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The Tryptase, Mouse Mast Cell Protease 7, Exhibits Anticoagulant Activity in Vivo and in Vitro Due to Its Ability to Degrade Fibrinogen in the Presence of the Diverse Array of Protease Inhibitors in Plasma

1997; Elsevier BV; Volume: 272; Issue: 50 Linguagem: Inglês

10.1074/jbc.272.50.31885

ISSN

1083-351X

Autores

Chifu Huang, G. William Wong, N. P. Ghildyal, Michael F. Gurish, Andrej Šali, Ryoji Matsumoto, Wen-Tao Qiu, Richard L Stevens,

Tópico(s)

Blood Coagulation and Thrombosis Mechanisms

Resumo

Mouse mast cell protease (mMCP) 7 is a tryptase of unknown function expressed by a subpopulation of mast cells that reside in numerous connective tissue sites. Because enzymatically active mMCP-7 is selectively released into the plasma of V3 mastocytosis mice undergoing passive systemic anaphylaxis, we used thisin vivo model system to identify a physiologic substrate of the tryptase. Plasma samples taken from V3 mastocytosis mice that had been sensitized with immunoglobulin (Ig) E and challenged with antigen were found to contain substantial amounts of four 34–55-kDa peptides, all of which were derived from fibrinogen. To confirm the substrate specificity of mMCP-7, a pseudozymogen form of the recombinant tryptase was generated that could be activated after its purification. The resulting recombinant mMCP-7 exhibited potent anticoagulant activity in the presence of normal plasma and selectively cleaved the α-chain of fibrinogen to fragments of similar size as that seen in the plasma of the IgE/antigen-treated V3 mastocytosis mouse. Subsequent analysis of a tryptase-specific, phage display peptide library revealed that recombinant mMCP-7 preferentially cleaves an amino acid sequence that is nearly identical to that in the middle of the α-chain of rat fibrinogen. Because fibrinogen is a physiologic substrate of mMCP-7, this tryptase can regulate clot formation and fibrinogen/integrin-dependent cellular responses during mast cell-mediated inflammatory reactions. Mouse mast cell protease (mMCP) 7 is a tryptase of unknown function expressed by a subpopulation of mast cells that reside in numerous connective tissue sites. Because enzymatically active mMCP-7 is selectively released into the plasma of V3 mastocytosis mice undergoing passive systemic anaphylaxis, we used thisin vivo model system to identify a physiologic substrate of the tryptase. Plasma samples taken from V3 mastocytosis mice that had been sensitized with immunoglobulin (Ig) E and challenged with antigen were found to contain substantial amounts of four 34–55-kDa peptides, all of which were derived from fibrinogen. To confirm the substrate specificity of mMCP-7, a pseudozymogen form of the recombinant tryptase was generated that could be activated after its purification. The resulting recombinant mMCP-7 exhibited potent anticoagulant activity in the presence of normal plasma and selectively cleaved the α-chain of fibrinogen to fragments of similar size as that seen in the plasma of the IgE/antigen-treated V3 mastocytosis mouse. Subsequent analysis of a tryptase-specific, phage display peptide library revealed that recombinant mMCP-7 preferentially cleaves an amino acid sequence that is nearly identical to that in the middle of the α-chain of rat fibrinogen. Because fibrinogen is a physiologic substrate of mMCP-7, this tryptase can regulate clot formation and fibrinogen/integrin-dependent cellular responses during mast cell-mediated inflammatory reactions. Tryptases are major constituents of the secretory granules of mast cells in the mouse (1Reynolds D.S. Stevens R.L. Lane W.S. Carr M.H. Austen K.F. Serafin W.