Evidence for a Pre-latent Form of the Serpin Plasminogen Activator Inhibitor-1 with a Detached β-Strand 1C
2006; Elsevier BV; Volume: 281; Issue: 47 Linguagem: Inglês
10.1074/jbc.m606851200
ISSN1083-351X
AutoresDaniel M. Dupont, Grant E. Blouse, Martin Duus Hansen, Lisa Mathiasen, Signe Kjelgaard, Jan K. Jensen, Anni Christensen, Ann Gils, Paul Declerck, Peter A. Andreasen, Troels Wind,
Tópico(s)Calpain Protease Function and Regulation
ResumoLatency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in β-sheet A and detachment of β-strand 1C (s1C) at the C-terminal hinge of the RCL. This is one of the largest structural rearrangements known for a folded protein domain without a concomitant change in covalent structure. Yet, the sequence of conformational changes during latency transition remains largely unknown. We have now mapped the epitope for the monoclonal antibody H4B3 to the cleft revealed upon s1C detachment and shown that H4B3 inactivates recombinant PAI-1 in a time-dependent manner. With fluorescence spectroscopy, we show that insertion of the RCL is accelerated in the presence of H4B3, demonstrating that the loss of activity is the result of latency transition. Considering that the epitope for H4B3 appears to be occluded by s1C in active PAI-1, this finding suggests the existence of a pre-latent conformation on the path from active to latent PAI-1 characterized by at least partial detachment of s1C. Functional characterization of mutated PAI-1 variants suggests that a salt-bridge between Arg273 and Asp224 may stabilize the pre-latent conformation. The binding of H4B3 and of a peptide targeting the cleft revealed upon s1C detachment was hindered by the glycans attached to Asn267. Conclusively, we have provided evidence for the existence of an equilibrium between active PAI-1 and a pre-latent form, characterized by reversible detachment of s1C and formation of a glycan-shielded cleft in the molecule. Latency transition of plasminogen activator inhibitor-1 (PAI-1) occurs spontaneously in the absence of proteases and results in stabilization of the molecule through insertion of its reactive center loop (RCL) as a strand in β-sheet A and detachment of β-strand 1C (s1C) at the C-terminal hinge of the RCL. This is one of the largest structural rearrangements known for a folded protein domain without a concomitant change in covalent structure. Yet, the sequence of conformational changes during latency transition remains largely unknown. We have now mapped the epitope for the monoclonal antibody H4B3 to the cleft revealed upon s1C detachment and shown that H4B3 inactivates recombinant PAI-1 in a time-dependent manner. With fluorescence spectroscopy, we show that insertion of the RCL is accelerated in the presence of H4B3, demonstrating that the loss of activity is the result of latency transition. Considering that the epitope for H4B3 appears to be occluded by s1C in active PAI-1, this finding suggests the existence of a pre-latent conformation on the path from active to latent PAI-1 characterized by at least partial detachment of s1C. Functional characterization of mutated PAI-1 variants suggests that a salt-bridge between Arg273 and Asp224 may stabilize the pre-latent conformation. The binding of H4B3 and of a peptide targeting the cleft revealed upon s1C detachment was hindered by the glycans attached to Asn267. Conclusively, we have provided evidence for the existence of an equilibrium between active PAI-1 and a pre-latent form, characterized by reversible detachment of s1C and formation of a glycan-shielded cleft in the molecule. The serpins constitute a family of proteins of which the majority are inhibitors of serine proteases (1Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (512) Google