Proteinwe-3 (PR-3): A Polymorphonuclear Leukocyte Serine Proteinase N. V. RAO," NANCY G. WEHNER," BRUCE C. MARSHALL," ANNE B. STURROCK," THOMAS P. HUECKSTEADT," GOPNA V. RAO," BEULAH H. GRAY,b AND JOHN R. HOIDAL".' aDepartment of Pulmonary Medicine University of Utah Medical Center Salt Lake City, Utah 84132 bDepartment of Microbiology University of Minnesota Minneapolis, Minnesota 55455
The current explanation for the development of emphysema is the proteinase pathogenesis hypothesis, which holds that progressive destruction of the interstitium is due to an excess of proteolytic enzymes (particularly elastase) in relation to the availability of proteolytic inhibitors. Inasmuch as a major endogenous source of elastase in the human lung is the polymorphonuclear leukocyte (PMNL), emphasis has been placed on the effects of PMNL-derived elastase and its inhibitors in regulating the degradation of lung elastin. Experimental emphysema has been produced by a single intratracheal injection of purified human PMNL elastase (HLE) in laboratory animals. Potent synthetic inhibitors have been developed for HLE, and several are effective in preventing emphysema in animal models of HLE-induced disease. To consider only HLE-mediated injury in examining the pathogenesis of emphysema in the human, however, is too narrow a focus. A direct relation of HLE to human disease remains to be demonstrated. Recent studies suggest that the increased risk of emphysema associated with cigarette smoking may be due partially to the effect of phagocyte- or smoke-derived oxidants in the lung. Another variable immediately tangent to the HLE pathogenesis concept is that other PMNL or alveolar macrophage (AM) proteinases may participate in matrix destruction. Cathepsin G and an AM cysteine proteinase(s1 both have elastolytic capabilities in uitro.'**Moreover, cathepsin G has been reported to act synergistically with HLE in the solubilization of elastin in ~ i t r oalthough ,~ results have been d i ~ c r e p a n t . Studies ~.~ to date that have failed to demonstrate that these proteinases can produce emphysema4do not exclude their participation in the induction of the disease.
Address for correspondence: John R. Hoidal, Pulmonary Division, Rm. 4R240, 50 North Medical Drive, University of Utah Medical Center, Salt Lake City, UT 84132. 60
RAO et al.: PROTEINASE-3
61
In preliminary studies, we marshalled evidence that another human PMNL serine proteinase, proteinase-3 (PR-3). also causes experimental emphysema.6 The studies to be described are aimed at elucidating the biochemical and functional properties of PR-3, an essential first step toward determining its role in physiologic and pathophysiologic processes.
METHODS Purification of Human PMNL Proteinases
PR-3, HLE, and cathepsin G were purified from an extract of PMNL granules using Matrex Gel Orange A chromatography followed by cation exchange chromatography on Bio-Rex 70 as previously described.6 The purity and molecular mass of each proteinase were determined by SDS-PAGE followed by silver staining of the gels. Purity was also ascertained by discontinuous nondenaturing gel electrophoresis followed by staining of the gels for esterase activity with a-naphthyl acetate as substrate. Protein concentration of purified enzymes was determined by the method of Hartree.'
Amino Acid Composition and N-Terminal Amino Acid Sequence of PR-3
Purified PR-3 was desalted and hydrolyzed in constant boiling HCI at 110°C in uacuo. Hydrolysates were dried and derivatized with phenyl isothiocyanate.
HPLC separations of derivatized samples were carried out at ambient temperature on Bio-Rad Biosil ODS-5S column using 30 mM of sodium phosphate buffer, pH 6.3, and acetonitrile as solvents.8 Peaks were assigned by comparison with standard amino acid mixtures. The N-terminal amino acid sequence of PR-3 was determined by sequential Edman degradation in a Beckman 890 D spinning cup sequenator, using 0.1 M of Quodral buffer and Polybrene ~ a r r i e r .Phenylthiohydantoin ~ derivatives were identified by HPLC on a Hewlett-Packard 1084B instrument using an Ultrasphere ODS column (0.46 x 15 cm, 5 pm particle size, endcapped) eluted with a gradient of acetonitrile in 0.05 M sodium acetate, pH 4.5.1° Search of the Swiss Prot 13 database (release 3.0, February 1990) for protein sequences homologous to PR-3 was carried out using PC/Gene software (Intelligenetics, Mountain View, California).
