characterization of proteinase-3 (pr-3), a neutrophil serine

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 266. No. 15, Issue of May 25, pp. 9540-9548,1991 Printed in U.S.A.

Characterization of Proteinase-3 (PR-3), a Neutrophil Serine Proteinase STRUCTURAL AND FUNCTIONAL PROPERTIES*

(Received for publication, October 17,1990)

Narayanam V. Rao$, Nancy G. Wehnere, Bruce C. Marshall$, William R. GrayT, Beulah H. Gray§, and John R. Hoidal$II From the Departments of $Pulmonary Medicine and TlBiology, the University of Utah Medical Center, Salt Lake City, Utah 84132 and the §Department of Microbiology, University of Minnesota, Minneapolis, Minnesota 55455

Proteinase 3 (PR-3) is a human polymorphonuclear leukocyte (PMNL) serine proteinase that degrades elastin in vitro and causes emphysema when administered by tracheal insufflation to hamsters (Kao, R. C., Wehner, N. G., Skubitz, K. M., Gray, B. H., and Hoidal, J. R. (1988) J. CZin. Invest. 82, 1963-1973). We have determined the primary structure of several PR-3 peptides and have analyzed catalytic properties of the enzyme. The enzyme has considerable amino acid sequence homology with two other well characterized PMNL neutral serine proteinases, elastase and cathepsin G. Furthermore, the NHz-terminal amino acid sequence of PR-3 is identical to that of the target antigen of the anti-neutrophil cytoplasmic autoantibodies associated with Wegener’s granulomatosis. PR-3 degrades a variety of matrix proteins including fibronectin, laminin, vitronectin, and collagen type IV. It shows no or minimal activity against interstitial collagens types I and 111, respectively.

The analysis of peptides generated by PR-3 digestion of insulin chains and the activity profile against a panel of chromogenic synthetic peptide substrates show that PR-3 prefers small aliphatic amino acids (alanine, serine, and valine) at the P, site. The elastase-like specificity of PR-3 is consistent with its striking sequence homology to elastase at substrate binding sites. PR-3 is inhibited by a,-proteinase inhibitor (k, = 8.1 X lo6 M” s-’; delay time = 25 ms) and a2-macroglobulin (k, = 1.1 X 10’ M” s-I; delay time = 114 ms) but not by a,-anti-chymotrypsin. In contrast to elastase and cathepsin G, PR-3 is not inhibited by secretory leukoprotease inhibitor and is weakly inhibited by eglin e. Thus, PR-3 is distinct from the other PMNL proteinases.

Serine proteinases play a critical role in numerous physiologic processes including digestion, blood coagulation, com- plement activation, fibrinolysis, reproduction, and develop- ment. These enzymes may contribute to the ability of poly-

* This research was supported in part by Clinical Investigator Award 1K08 HL 02370 (to B. C. M.) and by Grants HL-37615 and HL 07636 from the National Institute of Health and the Veterans Administration Research Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

11 To whom correspondence should be addressed Pulmonary Divi- sion, Rm. 4R240, 50 N. Medical Dr., University of Utah Medical Center, Salt Lake City, UT 84132.

morphonuclear leukocytes (PMNL)’ to migrate through the basement membrane as they exit the circulation (1) and to their ability to digest microorganisms and other ingested material within the phagolysosome (2). Human leukocyte elastase (HLE) and cathepsin G (Cat G) are two proteinases thought to be involved in these physiologic activities. Al- though the activity of these enzymes is precisely controlled in the healthy state, tissue destruction occurs when the enzymes released from PMNL overwhelm their natural inhibitors, al- proteinase inhibitor, az-macroglobulin (aZ”), and al-antichymotrypsin (al-Achy) at inflammatory sites. Diseases in which the action of PMNL serine proteinases has been im- plicated include pulmonary emphysema (3-7), the adult respiratory distress syndrome (8), rheumatoid arthritis (9, lo), and glomerulonephritis (11).

Recently we purified from human PMNL azurophilic granules a third neutral serine proteinase designated proteinase-3 (PR-3). Originally identified by Baggiolini et al. (12), PR-3 degrades elastin in vitro and causes extensive tissue damage and emphysema when administered to hamsters by tracheal insufflation (13). We now report on the structural and functional properties of PR-3.

EXPERIMENTAL PROCEDURES

Materials

Fibronectin was purified from human plasma by a two-step affinity chromatography procedure using gelatin-agarose (14) and heparin- agarose (15). EHS mouse tumor laminin, collagen type IV, and rat tail collagen type I were purchased from Collaborative Research Inc. Vitronectin purified from human serum by heparin-Sepharose affinity chromatography (16) was a gift of Dr. Charles Parker (Veterans Administration Medical Center, Salt Lake City, UT). Bovine skin collagen type 111 was a gift of Dr. Jerome Seyer (VA Medical Center, Memphis, TN). Oxidized insulin-B chain and sequencing grade trypsin were purchased from Boehringer Mannheim. Dansyl chloride, amino acids, and trifluoroacetic acid were from Pierce Chemical Co. HPLC solvents were from Burdick & Jackson Laboratories, Inc. (Muskegon, MI). Protected amino acidp-nitrophenyl ester substrates were from Bachem, Inc. (Torrance, CA). Synthetic peptide substrates with nitroanilide or 4-methyl-coumaryl-7-amide (MCA) leaving groups were from either Sigma or Peninsula Laboratories, Inc. (Bel-

l The abbreviations used are: PMNL, polymorphonuclear leukocyte; al-Achy, al-antichymotrypsin; a2-M, az-macroglobulin; Boc, t- butyloxycarbonyl; Cat G, cathepsin G; c-ANCA, cytoplasmic-staining anti-neutrophil cytoplasmic autoantibodies; dansyl, 5-dimethylami- nonaphthalene-1-sulfonyl; Glt, glutaryl; HLE, human leukocyte elastase; HPLC, high performance liquid chromatography; MCA, 4- methyl-coumaryl-7-amide; MeO, methoxy; NA, 4-nitroanilide; ONp, p-nitrophenylester; PR-3, proteinase-3; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; SUC, succinyl; Z, benzylox- ycarbonyl.