Scholar). Of decisive importance for the inhibitory mechanism is the surface-exposed reactive center loop (RCL) 5The abbreviations used are: RCL, reactive center loop; HBS, Hepes-buffered saline; HRP, horseradish peroxidase; LMW-uPA, low molecular weight uPA; PAI-1, plasminogen activator inhibitor-1; s, strand in a β-sheet; uPA, urokinase-type plasminogen activator; mAb, monoclonal antibody; ELISA, enzyme-linked immunosorbent assay; Ni-NTA, nickel-nitrilotriacetic acid. between β-strands s5A and s1C (Fig. 1). The scissile P1-P1′ bond in the RCL is attacked by the serine protease, but at the enzyme-acyl intermediate stage, where the active site serine of the protease and the P1 residue of the serpin are linked by an ester bond, the N-terminal part of the RCL inserts as s4A thereby pulling the protease to the opposite pole of the serpin and distorting its active site. The required energy stems from stabilization of the serpin in the "relaxed" conformation with an inserted RCL, as compared with the "stressed," active conformation with a surface-exposed RCL. The conformational changes associated with the insertion of s4A in serpins are well described by x-ray crystal structure analyses and include rearrangements in a region around s1A, s2A, hD, and hE referred to as the flexible joint region (reviewed in Ref. 2Gettins P.G. Chem. Rev. 2002; 102: 4751-4804Crossref PubMed Scopus (1003) Google Scholar). Plasminogen activator inhibitor-1 (PAI-1) and antithrombin III are unique among serpins as they can spontaneously adopt the relaxed conformation without prior cleavage of the RCL. This is referred to as latency transition and involves insertion of the N-terminal part of the intact RCL as s4A while the C-terminal part and the connected s1C is stretched out on the surface of the molecule (3Mottonen J. Strand A. Symersky J. Sweet R.M. Danley D.E. Geoghegan K.F. Gerard R.D. Goldsmith E.J. Nature. 1992; 355: 270-273Crossref PubMed Scopus (530) Google Scholar, 4Carrell R.W. Huntington J.A. Mushunje A. Zhou A. Thromb. Haemostasis. 2001; 86: 14-22Crossref PubMed Scopus (47) Google Scholar). Thus, during latency transition, full insertion of s4A requires the detachment of s1C and passage of the RCL through the gate region between the s3C/s4C and s3B/hG loops (Fig. 1). For PAI-1, latency transition is accelerated by the monoclonal antibody 33B8, which recognizes an epitope overlapping with part of the flexible joint region and binds preferentially to relaxed forms of PAI-1 (5Gorlatova N.V. Elokdah H. Fan K. Crandall D.L. Lawrence D.A. J. Biol. Chem. 2003; 278: 16329-16335Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Naessens D. Gils A. Compernolle G. Declerck P.J. Thromb. Haemostasis. 2003; 90: 52-58Crossref PubMed Scopus (26) Google Scholar, 7Verhamme I. Kvassman J.O. Day D. Debrock S. Vleugels N. Declerck P.J. Shore J.D. J. Biol. Chem. 1999; 274: 17511-17517Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Because latency transition is irreversible, the effect of 33B8 cannot be explained by stabilization of the latent conformation. It was therefore hypothesized that 33B8 accelerates latency transition by binding to and stabilizing an intermediate, pre-latent conformation existing on the path from active to latent PAI-1 (5Gorlatova N.V. Elokdah H. Fan K. Crandall D.L. Lawrence D.A. J. Biol. Chem. 2003; 278: 16329-16335Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 7Verhamme I. Kvassman J.O. Day D. Debrock S. Vleugels N. Declerck P.J. Shore J.D. J. Biol. Chem. 1999; 274: 17511-17517Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). This pre-latent form would exist in an unfavorable equilibrium with the active form and be characterized by sufficient insertion of N-terminal residues from the RCL at the top of β-sheet A for the high affinity 33B8 epitope to form (5Gorlatova N.V. Elokdah H. Fan K. Crandall D.L. Lawrence D.A. J. Biol. Chem. 2003; 278: 16329-16335Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Naessens D. Gils A. Compernolle G. Declerck P.J. Thromb. Haemostasis. 