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3230-3234Crossref PubMed Scopus (172) Google Scholar, 2Reynolds D.S. Gurley D.S. Austen K.F. Serafin W.E. J. Biol. Chem. 1991; 266: 3847-3853Abstract Full Text PDF PubMed Google Scholar, 3McNeil H.P. Reynolds D.S. Schiller V. Ghildyal N. Gurley D.S. Austen K.F. Stevens R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11174-11178Crossref PubMed Scopus (128) Google Scholar, 4Johnson D.A. Barton G.J. Protein Sci. 1992; 1: 370-377Crossref PubMed Scopus (66) Google Scholar), rat (5Braganza V.J. Simmons W.H. Biochemistry. 1991; 30: 4997-5007Crossref PubMed Scopus (36) Google Scholar, 6Ide H. Itoh H. Tomita M. Murakumo Y. Kobayashi T. Maruyama H. Osada Y. Nawa Y. J. Biochem. Tokyo. 1995; 118: 210-215Crossref PubMed Scopus (25) Google Scholar, 7Lützelschwab C. Pejler G. Aveskogh M. Hellman L. J. Exp. Med. 1996; 185: 13-29Crossref Scopus (137) Google Scholar), dog (8Vanderslice P. Craik C.S. Nadel J.A. Caughey G.H. Biochemistry. 1989; 28: 4148-4155Crossref PubMed Scopus (59) Google Scholar), gerbil (9Murakumo Y. Ide H. Itoh H. Tomita M. Kobayashi T. Maruyama H. Horii Y. Nawa Y. Biochem. J. 1995; 309: 921-926Crossref PubMed Scopus (21) Google Scholar), and human (10Schwartz L.B. Lewis R.A. Austen K.F. J. Biol. Chem. 1981; 256: 11939-11943Abstract Full Text PDF PubMed Google Scholar, 11Miller J.S. Westin E.H. Schwartz L.B. J. Clin. Invest. 1989; 84: 1188-1195Crossref PubMed Scopus (178) Google Scholar, 12Miller J.S. Moxley G. Schwartz L.B. J. Clin. Invest. 1990; 86: 864-870Crossref PubMed Scopus (154) Google Scholar, 13Vanderslice P. Ballinger S.M. Tam E.K. Goldstein S.M. Craik C.S. Caughey G.H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3811-3815Crossref PubMed Scopus (203) Google Scholar). In the mouse, two tryptases, designated mouse mast cell protease (mMCP) 1The abbreviations used are: mMCP, mouse mast cell protease; EK, enterokinase; FLAG, the peptide whose amino acid sequence is Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys; Ig, immunoglobulin; PAGE, polyacrylamide gel electrophoresis; pNA,p-nitroanilide; and pIII, the protein encoded by the phage gene designated gIII.-6 and mMCP-7, have been identified whose overall amino acid sequences are 71% identical. Although their physiologic substrates remain to be determined, the tryptase family of mouse mast cell proteases has been implicated in the pathobiology of certain airway responses elicited by the high affinity receptor for immunoglobulin (Ig) E. Tryptase inhibitors block antigen-induced airway constriction and tissue inflammatory responses in sheep sensitized with Ascaris suum(14Clark J.M. Abraham W.M. Fishman C.E. Forteza R. Ahmed A. Cortes A. Warne R.L. Moore W.R. Tanaka R.D. Am. J. Respir. Crit. Care Med. 1995; 152: 2076-2083Crossref PubMed Scopus (200) Google Scholar). In addition, linkage analysis (15De Sanctis G.T. Merchant M. Beier D.R. Dredge R.D. Grobholz J.K. Martin T.R. Lander E.S. Drazen J.M. Nat. Genet. 1995; 11: 150-154Crossref PubMed Scopus (264) Google Scholar) has implicated the region of chromosome 17 where the mMCP-6 and mMCP-7 genes reside (16Gurish M.F. Nadeau J.H. Johnson K.R. McNeil H.P. Grattan K.M. Austen K.F. Stevens R.L. J. Biol. Chem. 1993; 268: 11372-11379Abstract Full Text PDF PubMed Google Scholar, 17Gurish M.F. Johnson K.R. Webster M.J. Stevens R.L. Nadeau J.H. Mammal. Genome. 1994; 5: 656-657Crossref PubMed Scopus (19) Google Scholar) as one of the candidate loci for the inheritance of intrinsic airway hyper-responsiveness. mMCP-6 and mMCP-7 are stored in granules in their mature, enzymatically active forms ionically bound to the glycosaminoglycan side chains of serglycin proteoglycans (1Reynolds D.S. Stevens R.L. Lane W.S. Carr M.H. Austen K.F. Serafin W.E. