©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Purification and Characterization of Dog Mast Cell Protease-3, an Oligomeric Relative of Tryptases (*)

Wilfred W. Raymond (1), Elizabeth K. Tam (2)(§), John L. Blount (1), George H. Caughey (1)(¶)

From the (1) Cardiovascular Research Institute and Department of Medicine, University of California, San Francisco 94143-0911 and the (2) Departments of Medicine and Pharmacology, John A. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii 96813

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The existence of a protein 48% identical with mast cell tryptases was predicted previously from a dog mastocytoma cDNA. Antibodies raised against a peptide based on the deduced sequence suggested that the protein (dog mast cell protease-3, dMCP-3) is expressed in mast cells. In this report, characterization of the protein purified from mastocytomas reveals an N-glycosylated, high molecular weight, tryptic serine protease, which appears to be a tetramer of catalytic subunits, approximately half of which are linked by disulfide bonds. The oligomeric complex yields a single NH-terminal sequence, which is identical with that predicted by dMCP-3 cDNA. This finding, and the lack of closely related genes on blots of genomic DNA, predict that each subunit is the product of one gene. Although dMCP-3 binds to heparin, it is active and stable at low ionic strength in heparin's absence. It resists inactivation by inhibitors in plasma but is sensitive to small inhibitors, e.g. leupeptin and bis(5-amidino-2-benzimidazolyl)methane (BABIM). dMCP-3 hydrolyzes extended peptidyl p-nitroanilides ending in basic residues, with P1 arginine preferred to lysine; it hydrolyzes the Arg-Ser bond of calcitonin gene-related peptide but cleaves neither vasoactive intestinal peptide nor casein. These data suggest that dMCP-3 is a unique serine protease whose stability, formation of intersubunit disulfide bonds, inhibitor susceptibilities and substrate preferences differ from those of its closest relatives, the mast cell tryptases.


INTRODUCTION

The granule-associated mast cell and leukocyte serine proteases vary strikingly in specificity and function (1) . A subset of these enzymes, including mast cell tryptases, are tryptic in specificity, hydrolyzing target peptides on the COOH-terminal side of basic residues (2, 3). Tryptases, the expression of which is confined almost exclusively to mast cells in human tissues (4) , are the secretory granule's major protein (5, 6, 7) . They may regulate vascular and bronchomotor tone, act as local anticoagulants, and influence tissue remodeling in response to inflammation by activating matrix metalloproteinases and promoting the growth of fibroblasts (8, 9) . In humans and mice, there are two or more closely related tryptase genes (10-14). The existence of additional more distant relatives of mast cell tryptases was suggested by the serendipitous cloning from dog mastocytoma cells of a cDNA whose sequence predicted a protein with certain similarities to known tryptases (15) . RNA blots and immunohistochemical studies using antibodies raised against a portion of the predicted sequence suggested that the gene encoding this protein, termed dog mast cell protease-3 (dMCP-3),() is transcribed and translated in dog mastocytomas, normal dog tissue mast cells, and possibly neutrophils (16) . Immunoblotting of electrophoresed cell extracts confirmed the presence of immunoreactive material of approximately the expected size (30 kDa) in dog mastocytomas (16) . The work below describes the purification and characterization of dMCP-3, a novel member of the family of tryptic mast cell proteases.


MATERIALS AND METHODS

Enzyme Assays

Aliquots from purification steps were tested for amidolytic activity with 0.135 mMN-benzoyl-L-Val-Gly-Arg-p-nitroanilide (VGRpNA) in 60 mM Tris (pH 7.8) with 50 µg/ml bovine lung heparin. VGRpNA was dissolved in dimethyl sulfoxide to 20 mg/ml, then diluted in assay buffer. Release of p-nitroaniline at 37 °C was detected by monitoring change in A as described (17) ; moles of substrate hydrolyzed were calculated using an of 11,100 M cm. For convenience, column fractions were routinely monitored for N-benzyloxycarbonyl-L-Lys-thiobenzylester-hydrolyzing activity using modifications of a microplate assay (18) . For this assay, 50-µl aliquots of column fractions were mixed with 100 µl of 1 mM 5,5`-dithiobis(2-nitrobenzoic acid) (DTNB) in a solution of 10 mM Hepes (pH 7.2) containing 1 mM CaCl and 1 mM MgCl in single wells of U-Bottom Microtest III 96-well assay plates (Becton Dickinson). 50 µl of 2 mM substrate in assay buffer were added to the wells. A at 37 °C was measured on a Thermo microplate reader (Molecular Devices, Menlo Park, CA). Protein concentration in samples was determined by Bradford assay (Bio-Rad), using BSA as a standard, or by measuring the A of purified proteins. The of dMCP-3 was calculated to be 74,380 M cm based on tyrosine and tryptophan content deduced from dMCP-3 cDNA (15) .

