bonds. The N-terminal domain mediates the interaction of SLPI with heparin to increase its association with target serine proteases (6,7). The anti-protease activity is primarily associated with the C-terminal domain, where the amino acid residues surrounding the leucine at position 72 comprise the serine protease binding site (2). Recently, we reported the cloning and characterization of the murine form of SLPI (8). At the amino acid level, murine SLPI exhibits 58% identity to human SLPI with conservation of the cysteine residues. However, a methionine residue replaces leucine as the P1 residue involved in interaction with serine proteases. In order to evaluate the potential efficacy of human SLPI in animal models, it is crucial to know its potency against the corresponding serine proteases of the species of interest. For example, Virca et al. (9) reported similarities between human and rat leukocyte elastase and cathepsin G catalytic activities and their inhibition by the neutral protease inhibitors a 1-antiprotease and eglin C. In addition, the effect of human SLPI on immune complex-induced alveolitis in rats was examined (10) based on structural homologies between rat and human neutrophil serine proteases (11). In this study, we have evaluated the inhibitory activities of human and murine SLPI against elastase and cathepsin G from both species. MATERIALS AND METHODS Materials and reagents. Human neutrophil elastase, human neutrophil cathepsin G, and human plasma a 1-proteinase inhibitor were obtained from Calbiochem-Novabiochem International (San Diego, CA). Chromogenic protease substrates MeO-Suc-Ala-Ala-Pro-Val-pnitroanilide and MeO-Suc-Ala-Ala-Pro-Phe-p-nitroanilide were obtained from Sigma Chemical Company (St. Louis, MO). Aprotinin-
614
Vol. 254, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS TABLE 1
Characterization of Human and Murine Neutrophil Serine Proteases Protease
Substrate
K m (mM)
Human neutrophil elastase Murine neutrophil elastase Human neutrophil cathepsin G Murine neutrophil cathepsin G
MeO-Suc-Ala-Ala-Pro-Val-pNA MeO-Suc-Ala-Ala-Pro-Val-pNA N-Suc-Ala-Ala-Pro-Phe-pNA N-Suc-Ala-Ala-Pro-Phe-pNA
280 340 350 360
Sepharose 4B was obtained from Elastin Products Co., Inc. (Owensville, MO). Recombinant human and murine SLPIs were expressed and purified as previously described (2,8). Murine neutrophil serine protease preparation. Mice (BALB/c or CD1) were administered 4% thioglycollate medium solution (Sigma) by the intraperitoneal route in Dulbecco’s phosphate-buffered saline (GIBCO, Grand Island, NY). After ten hours, peritoneal exudates composed of 70 – 80% neutrophils were collected. Cell preparations were washed and resuspended in Hanks’ balanced salt solution (GIBCO). Contaminating erythrocytes were removed by hypotonic lysis. Neutrophils were resuspended in 50 mM Tris-HCl, pH 8.0 with 50 mM NaCl at 5 x 10 6 cells/ml. Cells were lysed by 3 freeze/thaw cycles with a 5 second sonication at 4°C after each cycle. Cellular debris was removed by centrifugation at 1400 x g for 15 minutes at 4°C. Murine elastase and cathepsin G were purified from the lysates by affinity chromatography using aprotinin-Sepharose according to the procedure of Baugh and Travis (12).