2003; 90: 52-58Crossref PubMed Scopus (26) Google Scholar, 7Verhamme I. Kvassman J.O. Day D. Debrock S. Vleugels N. Declerck P.J. Shore J.D. J. Biol. Chem. 1999; 274: 17511-17517Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The positioning of s1C at the C terminus of the RCL in the putative pre-latent PAI-1, however, remains elusive, although it has been suggested to be distorted by the strain imposed by inserting part of the RCL (8Hägglöf P. Bergström F. Wilczynska M. Johansson L.B. Ny T. J. Mol. Biol. 2004; 335: 823-832Crossref PubMed Scopus (36) Google Scholar, 9Stoop A.A. Eldering E. Dafforn T.R. Read R.J. Pannekoek H. J. Mol. Biol. 2001; 305: 773-783Crossref PubMed Scopus (38) Google Scholar). Here, the time-dependent loss of PAI-1 activity was found to be accelerated by the monoclonal antibody H4B3. To elucidate the underlying mechanism we characterized its effect in detail by fluorescence spectroscopy and site-directed mutagenesis. Binding of H4B3 was found to induce the conversion of PAI-1 to a form in which the RCL was inserted, consistent with an antibody-mediated acceleration of latency transition similar to what has been described for 33B8. The access of H4B3 to its binding area was demonstrated to require at least partial detachment of s1C, suggesting that this is a structural feature of pre-latent PAI-1. To obtain further information about the course of events during latency transition, we have isolated a peptide from phage displayed libraries with affinity exclusively for the latent conformation of human recombinant PAI-1. The binding sites for this peptide and for the monoclonal antibody MAI-12 were found to overlap with the epitope for H4B3. Considering that neither the peptide nor MAI-12 accelerated latency transition a map of their binding sites allowed us to clarify the features of H4B3 binding that govern its PAI-1-neutralizing effect. We also performed a functional analysis of the generated panel of PAI-1 variants with substitutions in the mapped binding regions and thereby identified a new region important for the functional stability of PAI-1. Finally, the binding profiles of the isolated peptide and antibodies to glycosylated and non-glycosylated PAI-1 in both stressed and relaxed conformations provide information about the orientation of the N-linked glycosylation on Asn267 in latent PAI-1. PAI-1—We will refer to amino acid residues in PAI-1 by the numbering system of Andreasen et al. (10Andreasen P.A. Riccio A. Welinder K.G. Douglas R. Sartorio R. Nielsen L.S. Oppenheimer C. Blasi F. Danø K. FEBS Lett. 1986; 209: 213-218Crossref PubMed Scopus (170) Google Scholar) starting at Ser1-Ala2-Val3. Recombinant PAI-1(W177F) was prepared from Escherichia coli expression cultures as described previously (11Blouse G.E. Perron M.J. Kvassman J.O. Yunus S. Thompson J.H. Betts R.L. Lutter L.C. Shore J.D. Biochemistry. 2003; 42: 12260-12272Crossref PubMed Scopus (35) Google Scholar). Other recombinant PAI-1 variants were expressed with an N-terminal His6 tag and purified from E. coli expression cultures as described previously (12Wind T. Jensen J.K. Dupont D.M. Kulig P. Andreasen P.A. Eur. J. Biochem. 2003; 270: 1680-1688Crossref PubMed Scopus (31) Google Scholar), except that expression cultures were incubated at 30 °C to minimize latency transition of the produced PAI-1. The cDNA for murine PAI-1 was PCR amplified and inserted into the expression vector pT7-PL as described for the human PAI-1 cDNA (12Wind T. Jensen J.K. Dupont D.M. Kulig P. Andreasen P.A. Eur. J. Biochem. 