Purification

Tumors of the BR and G dog mastocytoma lines propagated in athymic mice as described (19, 20) were harvested and frozen at -70 °C until extraction. Thawed tumors were washed in a solution of 150 mM NaCl in 10 mM Tris (pH 7.5), then homogenized in 10 ml of 10 mM Bis-tris (pH 6.1) per g of tissue. The supernatant generated by centrifuging the homogenate for 30 min at 27,500 g was brought to 20% glycerol and 1.6 M NaCl and applied to a 10-ml column of benzamidine-Sepharose 6B (Pharmacia Biotech) equilibrated with a solution of 8 mM Bis-tris (pH 6.1), 20% glycerol, and 1.6 M NaCl. After loading with supernatant, the column was washed with equilibration buffer and eluted with 30 ml of 0.15 M benzamidine in the same buffer. To remove NaCl and benzamidine, the column eluate was passed in 10-ml aliquots through 50 ml of Sephadex G-25 (Pharmacia) equilibrated with 20% glycerol in 8 mM Bis-tris. 3-ml aliquots of G-25 eluate, each containing 450 µg of protein, were applied to a 7.5 75 mm heparin-5PW high pressure liquid chromatography column (Toso-Haas, Montgomeryville, PA) equilibrated with 20% glycerol in 8 mM Bis-tris (pH 6.1). Fractions eluted with a gradient of 0 to 1.6 M NaCl in the glycerol/Bis-tris buffer were collected, assayed for tryptic esterase activity as above, and analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) on 12.5% gels stained with Coomassie Blue.

Column fractions also were assayed for dMCP-3 immunoreactivity by probing dot blots with anti-dMCP-3, a rabbit polyclonal antiserum raised against a synthetic peptide corresponding to residues 166-181 of catalytic domain sequence deduced from dMCP-3 cDNA (15, 16) . Aliquots of samples were transferred to prewetted nitrocellulose. Blots were blocked by incubation for 1 h with a solution of 1% BSA and 150 mM NaCl in 10 mM Tris (pH 7.5), then incubated for 1 h with a 1:1000 dilution of anti-dMCP-3 antiserum in a solution of 10 mM Tris (pH 7.5) containing 150 mM NaCl and 0.3% Tween 20. After washing of blots with detergent buffer alone, immunoreactive material was detected with a horseradish peroxidase-labeled goat anti-rabbit IgG (1:1000; Bio-Rad) incubated with 0.5 mg/ml 4-chloro-1-naphthol in 0.15% HO.

NH-terminal Sequencing

An 8-µg sample of purified dMCP-3 was subjected to automated NH-terminal sequence analysis on an Applied Biosystems gas-phase sequenator. The analysis was carried out by the University of California, San Francisco's Biomolecular Resource Center.

Thiol Titration

Free thiols were assayed using DTNB as described (21) . In brief, 0.01 ml of a solution of 4 mg/ml DTNB in 1% SDS, 40 mM NaPO (pH 8.0) was added to 0.5 ml of the same buffer containing dMCP-3, BSA, or carbonic anydrase. After 15 min, A was measured. The assay mixture was referenced against a mixture of buffer plus DTNB alone.

Deglycosylation

Purified dMCP-3 was deglycosylated with peptide N-glycosidase F (Genzyme). After boiling 2 µg of DMCP-3 for 5 min in the presence of 0.5% SDS and 0.3 M 2-mercaptoethanol, the mixture was diluted 3-fold with a solution containing 100 mM NaHPO4 (pH 7.5), 10 mM EDTA, and 1% Nonidet P-40 and was incubated for 16 h at 37 °C with 0.3 unit of glycosidase. A control aliquot of dMCP-3 was subjected to the same treatment in the absence of glycosidase. The reaction was stopped by the addition of an equal volume of SDS-PAGE sample buffer (4% SDS, 20% glycerol, 10% 2-mercaptoethanol in 0.125 M Tris-Cl (pH 6.8)), and the products were analyzed by SDS-PAGE.

Size Exclusion Chromatography

Purified dMCP-3 was injected onto a TSK-250 Bio-Sil column equilibrated with a solution of 10 mM Tris-Cl (pH 7.4), 2 M NaCl, and 20% glycerol. The elution volume of dMCP-3, as identified by peak esterase activity and A of the eluate, was compared to that of dog tryptase, cyanocobalamin, and globular protein standards eluted with the same solution. The M of dMCP-3 was estimated by extrapolation from a plot of molecular standard Kversus log M.

Stability of Amidolytic Activity

To compare the stability of dog tryptase and dMCP-3, purified preparations of each enzyme were incubated separately for 1 h in the presence or absence of bovine lung heparin (50 µg/ml). Incubations were carried out at 37 °C in polypropylene tubes containing 10 mM Tris-Cl (pH 7.4) and 150 mM NaCl. Aliquots of incubating enzyme solutions were withdrawn at intervals to assay residual 0.1 mMN-p-tosyl-Gly-L-Pro-Arg-pNA (GPRpNA)-hydrolyzing activity.

Substrate Specificity

To compare the substrate preferences of dMCP-3 and dog tryptase, the activity of the two enzymes was tested in parallel against a battery of peptidyl pNAs. Substrates were diluted from 20 mg/ml stocks in dimethyl sulfoxide to 0.2 mM in 60 mM Tris (pH 7.8) containing 50 µg/ml heparin. 30 ng of purified dog tryptase or dMCP-3 were mixed with substrate-containing assay buffer to a final volume of 200 µl in single wells of 96-well assay plates. Substrate hydrolysis at 37 °C was monitored at 405 nm in a microplate reader as described above. Assays were performed in duplicate in three separate experiments. All substrates were obtained from Sigma with the exception of N-benzoyl-L-Lys-Gly-Arg-pNA (KGRpNA), which was obtained as described (17) .

