A Urokinase-type Plasminogen Activator-inhibiting Cyclic Peptide with an Unusual P2 Residue and an Extended Protease Binding Surface Demonstrates New Modalities for Enzyme Inhibition
2005; Elsevier BV; Volume: 280; Issue: 46 Linguagem: Inglês
10.1074/jbc.m505933200
ISSN1083-351X
AutoresMartin Hansen, Troels Wind, Grant E. Blouse, Anni Christensen, Helle H. Petersen, Signe Kjelgaard, Lisa Mathiasen, Thor L. Holtet, Peter A. Andreasen,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoTo find new principles for inhibiting serine proteases, we screened phage-displayed random peptide repertoires with urokinase-type plasminogen activator (uPA) as the target. The most frequent of the isolated phage clones contained the disulfide bridge-constrained sequence CSWRGLENHRMC, which we designated upain-1. When expressed recombinantly with a protein fusion partner, upain-1 inhibited the enzymatic activity of uPA competitively with a temperature and pH-dependent Ki, which at 25 °C and pH 7.4 was ∼500 nm. At the same conditions, the equilibrium dissociation constant KD, monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was ∼400 nm. By an inhibitory screen against other serine proteases, including trypsin, upain-1 was found to be highly selective for uPA. The cyclical structure of upain-1 was indispensable for uPA binding. Alanine-scanning mutagenesis identified Arg4 of upain-1 as the P1 residue and indicated an extended binding interaction including the specificity pocket and the 37-, 60-, and 97-loops of uPA and the P1, P2, P3′, P4′, and the P5′ residues of upain-1. Substitution with alanine of the P2 residue, Trp3, converted upain-1 into a distinct, although poor, uPA substrate. Upain-1 represents a new type of uPA inhibitor that achieves selectivity by targeting uPA-specific surface loops. Most likely, the inhibitory activity depends on its cyclical structure and the unusual P2 residue preventing the scissile bond from assuming a tetrahedral geometry and thus from undergoing hydrolysis. Peptide-derived inhibitors such as upain-1 may provide novel mechanistic information about enzyme-inhibitor interactions and alternative methodologies for designing effective protease inhibitors. To find new principles for inhibiting serine proteases, we screened phage-displayed random peptide repertoires with urokinase-type plasminogen activator (uPA) as the target. The most frequent of the isolated phage clones contained the disulfide bridge-constrained sequence CSWRGLENHRMC, which we designated upain-1. When expressed recombinantly with a protein fusion partner, upain-1 inhibited the enzymatic activity of uPA competitively with a temperature and pH-dependent Ki, which at 25 °C and pH 7.4 was ∼500 nm. At the same conditions, the equilibrium dissociation constant KD, monitored by displacement of p-aminobenzamidine from the specificity pocket of uPA, was ∼400 nm. By an inhibitory screen against other serine proteases, including trypsin, upain-1 was found to be highly selective for uPA. The cyclical structure of upain-1 was indispensable for uPA binding. Alanine-scanning mutagenesis identified Arg4 of upain-1 as the P1 residue and indicated an extended binding interaction including the specificity pocket and the 37-, 60-, and 97-loops of uPA and the P1, P2, P3′, P4′, and the P5′ residues of upain-1. Substitution with alanine of the P2 residue, Trp3, converted upain-1 into a distinct, although poor, uPA substrate. Upain-1 represents a new type of uPA inhibitor that achieves selectivity by targeting uPA-specific surface loops. Most likely, the inhibitory activity depends on its cyclical structure and the unusual P2 residue preventing the scissile bond