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 3230-3234Crossref PubMed Scopus (172) Google Scholar). Although mMCP-6 and mMCP-7 both have an overall negative charge at neutral pH, the two exocytosed tryptases differ in their ability to dissociate from serglycin proteoglycans outside the mast cell (18Ghildyal N. Friend D.S. Stevens R.L. Austen K.F. Huang C. Penrose J.F. Šali A. Gurish M.F. J. Exp. Med. 1996; 184: 1061-1073Crossref PubMed Scopus (75) Google Scholar). Thus, they are metabolized quite differently in mice undergoing passive systemic anaphylaxis. Tongue, skin, spleen, and heart mast cells of normal BALB/c mice and spleen and liver mast cells of V3 mastocytosis mice all contain substantial amounts of mMCP-6 and mMCP-7 in their secretory granules. Ten min after antigen is administered to IgE-sensitized mice, protease-proteoglycan macromolecular complexes appear in the extracellular matrix adjacent to the activated tissue mast cells. These complexes can be readily stained by anti-mMCP-6 Ig but not by anti-mMCP-7 Ig. In V3 mastocytosis mice sensitized with IgE and challenged with antigen, exocytosed mMCP-7 rapidly makes its way into blood, where it circulates for >1 h. This plasma form of mMCP-7 has an intact N terminus. Moreover, it is properly folded, enzymatically active, and not degraded. Despite the fact that as much as 10% of the proteins in blood are protease inhibitors (19Diem K. Lentner C. Scientific Tables. 7th Ed. Ciba-Geigy Limited, Ardsley, NY1970: 579-580Google Scholar), plasma-localized mMCP-7 does not rapidly form covalent complexes with any protease inhibitor in the blood of V3 mastocytosis mice. Modeling and site-directed mutagenesis of recombinant pro-mMCP-7 suggested that the natural form of the tryptase selectively dissociates from the macromolecular complex when exocytosed into a pH 7.0 environment because the glycosaminoglycan-binding domain on its surface consists predominately of a cluster of His residues rather than predominately Lys and Arg residues (20Matsumoto R. Šali A. Ghildyal N. Karplus M. Stevens R.L. J. Biol. Chem. 1995; 270: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), as found in mMCP-6 (18Ghildyal N. Friend D.S. Stevens R.L. Austen K.F. Huang C. Penrose J.F. Šali A. Gurish M.F. J. Exp. Med. 1996; 184: 1061-1073Crossref PubMed Scopus (75) Google Scholar) and all mast cell chymases (21Šali A. Matsumoto R. McNeil H.P. Karplus M. Stevens R.L. J. Biol. Chem. 1993; 268: 9023-9034Abstract Full Text PDF PubMed Google Scholar). The prolonged retention of exocytosed mMCP-6 in the extracellular matrix around activated tissue mast cells suggests a local action, whereas the rapid dissipation of mMCP-7 from tissues and its poor ability to be inactivated by circulating protease inhibitors suggests that this tryptase cleaves proteins located at more distant sites. Although mast cell tryptases have been purified from different species in an attempt to deduce their protein substrates, the number of mature mast cells that can be isolated from a mouse is inadequate to obtain enough mMCP-7 for in-depth study. No mast cell has been found that expresses just mMCP-7. For example, the mMCP-7+ mast cells in the skin of the BALB/c mouse also express substantial amounts of similar sized mMCP-4, mMCP-5, mMCP-6, and carboxypeptidase A (22Stevens R.L. Friend D.S. McNeil H.P. Schiller V. Ghildyal N. Austen K.F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 128-132Crossref PubMed Scopus (117) Google Scholar). Because all of these proteases are catalytic at neutral pH, the nearly impossible task of removing 100% of the other mMCPs from the starting preparations of mast cell lysates has prevented definitive studies that address the function of mMCP-7. A contributing problem is the fact that serine proteases like mMCP-7 tend to undergo inactivation during their isolation. We now show that fibrinogen is preferentially degraded when mMCP-7 is released into the circulation of V3 mastocytosis mice that