k cat/K m (M 21 s 21) 2.1 3 10 4 1.6 3 10 4 8.4 3 10 3 2.4 3 10 4
Inhibition of human and murine elastase and cathepsin G by human and murine SLPI. The inhibitory activities of human and murine SLPIs versus neutrophil elastase and cathepsin G of both species are illustrated in Figs. 1 and 2, respectively and summarized in Table 2. Human SLPI inhibits human elastase and cathepsin G with a 4.7- and 9-fold increase in potency, respectively, compared with murine SLPI. In comparison, human inhibitor is also a more potent inhibitor of the murine elastase and cathepsin G (250- and 6-fold, respectively), than murine SLPI. In contrast, human plasma a 1-proteinase inhibitor was active against human elastase and cathepsin G with K i ’s of 6 and 16 nM,
Enzyme assays. Neutrophil elastase and cathepsin G were assayed using enzyme-specific peptide-p-nitroanilide (pNA) substrates. Human neutrophil elastase was assayed in a reaction mixture containing 7.5 nM enzyme and 0.3 mM MeO-Suc-Ala-Ala-Pro-Val-pNA in 100 mM Tris-HCl, pH 8.3 with 0.96 M NaCl and 1% BSA (13). Murine neutrophil elastase was assayed in the same buffer using 0.9 nM active enzyme and 0.5 mM elastase substrate. Human neutrophil cathepsin G was assayed in a reaction mixture containing 16 nM enzyme and 0.4 mM N-Suc-Ala-Ala-Pro-Phe-pNA in 625 mM TrisHCl, pH 7.5 with 2.5 mM MgCl 2 and 0.125% Brij 35 (14). Murine neutrophil cathepsin G was assayed in the same buffer using 0.7 nM active enzyme and 0.5 mM cathepsin G substrate. The p-nitroaniline product of proteolysis was quantified at 405 nm on a SpectraMAX 340 plate reader (Molecular Devices, Sunnyvale, CA). The K m for the each enzyme with its selective substrate was determined by iterative fitting of the initial velocities of substrate hydrolysis to the Michaelis-Menton equation using Sigma Plot (Jandel Scientific, San Rafael, CA). Data were plotted according to the Lineweaver-Burk transformation from initial velocities of substrate hydrolysis. The enzyme concentrations were determined by active site titration with human SLPI (1). Each protease was incubated with varying concentrations of SLPI or a 1-proteinase inhibitor for 15 minutes at 37°C in specific assay buffer. The residual protease activity was measured following addition of the respective substrate. The inhibition constants (K i’s) for human and murine SLPI versus each protease were determined as previously described (8). Proteases prepared from different mouse strains exhibited similar kinetics and inhibition profiles.
RESULTS Enzymatic characterization of human and murine neutrophil elastase and cathepsin G. The enzymatic characterization of human and murine neutrophil serine proteases is summarized in Table 1. Comparable K m and k cat/K m values were calculated for elastases and cathepsin G’s of both species.
FIG. 1. Comparison of the inhibition of human and murine elastase by purified recombinant human SLPI and recombinant murine SLPI. The residual protease activity was measured in the presence of varying concentrations of SLPI. The inhibition constants (K i’s) for human and murine SLPI against each protease were determined as previously described (8) assuming a tight-binding model of inhibition. The curves shown are representative of 3 replicate experiments performed with each protease.
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Vol. 254, No. 3, 1999
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
These results are in contrast to the report of Jin, et al. (15) that human SLPI failed to inhibit murine elastase. The demonstrated ability of human SLPI to inhibit murine neutrophil serine proteases supports the use of the inhibitor to assess its potential therapeutic utility in murine animal models. However, in vivo activity should also be evaluated in the context of the increased potency of human SLPI against murine leukocyte proteases as well as its potential breadth of inhibitory activity against additional murine serine proteases. Cellular effects of SLPI which are independent of its antiprotease activity should also be considered (16). While the amino acid sequence of murine SLPI exhibits 58% identity with the human analog, variations in the serine protease binding region may account for differences in their inhibitory activities. Comparison of the P5-P59 region of murine SLPI is shown below: P5 P4 P3 P2 P1 P19 P29 P39 P49 P59 Human SLPI Tyr 68-Gly 69-Gln 70-Cys 71-Leu 72-Met 73Leu 74-Asn 75-Phe 76-Phe 77 Murine SLPI Gln 68-Ala 69-Ala 70-Cys 71-Met 72-Met 73Leu 74-Asn 75-Phe 76-Phe 77
FIG. 2. Comparison of the inhibition of human and murine cathepsin G by purified recombinant human SLPI and recombinant murine SLPI. The residual protease activity was measured in the presence of varying concentrations of SLPI. The inhibition constants (K i’s) for human and murine SLPI against each protease were determined as previously described (8) assuming a tight-binding model of inhibition. The curves shown are representative of 3 replicate experiments performed with each protease.