from assuming a tetrahedral geometry and thus from undergoing hydrolysis. Peptide-derived inhibitors such as upain-1 may provide novel mechanistic information about enzyme-inhibitor interactions and alternative methodologies for designing effective protease inhibitors. Serine proteases of the trypsin family (clan SA) have many physiological and pathophysiological functions. There is therefore extensive interest in generating specific inhibitors to be used for pharmacological interference with their enzymatic activity. Moreover, serine proteases are classical subjects for studies of catalytic and inhibitory mechanisms. Serine protease-catalyzed peptide bond hydrolysis proceeds through a tetrahedral transition state formed by a nucleophilic attack on the carbonyl group of the substrate P1 amino acid by the hydroxyl group of Ser195 (using the chymotrypsin template numbering (1.Spraggon G. Phillips C. Nowak U.K. Ponting C.P. Saunders D. Dobson C.M. Stuart D.I. Jones E.Y. Structure. 1995; 3: 681-691Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar)), with His57 and Asp102 acting as a charge relay system. The protonated His57 functions as a general acid to facilitate collapse of the tetrahedral intermediate that is stabilized through interactions at the oxyanion hole and main chain β-strand-type hydrogen bonds between the P1–P3 and P2′ amino acids of the substrate and residues within the polypeptide binding cleft, as well as specific contacts within the S1, S2, S3, S1′, and S2′ pockets, which bind respective side chains of the P1, P2, P3, P1′, and P2′ residues (for reviews, see Refs. 2.Brandén C. Tooze J. Introduction to Protein Structure. Garland Publishing, New York1991: 231-246Google Scholar and 3.Hedström L. Chem. Rev. 2002; 102: 4501-4524Crossref PubMed Scopus (1336) Google Scholar). Substrate specificity is governed by the fit of the P residues into their corresponding S-pockets as well as protease-specific surface loops surrounding the active site. Serine proteases of the trypsin family are biosynthesized as inactive zymogens, with activation occurring by cleavage of the peptide bond between residues 15 and 16 to stabilize the oxyanion hole (4.Robertus J.D. Kraut J. Alden R.A. Birktoft J.J. Biochemistry. 1972; 11: 4293-4303Crossref PubMed Scopus (362) Google Scholar). One interesting member of the trypsin family is urokinase-type plasminogen activator (uPA), 2The abbreviations and trivial names used are: uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; aPC, activated protein C; BSA, bovine serum albumin; DFP, diisopropylfluorophosphate; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; mAb, monoclonal antibody; PAB, p-aminobenzamidine; PAI, plasminogen activator inhibitor; PN-1, protease nexin-1; S-2222, benzyl-Ile-Glu-Gly-Arg-p-nitroanilide; S-2238, H-d-Phe-piperidine-Arg-p-nitroaniline; S-2288, Ile-Pro-Arg-p-nitroanilide; S-2302, Pro-Phe-Arg-p-nitroanilide; S-2366, pyro-Glu-Pro-Arg-p-nitroanilide; S-2403, pyro-Glu-Phe-Lys-p-nitroanilide; S-2444, pyro-Glu-Gly-Arg-p-nitroanilide; S-2586, methyl-succinyl-Arg-Pro-Tyr-p-nitroanilide; S-2765, benzyloxycarbonyl-d-Arg-Gly-Arg-p-nitroaniline; SPD, serine protease domain; SpectrozymeFVIIa, methansulfonyl-d-cyclohexylalanyl-butyl-Arg-p-nitroaniline; tPA, tissue-type plasminogen activator; VLK-AMC, H-d-Val-Leu-Lys-7-amido-4-methylcoumarin; WT, wild type; fVIIa, factor VIIa; fXa, factor Xa.2The abbreviations and trivial names used are: uPA, urokinase-type plasminogen activator; uPAR, uPA receptor; aPC, activated protein C; BSA, bovine serum albumin; DFP, diisopropylfluorophosphate; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; mAb, monoclonal