9540

Characterization of PR-3 954 1

mont, CA). Peptide chloromethyl ketone inhibitors were from En- zyme Systems Products (Livermore, CA). Human al-proteinase inhibitor, aZ-M, and al-Achy were from Athens Research and Technol- ogy, Inc. (Athens, GA). Recombinant forms of secretory leukoprotease inhibitor and eglin c were gifts of Dr. Robert Thompson (Synergen, Boulder, CO) and Dr. Hans Peter Schnebli (Ciba-Geigy AG, Basel, Switzerland), respectively. All other chemicals not specifically men- tioned were high quality grade from Sigma.

Purification of Human PMNL Proteinases PR-3, HLE, and Cat 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 described previ- ously (13). The purity and molecular mass of each proteinase were determined by SDS-PAGE (17) followed by silver staining of the gels (18). Purity was also ascertained by discontinuous nondenaturing gel electrophoresis (19) followed by staining of the gels for esterase activity with a-naphthyl acetate as substrate (20). Protein concentration of purified enzymes was determined by the method of Hartree (21).

Primary Structure of PR-3 Deriuatization of PR-3-PR-3 was reduced and alkylated with 4-

vinylpyridine (22, 23). Briefly, 100 pg of PR-3 was suspended in 200 pl of 0.1 M Tris, 6 M guanidine HCl, pH 7.5. 2 pl of 0.65 M dithio- threitol in Tris guanidine buffer (about 50-fold excess over 24 nmol of cysteine in PR-3) was then added under nitrogen and incubated at 37 'C for 1 h in the dark. Then 2 pl of 4-vinylpyridine (300-fold excess over sulfhydryl groups) was added under nitrogen and incubated for an additional h a t 37 "C in the dark. The S-pyridylethylated PR-3 was precipitated by the addition of 9 volumes of cold methanol and kept a t -20 'C overnight. The precipitate was collected by centrifugation and washed with an additional 1 ml of cold methanol. The derivatized PR-3 was subjected to either trypsin digestion to obtain peptides or directly sequenced.

Trypsin Digestion of PR-3-Trypsin (5 pg) was added to the derivatized PR-3 suspended in 200 pl of 0.1 M NH4HC03, 0.1 M CaClz buffer, pH 8.5. The mixture was incubated for 2 h at 37 "C. The reaction was terminated by the addition of 5 pl of glacial acetic acid. The peptides from the digest were fractionated by reverse phase HPLC using a Vydac CIS column (3.9 X 300 mm, 5-pm particle size) equilibrated with 0.1% trifluoroacetic acid (solvent I). Peptides were eluted with a linear gradient of acetonitrile (80%) containing 0.1% trifluoroacetic acid (solvent 11) at a flow rate of 1 ml/min for 100 min. Fractions containing a mixture of peptides were repurified by HPLC and then subjected to amino acid sequence analysis.

NH2-terminal Amino Acid Sequence Analysis of PR-3 and Its Tryp- tic Peptides-The NH2-terminal amino acid sequence of derivatized PR-3 and the peptides obtained by trypsin digestion was determined by sequential Edman degradation in a Beckman 890 D spinning cup sequenator, using 0.1 M Quadrol buffer and Polybrene carrier (24). Phenylthiohydantoins were identified by HPLC on a Hewlett-Pack- ard 1084B instrument using an Ultrasphere ODS column (4.6 X 150 mm, 5-pm particle size, end capped) eluted with a gradient of acetonitrile in 0.05 M sodium acetate, pH 4.5 (25). Search of the Swiss Prot 13 data base (release 3.0, February 1990) for protein sequences homologous to PR-3 was carried out using PC/Gene software (Intel- ligenetics, Mountain View, CA).

Digestion of Matrix Proteins by PMNL Proteinases Digestion of purified matrix proteins by PR-3, HLE, or Cat G was

performed in 0.15 M NaCl, 0.05 M Tris-HC1 buffer, pH 7.4, with an enzyme/substrate (w/w) ratio of 1:lOO except for fibronectin (enzyme/substrate (w/w) ratio of 1:600). Fibronectin, laminin, vitronectin, and collagen type IV were digested at 37 "C for periods ranging from 15 min to 3 h. Digestion of collagen type I and type 111 was performed at room temperature for a period of 16 h. The reactions were stopped by rapid freezing followed by lyophilization. The samples were boiled in 2% SDS containing 8-mercaptoethanol and analyzed by SDS-PAGE (17). Gels were stained with either Coomassie Blue R-250 or silver nitrate (Bio-Rad silver staining kit).

Characterization of Peptide Bond Specificity of PR-3 Purification of Insulin C h i n Peptides Generated by PR-3-The

oxidized insulin-B chain, 250 pg (67 nmol), or insulin-A chain, 250 pg (90 nmol) was incubated with 10 pg (0.3 nmol) of PR-3 at 37 "C

for 20 min in a final volume of 250 pl of 50 mM Tris, pH 7.4. The reaction was terminated with 2 p1 of glacial acetic acid. The resulting insulin peptides were fractionated on a Waters C18 pBondapak reverse phase column (3.9 X 300 mm, 10-pm particle size) as follows. Solvents I and I1 were composed of 10% acetonitrile, 0.07% trifluoroacetic acid and 50% acetonitrile, 0.06% trifluoroacetic acid, respectively. The insulin-B digest was fractionated with a linear gradient from 0 to 100% solvent 11, and the insulin-A digest was fractionated with a linear gradient from 0 to 60% solvent 11. Each fractionation was performed over 60 min at a flow rate of 1 ml/min. Peptide- containing fractions detected by absorbance at 210 nm were collected, dried in a Speed-Vac concentrator, and stored at -20 "C until analysis.