Proteolytic Activity of PR-3
Elastin degradation was assayed by determining the ability of PR-3 to solubilize bovine ligament elastin over a pH range from 6.5 to 8.9 using the procedure of Stone et a/." Tritiated powdered elastin was washed and resuspended in 0.2 M sodium phosphate buffer, pH 6.5 or 7.4, o r sodium bicarbonate buffer, pH 8.9. The proteinase in physiologic saline solution was added to a 5-mg aliquot of 3Helastin and the reaction mixture was incubated at 37°C for 6 hours. After incubation, the contents of each tube were filtered through medium porosity filter paper
62
ANNALS NEW YORK ACADEMY OF SCIENCES
to remove the insoluble elastin. The rate of degradation was determined by measuring the radioactivity in the filtrate. Hydrolysis of synthetic peptides was determined using peptide substrates coupled to chromogenic or fluorogenic groups. The method used for these substrates was that described by Barrett,I2 with some modification. Briefly, 50 pl of substrate (10 mg/I. I mi DMSO) was added to 0.1 M Tris buffer, pH 7.0. The reaction was started by the addition of the proteinase for a final volume of 3.0 mi. The release of p-nitrophenol and p-nitroaniline was measured by the increase in optical density at 347.5 nm and 410 nm, respectively. The release of 7-amino-4methylcoumarin (AMC) from MCA substrates was monitored flurometrically with excitation and emission wavelength of 380 nm and 440 nm. respectively.
Inhibition of PR-3
The ability of natural inhibitors to prevent PR-3 activity was determined for alphal-proteinase inhibitor (al-PI), alphal-antichymotrypsin (a,-Achy), and alpha2-macroglobulin (az-M). For at-PI and aI-Achy, equimolar concentrations of PR-3 and inhibitor in a final volume of 975 pI of 0.05 M phosphate buffer, pH 7.5, were incubated for selected periods of time. Residual PR-3 activity was then determined by the addition of 25 pi of 20 mM Boc-ala-ONp in methanol. The inhibition of a2-M was measured according to Virca and Travis.I3 Briefly, PR-3 (25 pmol) was incubated with a*-M in 0.5 ml of 0.05 M phosphate buffer, pH 7.5, at 25°C for 10 minutes. Subsequently, 25 pi of aI-PI (0.5 nmol) were added to the reaction mixture. After a 5-minute incubation, the mixture was brought up to 975 p1 with phosphate buffer, and then 25 pl of 20 mM Boc-Ala-ONp in methanol was added. The aI-PI-resistantesterase activity of PR-3 (that bound to a*-M) was monitored at 347.5 nm.
Immunologic Properties of PR-3
Monoclonal antibodies were prepared by initially immunizing 6-week-old female BALB/c J mice with purified PR-3 (in complete Freunds’ adjuvant). After three boosts, spleen cells were harvested and fused with murine myeloma P3-X63Ag 8.653 cells. After 10-14 days of growth in hypoxanthine-aminopterinthymidine selection culture medium, supernatants from hybridomas were evaluated for anti-PR-3 activity. Dot blot analysisI4 using anti-PR-3 monoclonal antibodies was performed on purified PMNL proteinases. Indicated amounts of purified enzymes were spotted on nitrocellulose paper. The blots were blocked with 5% nonfat dry milk, 0.05% Tween 20, and phosphate-buffered saline solution, pH 7.4, overnight at 4°C. Blots were incubated with antibodies diluted in the blocking solution for 1 hour at room temperature followed by 16 hours at 4°C. After washing with 0.05% Tween 20 in phosphate-buffered saline solution. the blots were reacted with affinity-purified rabbit anti-mouse IgG (heavy and light chains) conjugated to alkaline phosphatase.