antibody; PAB, p-aminobenzamidine; PAI, plasminogen activator inhibitor; PN-1, protease nexin-1; S-2222, benzyl-Ile-Glu-Gly-Arg-p-nitroanilide; S-2238, H-d-Phe-piperidine-Arg-p-nitroaniline; S-2288, Ile-Pro-Arg-p-nitroanilide; S-2302, Pro-Phe-Arg-p-nitroanilide; S-2366, pyro-Glu-Pro-Arg-p-nitroanilide; S-2403, pyro-Glu-Phe-Lys-p-nitroanilide; S-2444, pyro-Glu-Gly-Arg-p-nitroanilide; S-2586, methyl-succinyl-Arg-Pro-Tyr-p-nitroanilide; S-2765, benzyloxycarbonyl-d-Arg-Gly-Arg-p-nitroaniline; SPD, serine protease domain; SpectrozymeFVIIa, methansulfonyl-d-cyclohexylalanyl-butyl-Arg-p-nitroaniline; tPA, tissue-type plasminogen activator; VLK-AMC, H-d-Val-Leu-Lys-7-amido-4-methylcoumarin; WT, wild type; fVIIa, factor VIIa; fXa, factor Xa. which catalyzes the conversion of the zymogen plasminogen to the active protease plasmin (5.Barret A.J. Rawlings N.D. Woessner J.F. Handbook of Proteolytic Enzymes. Academic Press, London1998: 177-184Google Scholar). uPA has a catalytic serine protease domain with surface-exposed loops around residues 37, 60, 97, 110, 170, and 185 and an N-terminal extension consisting of a kringle domain and an epidermal growth factor domain, which binds to the cell surface-anchored uPA receptor (uPAR) (6.Danø K. Møller V. Ossowski L. Nielsen L.S. Biochim. Biophys. Acta. 1980; 613: 542-555Crossref PubMed Scopus (50) Google Scholar). uPA-catalyzed plasmin generation participates in the turnover of extracellular matrix proteins in physiological and pathophysiological tissue remodeling (for a review, see Ref. 7.Danø K. Andreasen P.A. Grøndahl-Hansen J. Kristensen P. Nielsen L.S. Skriver L. Adv. Cancer Res. 1985; 44: 139-266Crossref PubMed Scopus (2299) Google Scholar). Abnormal expression of uPA is responsible for tissue damage in several pathological conditions, including rheumatoid arthritis, allergic vasculitis, and xeroderma pigmentosum, and in particular, is a key factor for the invasive capacity of malignant tumors (for reviews, see Refs. 8.Andreasen P.A. Egelund R. Petersen H.H. Cell. Mol. Life Sci. 2000; 57: 25-40Crossref PubMed Scopus (836) Google Scholar and 9.Andreasen P.A. Kjøller L. Christensen L. Duffy M.J. Int. J. Cancer. 1997; 72: 1-22Crossref PubMed Scopus (1445) Google Scholar). As with other serine proteases, there has been extensive interest in generating specific inhibitors of uPA. The plasminogen activation activity of uPA can be inhibited specifically by polyclonal (10.Danø K. Nielsen L.S. Møller V. Engelhart M. Biochim. Biophys. Acta. 1980; 630: 146-151Crossref PubMed Scopus (47) Google Scholar) and monoclonal antibodies (11.Kaltoft K. Nielsen L.S. Zeuthen J. Danø K. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 3720-3723Crossref PubMed Scopus (38) Google Scholar). Several inhibitory monoclonal antibodies have epitopes encompassing the 37- and 60-loops (12.Petersen H.H. Hansen M. Schousboe S.L. Andreasen P.A. Eur. J. Biochem. 2001; 268: 4430-4439Crossref PubMed Scopus (49) Google Scholar). They preclude access of the activation loop of plasminogen to the active site of uPA but do not inhibit the uPA-catalyzed hydrolysis of low molecular weight substrates comprising three amino acids and a p-nitroaniline leaving group, which interacts only with the S1–S3 binding area. Another type of protein protease inhibitor is the dimeric nonspecific serine protease inhibitor ecotin, binding two serine protease molecules per ecotin dimer through interactions with both the protease active site and an exosite. With some success, ecotin has been converted into a high affinity uPA inhibitor by engineering each of the two interaction sites (13.Laboissière M.C. Young M.M. Pinho R.G. Todd S. Fletterick R.J. Kuntz I. Craik C.S. J. Biol. Chem. 