Determination of Amino Acid Composition and the NH2-terminal Amino Acid of Insulin Chain Peptides-Amino acids were derivatized with dansyl chloride as described by De Jong and Hughes (26), a modification of Tapuhi et al. (27). In brief, dried peptides were hydrolyzed in constant boiling HCl at 110 "C in uacuo. Hydrolysates were dried and derivatized by adding 200 p1 of 40 mM LizC03 buffer, pH 9.5, and 100 pl of dansyl chloride (1.5 mg/ml, 5.56 mM) in acetonitrile and incubated at 37 "C for 60 min. The reaction was terminated by the addition of 10 pl of 2% methylamine HCl and incubation continued for another 5 min. Standard amino acid mix- tures were derivatized in a similar fashion and used to establish column retention times. The derivatized samples were kept at -20 "C until analysis.

To determine the NHz-terminal amino acid, the peptides were first dansylated, dried, and hydrolyzed with constant boiling HCl and redried. The samples were then dissolved in water/acetonitrile (2:1, v/v) and subjected to HPLC. All HPLC separations were carried out at ambient temperature on a Bio-Rad Bio-Si1 ODs-5S column (4 X 250 mm, 5-pm particle size) using 30 mM sodium phosphate buffer, pH 6.5, and acetonitrile as solvents. A complex multistep gradient was set up as described by Oray et af. (28). Fluorescence of dansylated amino acids was monitored by using a Kratos fluorescence detector, model Spectra Flow 980. An excitation wavelength of 250 nm and a 470-nm emission filter were employed.

Kinetics Substrate Kinetics-Kinetic constants K, and kat were calculated

from initial rates of hydrolysis by Hanes-Woolf plots using linear regression. The correlation coefficients were > 0.99 in all experiments. Hydrolysis of the 4-nitroanilide substrates was measured in a 1-ml final volume of 0.1 M Tris buffer, pH 7.5, containing 0.05% Triton X-100. Initial velocities were calculated based on an extinction coefficient of 8.8 X lo3 M" cm" for nitroaniline at 410 nm. Hydrolysis rates of the nitrophenyl ester substrates were measured in a similar fashion except the reactions were performed in 0.05 M sodium phosphate buffer, pH 7.5, and the initial velocities were calculated based on the extinction coefficient of 5.5 X lo3 M" cm" for p-nitrophenol a t 347.5 nm (29).

The release of 7-amino-4-methylcoumarin from the MCA substrates was monitored fluorometrically with excitation and emission wavelengths of 380 and 440 nm, respectively. The fluorometer was calibrated with known concentrations of 7-amino-4-methylcoumarin determined spectrophotometrically by using an extinction coefficient of 9.77 X lo3 M" cm" at 342.5 nm (30). Assays were performed in a total volume of 100 pl of 0.05 M Tris, pH 7.5, containing 0.05% Triton x-100.

Synthetic Inhibitor Kinetics-Inhibition of enzymes by peptide chloromethyl ketones was measured under pseudo-first order conditions by incubating 50-500-fold excess of inhibitor with enzyme. For example, 10 pl of 4 mM inhibitor dissolved in methanol was mixed with 10 pl of 40 pM PR-3 in a final volume of 1 ml of 0.1 M phosphate buffer, pH 7.5, and incubated at 25 "C. The time course of inactivation was followed by assaying aliquots (100 pl) from the reaction mixture for residual enzyme activity [E] using Boc-Ala-ONp as described (29). The pseudo-first order rate constant (hoba) was obtained from the slope by plotting In [E] against t (time of sampling) using the relationship In [ E ] = - k b s . t expressed in terms of the second order inhibition constant k,,h/[Il (31).

Natural Inhibitor Kinetics-The molar concentrations of proteinases and inhibitors were calculated based on the following published A% and molecular masses (in Da): porcine trypsin, 13.5 and 23,400 (32); bovine chymotrypsin, 20.5 and 25,600 (33); al-proteinase inhibitor, 5.3 and 52,000 (34); secretory leukoprotease inhibitor, 7.8 and 11,200 (35); a,", 8.93 and 720,000 (36); soybean trypsin inhibitor, 10.1 and 20,100 (37); al-Achy, 6.2 and 68,000 (38); eglin c, 8.9 and

9542 Characterization of PR-3 8,100 (39). Molar concentrations were corrected after active concentration determination performed as follows.

The active sites of porcine pancreatic trypsin and bovine pancreatic chymotrypsin were titrated with p-nitrophenyl 4'-guanidinobenzoate and Boc-Tyr-ONp ester, respectively (40, 41). They were found to be 74% (trypsin) and 85% (chymotrypsin) active with respect to total protein. These were then used as primary standards in determining active ,inhibitor concentrations. For example, a constant amount of trypsin (1 x 10"' mol) was incubated with varying amounts of a,- proteinase inhibitor (0.1-2.0 X 10"O mol) in 500 p1 of 0.05 M Tris buffer, pH 8.0, at 25 "C for 5 min. It was then diluted to a final volume of 990 pl with buffer, and 10 p1 of 100 mM benzoyl-arginine ethyl ester was added (dissolved in 10% dimethyl sulfoxide in acetonitrile). The residual trypsin activity was monitored at 253 nm. A plot of residual trypsin activity versus al-proteinase inhibitor concentration was used to determine the active inhibitor concentration. The active concentrations of a,-proteinase inhibitor and secretory leukoprotease inhibitor titrated against trypsin were found to be 62 and 52%, respectively, and those of a,-Achy and eglin c titrated against chymotrypsin were found to be 88 and loo%, respectively. The standardized al-proteinase inhibitor was used as secondary standard against purified PR-3 to determine the active enzyme concentration using Boc-Ala-ONp as the substrate.

Association rate constants of PR-3 with a,-proteinase inhibitor, al-Achy, secretory leukoprotease inhibitor, and eglin c were measured under second order conditions in which the rate was dependent on the time and concentrations of both enzyme and inhibitor. Equimolar concentrations of PR-3 and inhibitor were incubated in a final volume of 975 pl of 0.05 M phosphate buffer, pH 7.5, for selected periods of time. Residual PR-3 activity was then determined by the addition of 25 p1 of 20 mM Boc-Ala-ONp in methanol. The amount of inhibitors used in these experiments was as follows: a,-proteinase inhibitor, 8.25 pmol; al-Achy, 30 pmol; secretory leukoprotease inhibitor, 33 pmol; eglin c, 50 pmol. The association rate constant for PR-3 with a,-proteinase inhibitor was also measured by competition experiments, a method employing one inhibitor and two enzymes (bovine chymotrypsin was used as the second enzyme) (42,43).