are undergoing systemic anaphylaxis. We show that recombinant mMCP-7 is not readily inhibited by any protease inhibitor in plasma, that this tryptase degrades fibrinogen in a manner comparable to that seen in the V3 mastocytosis mouse, and that the enzymatic activity of this tryptase is not heparin-dependent. Based on these in vivo andin vitro findings, we conclude that fibrinogen is a physiologic substrate of mMCP-7. V3 mastocytosis mice were created and systemically sensitized intraperitoneally with ∼200 μg of anti-trinitrophenol IgE, as described (18Ghildyal N. Friend D.S. Stevens R.L. Austen K.F. Huang C. Penrose J.F. Šali A. Gurish M.F. J. Exp. Med. 1996; 184: 1061-1073Crossref PubMed Scopus (75) Google Scholar, 23Gurish M.F. Pear W.S. Stevens R.L. Scott M.L. Sokol K. Ghildyal N. Webster M.J. Hu X. Austen K.F. Baltimore D. Friend D.S. Immunity. 1995; 3: 175-186Abstract Full Text PDF PubMed Scopus (75) Google Scholar). Approximately 24 h later, ∼300 μl of Hank's balanced salt solution alone or containing 10–1000 μg of trinitrophenol-bovine serum albumin was injected intraperitoneally into each mouse. Twenty min after antigen administration, 100–500 μl of blood was obtained from the retroorbital plexus with a Pasteur pipette pretreated with an anticoagulant (either 25 USP units of heparin glycosaminoglycan (Elkins-Sinn, Cherry Hill, NC) or 10 mm EDTA). After a 4-min centrifugation step at ∼10,000 × g and at 4 °C, each 15-μl sample of plasma was subjected to SDS-polyacrylamide gel electrophoresis (PAGE). The four prominent ∼34–55-kDa peptides that preferentially appeared after the sensitization and antigen challenge were transferred to Immobilon-P membranes (Millipore, Bedford, MA) and subjected to N-terminal amino acid analysis. Using a polymerase chain reaction approach, an oligonucleotide (5′-GACGACGATGACAAG-3′) encoding the EK-susceptible peptide Asp-Asp-Asp-Asp-Lys was inserted into the mMCP-7 cDNA (3McNeil H.P. Reynolds D.S. Schiller V. Ghildyal N. Gurley D.S. Austen K.F. Stevens R.L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11174-11178Crossref PubMed Scopus (128) Google Scholar) between the domain that encodes the pro-peptide and the N terminus of the mature tryptase. EK is a highly specific enzyme that cleaves the Lys-Ile bond in its Asp-Asp-Asp-Lys-Ile recognition motif (24Light A. Janska H. Trends Biochem. Sci. 1989; 14: 110-112Abstract Full Text PDF PubMed Scopus (84) Google Scholar). Because Ile is the essential N-terminal amino acid of mature mMCP-7 and because EK is a relatively stable enzyme at pH 5.0, it was anticipated that the secreted recombinant pseudozymogen could be activated under conditions where the generated tryptase would have very little enzymatic activity until the pH is raised to 7.0. The FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys), which consists of the EK-cleavage sequence C-terminal of a 3-residue linker, has been used by many to epitope-tag the N or C terminus of recombinant proteins (25Hopp T.P. Prickett K.S. Price V.L. Libby R.T. March C.J. Cerretti D.P. Urdal D.L. Conlon P.J. Biotechnology. 1988; 6: 1204-1210Crossref Scopus (754) Google Scholar). To facilitate the purification of the recombinant pseudozymogen with an anti-FLAG IgG antibody (26Prickett K.S. Amberg D.C. Hopp T.P. Biotechniques. 1989; 7: 580-589PubMed Google Scholar, 27Brizzard B.L. Chubet R.G. Vizard D.L. Biotechniques. 