2002; 277: 26623-26631Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar). Moreover, several classes of low molecular weight organochemical inhibitors of uPA have been synthesized. The binding modes for many of these were studied by x-ray crystal structure analyses. An important feature of such inhibitors is an Arg analogue inserting into the S1 pocket of uPA. However, the challenge has been to achieve selectivity for uPA over other serine proteases with P1 Arg specificity by exploiting small variations in the subsite geometry of the S1 pocket and its surroundings (14.Mackman R.L. Katz B.A. Breitenbucher J.G. Hui H.C. Verner E. Luong C. Liu L. Sprengeler P.A. J. Med. Chem. 2001; 44: 3856-3871Crossref PubMed Scopus (90) Google Scholar, 15.Sperl S. Jacob U. Arroyo de Prada N. Sturzebecher J. Wilhelm O.G. Bode W. Magdolen V. Huber R. Moroder L. Proc. Natl. Acad. Sci. U. S. 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To isolate uPA inhibitors with a potential for providing mechanistic information, with specificity comparable with that of monoclonal antibodies and protein protease inhibitors and a size eventually allowing for chemical synthesis and modification, we screened phage displayed peptide libraries with uPA as bait. We have isolated a dodecapeptide that binds both to the active site and surface loops of uPA and have shown that it is a highly specific inhibitor of the enzymatic activity of uPA. uPA—Two-chain uPA, originating from human urine, was purchased from Wakamoto Pharmaceutical Co., Tokyo, Japan. Protease concentrations were determined from the absorbance at 280 nm using an extinction coefficient of 1.36 ml mg-1 cm-1 and a Mr value of 54,000. Natural human pro-uPA was purified from serum-free conditioned medium of HT-1080 cells (20.Egelund R. Petersen T.E. Andreasen P.A. Eur. J. Biochem. 2001; 268: 673-685Crossref PubMed Scopus (53) Google Scholar). Wild-type (WT) and mutant recombinant human uPA were expressed in and purified from HEK293T cells (12.Petersen H.H. Hansen M. Schousboe S.L. Andreasen P.A. Eur. J. Biochem. 2001; 268: 4430-4439Crossref PubMed Scopus (49) Google Scholar). When the cells were cultured under standard conditions, ∼50% of the uPA in the conditioned medium and the purified preparations was in the pro-form and the rest in the active form. Conditioned medium with uPA exclusively in the uncleaved single-chain pro-form was produced by expressing the cleavage-resistant K158/15A uPA variant (we will refer to amino acid residues in uPA by a double numbering system based on numbering from the N terminus of the native protein and on the chymotrypsin template numbering system (1.Spraggon G. Phillips C. Nowak U.K. Ponting C.P. Saunders D. Dobson C.M. Stuart D.I. Jones E.Y. Structure. 1995; 3: 681-691Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar)). uPA was purified from conditioned media by immunoaffinity chromatography as described previously (20.Egelund R. Petersen T.E. Andreasen P.A. Eur. J. Biochem. 2001; 268: 673-685Crossref PubMed Scopus (53) Google Scholar). Low molecular weight uPA (N-terminally truncated at Lys136), the amino-terminal fragment of uPA (Ser1–Lys135), uPA·PAI-1 complex, low molecular weight uPA·PAI-1 complex, and uPA·protease nexin-1 (PN-1) complex were prepared as described previously (20.Egelund R. Petersen T.E. Andreasen P.A. Eur. J. Biochem. 