The molar binding stoichiometry of proteinases to a,-M was determined by taking into account the characteristic feature that az-M- bound proteinases retain activity with low molecular weight substrates and resist inhibition by other high molecular weight inhibitors such as a,-proteinase inhibitor. A constant amount of az-M (50 pmol) was incubated with increasing amounts of trypsin (10-140 pmol, enzyme/inhibitor ratio of 0.2-2.8) in 965 p1 of 0.1 M Tris, 0.01 M CaC12 buffer, pH 8.0, containing 0.05% Triton X-100 at 25 "C for 5 min. Subsequently, 25 p1 (1 nmol) of soybean trypsin inhibitor was added to the reaction mixture to inhibit free trypsin. After 5 min, 10 p1 of 100 mM p-toluene-sulfonyl-L-arginine methyl ester substrate was added to the reaction mixture. The soybean trypsin inhibitor- resistant esterase activity of trypsin (that bound to az-M) was followed at 540 nm. The stoichiometry of aZ-M to trypsin was determined by plotting az-M-bound trypsin activity versus trypsin:az-M molar ratios.

Equivalence titration of PR-3 to a2-M was performed in a similar fashion by incubating a constant amount of PR-3 (25 pmol) with increasing amounts of az-M in 0.5 ml of 0.05 M phosphate buffer, pH 7.5, at 25 "C for 10 min. Subsequently, 25 pl of al-proteinase inhibitor (0.5 nmol) was added to the reaction mixture. After a 5-min incubation the mixture was brought to 975 pl with phosphate buffer, and then 25 pl of 20 mM Boc-Ala-ONp in methanol was added. The a,- proteinase inhibitor-resistant esterase activity of PR-3 (that bound to a,") was monitored at 347.5 nm. The stoichiometry of az-M to PR-3 was determined by plotting esterase activity versus 012-M concentration.

The association rate constant of PR-3 with aZ-M was measured competitively with a,-proteinase inhibitor according to published methods (42,44). Briefly, PR-3 (25 pmol) was added to a mixture of az-M (25 pmol) and al-proteinase inhibitor (25 pmol) in a final volume of 975 pl of 0.05 M phosphate buffer, pH 7.5, at 25 "C. After 10 min 25 pl of 20 mM Boc-Ala-ONp in methanol was added, and the esterase activity of PR-3 bound to az-M was followed at 347.5 nm.

RESULTS

Amino Acid Sequence-Leukocyte granules have been shown to consist of closely related proteinases. Table I shows a comparison of the first 40 residues of PR-3 with other serine

proteinases. The first 4 NH,-terminal residues (Ile' to Gly4) are identical to those of HLE (45) whereas the 8-residue stretch from Prog to Ala16 is identical to that of Cat G (46), murine granzymes A to F (47-50), rat mast cell proteinases (51-531, and human lymphocyte proteinase (54). Over the first 40 residues PR-3 has approximately 60% similarity to HLE (45), Cat G (46), and human lymphocyte proteinase (54). When compared with the granzyme family of serine proteinases, PR-3 exhibits strong similarity (57%) to granzyme B and less (48-55%) to granzyme A, C through F, and rat mast cell proteinases. The first 20 residues of the PR-3 sequence are identical to that published recently for the anti- neutrophil cytoplasmic autoantibodies (c-ANCA) antigen (55, 56) and AGP7/p29b, a PMNL serine proteinase of azurophil granules (except for Gln" instead of Glu in AGP7) (57, 58). In addition, residues 15-40 of PR-3 are very similar to the first 26 deduced amino acids of myeloblastin (except for uncertainity at residue 35 and a mismatch at residue 37) (59).

Two peptides (p-I and p-11) generated by trypsin digestion appear to come from the substrate binding region (Table 11). Four of the 8 residues of p-I and 5 of the 9 residues of p-I1 are identical to regions near the COOH terminus of HLE (Leulgs to Prozo5 and to Asp214, HLE numbering). Res- idues forming the cental core of the substrate binding pocket are thought to be at positions -6, +15 to +17, and +28 relative to the catalytic serine residue (60). By combining the sequence of the active site peptide of AGP7 (57) and the tryptic fragments of PR-3 (on the assumption that AGP7 and PR-3 are the same protein) we found that the amino acid residues at positions -6, +15, +16, and +28 in PR-3 are identical to those of HLE.

Action on Extracellular Matrix Proteins-In previous work we showed that PR-3 degrades elastin in uitro (13). In the present study we evaluated further the in uitro catalytic activity of PR-3 against the following extracellular matrix macromolecules: fibronectin, laminin, vitronectin, and collagen types I, 111, and IV (Fig. 1). PR-3 cleaved fibronectin into seven distinct fragments, two of which were between 200 and 220 kDa. Under the conditions used these two peptides were not observed after digestion by HLE or Cat G. Enzyme digests of laminin showed that PR-3 partially degraded both a and p subunits. By comparison, HLE digested both a and p subunits completely whereas Cat G attacked only the a subunit. PR-3 partially degraded the 75-kDa polypeptide of vitronectin whereas HLE degraded both the 75- and 65-kDa polypeptides, and Cat G completely digested the 75-kDa polypeptide. All three proteinases cleaved collagen type IV. On 16-h incubation, PR-3 did not degrade collagen type I but produced a faintly staining peptide fragment from collagen type 111, similar to that formed when HLE was incubated with collagen type 111. Preincubation of PR-3 either with an inhibitor of HLE or Cat G, secretory leukoprotease inhibitor (see below), or with an inhibitor of metalloproteinases, 1,lO-phenanthro- line, did not effect digestion of fibronectin or vitronectin. Thus, degradation of these protein substrates was not caused by contaminating HLE, Cat G, collagenase, or gelatinase in the enzyme preparations.