1994; 16: 730-735PubMed Google Scholar), a second construct (pro-EK-mMCP-7-FLAG) was created that also contained the 8-residue FLAG peptide at its C terminus. These two cDNAs were inserted in the correct orientation into the multiple cloning site of pVL1393 (PharMingen, San Diego, CA) downstream of the promoter of the polyhedrin gene, as described for the expression of recombinant pro-mMCP-7 (20Matsumoto R. Šali A. Ghildyal N. Karplus M. Stevens R.L. J. Biol. Chem. 1995; 270: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In each instance, purified plasmid DNA (∼5 μg) was mixed with 0.5 μg of linearized BaculoGoldTM DNA (PharMingen) and calcium phosphate. The resulting DNA solution was added to 3 × 106 adherent Spodoptera frugiperda 9 insect cells (Invitrogen, San Diego, CA) that were in their log phase of growth, and infected cells were cultured for 7 days at 27 °C in medium (Invitrogen) supplemented with 10% heat-inactivated (56 °C, 30 min) fetal calf serum (Sigma). Recombinant virus particles (≥3 × 107) were added to a culture dish containing 6 × 106 Trichoplusia ni High FiveTMinsect cells (Invitrogen) in their log phase of growth, and the infected cells were cultured in serum-free, Xpress medium (BioWhittaker, Walkersville, MD). Four days later, the conditioned medium was centrifuged at 1500 × g for 15-min at room temperature. Under these conditions, recombinant pro-EK-mMCP-7 and pro-EK-mMCP-7-FLAG were recovered in the supernatants as soluble proteins. Recombinant pro-EK-mMCP-7 and pro-EK-mMCP-7-FLAG were purified by heparin-Sepharose chromatography, as described for pro-mMCP-7 (20Matsumoto R. Šali A. Ghildyal N. Karplus M. Stevens R.L. J. Biol. Chem. 1995; 270: 19524-19531Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Alternatively, recombinant pro-EK-mMCP-7-FLAG was purified with a 2-ml column containing the mouse anti-FLAG M2 monoclonal antibody (International Biotechnol Inc., New Haven, CT). This anti-FLAG IgG affinity column was washed with 10 ml of 0.1m glycine, pH 3.5, followed by 50 ml of 50 mmTris-HCl and 150 mm NaCl, pH 7.4. After ∼200 ml of insect cell-conditioned medium was passed through the affinity column, the resin was washed with 50 ml of the same pH 7.4 buffer. Bound pro-EK-mMCP-7-FLAG was eluted by washing the column with 0.1m glycine, pH 3.5. The eluate was collected into tubes that contained 0.1 m Tris-HCl, pH 7.0, to minimize acid-mediated denaturation of the recombinant proteins. The final concentration of each recombinant protein was estimated by measuring the absorbence at 280 nm. Purified pro-EK-mMCP-7 and pro-EK-mMCP-7-FLAG (∼100 μg) was separately suspended in ∼100 μl of 50 mm sodium acetate and 5 mm calcium chloride, pH 5.2. One μl of a solution containing 310 units of calf intestinal EK (Biozyme Laboratories, San Diego, CA) was added to each, and the mixture was incubated at 37 °C for ∼3 h to allow EK to activate the zymogen. The spectrophotometric method of Svendsen and co-workers (28Svendsen L. Blombäck B. Blombäck M. Olsson P.I. Throm. Res. 1972; 1: 267-278Abstract Full Text PDF Scopus (209) Google Scholar) was used to determine whether or not recombinant mMCP-7 and mMCP-7-FLAG were enzymatically active. A 1-μl sample of each activation mixture was placed in 1 ml of assay buffer (pH 7.4 buffer containing 25 mm sodium phosphate, 1 mm EDTA, and 50 μg/ml of a p-nitroanilide (pNA) substrate such as tosyl-Gly-Pro-Lys-pNA (Sigma)). The change in optical density at 405 nm was determined after a 3–5-min incubation at room temperature. In this assay, 1 unit of enzymatic activity is defined as a change in optical density at 405 nm of 0.001 per min. As noted under "Results," mMCP-7 exhibits an enzymatic activity of 175 to 50 units/μg when tosyl-Gly-Pro-Lys-pNA is the test substrate. However, analysis of a phage display peptide library revealed that tosyl-Gly-Pro-Lys-pNA is not the optimal substrate of mMCP-7. Thus, it is likely that the recombinant tryptase exhibits greater enzymatic activity against its physiologic substrate(s). One μg of bovine pancreatic trypsin (Sigma) is equivalent to ∼350 units when tosyl-Gly-Pro-Lys-pNA is the test substrate. With the more widely used substrateN α-benzoyl-l-Arg-ethyl ester, the reference