Hydrolysis of Azocasein and Bioactive Peptides

To test the general proteinase activity of dMCP-3, aliquots of dMCP-3, tryptase, or trypsin stocks were added to a solution of azocasein (final concentration 14 mg/ml; Sigma) in 60 mM Tris-Cl (pH 7.8). The final concentration of dMCP-3, tryptase, and trypsin in their respective incubation solutions was 5.0, 3.6, and 5.4 µM, respectively. Unhydrolyzed azocasein remaining after 10 min of incubation at 40 °C was precipitated by addition of a 5-fold greater volume of 5% trichloroacetic acid. The resulting mixture was centrifuged twice for 5 min at 16000 g to obtain a supernatant, the A of which was measured after zeroing against a solution of azocasein incubated and acidified in the same manner, without added protease. To test the ability of dMCP-3 to hydrolyze selected bioactive peptides, dMCP-3 was incubated in separate experiments with substance P, vasoactive intestinal peptide, and calcitonin gene-related peptide (Sigma). As controls, the same peptides were incubated with human lung tryptase (purified as described previously (22) , with minor modifications) or with buffer alone. In each incubation mixture, the concentration of peptide was 0.1 mM and that of dMCP-3 or tryptase (based on protein measurement) was 0.1 µM and 0.2 µM, respectively. As tested in a microplate-based assay using VGRpNA as a substrate, the specific activity of dMCP-3 used in these experiments was approximately twice that of the human lung tryptase. Protease stocks contained 2 µg of bovine lung heparin per µg of dMCP-3 or tryptase. Solutions formed by addition of protease and peptide to 120 mM Tris (pH 7.4) containing 140 mM NaCl were incubated for 30 min at 37 °C. Aliquots withdrawn at intervals were injected onto a Vydac C18 reverse phase column (The Separations Group; Hesperia, CA) and eluted using a linear gradient of 10-40% acetonitrile in 0.1% trifluoroacetic acid, as described (23) with monitoring of A.

Enzyme pH Activity Curve

Activity of purified dMCP-3 (94 ng/ml) was measured at 37 °C using 0.3 mM GPRpNA in 100 mM Bis-tris (pH 5.5-6.5) or Tris-Cl (pH 7.0-9.0).

Inhibitor Profile

Purified dMCP-3 (3 µg/ml; 100 nM) was preincubated in 60 mM Tris (pH 7.8) with 50 µg/ml bovine lung heparin for 10 min at 37 °C with each of a panel of inhibitors. Activity remaining after preincubation was determined in an assay for amidolytic activity initiated by the addition of VGRpNA as described above. In control assays, dMCP-3 was preincubated under identical conditions in the absence of inhibitor. To improve solubility, some inhibitors were incubated with dMCP-3 and assay buffer in the presence of 1% dimethyl sulfoxide or dimethylformamide. Pilot studies established that neither solvent at this concentration alters dMCP-3 amidolytic activity. All inhibitors were obtained from Sigma except for leupeptin (Boehringer Mannheim), BABIM (from R. Tidwell (24) ), secretory leukocyte protease inhibitor (from J. Kramps), and dog plasma, which was prepared from heparinized blood.

Genomic DNA Blotting

Genomic DNA was purified from dog and human circulating mononuclear cells, which were purified in each case from 10 ml of fresh blood. Blood diluted with 12 ml of Ca- and Mg-free Hank's balanced salt solution was overlaid with 15 ml of Histopaque-1077 (Sigma) and centrifuged for 30 min at 400 g. Cells from the mononuclear band were lysed and digested in 10 mM Tris-Cl (pH 8) containing 100 mM NaCl, 25 mM EDTA, 0.5% SDS, and 0.1 mg/ml proteinase K. The resulting suspension was shaken for 12-18 h at 50 °C. DNA was isolated by phenol-chloroform extractions followed by ethanol precipitation. After digestion with EcoRI or HindIII, the resulting dog and human DNA was electrophoresed in 1% agarose and transferred to a nylon membrane (Nytran Plus; Schleicher and Schuell). The membrane was washed for 5 min in 75 mM sodium citrate (pH 7.0) containing 0.75 M NaCl, air-dried, baked for 2 h in a vacuum oven at 80 °C, then prehybridized in 90 mM sodium citrate (pH 7.0) containing 50% formamide, 0.9 M NaCl, 0.5% SDS, 1 g/liter each of Ficoll 400, polyvinylpyrrolidone, and BSA, and 100 µg/ml salmon sperm DNA. Hybridization was initiated by the addition of a P-labeled cDNA probe to the above solution. The probe was generated by random priming of previously cloned and sequenced 1174-base pair dMCP-3 cDNA (15) (excised from pBluescript using EcoRI) to a specific activity of 2 10 cpm/µg (using [-P]dCTP, [-P]dGTP, and a Megaprime kit, Amersham). After 15 h of hybridization, the membrane was subjected to washes of escalating stringency, followed by autoradiography.