Action on Insulin Chains-To gain insight into the peptide bonds susceptible to scission by PR-3 we analyzed the peptides produced by catalytic activity of PR-3 against oxidized insulin-B and -A chains. When the oxidized insulin-B chain was incubated with PR-3 and the digest fractionated by HPLC, five peptides were observed (Fig. 2 A ) . At the end of the 20-min incubation period the five peptides constituted approximately 85% of the initial amount of the insulin-B chain. The amino acid composition and NH2-terminal amino

9543 Characterization of PR-3 TABLE I

Amino-terminal sequence similarity of PR-3 to other granule serine proteinases The single-letter amino acid code is used (X = uncertain). Identical residues are indicated by (:) and conserved

residues by (.). A gap (-) in HLE was introduced to maximize homology. Residues are numbered starting with the first amino acid residue of the active proteinase and is given at the end of each line. Residue numbering for myeloblastin starts at methionine.

10 PR-3

20 I V G G H E A Q P H S R P Y M A S L Q M R G N P G S H F C G G T L I X P D F V L 40

30 40

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Myeloblastin" M A S L Q M R G N P G S H F C G G T L I H P S F V L 26

c-ANCA antigenb I V G G H E A Q P H S X P Y M A S L Q M 20

AGP7/p29b' I V G G H E A Q P H S R P Y M A S L E M 20

HLEd I V G G R R A R P H A W P F M V S L Q L R G - - G - H F C G A T L I A P N F V M 37

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

First 26 residues deduced from cDNA (Ref. 59). From Ref. 55.

From Ref. 45. e From Refs. 57 and 58.

TABLE I1 Active site and substrate binding site regions of PR-3/AGP7 and HLE

The single-letter amino acid code is used ( X = uncertain). Identical residues are indicated by (:) and conserved residues by (.).

4 170 1 SO 190 200 210

t 4 + 4 4 HLE" A G V C F G D S G S P L V C N G L I H G I A S F V R G G C A S G L Y P D A F A P V A Q F V N W I D

AGP7' . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A G I C F G D S G G P L I X D G I I Q G X D S F

. . . . . . . . . . . . . . . . . . . . . . . .

PR-3' L F P D F F T R V A L Y V D W I R i( P-1 * p-I1

From Ref. 45. Residue numbering starts with the first amino acid residue of active HLE and is given on the -

top. Residues forming the structure of the binding site relative to active site serine (*) are marked (+). - - From Ref. 57. Peptides p-I and p-I1 are tryptic peptides of PR-3.

acid residues of each peptide indicated that the initial cleavage sites were the Ala14-Leu's and Leu17-Va118 bonds (Fig. 2B). Based on peptide recovery it appears that cleavage of the Vall8-Cydg bond was the result of further cleavage of peptide B-3. Under similar conditions the insulin-A chain was degraded into six peptides (Fig. 2C). The initial cleavage sites were found to be the Va13-Glu4, Serg-ValIo, Ser12-Leu'3, and Gln's-Leu'6 bonds (Fig. 20 ) . These results suggest that PR-3 preferentially attacks peptide bonds having small aliphatic amino acids (alanine, valine, leucine, and serine) at the PI and P: sites.'

Action on Synthetic Substrates-Given these findings, we assessed the esterolytic and amidolytic activity of PR-3 against selected chromogenic and fluorogenic substrates. A representative Hanes-Woolf plot of the kinetic data is shown in Fig. 3. The data are summarized in Table 111. PR-3 hydrolyzed p-nitrophenyl esters of alanine and valine. In this respect it is similar to HLE. However, the catalytic efficiency of PR-3 (kca,/Km = 15.2 X lo3 M" s-I) for the alanine ester was substantially lower than that reported for HLE (kCat/Km = 77 X lo3 M" s-') by Baici et al. (30). PR-3 showed no activity against amino acid ester substrates containing either an aromatic (tyrosine) or hydrophobic (leucine, methionine) amino acid at the Pl site. For full functioning of the serine

'The nomenclature introduced by Schechter and Berger (61) is used to describe the position of amino acid in a peptide substrate. P1, Pz, etc. refer to amino acid residues of the substrate (or inhibitor) in the amino-terminal direction from the scissile bond and Pi, P;, etc. refer to amino acid residues in the carboxyl-terminal direction from the scissile bond. The corresponding subsites of the enzyme refer to SI, Sp, etc. and Si, Si, etc., respectively.

proteinase catalytic triad, tri- and tetrapeptide substrates have proven to be more efficacious, and a proline residue at the Pz position has been shown to restrict multiple binding modes of substrates to serine proteinases (62-64). Therefore, we tested PR-3 against four other HLE-specific extended oligopeptide substrates; of the three tripeptide substrates (Suc-Ala-Ala-Ala-NA, Suc-Ala-Ala-Val-NA, and Suc-Ala- Pro-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 with kcat/Km values of 1 and 3,050 M" s-l, respectively. The corresponding value reported for HLE-catalyzed hydrolysis of MeO-Suc- Ala-Ala-Pro-Val-NA (120 x lo3 M" s-') is 40-fold higher than that of PR-3 (65). PR-3 did not cleave Suc-Ala-Ala-Ala-NA at the Pz or P3 alanine position (data not shown). PR-3 showed no activity against oligomeric amide substrates having either leucine, phenylalanine, or tyrosine at the P, site, substrates cleaved by Cat G and/or chymotrypsin. Finally, we tested PR-3 against MeO-Suc-Ala-Ile-Pro-Met-NA, a substrate that has the reactive site sequence of al-proteinase inhibitor. This substrate is cleaved at the methionine residue by HLE (kcat/ K,,, = 4,000 M" s-') and Cat G (kcat/K,,, = 710 M" s-') (65), both known to be inhibited by al-proteinase inhibitor in vitro. PR-3 showed activity against this substrate with a k,.,/K,,, of 1,230 M-' s-l, a value intermediate to that reported for HLE and Cat G. The hydrolysis of synthetic peptides confirmed that PR-3 prefers small aliphatic residues (alanine, valine, methionine) at the P1 site. In addition, it appears to prefer substrates containing proline at the P2 site.