preparation of trypsin employed in our study for comparative purposes has 11 units/μg. The ability of recombinant mMCP-7 and mMCP-7-FLAG to cleave the trypsin-susceptible substrates tosyl-Gly-Pro-Arg-pNA, benzoyl-Ile-Glu-Gly-Arg-pNA, benzoyl-Phe-Val-Arg-pNA, benzoyl-Pro-Phe-Arg-pNA, acetyl-Ile-Glu-Ala-Arg-pNA, benzoyl-Val-Gly-Arg-pNA, andd-Ile-Phe-Lys-pNA (Sigma) were also evaluated. Insect cell-conditioned medium (∼20 μl) containing pro-mMCP-7, pro-EK-mMCP-7, pro-EK-mMCP-7-FLAG, or purified EK-activated mMCP-7 (∼1 μl) was diluted in SDS-PAGE buffer (1% SDS, 5% β-mercaptoethanol, 0.1% bromphenol blue, and 500 mmTris-HCl, pH 6.8) and boiled for 5 min before being loaded onto 12% polyacrylamide gels. After electrophoresis, the gels were stained with Coomassie Blue or placed in a Bio-Rad (Richmond, CA) immunoblotting apparatus, and the resolved proteins were transferred for 2–4 h at 200 mA to Immobilon-P membranes in a solution consisting of 20% methanol, 16 mm Tris-HCl, and 120 mm glycine, pH 8.3. For analysis of the resulting protein blots, each membrane was incubated for 1 h in 5% non-fat milk and then for 1 h with a 1:500 dilution of affinity-purified rabbit anti-mMCP-7 Ig (29Ghildyal N. Friend D.S. Freelund R. Austen K.F. McNeil H.P. Schiller V. Stevens R.L. J. Immunol. 1994; 153: 2624-2630PubMed Google Scholar) in Tris-buffered saline with 0.01% Tween 20 (TBST buffer). After 3 washes in TBST buffer, the blots were incubated for 1 h in a 1:1000 dilution of anti-rabbit IgG alkaline phosphatase conjugate (∼1 ng/ml final concentration) in TBST buffer. Immunoreactive proteins were visualized with nitro blue tetrazolium (0.2 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.1 mg/ml) as substrates. For N-terminal amino acid analysis, SDS-PAGE-resolved proteins were electroblotted onto membranes and briefly stained with 0.5% Ponceau S red (Sigma), and the relevant proteins/peptides were subjected to automated Edman degradation by the Harvard Microchemistry Facility (Harvard Biological Laboratories, Cambridge, MA). Samples (50 μg or more) of purified mouse fibrinogen (Sigma) were suspended in 1 mmEDTA and 25 mm sodium phosphate, pH 7.4, containing 0.2–0.5 μg of recombinant mMCP-7-FLAG (10–25 units corresponding to 0.007–0.014 nmol of tryptase activated with 0.01 units of EK), ∼0.5 μg of recombinant pro-EK-mMCP-7-FLAG, or 0.01 units of EK and then incubated at 37 °C for various time periods. The resulting digests were subjected to SDS-PAGE. In one experiment, the N-terminal amino acid sequences of the major fibrinogen fragments in an exhaustive digest were determined after a 3-h treatment of 100 μg of fibrinogen with 120 units of mMCP-7-FLAG. The ability of mMCP-7-FLAG, bovine trypsin, and bovine α-chymotrypsin (Sigma) to digest mouse fibrinogen in the presence and absence of the protease inhibitors found in normal mouse plasma was also evaluated. In these latter experiments, mMCP-7-FLAG (10 units), trypsin (10 units), and chymotrypsin (0.03 μg; an amount equal to that of trypsin on a weight basis) were suspended separately in 4 μl of 1 mm EDTA and 25 mm sodium phosphate, pH 7.4, lacking or containing 2.0–4.0% (v/v) mouse plasma for 30 min at room temperature. Thirty μl of reaction buffer containing 15 μg of fibrinogen was added and the samples were incubated for 1 h at 37 °C. The final fibrinogen:enzyme ratio in each of these experiments was at least 75:1. Comparable amounts of plasma was added to those samples that had not been exposed previously to plasma, and then the digests were subjected to SDS-PAGE. The resulting gels were stained with Coomassie Blue and the extent of digestion of each chain of fibrinogen by mMCP-7 in the presence and absence of plasma was evaluated by measuring the decrease in the optical density of SDS-PAGE separated, Coomassie Blue-stained α-, β-, and γ-chains by means of a densitometer (Molecular Dynamics, Sunnyvale, CA). To demonstrate that the α-chain of mouse fibrinogen is much more susceptible to digestion by mMCP-7-FLAG than either the β- or γ-chains, kinetic experiments also were carried in which 70 units of mMCP-7-FLAG was incubated at 37 °C for 1 min to 17 h with 15 μg of mouse fibrinogen in the presence or absence of 2% normal mouse plasma prior to the SDS-PAGE analysis. A standard fibrinogenolysis assay (30Brown B. Hematology: Principles and Procedures. 