RESULTS

Purification

The results of a single, representative set of chromatographic steps leading to the purification of 7.1 mg of dMCP-3 from G mastocytoma extract are summarized in . The benzamidine-Sepharose step effected 62-fold purification of VGRpNA-hydrolyzing activity from the crude, low ionic strength extract. The VGRpNA-hydrolyzing activity of the benzamidine-Sepharose eluate is due to the presence of both dMCP-3 and tryptase, which are separated in the heparin affinity step. The -fold purification of dMCP-3 falls slightly from the benzamidine-Sepharose to the heparin affinity step because of the removal of tryptase, whose specific activity toward VGRpNA is higher than that of dMCP-3 (see below). As shown in Fig. 1, heparin affinity chromatography reveals two widely separated peaks of A, the first of which is due to dMCP-3 and the second of which is due to tryptase. Heparin-column fractions spanning these two peaks are the only fractions with VGRpNA-hydrolyzing activity. Neither A nor amidolytic activity was detected in flow-through fractions. Fig. 1also suggests that the concentration of dMCP-3 is higher in G than in BR mastocytoma extracts and demonstrates a difference between BR and G mastocytomas in relative content of dMCP-3 and tryptase. Although dMCP-3 is the principal VGRpNA-hydrolyzing enzyme in low ionic strength extracts of both mastocytoma lines, only BR extracts contain substantial tryptase. Prior work in this laboratory demonstrated that most tryptase and most tryptic amidolytic activity in BR homogenates is soluble only when extracted using buffer of high ionic strength (17, 25) . High ionic strength extracts of G cells, on the other hand, contain little tryptic activity (25) and negligible dMCP-3 (not shown). Therefore, dMCP-3 is the major tryptic protease of G mastocytomas. BR mastocytomas contain substantial amounts of both dMCP-3 and tryptase, with most of the former extracting into low ionic strength buffer and most of the latter into high ionic strength buffer.


Figure 1: Heparin affinity chromatography of mastocytoma tryptase and dog mast cell protease-3 (dMCP-3). Tryptic activity extracted from mastocytoma cell homogenates was purified initially on benzamidine-Sepharose, loaded onto a heparin-5PW column, and eluted with a gradient of NaCl (as shown by the dotted lines) to separate dMCP-3 from tryptase. A and B show the A of protein derived from low ionic strength extracts of ``BR'' and ``G'' mastocytoma cells, respectively. Tryptic activity (not shown) in column fractions corresponded to the peaks of A. As reflected in the relative peak sizes in the chromatograms in the two panels, dMCP-3 is by far the major tryptic protease in cell extracts and benzamidine-Sepharose eluates derived from G mastocytomas.



NH-terminal Sequence

dMCP-3 corresponding to the ``dMCP-3'' peak of Fig. 1A yielded the following sequence: IVGG()KVPARRY, which corresponds exactly to the sequence of the catalytic domain (after removal of signal and activation peptide) deduced from nucleotide sequence of cloned dMCP-3 cDNA (15) . The fifth cycle was blank, which is consistent with modification of the cysteine predicted to occupy this position.

Free Thiols

An 8 µM solution of purified dMCP-3 (molarity based on moles of monomer) yielded 9.3 µM DTNB-titratable ``free'' thiol, an average of 1.2 thiols per monomer. Under the same assay conditions, bovine carbonic anhydrase (which lacks cysteines), yielded 0.05 mol of thiols per molecule; BSA, with 17 disulfide linkages and 1 unpaired cysteine, yielded 0.5 thiol per molecule.

Electrophoretic Behavior

The results of SDS-PAGE of proteins purified from mastocytoma extracts are seen in Fig. 2 , which emphasizes the dramatic increase in purity achieved in the benzamidine-Sepharose step. The modest further increase in homogeneity achieved by the application of the heparin affinity step to the G mastocytoma material is due to the removal of contaminating tryptase. A comparison of BR mastocytoma-derived material prepared from the two heparin affinity peaks shown in Fig. 1A demonstrates a clear difference in electrophoretic behavior between dMCP-3 and tryptase, with the former migrating as a broad band centered around 37 kDa. The gel in Fig. 3illustrates the difference between reduced and unreduced dMCP-3. In the absence of 2-mercaptoethanol, purified dMCP-3 separates into two bands, one approximately twice the apparent M of the other, consistent with the higher band being a disulfide-linked dimer. As is often the case of proteins containing intramolecular Cys-Cys linkages, the fully reduced monomer migrates at a higher apparent M than that of the unreduced monomer.


Figure 2: SDS-PAGE of mastocytoma proteins. Samples were electrophoresed in a gel containing 12.5% acrylamide. All samples were reduced with 2-mercaptoethanol. Lanes 1 and 7 (MW) contain marker proteins, whose size (in kDa) are indicated to the left of the gel. These proteins are phosphorylase B (97.4), BSA (66.2), ovalbumin (42.7), carbonic anhydrase (31.0), and soybean trypsin inhibitor (21.5). Lane 2 (G LSE) contains 20 µg of protein from crude low salt extract of G mastocytomas, the starting material for purification of dMCP-3. Lane 3 (Bz eluate) contains 5 µg of G mastocytoma-derived protein retained by, and then eluted from, benzamidine-Sepharose. Lane 4 (dMCP-3) contains 2 µg of protein from the dMCP-3 peak (see Fig. 1) after further purification by heparin affinity chromatography. Lane 5 (dMCP-3 + PNGase F) contains 2 µg of protein from the same dMCP-3 peak preincubated with 0.3 unit of peptide N-glycosidase F to remove asparagine-linked sugars. Lane 6 (PNGase F) contains 0.3 unit of glycosidase alone, which is a faint band just above that of dMCP-3. Lane 8 (BR tryptase) contains 2 µg of purified, BR mastocytoma-derived dog tryptase from the tryptase peak seen in A in Fig. 1. Lane 9 (BR dMCP-3) contains 2 µg of purified, BR mastocytoma-derived dMCP-3 from the dMCP-3 peak seen in A in Fig. 1.