2003; 270: 1680-1688Crossref PubMed Scopus (31) Google Scholar). The DNA coding for a PAI-1 murine/human chimera consisting of the murine sequence from the N terminus to residue 122, the conserved human and murine sequence between residues 123 and 142, and the human sequence from residue 143 to the C terminus was constructed using overlap extension PCR (13Horton R.M. Hunt H.D. Ho S.N. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 61-68Crossref PubMed Scopus (2648) Google Scholar). The inverse human/murine chimera was also constructed. The stable, active PAI-114-1B variant carries the four amino acid substitutions described by Berkenpas et al. (14Berkenpas M.B. Lawrence D.A. Ginsburg D. EMBO J. 1995; 14: 2969-2977Crossref PubMed Google Scholar). PAI-1 cleaved at the P4-P3 peptide bond was prepared by incubating 425 μg/ml PAI-1 with 12.5 μg/ml elastase (Roche) in HBS (10 mm Hepes, 140 mm NaCl, pH 7.4). Elastase activity was irreversibly inhibited after 5 min at 37 °C by adding 4 mm Pefablock SC (Roche). The same conditions were used to prepare the uPA·PAI-1 complex, except that the concentration of PAI-1 was reduced to 225 μg/ml and elastase was replaced with 500 μg/ml uPA (Wakamoto Pharmaceutical Co., Japan). Natural glycosylated human PAI-1 was purified by immunoaffinity chromatography from serum-free conditioned medium of dexamethasone-treated HT-1080 cells (15Munch M. Heegaard C.W. Andreasen P.A. Biochim. Biophys. Acta. 1993; 1202: 29-37Crossref PubMed Scopus (97) Google Scholar). Recombinant glycosylated human PAI-1, PAI-1(N211Q), and PAI-1(N267Q) were expressed by transiently transfected HEK293T cells and purified from the conditioned medium by immunoaffinity chromatography (16Gils A. Pedersen K.E. Skottrup P. Christensen A. Naessens D. Deinum J. Enghild J.J. Declerck P.J. Andreasen P.A. Thromb. Haemostasis. 2003; 90: 206-217Crossref PubMed Google Scholar). Latent PAI-1 from HT-1080 and HEK293T cells was activated by denaturation with 4 m guanidinium chloride and dialysis against HBS (15Munch M. Heegaard C.W. Andreasen P.A. Biochim. Biophys. Acta. 1993; 1202: 29-37Crossref PubMed Scopus (97) Google Scholar). Antibodies—The monoclonal anti-PAI-1 antibodies were described previously as follows: mAb-1 (17Nielsen L.S. Andreasen P.A. Grøndahl-Hansen J. Huang J.Y. Kristensen P. Danø K. Thromb. Haemostasis. 1986; 55: 206-212Crossref PubMed Scopus (94) Google Scholar, 18Bødker J.S. Wind T. Jensen J.K. Hansen M. Pedersen K.E. Andreasen P.A. Eur. J. Biochem. 2003; 270: 1672-1679Crossref PubMed Scopus (15) Google Scholar), mAb-2 (17Nielsen L.S. Andreasen P.A. Grøndahl-Hansen J. Huang J.Y. Kristensen P. Danø K. Thromb. Haemostasis. 1986; 55: 206-212Crossref PubMed Scopus (94) Google Scholar, 19Wind T. Jensen M.A. Andreasen P.A. Eur. J. Biochem. 2001; 268: 1095-1106Crossref PubMed Scopus (52) Google Scholar), mAb-5 (20Stoop A.A. Jespers L. Lasters I. Eldering E. Pannekoek H. J. Mol. Biol. 2000; 301: 1135-1147Crossref PubMed Scopus (51) Google Scholar, 21Munch M. Heegaard C. Jensen P.H. Andreasen P.A. FEBS Lett. 1991; 295: 102-106Crossref PubMed Scopus (52) Google Scholar), mAb-6 (19Wind T. Jensen M.A. Andreasen P.A. Eur. J. Biochem. 2001; 268: 1095-1106Crossref PubMed Scopus (52) Google Scholar), mAb-7 (16Gils A. Pedersen K.E. Skottrup P. Christensen A. Naessens D. Deinum J. Enghild J.J. Declerck P.J. Andreasen P.A. Thromb. Haemostasis. 2003; 90: 206-217Crossref PubMed Google Scholar), 33B8 (5Gorlatova N.V. Elokdah H. Fan K. Crandall D.L. Lawrence D.A. J. Biol. Chem. 2003; 278: 16329-16335Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 6Naessens D. Gils A. Compernolle G. Declerck P.J. Thromb. Haemostasis. 