5th Ed. Lea and Febiger, Philadelphia, PA1988: 219-222Google Scholar) was used to detect mMCP-7 anticoagulant activity in vitro. Sodium citrate-treated, normal mouse plasma (100 μl/assay) was incubated for 30 min to 1 h at 37 °C in the absence or presence of ∼4 μg (∼200 units or 0.12 nmol) of EK-activated mMCP-7-FLAG or 10 USP units of heparin (∼100 μg of the glycosaminoglycan). The time required for thrombin to clot the samples was then determined with a fibrometer. The plasma concentration of fibrinogen is ∼3 mg/ml. Thus, even if 100% of the recombinant pseudozymogen was converted to active enzyme by EK treatment, there is ∼75-fold more fibrinogen than mMCP-7 in the assay. The genome of the Ff bacteriophage consists of the 11 genes designated gI to gXI. Although the protein (pIII) encoded by gIII is chymotrypsin-, thermolysin-, and subtilisin-susceptible, it is trypsin-resistant (31Grant R.A. Lin T.-C. Konigsberg W. Webster R.E. J. Biol. Chem. 1981; 256: 539-546Abstract Full Text PDF PubMed Google Scholar). Thus, phage display peptide libraries can give insight into the substrate specificities of certain proteases (32Mathews D.J. Wells J.A. Science. 1993; 260: 1113-1117Crossref PubMed Scopus (318) Google Scholar, 33Smith M.M. Shi L. Navre M. J. Biol. Chem. 1995; 270: 6440-6449Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The N terminus of pIII extends out from the surface of the body of the filamentous phage. By taking advantage of the fact that pIII is trypsin-resistant and exhibits low valency, a phage display peptide library specific for tryptases was generated that encodes a pIII fusion protein containing at its N terminus the FLAG peptide followed by an 8-residue hypervariable peptide. The FLAG peptide was selected as the "tether" ligand so that those phage producing a bioengineered pIII could be readily isolated with the monoclonal anti-FLAG M1 antibody. To create the tryptase-specific library, two complementary single-stranded oligonucleotides (5′-CGGCCGACTACAAGGACGACGATGACAAG(X)12A(A/G)G(X)9GC-3′ and 5′-GGCCGC(X)9C(T/C)T(X)12CTTGTCATCGTCGTCCTTGTAGTCGGCCGGCT-3′, where "X" indicates a random nucleotide) were synthesized such that they could be annealed to one another in vitro to form short double-stranded DNAs that each containedSfiI and NotI restriction sites at their 5′- and 3′-ends, respectively. Because it was found that recombinant mMCP-7 cleaves tosyl-Gly-Pro-Lys-pNA, the library was created such that the fifth residue in the hypervariable domain would be either Arg or Lys. The single-stranded oligonucleotides were mixed in approximately equal concentrations, heated to 94 °C for 1 min, and cooled to room temperature. The resulting doublestranded oligonucleotides were ligated intoSfiI/NotI-digested phagemid vector pCANTAB-5E (International Biotechnology). Escherichia coli (strain TG1), transformed by electroporation with the resulting constructs, were incubated for 1 h at 37 °C in 2 × YT medium (0.09m NaCl containing 1.7% Bacto-tryptone (Difco Labs, Detroit, MI), and 1% Bacto-yeast extract (Difco Labs), pH 7.0) and 2% glucose. Ampicillin (50 μg/ml) and the M13 helper phage K (∼10 phage/bacteria) were added, and the bacteria were incubated at 37 °C for another 1 h to induce the formation of recombinant