Interaction with Synthetic Inhibitors-The cleavage speci-

9544 Characterization of PR-3

A k Da

200-

116- 93 - 66 -

45-

rn 200- rr) 116- 93- 66-

45-

C D k Da kDa 93- 66- pi- CI. ” 200- I

45- 116- 93

3 l- 66-

45-

31 - 22-

14-

FIG. 1. A, digestion of fibronectin by PMNL serine proteinases. Fibronectin was digested by PMNL serine proteinases a t an enzyme/ substrate (w/w) ratio of 1:600 at 37 “C for 1 h. The digestion products were separated on a 7.5% SDS-PAGE gel under reduced conditions and visualized by Coomassie staining. From left to right, the first lane shows fibronectin alone. The second through the fourth lanes show the products from digests formed by PR-3, HLE, and Cat G , respectively. B, digestion of laminin by PMNL serine proteinases. Laminin was digested by PMNL serine proteinases a t an enzyme/substrate (w/w) ratio of 1:lOO a t 37 “C for 30 min. The digestion products were separated on a 7.5% SDS-PAGE gel under reduced conditions and visualized by Coomassie staining. From left to right, the first lane shows the (Y (400-kDa) and B (200-kDa) subunits of laminin. The second through the fourth lanes show digests formed by PR-3, HLE, and Cat G, respectively. C, digestion of vitronectin by PMNL serine proteinases. Vitronectin was digested by PMNL serine proteinases a t an enzyme/substrate (w/w) ratio of 1: lOO a t 37 “C for 15 min. The digestion products were separated on a 12% SDS-PAGE gel under reduced conditions and visualized by Coomassie staining. From left to right, the first lane shows vitronectin alone. The second through the fourth lanes show digests formed by PR-3, HLE, and Cat G, respectively. D, digestion of collagen type IV by PMNL serine proteinases. Collagen type IV was digested by PMNL serine proteinases a t an enzyme/substrate (w/w) ratio of 1: lOO a t 37 “C for 3 h. The digestion products were separated on a 4-12% gradient SDS-PAGE gel under reduced conditions and visualized by silver staining. From left to right, the first lane shows collagen alone. The second through the fourth lanes show digests formed by PR-3, HLE, and Cat G, respectively.

ficity for alanine and valine at PI and proline a t Pi! was confirmed further with peptide chlormethyl ketone inhibitors. PR-3 was inhibited by MeO-Suc-Ala-Ala-Pro-Val-CH2Cl with kOl,,/[fl of 10 M” s-’, a value 160-fold less than that reported for HLE (1,560 M” s-’) (66). PR-3 was not inhibited by MeO- Suc-Ala-Ala-Pro-Ala-CH&l, but HLE was inhibited ( h o h s / [ f l

= 20.8 M” s-I). Thus, the rate of inhibition by chloromethyl ketones paralleled the relative activities of PR-3 or HLE to the respective chromogenic substrates.

Interaction with Natural Inhibitors-We used Boc-Ala- ONp, the most efficiently hydrolyzed substrate, to test the

- ” ”

I I I I I

20 30 40 50 60 MINUTES

B t t t

F V N Q H L C C S H L V E A L Y L V C C E R G F F Y T P K A I . , I ., 8 . i . .I ,,, I I _ . I t i, I.. 11, i. I. I., 1,s I, ?_. _I, 2, .I,. ..,. :- .I” ...I ”I

I- P

81 4 85

84 83 82

4 -I c

C 1.01

u

FIG. 2. A, reverse phase HPLC fractionation of oxidized insulin- B chain peptides obtained by digestion with PR-3. Peptides are denoted as BI-B5. B, initial cleavage sites of PR-3 on oxidized insulin-B chain. Peptides corresponding to Fig. 2A and were analyzed as described under “Experimental Procedures.” The cleavage sites are indicated by arrows. C, reverse phase HPLC fractionation of oxidized insulin-A chain peptides obtained by digestion with PR-3. Peptides are denoted as Al-A6. D, initial cleavage sites of PR-3 on oxidized insulin-A chain. Peptides correspond to Fig. 2C and were analyzed as described under “Experimental Procedures.” The cleavage sites are indicated by arrows.

ability of the human proteinase inhibitors al-proteinase inhibitor, a?”, @,-Achy, and secretory leukoprotease inhibitor to interact with PR-3 (Table IV). The in vitro association constants for PR-3 with al-proteinase inhibitor measured under second order conditions and by competition experiments were 8.1 X lo” M” s” and 5.2 X lo6 M-l s-’, respectively. These values were 1 order of magnitude less than that of HLE (6.5 X lo7 M” s-’) (43). Using the k, value for PR-3 with cyI-

proteinase inhibitor we determined the association constant of n2-M for PR-3 by a competition assay as described previ- ously (42,44). The 12, for PR-3 with a2-M was found to be 1.1 X 10’ M“ s-’, a value similar to that reported for HLE (4.1 X

Characterization of PR-3 9545

lo7 M-' s-l) and 10-fold higher than that of Cat G (3.7 X lo6 M" S" ) (44). PR-3 was not inhibited by either al-Achy or secretory leukoprotease inhibitor under the conditions employed. We also examined the interaction of PR-3 with eglin c, the leech product that has been demonstrated to be an effective inhibitor of HLE and Cat G. The h,, value for PR-3 with eglin c was 4.2 X lo4 M-' s-I, 1,000- and 100-fold lower t.han that of HLE (1.3 X 10' M-' s-I) and Cat G (2.0 X 10" M" s-'), respectively (67). This suggests that eglin c is a weak inhibitor of PR-3.

We evaluated the in vivo significance of the interaction of the inhibitors with PR-3 by calculating the delay time of inhibition as described by Bieth (68), based on the determined in vitro k, values of a,-proteinase inhibitor, and 0.-M and the published plasma concentrations for these inhibitors (44, 69). The calculated delay times were 25 and 114 ms, respectively,

y = 99.847 + 0.60008X R"2 = 0.999 900

700 1

:::: 100 0 0 2 0 0 4 0 0 6 0 0 8 0 0 1000 1200

IS1 [P M)

FIG. 3. Hanes-Woolf plot for PR-3 with MeO-Suc-Ala-Ile- Pro-Met-NA as a substrate. Initial rates were determined as described under "Experimental Procedures." The values for K,, ( x intercept) and V,,,,, (slope = l /VmJ were obtained from t.he linear regression equation.

suggesting that both are effective inhibitors of PR-3 and likely regulate the activity of PR-3 in vivo.