RAO et af.: PROTEINASE-3
63
RESULTS SDS polyacrylamide gel electrophoresis of PR-3 revealed a major band of 26.8
kD and two minor bands of slightly larger molecular mass, the largest estimated at 28.6 kD. Values calculated after SDS polyacrylamide gel electrophoresis with nonreducing conditions were identical to those determined with reducing conditions. The amino acid composition of PR-3 is shown in TABLE I and compared to that of HLE and cathepsin G as reported by Travis et a/.'?PR-3 consisted of 224 amino
TABLE 1. Amino Acid Composition of Purified Proteinases
ResidueslMolecule PR-3 CysI2
Asx Thr Ser Glx
Pro GlY Ala Val Met Ile Leu T Y ~ Phe His LYS Arg TrP
8 20
II 7 20 16 32 17 19 2
HLEl' 6 24 7 13 18 10 28 24 25
II
2 II
19 4
20 3
12
9 4 I
9 4 I/ 2
22 2h
Cat Gi4 6 17 12 15 23 13 21 13 14 4
II 16 5 10 6
3
31 nd'
From ref. 15. Determined spectrophotometrically. nd = not determined.
acid residues per molecule. The composition was similar to that of HLE and cathepsin G, the greatest difference being the comparatively lower amount of arginine. The arginine content of PR-3 was one half that of HLE and approximately one third that of cathepsin G. A comparison of the first 20 residues of PR-3I6 with other serine proteinases shows that the first four N-terminal residues (ile' to gly4) were identical to that of HLE,I7 whereas the eight-residue stretch from pro9 to alaI6 was identical to that of Cat G,I8 murine granzymes A to F,is22 rat mast cell pro tease^,^^.^^ and human lymphocyte pro tease^.^^ Over the first 20 residues, PR-3 has approximately 60% homology to HLE,I7 70% to Cat G,I8 and 75% to human lymphocyte pro tease^.^^
ANNALS NEW YORK ACADEMY OF SCIENCES
64
When compared with the granzyme family of serine proteases, PR-3 exhibited strong homology to granzyme B and less to granzymes A and C through F and to rat mast cell proteases. The first 20 residues of the PR-3 sequence are essentially identical to those recently published for c-ANCA antigen and AGP7.26*27 In light of the initial studies suggesting that PR-3 could induce emphysema, its elastinolytic activity was evaluated. Assays employing 3H-elastin (TABLE2) demonstrated that PR-3 hydrolyzed more elastin than did HLE (w/w) at pH 6.5, that its activity at pH 7.4 was -50% that of HLE, and that it was much less active than was HLE at pH 8.9. We assessed the esterolytic and amidolytic activity of PR-3 against selected chromogenic and fluorogenic substrates. The data are summarized in TABLE3. Using monomeric p-nitrophenyl ester substrates, PR-3 hydrolyzed the monomeric p-nitrophenyl esters of alanine and valine. PR-3 showed no activity against ester substrates containing leucine at the p l site. We next tested the amidolytic activity of PR-3 against four other HLE-specific oligopeptide substrates. Of the three tripeptide substrates (Suc-Ala-Ala-Ala-NA, Suc-Ala-Ala-Val-NA, and Suc-AlaPro-Ala-MCA) and one tetrapeptide substrate (MeO-Suc-Ala-Ala-Pro-Val-NA) tested, only Suc-Ala-Pro-Ala-MCA and MeO-Suc-Ala-Ala-Pro-Val-NA were hydrolyzed. PR-3 showed no activity against other oligomeric amide substrates having Leu at the p l site. We next focused on the interaction of PR-3 with the human plasma proteinase inhibitors al-PI, Q-M, and al-Achy utilizing the most efficiently hydrolyzed substrate Boc-ala-ONp. PR-3 was inhibited by a,-PI and a*-M under the experimental conditions employed: PR-3 was not inhibited by a,-Achy. The uniqueness of PR-3 was further documented when purified PR-3, HLE, and cathepsin G were tested for their ability to react with monoclonal antibodies raised against PR-3. A representative result using anti-HPR-3 monoclonal antibody H-66 is shown in FIGURE1. Only PR-3 reacted with H-66. Similar results were obtained using three other monoclonal antibodies. DISCUSSION PR-3 was originally described by Baggiolini et a1.2sas an a-naphthyl acetate esterase present in azurophilic granules of human PMNL. Its electrophoretic
TABLE 2. Degradation of Elastin by Purified PMNL Proteinaseu "-Elastin (cpm released/5 rng 3H-Elastin/6 h)
PR-3 HLE Cathemin G
pH 6.5 1710 t 50" 1.190 k 240 nd'
pH 7.4 2,250 t 30 4,470 t 300 408"
pH 8.9 2,100 t 120 18,770 2 640 890 t 50
From ref. 6.