Figure 3: SDS-PAGE of dMCP-3. Protein contained in the heparin affinity chromatographic peaks corresponding to dMCP-3, as identified in Fig. 1, was subjected to 12.5% SDS-PAGE then stained with Coomassie Blue. Lanes 1 and 2 contain 2.6 µg of G mastocytoma dMCP-3 prepared for electrophoresis in the absence and presence, respectively, of 5% 2-mercaptoethanol. The molecular weights ( 10) and migration positions of marker proteins (see Fig. 2 legend) are indicated to the left of the figure.



Post-translational Modification

As seen in Fig. 2 , incubation of dMCP-3 with N-glycosidase narrows the protein band and increases mobility. This suggests that most, and perhaps all, of the heterogeneity of dMCP-3 is due to variable N-glycosylation at one or both of the consensus sites identified in the catalytic domain sequence deduced from dMCP-3 cDNA (15) .

Oligomerization

Protein and esterase activity of dMCP-3 subjected to analytical TSK-250 size exclusion chromatography elutes at an apparent M of 196,000, which is higher than the apparent M of dog tryptase (133, 0) eluted under the same conditions. The size of the tryptase complex predicted by these experiments agrees with the value of 132,000 estimated previously by analytical gel filtration in buffer without glycerol (17) . In additional experiments, the elution behavior of dMCP-3 under identical conditions, except for the presence of 1 mM dithiothreitol, was similar (results not shown). Pilot gel filtration runs established the need to include glycerol in the eluting buffer to prevent nonspecific binding of dMCP-3 to the chromatography matrix. 2 M NaCl was included in the elution buffer to maintain parity of elution conditions with tryptase, which, if chromatographed in pure form, loses activity rapidly at low ionic strength. The resulting gel filtration data suggest that native dMCP-3, like dog tryptase (25, 26) , is oligomeric, and that productive interactions between subunits are more likely to be hydrophobic than electrostatic in nature (Fig. 4).


Figure 4: Analytical gel filtration of dMCP-3. 20 µl of a 1.5 mg/ml solution of purified dMCP-3 were loaded onto a Bio-Sil TSK-250 column (7.5 300 mm; Bio-Rad) equilibrated and eluted with buffer containing 10 mM Tris-Cl (pH 7.4), 2 M NaCl, and 20% glycerol, at a flow rate of 0.5 ml/min. A of the eluate was monitored continuously (A, solid line), and fractions were collected for determination of N-benzyloxycarbonyl-L-Lys-thiobenzylester-hydrolyzing activity (Esterase activity, --) in wells of a microtiter plate. The column was calibrated under the same conditions with molecular standards (M), including thyroglobulin (670,000), -globulin (158,000), ovalbumin (44,000), horse myoglobin (17,000), and cyanocobalamin (1,350), whose molecular weights ( 10) and elution positions are indicated by open arrows. The elution position of dog tryptase is indicated by the closed arrow. The estimated M of the peak of dMCP-3 A and catalytic activity is 195,000. Esterase activity is the change in A/min.



Stability

Comparison of the stability of dMCP-3 and dog tryptase catalytic activity reveals striking differences, as illustrated in Fig. 5. At low ionic strength, tryptase's amidolytic activity rapidly declines. After 20 min, <10% of activity remains. Loss of activity is almost entirely prevented by the inclusion of heparin in the incubation solution. The activity of dMCP-3, on the other hand, is stable at low ionic strength in heparin's absence. In other experiments, dMCP-3 was found to be stable without heparin after 5 h of incubation at 37 °C, 16 h at 25 °C, or 3 months at 4 °C. SDS-PAGE of dMCP-3 after prolonged incubation fails to reveal evidence of autodegradation (data not shown). These findings suggest that purified dMCP-3 is more stable than purified dog tryptase.


Figure 5: Stability of tryptase versus dMCP-3. Purified dog tryptase (178 nM) and dMCP-3 (175 nM) were incubated separately for 1 h in the presence or absence of 50 µg/ml heparin. Incubations were carried out at 37 °C in 10 mM Tris-Cl (pH 7.4) containing 150 mM NaCl. Aliquots of protease solution were withdrawn at intervals during incubation for immediate assay of residual tosyl-Gly-L-Pro-Arg-p-nitroanilide-hydrolyzing activity. Observed residual activity is plotted on a log scale as a fraction of activity measured at time zero. Tryptase, in the absence of heparin (--), loses >90% of catalytic activity after 10 min of incubation, but is stable when incubated with heparin (--). In contrast, dMCP-3 (closed squares), is stable in the absence of heparin.