2003; 90: 52-58Crossref PubMed Scopus (26) Google Scholar, 7Verhamme I. Kvassman J.O. Day D. Debrock S. Vleugels N. Declerck P.J. Shore J.D. J. Biol. Chem. 1999; 274: 17511-17517Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), H4B3 (16Gils A. Pedersen K.E. Skottrup P. Christensen A. Naessens D. Deinum J. Enghild J.J. Declerck P.J. Andreasen P.A. Thromb. Haemostasis. 2003; 90: 206-217Crossref PubMed Google Scholar, 22Declerck P.J. Verstreken M. Collen D. Thromb. Haemostasis. 1995; 74: 1305-1309Crossref PubMed Scopus (105) Google Scholar), and MAI-12 (also known as MA7D4B7) (16Gils A. Pedersen K.E. Skottrup P. Christensen A. Naessens D. Deinum J. Enghild J.J. Declerck P.J. Andreasen P.A. Thromb. Haemostasis. 2003; 90: 206-217Crossref PubMed Google Scholar, 23Björquist P. Ehnebom J. Deinum J. Biochim. Biophys. Acta. 1999; 1431: 24-29Crossref PubMed Scopus (7) Google Scholar, 24Björquist P. Ehnebom J. Inghardt T. Deinum J. Biochim. Biophys. Acta. 1997; 1341: 87-98Crossref PubMed Scopus (26) Google Scholar, 25Keijer J. Linders M. van Zonneveld A.J. Ehrlich H.J. de Boer J.P. Pannekoek H. Blood. 1991; 78: 401-409Crossref PubMed Google Scholar, 26Declerck P.J. Alessi M.C. Verstreken M. Kruithof E.K. Juhan-Vague I. Collen D. Blood. 1988; 71: 220-225Crossref PubMed Google Scholar). Rabbit polyclonal anti-PAI-1 antibodies were those described (27Offersen B.V. Nielsen B.S. Høyer-Hansen G. Rank F. Hamilton-Dutoit S. Overgaard J. Andreasen P.A. Am. J. Pathol. 2003; 163: 1887-1899Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). A horseradish peroxidase (HRP)-conjugated monoclonal anti-M13 phage antibody was from Amersham Biosciences. Measurements of the Specific Inhibitory Activity of PAI-1—To measure the specific inhibitory activity of PAI-1, i.e. the fraction of PAI-1 capable of inhibiting the proteolytic activity of uPA, PAI-1 was serially diluted in HBS supplemented with 0.25% gelatin (HBS-G), resulting in PAI-1 concentrations between 0.01 and 20 μg/ml (0.22 nm and 0.45 μm). One volume of 0.25 μg/ml (4.6 nm) uPA in HBS-G was added to each well, followed by incubation for at least 5 min. The remaining uPA activity was determined by adding the chromogenic substrate S-2444 (pyro-Glu-Gly-Arg-p-nitroanilide, Chromogenix, Sweden) for 30 min and measuring the absorbance at 405 nm. The specific inhibitory activity of PAI-1 was calculated from the amount of PAI-1 that inhibited 50% of the uPA. Determining the Rate of PAI-1 Latency Transition—PAI-1 was incubated at 37 °C in HBS-G at a concentration of 20 μg/ml. At various time points, the specific inhibitory activity of PAI-1 was determined as described above and the half-life for latency transition was calculated from semilogarithmic plots of the specific inhibitory activity versus time. The effect of H4B3 and paionin-3 on latency transition was assessed in a similar assay with the following modifications. The temperature was lowered to 23 °C to reduce the rate of latency transition; the concentration of PAI-1 was 5 μg/ml (112 nm); H4B3 (200 nm)or paionin-3 (10 μm) were present during the incubation; and the concentration of uPA was 0.1 μg/ml (1.9 nm). Fluorescence Labeling of Recombinant PAI-1—Labeling of the P9-Cys in PAI-1(S340C) with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (Molecular Probes) and the P9-Cys in PAI-1(W177F/S340C) with N,N′dimethyl-N-(acetyl)-N′-methyl(7-nitrobenz-2-oxa-1,3-diazol-4-yl)ethylene diamine (Molecular Probes) created the fluorescent molecules PAI-1P9-NBD and PAI-1(W177F)P9-NBD and was carried out essentially as described (11Blouse G.E. Perron M.J. Kvassman J.O. Yunus S. Thompson J.H. Betts R.L. Lutter L.C. Shore J.D. Biochemistry. 