* All values are corrected for background
activity by subtracting radioactivity in buffer controls. Background activity was always
The current explanation for the development of emphysema is the proteinase pathogenesis hypothesis, which holds that progressive destruction of the interstitium is due to an excess of proteolytic enzymes (particularly elastase) in relation to the availability of proteolytic inhibitors. Inasmuch as a major endogenous source of elastase in the human lung is the polymorphonuclear leukocyte (PMNL), emphasis has been placed on the effects of PMNL-derived elastase and its inhibitors in regulating the degradation of lung elastin. Experimental emphysema has been produced by a single intratracheal injection of purified human PMNL elastase (HLE) in laboratory animals. Potent synthetic inhibitors have been developed for HLE, and several are effective in preventing emphysema in animal models of HLE-induced disease. To consider only HLE-mediated injury in examining the pathogenesis of emphysema in the human, however, is too narrow a focus. A direct relation of HLE to human disease remains to be demonstrated. Recent studies suggest that the increased risk of emphysema associated with cigarette smoking may be due partially to the effect of phagocyte- or smoke-derived oxidants in the lung. Another variable immediately tangent to the HLE pathogenesis concept is that other PMNL or alveolar macrophage (AM) proteinases may participate in matrix destruction. Cathepsin G and an AM cysteine proteinase(s1 both have elastolytic capabilities in uitro.'**Moreover, cathepsin G has been reported to act synergistically with HLE in the solubilization of elastin in ~ i t r oalthough ,~ results have been d i ~ c r e p a n t . Studies ~.~ to date that have failed to demonstrate that these proteinases can produce emphysema4do not exclude their participation in the induction of the disease.
Address for correspondence: John R. Hoidal, Pulmonary Division, Rm. 4R240, 50 North Medical Drive, University of Utah Medical Center, Salt Lake City, UT 84132. 60
RAO et al.: PROTEINASE-3
61
In preliminary studies, we marshalled evidence that another human PMNL serine proteinase, proteinase-3 (PR-3). also causes experimental emphysema.6 The studies to be described are aimed at elucidating the biochemical and functional properties of PR-3, an essential first step toward determining its role in physiologic and pathophysiologic processes.
METHODS Purification of Human PMNL Proteinases
PR-3, HLE, and cathepsin G were purified from an extract of PMNL granules using Matrex Gel Orange A chromatography followed by cation exchange chromatography on Bio-Rex 70 as previously described.6 The purity and molecular mass of each proteinase were determined by SDS-PAGE followed by silver staining of the gels. Purity was also ascertained by discontinuous nondenaturing gel electrophoresis followed by staining of the gels for esterase activity with a-naphthyl acetate as substrate. Protein concentration of purified enzymes was determined by the method of Hartree.'
Amino Acid Composition and N-Terminal Amino Acid Sequence of PR-3
Purified PR-3 was desalted and hydrolyzed in constant boiling HCI at 110°C in uacuo. Hydrolysates were dried and derivatized with phenyl isothiocyanate.
HPLC separations of derivatized samples were carried out at ambient temperature on Bio-Rad Biosil ODS-5S column using 30 mM of sodium phosphate buffer, pH 6.3, and acetonitrile as solvents.8 Peaks were assigned by comparison with standard amino acid mixtures. The N-terminal amino acid sequence of PR-3 was determined by sequential Edman degradation in a Beckman 890 D spinning cup sequenator, using 0.1 M of Quodral buffer and Polybrene ~ a r r i e r .Phenylthiohydantoin ~ derivatives were identified by HPLC on a Hewlett-Packard 1084B instrument using an Ultrasphere ODS column (0.46 x 15 cm, 5 pm particle size, endcapped) eluted with a gradient of acetonitrile in 0.05 M sodium acetate, pH 4.5.1° Search of the Swiss Prot 13 database (release 3.0, February 1990) for protein sequences homologous to PR-3 was carried out using PC/Gene software (Intelligenetics, Mountain View, California).