respectively, while inhibiting murine enzymes with K i’s of 0.6 and 2 nM. Human and murine SLPI are also be distinguished by their relative potencies against serine proteases of the respective species. Human SLPI has a 33-fold selectivity for human elastase versus human cathepsin G, while murine SLPI has a 42-fold selectivity for murine cathepsin G versus murine elastase. These results demonstrate differences in potency and selectivity for human and murine SLPI. DISCUSSION Murine and human forms of SLPI are shown to inhibit neutrophil elastase and cathepsin G from both species.
Residues of the serine protease binding site have been shown to contribute to the selectivity and potency of human SLPI for serine proteases. Mutagenesis studies with human SLPI (2) demonstrated that substitution of leucine at residue 72 with the polar cationic amino acids arginine or lysine results in increased potency and selectivity of the inhibitor for trypsin. In contrast, substitution with the aromatic amino acid phenylalanine increases the inhibitory activity versus trypsin and chymotrypsin while reducing its activity against elastase. Data presented in this report suggests that presence of a methionine residue at position 72 in murine SLPI may contribute to increased activity against chymotryptic enzymes such cathepsin G. The relative contributions of P5-P3 substitutions are not known. Little has been reported regarding the in vivo pharmacologic activity of human SLPI in murine models. We have found that eosinophil and lymphocyte infiltration into airways of mice following chronic allergen challenge is reduced by i.t. administration of human SLPI (17). In rats, human SLPI was effective in preventing immune complex-induced acute alveolitis (10) as well as the proteolytic activation of influenza A and Sendai virus infectivity by the rat serine protease, tryptase Clara (18).
TABLE 2
Inhibition of Neutrophil Serine Proteases by Human and Murine SLPI (K i, nM) Inhibitor
Human neutrophil elastase
Murine neutrophil elastase
Human neutrophil cathepsin G
Murine neutrophil cathepsin G
Human SLPI Murine SLPI
0.3 1.4
0.02 5.0
10 90
0.02 0.12
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pharmacologic activity of human SLPI in hamsters (19), guinea pigs (20), and sheep (5,21,22) has also been reported. The inhibitory activity of human SLPI against of murine leukocyte serine proteases by human SLPI supports its use in murine models of inflammation. REFERENCES 1. Thompson, R. C. and Ohlsson, K. (1986) Proc. Natl. Acad. Sci. USA 83, 6692– 6696. 2. Eisenberg, S. P., Hale, K. K., Heimdal, P., and Thompson, R. C. (1990) J. Biol. Chem. 265, 7976 –7981. 3. Vogelmeier, C., Hubbard, R. C., Fells, G. A., Schnebli, H-P., Thompson, R. C., Fritz, H., and Crystal, R. G. (1991) J. Clin. Invest. 87, 482– 488. 4. Walter, M., Plotnick, M., and Schechter, N. M. (1996) Arch. Biochem. Biophys. 327, 81– 88. 5. Abraham, W. M., Forteza, R., Ahmed, A., Cortes, A., Kashem, M., and and Wright, C. (1997) Am. Rev. Respir. Crit. Care Med. 155, A880. 6. Mellet, P., Ermolieff, J., and Bieth, J. G. (1995) Biochemistry 34, 2645–2652. 7. Fath, M. A., Wu, X., Hileman, R. E., Linhardt, R. J., Kashem, M. A., Nelson, R. M., Wright, C. D., and Abraham, W. M. (1998) J. Biol. Chem. 273, 13563–13569. 8. Zitnik, R. J., Zhang, J., Kashem, M. A., Kohno, T., Lyons, D. E., Wright, C. D., Rosen, E., Goldberg, I., and Hayday, A. C. (1997) Biochem. Biophys. Res. Commun. 232, 687– 697. 9. Virca, G. D., Metz, G., and Schnebli, H-P. (1984) Eur. J. Biochem. 144, 1–9.
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