Preferences for Peptidyl pNAs

Fig. 6 shows a comparison of peptidyl pNA substrate preferences of dMCP-3 versus those of dog tryptase. The substrate most rapidly hydrolyzed by dMCP-3 is GPRpNA. The rate of dMCP-3-catalyzed hydrolysis of N-p-tosyl-Gly-L-Pro-Lys-pNA (GPKpNA) proceeds at slightly more than half that of GPRpNA, suggesting a modest preference for P1 arginine over P1 lysine. Both GPRpNA and GPKpNA are more rapidly cleaved than the other peptidyl pNAs examined, each of which contains P1 arginine and P2 glycine. Thus, substrates with P2 proline may be preferred over substrates with P2 glycine. Among the 3 Xaa-Gly-Arg substrates, KGRpNA is cleaved slightly faster than N-benzoyl-L-Ile-Glu-Gly-Arg-pNA, which is cleaved somewhat faster than VGRpNA, indicating a sensitivity to variations in the P3 residue, with P3 lysine preferred to P3 glutamic acid, which is preferred to P3 valine. The best of the pNA substrates for tryptase (i.e. VGRpNA) is among the worst for dMCP-3. Compared in terms of specific activity, the rate of VGRpNA hydrolysis per ng of tryptase is 50-fold greater than that per ng of dMCP-3. Neither dMCP-3 nor tryptase cleaves the standard trypsin substrate N-benzoyl-DL-Arg-pNA with much alacrity, reinforcing the importance of subsite interactions suggested by the variation in hydrolysis rates among the more extended pNA substrates. Not shown are results of assays using substrates lacking a basic residue in the P1 position, including the chymase/cathepsin G substrates succinyl-L-Val-Pro-Phe-pNA, succinyl-L-Ala-Ala-Pro-Phe-pNA, and succinyl-L-Phe-pNA, the elastase substrate succinyl-L-Ala-Ala-Ala-pNA, and the acidic dipeptide derivative benzoyl-L-Glu-Glu-pNA. In each case, there is no detectable hydrolysis by dMCP-3. Thus, dMCP-3 is a peptidyl amidase with a preference for certain extended substrates with P1 basic residues. The selectivity for particular tri- and tetrapeptide substrates with P1 arginine is evidence of an extended substrate binding pocket.


Figure 6: Substrate preferences of tryptase versus dMCP-3. Substrates are N-p-tosyl-Gly-L-Pro-Arg-p-nitroanilide (GPR), N-p-tosyl-Gly-L-Pro-Lys-p-nitroanilide (GPK), N-benzoyl-L-Ile-Glu-Gly-Arg-p-nitroanilide (IEGR), N-benzoyl-L-Lys-Gly-Arg-p-nitroanilide (KGR), N-benzoyl-L-Val-Gly-Arg-p-nitroanilide (VGR), and N-benzoyl-DL-Arg-p-nitroanilide (R). All assays were carried out at 37 °C using 0.2 mM substrate in 60 mM Tris-Cl (pH 7.8) containing 50 µg/ml bovine lung heparin. The concentration of each enzyme was 150 ng/ml (5 nM). The activity unit (A) is the observed change in mA/min. Error bars represent standard error of the mean of 3 independent determinations.



Hydrolysis of Casein

The caseinolytic activity of dMCP-3 was compared to that of trypsin and dog tryptase in duplicate in two separate experiments. Although the concentration of dMCP-3 in the incubation solution was similar to that of trypsin and tryptase, the rate of hydrolysis of azocasein by dMCP-3 was undetectable, i.e. <0.5% of that of hydrolysis by trypsin. Under these conditions, however, the rate of azocasein hydrolysis by tryptase was 13% that of trypsin. Therefore, dMCP-3 possesses minimal general proteinase activity.

Hydrolysis of Bioactive Peptides

The hydrolysis of bioactive peptides by dMCP-3 also is limited. Although substance P contains a potential tryptic hydrolysis site at the Lys-Pro bond, it is not hydrolyzed by dMCP-3. Nor is it cleaved by human lung tryptase, a finding which is consistent with prior studies (23, 27) . Vasoactive intestinal peptide also completely resists degradation by dMCP-3, but is rapidly hydrolyzed by tryptase, with complete loss of the parent peptide peak within 5 min of the start of incubation. In contrast, within 30 min, both dMCP-3 and tryptase hydrolyze all of the calcitonin gene-related peptide in the incubation solution. dMCP-3 generated just two product peaks, suggesting a single site of hydrolysis. Amino acid analysis of the earlier eluting product peak revealed the composition expected of calcitonin gene-related peptide fragment 19-37 (i.e. 3 Asx, 1 Thr, 2 Ser, 1 Pro, 3 Gly, 1 Ala, 3 Val, 2 Phe, 2 Lys). Therefore, dMCP-3 hydrolyzes the Arg-Ser bond of calcitonin gene-related peptide. Tryptase, as noted previously, generates multiple peaks, consistent with two sites of hydrolysis (23) . No hydrolysis is detected in solutions of peptide incubated with buffer alone. Thus, dMCP-3 differs from human lung tryptase in its ability to hydrolyze bioactive peptides.

pH-Activity Relationship

The amidolytic activity of dMCP-3 is greatest at alkaline pH. Peak activity plateaus between pH 8 and 8.5 and falls sharply at pH 9 and below pH 7. Activity at pH 5.5, 6.0, 6.5, 7.0, 7.5, and 9.0 is 15%, 21%, 42%, 69%, 92%, and 71%, respectively, of maximal activity. In the acidic environment of the mast cell secretory granule, therefore, the activity of dMCP-3 will be a small fraction of its activity upon secretion into the alkaline extracellular milieu of the degranulating mast cell.