phage. After the mixture was centrifuged at 2,000 × g for 20 min, the pellet was resuspended in 20 ml of 2 × YT medium containing 50 μg/ml ampicillin and 50 μg/ml kanamycin. Infected bacteria were incubated overnight at 37 °C and then subjected to a 20-min centrifugation at 2,000 × g to obtain the phage-enriched supernatant. The resulting phage display peptide library was screened with bovine pancreatic trypsin to determine its suitability for substrate specificity studies. Because phage clones were obtained after two rounds of trypsin treatment that possessed different peptide sequences in the random portion of the pIII fusion protein, the library was screened with recombinant mMCP-7-FLAG. To purify the recombinant phage, 10 ml of the phage-enriched supernatant was added to 2 ml of 20% polyethylene glycol (8 kDa; Sigma) and 2.5 m NaCl and the mixture was incubated at 4 °C for 30 min. After a 30-min centrifugation of the mixture at 10,000 × g, the recombinant phage in the pellet were resuspended in 2 ml of 150 mm NaCl, 1 mm CaCl2, and 10 mm sodium phosphate, pH 7.0, and applied to a 1-ml affinity column containing the anti-FLAG M1 monoclonal antibody. The column was washed three times with 10 ml of the same pH 7.0 buffer to remove unbound phage. Recombinant mMCP-7-FLAG or bovine pancreatic trypsin (∼50 μg in 200 μl of the pH 7.0 buffer) was added, and the column was sealed and incubated at room temperature for 90 min. After treatment with protease, the column was washed with 2 ml of the pH 7.0 buffer to recover those phage that possessed protease-susceptible pIII fusion proteins. Log phase E. coli were infected with the obtained phage to produce phagemid. Bacteria were again grown in 2 × YT medium containing 2% glucose and the phagemid in the bacteria were converted to phage with the addition of helper phage. The selection procedure was repeated one to three additional times to isolate those phage that possessed the most protease-susceptible pIII fusion proteins. E. coli were infected with phage that were susceptible to either trypsin or mMCP-7-FLAG to generate phagemids. The infected bacteria were seeded onto a plate containing 1.5% agar, 2% Bacto-tryptone, 0.5% Bacto-yeast extract, 2% glucose, 90 mm NaCl, 10 mm MgCl2, and 50 μg/ml ampicillin. Individual clones were isolated and grown overnight at 37 °C in 2 ml of 2 × YT medium containing 2% glucose with 50 μg/ml ampicillin. Samples (50 μl) of the overnight cultures were centrifuged at ∼12,000 × g for 5 min. The bacteria in the pellets were resuspended in 50 μl of water, boiled for 10 min, and again centrifuged. Each polymerase chain reaction was carried out on 2-μl samples of the supernatant with sense (5′-CCCAGCCGGCCGACTACAAGGACG-3′) and antisense (5′-TGTTCCTTTCTATGCGGCCCAGC-3′) primers. Each of the 35 cycles of the polymerase chain reaction consisted of a 1-min denaturing step at 94 °C, a 1-min annealing step at 60 °C, and a 1-min extension step at 72 °C. The polymerase chain reaction products were subjected to electrophoresis on a 1% agarose gel, and the nucleotide sequences that encode the 8-mer, protease-susceptible peptide domains in the pIII fusion proteins were determined. Relative to V3 mastocytosis mice sensitized with IgE but not challenged with antigen, the plasma from IgE/antigen-treated V3 mastocytosis mice contained large amounts of ∼34-, 40-, and 55-kDa peptides, and lesser amounts of an ∼42-kDa peptide (Fig.1). Changes were also seen in at least three proteins whose molecular masses ranged from ∼100 to 200 kDa. While we were not able to resolve the larger sized proteins well enough to determine their N termini, the ∼34-, 40-, and 42-kDa peptides possessed the same N-terminal amino acid sequence of Thr-Asp-Thr-Glu-Asp-Lys-Gly-Glu-Ph

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