2001; 268: 673-685Crossref PubMed Scopus (53) Google Scholar). Miscellaneous Proteases and Inhibitors—Human activated protein C (aPC) was a gift from Maxygen, Hørsholm, Denmark. Human fXa was a gift from Borean Pharma, Aarhus, Denmark. Human plasmin was purchased from American Diagnostica. Human plasma kallikrein was a gift from Dr. Inger Schousboe, University of Copenhagen, Denmark. Human thrombin was a gift from Dr. John Fenton, Albany, NY. Human fVIIa was purchased from Enzyme Research Laboratories. Human recombinant tPA (activase) was provided by Genentech Inc.; the single-chain form of this tPA preparation was converted to the fully active two-chain form via activation on immobilized plasmin (21.Kvassman J.O. Verhamme I. Shore J.D. Biochemistry. 1998; 37: 15491-15502Crossref PubMed Scopus (46) Google Scholar). Bovine β-trypsin was purified from a tosyl-Phe-chloromethylketone-treated commercial preparation (Roche Applied Science) by chromatography on soybean trypsin inhibitor-Sepharose (22.Olson S.T. Bock P.E. Kvassman J. Shore J.D. Lawrence D.A. Ginsburg D. Bjork I. J. Biol. Chem. 1995; 270: 30007-30017Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). The functional concentration of β-trypsin in the preparation was determined by active site titration using fluorescein mono-p-guanidinobenzoate as described previously (22.Olson S.T. Bock P.E. Kvassman J. Shore J.D. Lawrence D.A. Ginsburg D. Bjork I. J. Biol. Chem. 1995; 270: 30007-30017Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Preparation and purification of recombinant human wild-type PAI-1 in the pET24d vector (Merck Novagen, Nottingham, UK) has been described previously (23.Blouse G.E. Perron M.J. Thompson J.H. Day D.E. Link C.A. Shore J.D. Biochemistry. 2002; 41: 11997-12009Crossref PubMed Scopus (20) Google Scholar). Antibodies—The monoclonal anti-uPA antibodies mAb 2, mAb 5, mAb 6, mAb 12, mAb 16, American Diagnostica mAbs 390, 394, and 3689, and the monoclonal anti-PAI-1 antibody mAb 2 were those described previously (12.Petersen H.H. Hansen M. Schousboe S.L. Andreasen P.A. Eur. J. Biochem. 2001; 268: 4430-4439Crossref PubMed Scopus (49) Google Scholar, 24.Christensen L. Wiborg Simonsen A.C. Heegaard C.W. Moestrup S.K. Andersen J.A. Andreasen P.A. Int. J. Cancer. 1996; 66: 441-452Crossref PubMed Scopus (105) Google Scholar). A horseradish peroxidase (HRP)-conjugated monoclonal anti-M13 phage antibody was from Amersham Biosciences. Miscellaneous Materials for DNA Technology—Unless stated otherwise, enzymes used for DNA technology were from New England Biolabs. Oligonucleotides were obtained from DNA Technology or MWG Biotech. In all of the DNA constructs described, the region coding for the recombinant protein was validated by sequencing with the DYEnamic ET Terminator Cycle Sequencing Kit (Amersham Biosciences) and an ABI PRISM 3100 genetic analyzer (Applied Biosystems). Miscellaneous Reagents—S-2222 (benzyl-IEGR-p-nitroaniline), S-2238 (H-d-Pro-piperidine-Arg-p-nitroaniline), S-2288 (H-d-IPR-p-nitroaniline), S-2302 (H-d-PFR-p-nitroaniline), S-2366 (pyro-EPR-p-nitroaniline), S-2403 (pyro-EFK-p-nitroaniline), S-2444 (pyro-EGR-p-nitroaniline), S-2586 (methyl-succinyl-RPY-p-nitroaniline), and S-2765 (benzyloxycarbonyl-d-RGR-p-nitroaniline) were from Chromogenix, Mölndal, Sweden. H-d-VLK-7-amido-4-methylcoumarin (VLK-AMC) was from Bachem, Bubendorf, Switzerland. SpectrozymeFVIIa (methansulfonyl-d-cyclohexylalanyl-butyl-Arg-p-nitroaniline) was from American Diagnostica, Greenwich, CT. The synthetic peptides CSWRGLENHRMC, with a disulfide bridge between the two Cys residues, and SSWRGLENHRMS were purchased from Biopeptides, San Diego, CA. Bovine pancreatic trypsin inhibitor was purchased from Sigma. Screening of a Phage-displayed Peptide Library for uPA Binding Peptides—Monoclonal antibodies (5 μg/ml) against uPA (mAb 6, mAb 12, or mAb 390) were immobilized in MaxiSorp Nunc-Immuno Tubes (Nunc, Roskilde, Denmark) overnight at 4 °C. Nonspecific binding was blocked by incubation with phosphate-buffered saline (PBS: 10 mm sodium phosphate, pH 7.4, 145 mm NaCl) containing 5% skim milk powder. Fifty nm uPA was then incubated for 1 h at room temperature in blocking buffer with 1011 colony-forming units from each of the four phage-displayed peptide libraries, in the formats X7, CX7C, CX10C, and CX3CX3CX3C, obtained from Dr. Erkki Koivunen, University of Helsinki, Finland (25.Koivunen E. Wang B. Dickinson C.D. Ruoslahti E. Methods Enzymol. 