Proteolytic Activity of PR-3
Elastin degradation was assayed by determining the ability of PR-3 to solubilize bovine ligament elastin over a pH range from 6.5 to 8.9 using the procedure of Stone et a/." Tritiated powdered elastin was washed and resuspended in 0.2 M sodium phosphate buffer, pH 6.5 or 7.4, o r sodium bicarbonate buffer, pH 8.9. The proteinase in physiologic saline solution was added to a 5-mg aliquot of 3Helastin and the reaction mixture was incubated at 37°C for 6 hours. After incubation, the contents of each tube were filtered through medium porosity filter paper
62
ANNALS NEW YORK ACADEMY OF SCIENCES
to remove the insoluble elastin. The rate of degradation was determined by measuring the radioactivity in the filtrate. Hydrolysis of synthetic peptides was determined using peptide substrates coupled to chromogenic or fluorogenic groups. The method used for these substrates was that described by Barrett,I2 with some modification. Briefly, 50 pl of substrate (10 mg/I. I mi DMSO) was added to 0.1 M Tris buffer, pH 7.0. The reaction was started by the addition of the proteinase for a final volume of 3.0 mi. The release of p-nitrophenol and p-nitroaniline was measured by the increase in optical density at 347.5 nm and 410 nm, respectively. The release of 7-amino-4methylcoumarin (AMC) from MCA substrates was monitored flurometrically with excitation and emission wavelength of 380 nm and 440 nm. respectively.
Inhibition of PR-3
The ability of natural inhibitors to prevent PR-3 activity was determined for alphal-proteinase inhibitor (al-PI), alphal-antichymotrypsin (a,-Achy), and alpha2-macroglobulin (az-M). For at-PI and aI-Achy, equimolar concentrations of PR-3 and inhibitor in a final volume of 975 pI of 0.05 M phosphate buffer, pH 7.5, were incubated for selected periods of time. Residual PR-3 activity was then determined by the addition of 25 pi of 20 mM Boc-ala-ONp in methanol. The inhibition of a2-M was measured according to Virca and Travis.I3 Briefly, PR-3 (25 pmol) was incubated with a*-M in 0.5 ml of 0.05 M phosphate buffer, pH 7.5, at 25°C for 10 minutes. Subsequently, 25 pi of aI-PI (0.5 nmol) were added to the reaction mixture. After a 5-minute incubation, the mixture was brought up to 975 p1 with phosphate buffer, and then 25 pl of 20 mM Boc-Ala-ONp in methanol was added. The aI-PI-resistantesterase activity of PR-3 (that bound to a*-M) was monitored at 347.5 nm.
Immunologic Properties of PR-3
Monoclonal antibodies were prepared by initially immunizing 6-week-old female BALB/c J mice with purified PR-3 (in complete Freunds’ adjuvant). After three boosts, spleen cells were harvested and fused with murine myeloma P3-X63Ag 8.653 cells. After 10-14 days of growth in hypoxanthine-aminopterinthymidine selection culture medium, supernatants from hybridomas were evaluated for anti-PR-3 activity. Dot blot analysisI4 using anti-PR-3 monoclonal antibodies was performed on purified PMNL proteinases. Indicated amounts of purified enzymes were spotted on nitrocellulose paper. The blots were blocked with 5% nonfat dry milk, 0.05% Tween 20, and phosphate-buffered saline solution, pH 7.4, overnight at 4°C. Blots were incubated with antibodies diluted in the blocking solution for 1 hour at room temperature followed by 16 hours at 4°C. After washing with 0.05% Tween 20 in phosphate-buffered saline solution. the blots were reacted with affinity-purified rabbit anti-mouse IgG (heavy and light chains) conjugated to alkaline phosphatase.
RAO et af.: PROTEINASE-3
63
RESULTS SDS polyacrylamide gel electrophoresis of PR-3 revealed a major band of 26.8
kD and two minor bands of slightly larger molecular mass, the largest estimated at 28.6 kD. Values calculated after SDS polyacrylamide gel electrophoresis with nonreducing conditions were identical to those determined with reducing conditions. The amino acid composition of PR-3 is shown in TABLE I and compared to that of HLE and cathepsin G as reported by Travis et a/.'?PR-3 consisted of 224 amino
TABLE 1. Amino Acid Composition of Purified Proteinases
ResidueslMolecule PR-3 CysI2
Asx Thr Ser Glx
Pro GlY Ala Val Met Ile Leu T Y ~ Phe His LYS Arg TrP
8 20
II 7 20 16 32 17 19 2
HLEl' 6 24 7 13 18 10 28 24 25
II
2 II
19 4
20 3
12
9 4 I
9 4 I/ 2
22 2h
Cat Gi4 6 17 12 15 23 13 21 13 14 4
II 16 5 10 6
3
31 nd'
From ref. 15. Determined spectrophotometrically. nd = not determined.