2003; 42: 12260-12272Crossref PubMed Scopus (35) Google Scholar). The labeling efficiency was ∼0.8-1.0 mol of probe/mol of PAI-1. Incorporation of the fluorescent probes at this position on PAI-1 was shown previously to have no adverse effects on PAI-1 activity for either variant (11Blouse G.E. Perron M.J. Kvassman J.O. Yunus S. Thompson J.H. Betts R.L. Lutter L.C. Shore J.D. Biochemistry. 2003; 42: 12260-12272Crossref PubMed Scopus (35) Google Scholar, 29Shore J.D. Day D.E. Francis-Chmura A.M. Verhamme I. Kvassman J. Lawrence D.A. Ginsburg D. J. Biol. Chem. 1995; 270: 5395-5398Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Latent PAI-1, which typically accumulated during the labeling reaction, was subsequently removed by affinity chromatography on immobilized β-anhydrotrypsin as described (11Blouse G.E. Perron M.J. Kvassman J.O. Yunus S. Thompson J.H. Betts R.L. Lutter L.C. Shore J.D. Biochemistry. 2003; 42: 12260-12272Crossref PubMed Scopus (35) Google Scholar). Latent PAI-1P9-NBD was prepared by incubation of a 0.5 μm concentration at 37 °C for 24 h in a buffer containing 30 mm Hepes, 135 mm NaCl, 1 mm EDTA, 0.1% PEG 8000, pH 7.4, with the addition of 0.02% NaN3. Complete conversion to the latent state was verified by the absence of complex formation when the latent preparation was reacted with a molar excess of uPA and analyzed by SDS-PAGE. Fluorescence Emission Spectroscopy—Emission spectra of the binding interactions between labeled PAI-1 variants and H4B3 were carried out in a SPEX-3 spectrofluorimeter equipped with a Peltier temperature controller maintaining the measurement and incubation temperatures at 25 °C. Fluorescence experiments were carried out using semi-micro (0.2 × 1.0-cm) quartz cuvettes and in a reaction buffer containing 30 mm Hepes, 135 mm NaCl, 1 mm EDTA, 0.1% PEG 8000, pH 7.4. The excitation wavelength used for studying the fluorescence of PAI-1P9-NBD was 480 nm, and the emission spectra were scanned from 500 to 650 nm using a bandwidth of 5 nm for both the excitation and emission beams. Emission spectra for PAI-1P9-NBD (50 nm) were recorded prior to and after the addition of H4B3 (500 nm) and a 40-60-min incubation at 25 °C. Results are presented as the averaged spectra of three to nine independent acquisitions. All individual emission spectra were collected as averages of three emission scans using a 0.5-s integration over a 1.0-nm step resolution and corrected for background fluorescence in the absence of PAI-1P9-NBD and dilution effects, which were typically less than 2%. Spectra of latent PAI-1P9-NBD (50 nm) were collected as described above, whereas reactions of PAI-1P9-NBD (50 nm) with uPA (0.5 μm) were recorded after a 10-min incubation with the protease. Kinetic experiments following the time-dependent fluorescence change in NBD fluorescence were carried out with PAI-1P9-NBD (100 nm) or PAI-1(W177F)P9-NBD (100 nm) and H4B3 (500 nm). Single emission spectra were collected rapidly to limit acquisition time by using modified excitation and emission bandwidths of 10 and 5 nm, respectively, while maintaining the 0.5-s integration over a 1.0-nm step resolution. Integrated fluorescence data were subsequently normalized as ΔFobs/ΔFmax, where ΔFobs is the observed integrated fluorescence change at each indicated time point and ΔFmax represents the total change in fluorescence following reaction with a 2-fold molar excess of uPA. Integrated fluorescence results are presented as the average of two independent experiments. H4B3 Binding to PAI-1 Captured on Vitronectin—Maxisorp wells were coated with vitronectin (0.5 μg/ml in 100 mm NaHCO3/Na2CO3, pH 9.6) and blocked with HBS supplemented with 1% bovine serum albumin (HBS-B). Active PAI-1, purified on immobilized β-anhydrotrypsin as described (11Blouse G.E. Perron M.J. Kvassman J.O. Yunus S. Thompson J.H. Betts R.L. Lutter L.C. Shore J.D. Biochemistry. 