1994; 245: 346-369Crossref PubMed Scopus (60) Google Scholar). Subsequent to 10 washes with PBS, the bound phage particles were eluted by adding 1 ml of 100 mm glycine adjusted to pH 2.2 with HCl and incubation for 10 min at room temperature. The eluted phage particles were neutralized by addition of 400 μl 0.1 m Tris, pH 9.0, and propagated in E. coli TG-1 cells overnight. After removal of bacterial cells by centrifugation, the secreted phage particles were precipitated by addition of 0.25 volumes of 2.5 m NaCl, 20% polyethylene glycol 6000, followed by incubation for 1 h at 0 °C and centrifugation. The resulting phage pellet was dissolved in PBS with 10% glycerol. Four successive rounds of selection were performed. Alternating antibodies were used for subsequent rounds of selection in order to avoid enrichment of antibody-binding phages. Expression of the Upain-1 Peptide Sequence and Derivatives thereof, Fused to the N-terminal Domains of the Phage Coat Protein g3p—A DNA fragment encoding the two SfiI cloning sites plus the first two domains of the phage coat protein g3p (D1 and D2, residues 1–219) was amplified from the phage fUSE5 (26.Scott J.K. Smith G.P. Science. 1990; 249: 386-390Crossref PubMed Scopus (1892) Google Scholar) with the PCR primers fUSEfwd (5′-CATGCCATGGGCTCGGCCGACGGGGC-3′) and fUSEbck2 (5′-GTACCTCGAGGCCGCCAGCATTGACAGG-3′) using the PfuTurbo DNA polymerase (Stratagene, La Jolla, CA, USA) according to the manufacturer's instructions. The generated PCR product was purified with the Qiaquick PCR Purification kit (Qiagen, Hilden, Germany) and ligated into the E. coli expression vector pET20b(+) (Merck Novagen, Nottingham, UK) via NcoI and XhoI restriction sites. The resulting vector is referred to as pETD1D2. Using the same approach, but with the upain-1 phage as template for the PCR reaction, a vector was created for expression of the upain-1 peptide sequence fused to D1D2 of g3p (MGSADGACSWRGLENHRMCGAAG-g3p1–219-LEHHHHHH, the upain-1 sequence is underlined). This fusion protein will be referred to as upain-1-D1D2. Expression vectors for derivatives of upain-1 fused to D1D2 were created by ligating suitable oligonucleotide cassettes into SfiI-digested pETD1D2. Likewise, a vector for expression of the peptide-flanking sequences fused to D1D2 (MGSADGAGAAG-g3p1–219-LEHHHHHH) was constructed. Individual fusion proteins were expressed from cultures of E. coli BL21(DE3)pLysS (Merck Novagen, Nottingham, UK) containing the relevant plasmids and purified by immobilized metal affinity chromatography as previously described for His6-tagged PAI-1 (27.Wind T. Jensen J.K. Dupont D.M. Kulig P. Andreasen P.A. Eur. J. Biochem. 