acid residues per molecule. The composition was similar to that of HLE and cathepsin G, the greatest difference being the comparatively lower amount of arginine. The arginine content of PR-3 was one half that of HLE and approximately one third that of cathepsin G. A comparison of the first 20 residues of PR-3I6 with other serine proteinases shows that the first four N-terminal residues (ile' to gly4) were identical to that of HLE,I7 whereas the eight-residue stretch from pro9 to alaI6 was identical to that of Cat G,I8 murine granzymes A to F,is22 rat mast cell pro tease^,^^.^^ and human lymphocyte pro tease^.^^ Over the first 20 residues, PR-3 has approximately 60% homology to HLE,I7 70% to Cat G,I8 and 75% to human lymphocyte pro tease^.^^
ANNALS NEW YORK ACADEMY OF SCIENCES
64
When compared with the granzyme family of serine proteases, PR-3 exhibited strong homology to granzyme B and less to granzymes A and C through F and to rat mast cell proteases. The first 20 residues of the PR-3 sequence are essentially identical to those recently published for c-ANCA antigen and AGP7.26*27 In light of the initial studies suggesting that PR-3 could induce emphysema, its elastinolytic activity was evaluated. Assays employing 3H-elastin (TABLE2) demonstrated that PR-3 hydrolyzed more elastin than did HLE (w/w) at pH 6.5, that its activity at pH 7.4 was -50% that of HLE, and that it was much less active than was HLE at pH 8.9. We assessed the esterolytic and amidolytic activity of PR-3 against selected chromogenic and fluorogenic substrates. The data are summarized in TABLE3. Using monomeric p-nitrophenyl ester substrates, PR-3 hydrolyzed the monomeric p-nitrophenyl esters of alanine and valine. PR-3 showed no activity against ester substrates containing leucine at the p l site. We next tested the amidolytic activity of PR-3 against four other HLE-specific oligopeptide substrates. Of the three tripeptide substrates (Suc-Ala-Ala-Ala-NA, Suc-Ala-Ala-Val-NA, and Suc-AlaPro-Ala-MCA) and one tetrapeptide substrate (MeO-Suc-Ala-Ala-Pro-Val-NA) tested, only Suc-Ala-Pro-Ala-MCA and MeO-Suc-Ala-Ala-Pro-Val-NA were hydrolyzed. PR-3 showed no activity against other oligomeric amide substrates having Leu at the p l site. We next focused on the interaction of PR-3 with the human plasma proteinase inhibitors al-PI, Q-M, and al-Achy utilizing the most efficiently hydrolyzed substrate Boc-ala-ONp. PR-3 was inhibited by a,-PI and a*-M under the experimental conditions employed: PR-3 was not inhibited by a,-Achy. The uniqueness of PR-3 was further documented when purified PR-3, HLE, and cathepsin G were tested for their ability to react with monoclonal antibodies raised against PR-3. A representative result using anti-HPR-3 monoclonal antibody H-66 is shown in FIGURE1. Only PR-3 reacted with H-66. Similar results were obtained using three other monoclonal antibodies. DISCUSSION PR-3 was originally described by Baggiolini et a1.2sas an a-naphthyl acetate esterase present in azurophilic granules of human PMNL. Its electrophoretic
TABLE 2. Degradation of Elastin by Purified PMNL Proteinaseu "-Elastin (cpm released/5 rng 3H-Elastin/6 h)
PR-3 HLE Cathemin G
pH 6.5 1710 t 50" 1.190 k 240 nd'
pH 7.4 2,250 t 30 4,470 t 300 408"
pH 8.9 2,100 t 120 18,770 2 640 890 t 50
From ref. 6.
* All values are corrected for background
activity by subtracting radioactivity in buffer controls. Background activity was always