Inhibitor Susceptibility

As shown in , dMCP-3 resists inactivation by most serine protease inhibitors that are proteins, including -proteinase inhibitor, soybean trypsin inhibitor, ovoinhibitor, and secretory leukocyte protease inhibitor. It also retains most of its activity after incubation in 10% dog plasma, suggesting resistance to anti-proteases in the circulation. The most significant difference between dMCP-3 and dog tryptase detected in these experiments is in susceptibility to aprotinin, which dMCP-3 resists and dog tryptase does not (17) . Like tryptases (6, 17, 24, 28, 29) , dMCP-3 is virtually completely inactivated by leupeptin and by BABIM, and its activity is reduced by high concentrations of NaCl and CaCl. Despite the abundance of cysteine residues predicted by the dMCP-3 cDNA sequence and the evidence of disulfide involvement in oligomerization, 2 mM dithiothreitol does not diminish dMCP-3 activity.

Genomic Blots

As shown in Fig. 7, hybridization of dMCP-3 cDNA to dog genomic DNA reveals strong binding to one major band in electrophoresed digests of EcoRI- and HindIII-digested DNA, consistent with the hypothesis that the subunits constituting the dMCP-3 oligomer are the products of a single gene. Weaker recognition of one or more bands of similarly digested human genomic DNA supports the existence of a human homolog.


Figure 7: Hybridization of a dMCP-3 probe to dog and human genomic DNA. Restriction digests of dog and human genomic DNA (10 µg/lane) were electrophoresed in 1% agarose, transferred to a nylon membrane, hybridized to an 1174-base pair P-labeled dMCP-3 cDNA probe. The membrane then was subjected to a series of washes of increasing stringency. The autoradiogram shown was obtained after washing in 15 mM sodium citrate (pH 7.0), 150 mM NaCl, 0.5% SDS at 65 °C. Lanes 1 and 2 contain EcoRI-restricted dog and human genomic DNA, respectively. Lanes 3 and 4 contain HindIII-restricted dog and human DNA, respectively. The size (in kilobases) and elution positions of HindIII-restricted marker DNAs are indicated to the left of the figure.




DISCUSSION

This report describes the purification and properties of an oligomeric, trypsin-like protease isolated from dog mastocytoma cells. The findings establish that the protein predicted from a previously sequenced ``orphan'' cDNA cloned from a dog mastocytoma library (15) is translated and processed into a catalytically active serine protease, dMCP-3, which is abundantly expressed in the G line of dog mastocytomas. RNA blotting and immunohistochemical studies suggest that a similar or identical enzyme is present in normal dog mast cells (16) . Several features ally dMCP-3 with the group of known mast cell tryptases, although there are potentially key differences, such as stabilization by heparin, formation of intersubunit disulfide bonds, substrate preferences, and inhibitor susceptibility.

dMCP-3's gel filtration behavior suggests an oligomeric structure in the native, active state. The M = 196,000 deduced from dMCP-3's elution position on size exclusion chromatography is nominally consistent with a pentamer of the M = 37,000 subunit predicted by SDS-PAGE. More likely, native dMCP-3 is a tetramer, as dog and human tryptase appear to be (6, 17, 25, 30) . On gel filtration, the larger apparent size of dMCP-3 oligomer may be due to more extensive attachmentof asparagine-linked glycans, which, because of extensive hy-dration, occupy more volume in solution per unit weight than do unmodified globular proteins. dMCP-3 contains two consensus glycosylation sites, compared to a single site in dog tryptase. The results of SDS-PAGE also support the concept of dMCP-3 as an oligomer. Under nonreducing conditions, approximately half of the purified protein migrates at an apparent M twice that of the fully reduced monomer. Thus, approximately half of the subunits of dMCP-3 form intersubunit disulfide bonds.

Subunit association in dMCP-3, as in dog and human tryptase, may be sustained in part by noncovalent hydrophobic interactions. Tryptase tetramers derive additional stability through largely electrostatic interactions with heparin and other sulfated proteoglycans (17, 31) , but do not form covalent attachments between subunits and do not contain free sulfhydryl groups in the nonreduced state (32) . The dMCP-3 oligomer, being stable in heparin's absence, may be supplementally stabilized by the formation of disulfide linkages between some subunits. The cDNA-derived amino acid sequence of dMCP-3 predicts the presence of 4 more cysteines (Cys, Cys, Cys, and Cys, according to numbering in Ref. 15) than are present in mouse and human mast cell tryptases. Compared to mouse and human tryptases, dog tryptase has one extra cysteine, which does not correspond to any of the cysteines of dMCP-3. Mapping of the dMCP-3 sequence onto an existing model of human tryptase I (33) predicts that these supernumerary cysteines lie at or near the subunit surface and that none lies sufficiently close to another to form an intramolecular Cys-Cys linkage. One or more of these residues may form linkages between subunits. Of the four cysteines, Cys is the least likely to form a disulfide bond because it is next to a predicted site of N-glycosylation (Asn) and because it is the most remote from the hydrophobic, noncatalytic face of the enzyme thought to be involved in subunit interactions (33) . Of the other extra cysteines, Cys is the most likely to form an intersubunit disulfide bond because it appears to lie adjacent to, and between, the tryptophan- and proline-rich patches, which are conserved in dMCP-3 and tryptases and are candidate surface sites for contact between subunits (33). The detection of an average of 1 titratable thiol per subunit confirms that some of the cysteines of the dMCP-3 oligomer do not form disulfide bonds. These data, together with the electrophoretic evidence of subunit dimerization and insights derived from modeling, suggest that most of the cysteines in the dMCP oligomer are tied up in intrasubunit Cys-Cys linkages, that some cysteines (one or more pairs) form intersubunit linkages, and that some are free thiols. Other cysteines not involved in disulfide linkages may be modified in some manner (e.g. by oxidation) so that they are not detected in the free thiol assay.