2003; 270: 1680-1688Crossref PubMed Scopus (31) Google Scholar,28.Jensen J.K. Wind T. Andreasen P.A. FEBS Lett. 2002; 521: 91-94Crossref PubMed Scopus (54) Google Scholar), subjected to size exclusion chromatography on Superdex 75 (Amersham Biosciences) equilibrated in HEPES-buffered saline (HBS, 10 mm Hepes, pH 7.4, 150 mm NaCl), and finally concentrated with Centricon Centrifugal Filter Devices (Millipore Corp., Glostrup, Denmark). Expression of the Upain-1 Peptide Sequence Fused to the Coiled-coil Domain of Tetranectin—The upain-1 sequence was produced recombinantly in fusion with the trimerizing coiled-coil domain of tetranectin (EPPTQKPKKIVNAKKDVVNTKMFEELKSRLDTLAQEVALLKEQQALQTVGS) (29.Holtet T.L. Graversen J.H. Clemmensen I. Thøgersen H.C. Etzerodt M. Protein Sci. 1997; 6: 1511-1515Crossref PubMed Scopus (43) Google Scholar). The construct was expressed and purified as follows. Six liters of E. coli BL21 cells were grown to OD600 0.8 before induction of protein expression by addition of bacteriophage λCE6 at a multiplicity of infection of ∼5. Bacterial cultures were incubated for 4 h before harvesting the cells by centrifugation at 5000 rpm for 10 min. The bacterial pellet was resuspended in 100 ml of a buffer of 100 mm Tris-HCl, pH 8.0, 500 mm NaCl, 2 mm EDTA, 50 mm dithiothreitol. Phenol (150 ml), adjusted to pH 8.0, was added and the mixture sonicated to extract all protein. The mixture was centrifuged at 8,500 rpm for 30 min to isolate the phenol phase. Protein was precipitated from the phenol phase by addition of 2.5 volumes of ethanol and harvested by centrifugation at 5000 rpm for 10 min. Following resuspension in 6 m guanidinium chloride, 50 mm Tris-HCl, pH 8.0, the protein solution was gel filtered on a Sephadex G-25 column into 8 m urea, 500 mm NaCl, 50 mm Tris-HCl, 5 mm β-mercaptoethanol, pH 8.0, and loaded on a Ni2+-charged nitrilotriacetic acid column. The expressed protein was subjected to a patented cyclic in vitro refolding procedure (30.Thøgersen, H. C., Etzerodt, M., and Holtet, T. L. (August 18, 1994) International Patent WO94/18227, World Intellectual Property Organization PCT/DK94/00054Google Scholar). This construct will be referred to as upain-1-tetranectin. ELISA for Measuring uPA-Phage Particle Binding—Unless otherwise stated, the buffer used was HBS supplemented with 0.2% BSA. The concentration of uPA variants in conditioned medium was determined by a quantitative sandwich ELISA using the monoclonal antibody mAb 6 for capture and a polyclonal rabbit anti-uPA antibody for detection (12.Petersen H.H. Hansen M. Schousboe S.L. Andreasen P.A. Eur. J. Biochem. 2001; 268: 4430-4439Crossref PubMed Scopus (49) Google Scholar). Activation of pro-forms of uPA variants was achieved by incubation at 37 °C for 16 h at pH 7.4 in the presence of 2 μg/ml plasmin followed by addition of 1 μg/ml bovine pancreatic trypsin inhibitor. For the ELISAs, the antibody to be immobilized on the solid phase (5μg/ml in 100 mm NaHCO3/Na2CO3, pH 9.6) was coated in the wells of a 96-well Maxisorp plate (Nunc) followed by blocking with 5% BSA in HBS. The wells were incubated with 10 nm uPA for 1 h. Excess uPA was removed by washing before incubation with phage particles (∼109 colony-forming units/ml) for 1 h. For competition studies, up to 15 μm peptide-D1D2 fusion proteins were added along with the phage particles. The wells were washed and incubated for 1 h with a 5,000-fold dilution of HRP-conjugated anti-M13 monoclonal antibody directed against the major phage coat protein g8p (Amersham Biosciences). After a final wash, the wells were developed by the addition of 0.5 mg/ml ortho-phenylenediamine (100 μl) (KemEnTech, Denmark) in 50 mm citric acid, pH 5.0, supplemented with 0.03% H2O2. When suitable color had developed, the reactions were quenched with 50 μl 1 m H2SO4. The A492 of the wells was read in a microplate reader. When testing the binding of upain-1 phage to different uPA variants, the presence of equal amounts of the uPA variants on the solid phase was assured by a parallel ELISA with 1 μg/ml polyclonal rabbit anti-uPA antibody instead of phage particles and with HRP-conjugated swine-anti-rabbit serum (DAKO, Glostrup, Denmark, diluted 2,000-fold) instead of