DISCUSSION

PR-3 was originally described by Baggiolini et al. (12) as an a-naphthyl acetate esterase present in azurophilic granules of human PMNL. We subsequently purified PR-3 and demonstrated that it was an elastinolytic neutral serine proteinase (13). The molecular mass of the major band of PR-3 is 26.8 kDa with two minor bands having slightly larger molecular masses. PR-3 caused extensive tissue damage and emphysema after intratracheal instillation into hamsters. The present study further defines st,ructural and functional feat.ures of

The partial primary structure of PR-3 reported herein provides insight into the possible role of PR-3 in vivo and its mechanism of action. PR-3 shows extensive NH2-terminal sequence similarity to proteinases of the trypsin superfamily with strongest homology to HLE (45), Cat G (46), and human lymphocyte protease (54). The first 20 residues of PR-3 are identical (except for Gln'" instead of Glu) to those published recently for AGP7/p29b, suggesting that one role of PR-3 may be that of an antimicrobial protein (58). Of note, the antimicrobial activity of AGP7/p29b, similar to that of Cat G, was not inhibited by diisopropyl fluorophosphate, implying a nonproteolytic mechanism for the microbicidal action (58, 70).

Residues 15-40 of PR-3 are very similar (except for ambi- guity a t residue 35 and mismatch at residue 37) to the first 26 amino acids deduced from a cDNA clone recently isolated from HL-60 cells which encodes a serine proteinase, myeloblastin (59). The sequence of the peptides generated by tryptic digestion (Table 11) is identical to regions near the COOH terminus of the deduced myeloblastin sequence (Leu"" to

et al. (59) suggest that myeloblastin is part of a process regulating proliferation and differentiation of HL-60 cells and perhaps plays a role in the cellular differentiation and growth

PR-3.

Arg1% and Vall!#li to Arg204, myeloblastin numbering). Bories

TABLE 111 PR-3 activity on synthetic substrates

Substrate" Suhstrate concentration P, P, P,, Py P , P, '

K,,, kc,,,'' k, , d K t ,

Boc-Ala-ONp Boc-Val-ONp Boc-Leu-ONp Boc-Met-ONp Boc-Tyr-ONp Suc-Ala-NA

Suc-Ala-Ala-Ala-NA Suc-Ala -Ala -Va l -NA

MeO-Suc-Ala-Ala-Pro-Val-NA Z-Gly-Gly-Leu-NA

Suc-Ala -Ala -Pro -Leu-NA Suc-Ala -Ala -Pro -Phe -NA

MeO-Suc-Ala-Ile-Pro-Met-NA Lys-Ala-MCA

Suc-Ala-Pro-Ala-MCA Leu -MCA

Glt-Gly-Gly-Phe-MCA Suc-Leu-Leu-Val-Tyr-MCA

m M 0.1-0.5 0.1-0.25 0.1 0.1 0.1 1.0 1.0 0.3 0.1-3.33 0.2 1 .0 2.0 0.1-1.0 0.2 0.1-1.25 0.2 0.2 0.2

0.27

0.17

2.2

0.82

0.21

0.002

mM 5 ~ ' "1 ,s-l

0.66 10.0 15,150 0.42 2.3 5,600

N R N R N R N R NR N R

N R 3,050

NH N R

NR 1,230

N R 1

NR NR

" The 4-nitroanilide and MCA substrates were dissolved in Me2S0. Nitrophenyl ester substrates were dissolved

"The value of kc<<, (V,,,.,/[E]) was estimated from the V,,,:,, obtained from a Hanes-Wool1 plot and the active

' No reaction was observed with 0.25-4 pg (the amounts used initially to test all the suhstrates) of P I " Under

in methanol.

concentration of PR-3.

the assay conditions, detectable activity was observed only when >12 pg of I'R-:3 was used.

9546 Characterization of PR-3

TABLE IV PR-3 interaction with natural inhibitors

Inhibitor k. Delay time" "1 s - L ms

al-PIb 8.1 X lo6 25' a*" 1.1 X 107 114' al-Achy No inhibitiond SLPI' No inhibitiond Eglin c 4.2 X 10' 2 x lo6'

The delay time d, is the time required for nearly complete inhibition of a protease in uiuo and is calculated using the formula d, = 5/%. [inhibitor] (68).

PI, proteinase inhibitor. Value obtained in a competition experi- ment was 5.2 X lo6 M" s-'.

e [Inhibitor] from published values for plasma (44, 69). Inhibitor was incubated with PR-3 (1:l molar ratio) for 5 min,

and no change in the enzyme activity was observed. e SLPI, secretory leukoprotease inhibitor. ' [Inhibitor] from "therapeutic" concentrations (67).

arrest of normal cells. Whether the myeloblastin sequence represents an incomplete cDNA for PR-3 or will ultimately prove to be a separate enzyme awaits purification of myeloblastin. Preliminary studies in our laboratory demonstrate that PR-3 is present in uninduced HL-60 cells. Cell proliferation and differentiation are known to require a series of proteolytic events (71), and the above information suggests that PR-3 may be important in these processes.

The first 20 residues of PR-3 are also identical to the 29- kDa target antigen for c-ANCA associated with Wegener's granulomatosis, a disorder characterized by systemic necro- tizing vasculitis (72-74). We have further established the specificity of c-ANCA for PR-3 by showing that Wegener's granulomatosis sera reacted specifically with purified PR-3 in an enzyme-linked immunosorbent assay and that the c-ANCA staining pattern of PMNL observed by indirect immunofluo- rescence microscopy was blocked by PR-3-specific mono- clonal antibodies (75). The role of PR-3 in the pathobiochem- istry of this devastating disorder is currently not known.