2003; 42: 12260-12272Crossref PubMed Scopus (35) Google Scholar), or latent PAI-1 at the indicated concentrations were incubated in the wells and detected with either H4B3 (1 μg/ml in HBS-B) and a HRP-conjugated rabbit anti-mouse serum (DAKO, diluted 2,000-fold in HBS-B), or a polyclonol rabbit anti-PAI-1 antibody and a HRP-conjugated swine anti-rabbit serum (DAKO, diluted 2,000-fold in HBS-B). Antibody Epitope Mapping—Monoclonal anti-PAI-1 antibody H4B3 or MAI-12 was coated in 96-well plates (5 μg/ml in 100 mm NaHCO3/Na2CO3, pH 9.6) followed by blocking with HBS supplemented with 5% skimmed milk powder (HBS-M). Dilution series of PAI-1 variants, reactive center-cleaved PAI-1, or PAI-1-uPA complex (0.01-150 nm in HBS-M) were incubated for 1 h and bound PAI-1 was detected with a rabbit polyclonal anti-PAI-1 antibody as described above. All assays were done in triplicate and the obtained binding curves fitted to a one-site binding hyperbola using Prism 4.01 (GraphPad Software Inc., San Diego, CA) to calculate the EC50 value. Isolation and Identification of PAI-1-binding Peptides by Screening of Phage-displayed Peptide Libraries—PAI-1-binding peptides were selected from phage-displayed peptide repertoires in the formats X7, CX7C, CX10C, and CX3CX3CX3C (30Koivunen E. Wang B. Dickinson C.D. Ruoslahti E. Methods Enzymol. 1994; 245: 346-369Crossref PubMed Scopus (60) Google Scholar), kindly provided by Dr. E. Koivunen, University of Helsinki, Finland. Monoclonal anti-PAI-1 antibodies mAb-1 or mAb-5 (5 μg/ml in 100 mm NaHCO3/Na2CO3, pH 9.6) were coated in Immunotubes (Maxisorp, Nunc, Denmark) and the tubes were blocked with HBS-M. Alternation of antibodies for the immobilization of PAI-1 was used to prevent enrichment of antibody-binding peptides. PAI-1 expressed from E. coli was immobilized in the antibody-coated tubes and ∼1011 colony-forming units from each peptide repertoire added and incubated for 1 h at room temperature in HBS-M supplemented with 10% glycerol. After extensive washing, bound phage was eluted with an HCl/glycine buffer, pH 2.2, and neutralized (30Koivunen E. Wang B. Dickinson C.D. Ruoslahti E. Methods Enzymol. 1994; 245: 346-369Crossref PubMed Scopus (60) Google Scholar). The eluted phage was propagated in E. coli TG-1 cells and concentrated from the culture supernatant by precipitation with NaCl and polyethylene glycol. Peptides displayed on individual phage were identified by DNA sequencing as described previously (31Hansen M. Wind T. Blouse G.E. Christensen A. Petersen H.H. Kjelgaard S. Mathiasen L. Holtet T.L. Andreasen P.A. J. Biol. Chem. 2005; 280: 38424-38437Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). ELISAs for Measuring PAI-1-Phage Binding—Before the ELISA, PAI-1 variants other than PAI-114-1B and PAI-1(W177F) were incubated for 16-20 h at 37 °C to obtain latent material. In one type of ELISA, His6-tagged PAI-1 expressed from E. coli was immobilized in wells of Ni-NTA HisSorb Strips (Qiagen, Germany). PAI-1 at the indicated concentrations was immobilized and incubated with ∼109 colony-forming units/ml of phage in HBS supplemented with 0.2% bovine serum albumin in the absence or presence of monoclonal antibodies as stated for each experiment. The wells were washed with HBS supplemented with 0.05% Tween 20 (HBS-T) and bound phage was detected with a HRP-conjugated anti-M13 monoclonal antibody (Amersham Biosciences; diluted 5,000-fold in HBS-B). After a final wash, wells were developed by adding 0.5 mg/ml ortho-phenylenediamine (KemEnTech, Denmark) in 50 mm citric acid, pH 5, supplemented with 0.03% H2O2. When suitable color had developed, the reactions were quenched