The predicted net charge of dMCP-3 ((Arg + Lys) - (Asp + Glu)) is -11, compared to dog tryptase, which is -3 (15) . Both enzymes contrast strikingly with dog chymase (predicted net charge of +16). Despite its strong predicted negative charge, dMCP-3 binds to polyanionic heparin (albeit less strongly than does tryptase), as reflected by its binding to a heparin affinity column. 5 units of the net charge difference between dMCP-3 and tryptase are due to a disparity in the number of positively charged residues (13 versus 18). The remaining 3 units of difference are due to a disparity in negatively charged residues (24 versus 21). Thus, the ability of oligomeric tryptase-like serine proteases to bind to heparin appears to be conferred by a relatively small number of critically positioned basic residues. This contrasts with cationic chymases and human cathepsin G (net charge +22), whose ability to bind to heparin is thought to be a function of a considerably larger number of cationic amino acid side chains concentrated in two large surface patches (34, 35, 36) .

The results of the peptidyl pNA studies establish differences in substrate preferences between dMCP-3 and dog tryptase. The difference in the rate of hydrolysis of VGRpNA is particularly striking. It is reasonable to hypothesize from the contrasting rates of synthetic substrate hydrolysis that physiological targets of dMCP-3 and tryptase differ and that the two enzymes serve different functions. In the case of tryptases, several natural peptide and protein targets have been identified in vitro, although the importance of these targets awaits validation in vivo. In the case of dMCP-3, one putative physiological substrate identified in the current study is calcitonin gene-related peptide. Several studies suggest that the flare reaction caused by this neuropeptide in the setting of cutaneous neurogenic inflammation is limited by mast cell proteases (23, 37, 38) . dMCP-3 may be among these proteases. On the other hand, in contrast to tryptases, dMCP-3 does not degrade vasoactive intestinal peptide and will not directly influence bronchodilation and other consequences of this peptide's neural release. This finding, and the lack of caseinolytic activity, predict that dMCP-3 is highly selective for its targets. Identification of additional natural targets requires more investigation.

Like mast cell tryptases, dMCP-3 resists inactivation by large, natural inhibitors of most trypsin-like serine proteases. In tryptases, this idiosyncrasy is poorly understood in molecular terms. The major distinction between dMCP-3 and dog tryptase in inhibitor susceptibility is the former's resistance to inhibition by aprotinin. In this respect, dMCP-3 is similar to human tryptases (6, 30) , even though dog tryptase is more closely related to human tryptases in primary structure than dMCP-3 is to any known tryptase. Nonetheless, dMCP-3 and tryptases alike are inactivated by several low molecular weight trypsin-like serine protease inhibitors, which, because of their small size, do not make extensive contacts with amino acid residues in the vicinity of the substrate binding cleft. An especially effective dMCP-3 inhibitor is BABIM (24) , which, by analogy to benzamidine (a related aromatic amidine), is thought to reversibly inactivate dMCP-3, tryptases, and trypsin by occupying the portion of the active site that accommodates the substrate P1 lysine or arginine side chain, whose protonated amino group forms a salt bridge with the carboxyl anion of an internal residue (Asp, using chymotrypsinogen numbering (39) ; Asp of dMCP-3 (15) ). Another effective inhibitor is leupeptin, a small peptidyl aldehyde, which in trypsin forms a reversible covalent complex between the nucleophilic hydroxyl of the catalytically active serine (Ser, by chymotrypsin numbering; Ser of dMCP-3) and the aldehyde carbonyl (40). This suggests that key elements of dMCP's catalytic apparatus behave as they do in trypsin. However, the resistance of dMCP-3 and tryptases to most large, natural inhibitors of serine proteases suggests that the conformations of their extended substrate and inhibitor binding site are atypical of trypsin-like serine proteases. For dMCP-3, an implication of its exceptional stability and resistance to known antiproteases in plasma is that it may remain active for extended periods after escape or secretion from mast cells.

  
Table: Purification of dMCP-3


  
Table: Inhibition of dog mast cell protease-3 (dMCP-3)



FOOTNOTES

*
This work supported in part by Grant HL-24136 from the National Institutes of Health, the Leahi Trust of the Hawaii Community Foundation, and the Robert C. Perry Fund. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of New Investigator Award 1KT53 of the University of California Tobacco-related Disease Program.

Recipient of an American Lung Association Career Investigator award. To whom correspondence and reprint requests should be addressed. Tel.: 415-476-9920; Fax: 415-476-9749.

The abbreviations used are: dMCP-3, dog mast cell protease-3; VGRpNA, N-benzoyl-L-Val-Gly-Arg-p-nitroanilide; Bis-tris, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis; GPRpNA, N-p-tosyl-Gly-L-Pro-Arg-p-nitroanilide; KGRpNA, N-benzoyl-L-Lys-Gly-Arg-p-nitroanilide; BABIM, bis(5-amidino-2-benzimidazolyl)methane; GPKpNA, N-p-tosyl-Gly-L-Pro-Lys-pNA; DTNB, 2,2`-dithiobis(2-nitrobenzoic acid).


ACKNOWLEDGEMENTS

The technical assistance of Jeanne Aufderheide and Lianjie Du in the purification of lung tryptase and of Barbara A. Kirby in the peptide hydrolysis experiments is greatly appreciated.


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