Several investigators have shown that the neutrophil proteinases HLE and Cat G can degrade purfied matrix proteins and complex matrix models (11, 76-81). In our previous work we showed that PR-3 can degrade elastin, a major structural protein of the lung (13). The current investigation demon- strates that PR-3 can degrade several other matrix proteins. Further studies are needed to determine whether the differences in proteolytic fragments observed for PR-3 as compared with HLE or Cat G represent different sites of initial attack on the specific matrix molecule or can be explained by kinetic differences in the interaction of the proteinases with the matrix molecules. Studies to assess PR-3 proteolysis of a complex matrix model will provide additional insight into the role of PR-3 in degrading matrix proteins i n uivo.

Studies on the primary specificity of PR-3 against the insulin-B chain showed that the major sites of cleavage are between Ala14 and Leu1', Led7 and Val", and V a P and CysIg. These major sites are also cleaved by porcine pancreatic elastase (82). HLE also cleaved the insulin-B chain after Val" and Ala14 but not after Leu17 (83, 84). Out of four cleavage points in insulin-A chain produced by PR-3 (Fig. 2 0 ) only the site between Ser" and Led3 was cleaved by porcine pancreatic elastase (82). In contrast to pancreatic elastase, PR-3 did not hydrolyze the bond next to Ala8 but rather cleaved the bond between Serg and Val". Despite these differences the results demonstrate that like other elastases, PR- 3 preferentially hydrolyzes bonds adjacent to small aliphatic amino acids (alanine, serine, valine, and leucine).

Extension of our analysis to protected amino acid substrates

confirmed that PR-3 prefers either alanine or valine at the P, site. Studies of PR-3 specificity for alanine and valine at PI on extended peptides substrates of HLE (Suc-Ala-Ala-Ala- NA and Suc-Ala-Ala-Val-NA) showed that PR-3 had no activity against these substrates. Since a proline residue at the Pz position has been reported to orient substrate binding to serine proteinases in a productive mode (62-64), we tested the specificity of PR-3 against substrates containing proline at the Pz position. MeO-Suc-Ala-Ala-Pro-Val-NA was hydrolyzed by PR-3 but with less catalytic efficiency than HLE. Consistent with this observation, the chloromethyl ketone inhibitor analog MeO-Suc-Ala-Ala-Pro-Val-CH&l inhibited PR-3, but again less effectively than HLE. Suc-Ala-Pro-Ala- MCA was very weakly hydrolyzed by PR-3. Consistent with this finding, the inhibitor analog MeO-Suc-Ala-Ala-Pro-Ala- CH&l did not inhibit PR-3. The limited information available on the substrate specificity of the proteinase AGP7/p29b (57, 85) is similar to what we found for PR-3.

Comparison of the putative active site region of PR-3 with that of HLE reveals striking homology between the two enzymes which may account for their similar substrate spec- ificities. The residues forming the specificity determinants of the primary binding pocket are at positions -6, +15, +16, and +28 relative to the catalytic serine residue (60). These were strictly conserved between PR-3 and HLE but not Cat G. X- ray crystallograpic studies have provided a more complete definition of the primary binding site of HLE (86). These studies have shown that the binding pocket is made up of peptide regions G l ~ l ~ ~ - S e r l ~ ~ , Ala'87-Va1'90, AspZo1-Phezo3, and PheZ5. There are differences in these peptide regions between HLE and the assumed counterparts in PR-3 (see Table 11). Va1"j8 and Alals7 of HLE which are near the bottom of the primary binding pocket (S , ) are replaced by isoleucine and aspartic acid in PR-3, respectively. In addition PheZ5, which is part of the Si and Si subsites in HLE, remains unchanged as PheZ8 in PR-3 (Table I). However, in this region there are two insertions in PR-3 when compared with HLE, a two- amino acid insertion between GlyzZ and GlyZ3 and a single amino acid insertion between GlyZ3 and Hisz4 (see Table I). Such changes in primary binding pocket residues, in addition to those introduced by insertions, may alter the geometry of the binding pocket and therefore explain, in part, the differences in reactivity of PR-3 toward HLE substrates. Altera- tions in remote binding sites may also account for the observed differences.

HLE-mediated destruction is abrogated by the plasma proteinase inhibitors al-proteinase inhibitor and aZ-M and by the inhibitor present in mucous secretions and interstitial fluid, secretory leukoprotease inhibitor. The kinetic data on the interaction of these inhibitors with PR-3 suggest that al. proteinase inhibitor and cyz" are effective inhibitors in vitro, but secretory leukoprotease inhibitor is not. The i n vivo physiologic role of az-M in the lung is uncertain for two reasons: 1) its concentration in plasma is much lower than a,-proteinase inhibitor (69,87), and 2) its large molecular size retards its flux into the interstitial and bronchoalveolar space. Therefore, it is likely that al-proteinase inhibitor is the primary physiologically relevant inhibitor protecting the lower respiratory tract against both of the emphysema-inducing PMNL proteinases, HLE and PR-3. Although cy,-proteinase inhibitor is 1 order of magnitude less potent in inhibiting PR- 3 than in inhibiting HLE, its distribution between the two enzymes can be predicted, according to Vincent and Lazdun- ski (42), from the k, values and its concentration in lung secretions (88). One would predict from this that if both HLE and PR-3 are liberated in a similar fashion in the lower

Characterization of PR-3 9547

respiratory tract, a,-proteinase inhibitor will distribute such that 89% of it would be bound to HLE and 11% bound to PR- 3. Since Pr-3 is not inhibited by secretory leukoprotease inhibitor the action of PR-3 in the upper respiratory tract may be relatively unopposed, suggesting a potential role for PR-3 in inflammatory diseases of the upper airways.

In summary, the present study describes the partial primary structure and catalytic properties of PR-3, a PMNL neutral serine proteinase that may play an important role in tissue injury. PR-3 is capable of degrading a broad range of matrix proteins. Its catalytic properties, partial sequence, and sus- ceptibility to inhibitors demonstrate that it is a distinct PMNL proteinase. The role of PR-3 in destructive diseases such as pulmonary emphysema, the adult respiratory distress syndrome, rheumatoid arthritis, and glomerulonephritis remains to be defined.

Acknowledgments-We thank Dr. Martin C. Rechsteiner, Dr. Ed- ward J . Campbell, and Dr. John R. Michael for their critical review of the manuscript.

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