with 1 volume of 1 m H2SO4 and the plates read at 492 nm in a microplate reader. In a second type of ELISA, PAI-1 expressed from E. coli, HEK293T cells, or HT-1080 cells was immobilized on the solid phase by the use of monoclonal antibodies. Antibody (5 μg/ml in 100 mm NaHCO3/Na2CO3, pH 9.6) was coated in wells of a 96-well Maxisorp plate (Nunc, Denmark) followed by blocking with HBS-M. PAI-1 at the indicated concentrations and phage (∼109 colony-forming units/ml) were incubated for 1 h each in the antibody-coated wells, and bound phage was detected as described above. To ensure that equal amounts of the individual PAI-1 variants were bound to the wells, a parallel ELISA was performed with 1 μg/ml polyclonal rabbit anti-PAI-1 antibody instead of phage and with HRP-conjugated swine anti-rabbit serum (DAKO, Denmark, diluted 2,000-fold in HBS-B) instead of anti-M13 antibody. SDS-PAGE—PAI-1 variants (150 μg/ml in HBS) were incubated for 15 min at 37 °C in the presence or absence of H4B3 (300 μg/ml) before addition of 300 μg/ml LMW-uPA (Abbott, Denmark). The reaction products were separated by SDS-PAGE (12% acrylamide, 1.5 μg PAI-1 per lane) and stained with Coomassie Blue. The Monoclonal Antibody H4B3 Neutralizes PAI-1 by Accelerating Latency Transition—Incubating either human or murine PAI-1 with H4B3 accelerated the conversion of PAI-1 to an inactive form unable to inhibit the proteolytic activity of uPA (Fig. 2A). The half-life of inactivation mediated by 200 nm H4B3 at 23 °C was 26 ± 4 and 18 ± 2 min (n = 3) for human and murine PAI-1, respectively, compared with >300 min for both in the absence of antibody. Similar to the reported observations using the monoclonal antibody 33B8 (7Verhamme I. Kvassman J.O. Day D. Debrock S. Vleugels N. Declerck P.J. Shore J.D. J. Biol. Chem. 1999; 274: 17511-17517Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), the rate of H4B3-induced activity loss was concentration dependent as the half-life in the presence of 100 nm H4B3 was 39 ± 3 and 33 ± 9 min (n = 3) for human and murine PAI-1, respectively (data not shown). For human PAI-1, these values correspond to rate constants for latency transition of <0.002, 0.018, and 0.027 min-1 in the presence of 0, 100, and 200 nm H4B3, respectively. To determine whether H4B3 neutralizes PAI-1 by accelerating the latency transition in a manner analogous to that found for the monoclonal antibody 33B8 (7Verhamme I. Kvassman J.O. Day D. Debrock S. Vleugels N. Declerck P.J. Shore J.D. J. Biol. Chem. 1999; 274: 17511-17517Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), we took advantage of previous work demonstrating that labeling of the P9 position of PAI-1 (Ser340) with the environmentally sensitive fluorescent NBD probe produces a PAI-1 variant (PAI-1P9-NBD) in which the RCL insertion occurring during latency transition may be followed by fluorescence spectroscopy (29Shore J.D. Day D.E. Francis-Chmura A.M. Verhamme I. Kvassman J. Lawrence D.A. Ginsburg D. J. Biol. Chem. 1995; 270: 5395-5398Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Full insertion of the P9-NBD-labeled RCL as s4A thus results in a sizable fluorescence increase, which is consistent with transfer of the probe from a solvent exposed to a more hydrophobic environment. Fig. 2B illustrates the fluorescence emission spectrum of the PAI-1P9-NBD variant prior to, and following the binding of a molar excess of H4B3 for 40-60 min. Association of PAI-1P9-NBD and H4B3 resulted in a maximal enhancement of the fluorescence for the NBD probe by ∼35% with an associated 12-nm blue shift in the peak emission from 539 nm in the absence of H4B3 to 527 nm in its presence. A comparable enhancement in
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