anti-M13 antibody. Determination of the Mode of Inhibition of uPA by Upain-1—Four nm uPA was incubated with various concentrations of upain-1 peptide (0–400 μm) or upain-1-D1D2 (0–20 μm) in HBS with 0.1% BSA at 37 °C for 15 min prior to the addition of the chromogenic substrate, S-2444. Each inhibitor concentration was combined with a series of S-2444 concentrations in the range 10–2000 μm. The initial reaction velocity was monitored at an absorbance of 405 nm. The following equation is expected to apply for competitive inhibition according to Michaelis-Menten kinetics, V=(Vmax[S]0)/([S0]+Km(1+[I]0/Ki))eq 1 where [S]0 and [I]0 are the total substrate and inhibitor concentrations, respectively; Ki is the inhibition constant; Km is the Michaelis constant for S-2444 under the assay conditions. In Equation 1, one can define a Kmapp as Kapp=Km(1+[I]0/Ki)eq 2 The Kmapp values for S-2444 hydrolysis by uPA were determined by standard Michaelis-Menten kinetics at each inhibitor concentration by a nonlinear fit to the equation V=(Vmax[S]0/)([S]0+Kapp)eq 3 In the case of competitive inhibition, Kmapp, according to Equation 2, is expected to have a linear relationship to [I]0, whereas Vmax will be independent of [I]0. The Ki values could thus be estimated as the slope of the line relating Kmapp to [I]0. Determination of the Inhibition Constants (Ki) for the Inhibition of uPA and uPA Variants by Upain-1 Peptide and Upain-1-D1D2—For routine determination of inhibition constants (Ki) for the inhibition of purified uPA under equilibrium inhibition conditions, a fixed concentration of uPA (4.0 nm) was preincubated in a 200-μl volume of HBS with 0.1% BSA at 37 °C, at pH values of 6.0, 7.4, and 8.1, with various concentrations of upain-1 peptide (0–400 μm) or upain-1-D1D2 (0–20 μm) for 15 min prior to the addition of the chromogenic substrate, S-2444 (46.9 μm). The initial reaction velocities were monitored at an absorbance of 405 nm. Apparent equilibrium inhibition constants (Kiapp) were subsequently determined from the nonlinear regression analyses of plots for Vi/V0 versus [I]0 using Equation 4 (31.Knight C.G. Barret A.J. Salvesen G. Proteinase Inhibitors. Elsevier, Amsterdam1986: 23-51Google Scholar), vi/v0=1-([E]0+[I]0+Kiapp)-([E]0+[I]0+Kiapp)2-4[E]0[I]02[E]0eq 4 where Vi and V0 are the reaction velocities in the presence and absence of inhibitor, respectively, and [E]0 and [I]0 are the total protease and inhibitor concentrations, respectively. Kiapp represents the apparent inhibition constant in the presence of chromogenic substrate. The true Ki was subsequently determined by correcting for the competitive effect of the substrate, S, using the relationship, Ki=Kiapp/(1+[S]0/Km)eq 5 Experimental determinations of the Ki values for inhibition of the uPA variants by upain-1-D1D2 were accomplished essentially as described above except for the following modifications: 20 μl of conditioned HEK293T cell medium was used for each uPA variant instead of the purified protease, the tested upain-1 concentrations ranged from 0 to 150 μm, and the concentration of S-2444 used was increased to 100 μm. In these experiments, the exact concentration of uPA was not readily known, and therefore these data were analyzed by the simplified expression of Equation 6 (31.Knight C.G. Barret A.J. Salvesen G. Proteinase Inhibitors. Elsevier, Amsterdam1986: 23-51Google Scholar), Vi/V0=1/(1+[I]0/Kiapp)eq 6 where Vi and V0 are the reaction velocities in the presence and absence of inhibitor, respectively, [I]0 is the total inhibitor concentration, and Kiapp is the apparent inhibition constant with the final value of Ki being d
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