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• W2078969730 abstract "Neutrophil elastase and cathepsin G are abundant intracellular neutrophil proteinases that have an important role in destroying ingested particles. However, when neutrophils degranulate, these proteinases are released and can cause irreparable damage by degrading host connective tissue proteins. Despite abundant endogenous inhibitors, these proteinases are protected from inhibition because of their ability to bind to anionic surfaces. Plasminogen activator inhibitor type-1 (PAI-1), which is not an inhibitor of these proteinases, possesses properties that could make it an effective inhibitor of neutrophil proteinases if its specificity could be redirected. PAI-1 efficiently inhibits surface-sequestered proteinases, and it efficiently mediates rapid cellular clearance of PAI-1-proteinase complexes. Therefore, we examined whether PAI-1 could be engineered to inhibit and clear neutrophil elastase and cathepsin G. By introducing specific mutations in the reactive center loop of wild-type PAI-1, we generated PAI-1 mutants that are effective inhibitors of both proteinases. Kinetic analysis shows that the inhibition of neutrophil proteinases by these PAI-1 mutants is not affected by the sequestration of neutrophil elastase and cathepsin G onto surfaces. In addition, complexes of these proteinases and PAI-1 mutants are endocytosed and degraded by lung epithelial cells more efficiently than either the neutrophil proteinases alone or in complex with their physiological inhibitors, α1-proteinase inhibitor and α -antichymotrypsin. Finally, the PAI-1 mutants were 1more effective in reducing the neutrophil elastase and cathepsin G activities in an in vivo model of lung inflammation than were their physiological inhibitors. Neutrophil elastase and cathepsin G are abundant intracellular neutrophil proteinases that have an important role in destroying ingested particles. However, when neutrophils degranulate, these proteinases are released and can cause irreparable damage by degrading host connective tissue proteins. Despite abundant endogenous inhibitors, these proteinases are protected from inhibition because of their ability to bind to anionic surfaces. Plasminogen activator inhibitor type-1 (PAI-1), which is not an inhibitor of these proteinases, possesses properties that could make it an effective inhibitor of neutrophil proteinases if its specificity could be redirected. PAI-1 efficiently inhibits surface-sequestered proteinases, and it efficiently mediates rapid cellular clearance of PAI-1-proteinase complexes. Therefore, we examined whether PAI-1 could be engineered to inhibit and clear neutrophil elastase and cathepsin G. By introducing specific mutations in the reactive center loop of wild-type PAI-1, we generated PAI-1 mutants that are effective inhibitors of both proteinases. Kinetic analysis shows that the inhibition of neutrophil proteinases by these PAI-1 mutants is not affected by the sequestration of neutrophil elastase and cathepsin G onto surfaces. In addition, complexes of these proteinases and PAI-1 mutants are endocytosed and degraded by lung epithelial cells more efficiently than either the neutrophil proteinases alone or in complex with their physiological inhibitors, α1-proteinase inhibitor and α -antichymotrypsin. Finally, the PAI-1 mutants were 1more effective in reducing the neutrophil elastase and cathepsin G activities in an in vivo model of lung inflammation than were their physiological inhibitors. Neutrophils are the first defensive cells to extravasate from the circulation into infected areas where their primary role is to ingest foreign particles and eliminate them by using an arsenal of bactericidal, hydrolytic, and oxidative agents (1Linzmeier R. Michaelson D. Liu L. Ganz T. FEBS Lett. 1993; 321: 267-273Crossref PubMed Scopus (59) Google Scholar, 2Bullen J.J. Armstrong J.A. Immunology. 1979; 36: 781-791PubMed Google Scholar, 3Hampton M.B. Kettle A.J. Winterbourn C.C. Blood. 1998; 92: 3007-3017Crossref PubMed Google Scholar). The direct action of neutrophils is temporary because they degranulate soon after reaching the affected area, but their intracellular proteinases can have a lasting effect. In many chronic inflammatory disorders where there is a persistent influx and degranulation of neutrophils, their proteinases can overwhelm endogenous proteinase inhibitors, cause tissue degradation, and augment the inflammatory response. The broad substrate specificities of neutrophil elastase and cathepsin G are similar to the digestive proteinases, pancreatic elastase and chymotrypsin, respectively. Like their pancreatic counterparts, the neutrophil proteinases can degrade most proteins, including cross-linked extracellular matrix proteins such as collagens and elastin. In addition to degrading extracellular matrix proteins, these neutrophil proteinases can also intensify the host inflammatory response by both proteolytic and non-proteolytic mechanisms (4Si-Tahar M. Pidard D. Balloy V. Moniatte M. Kieffer N. Van Dorsselaer A. Chignard M. J. Biol. Chem. 1997; 272: 11636-11647Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 5Belyavsky A. Vinogradova T. Rajewsky K. Nucleic Acids Res. 1989; 17: 2919-2932Crossref PubMed Scopus (198) Google Scholar, 6Kiss I. Deak F. Holloway R.G. Delius H. Mebust K.A. Frimberger E. Argraves W.S. Tsonis P.A. Winterbottom N. Goetinck P.F. J. Biol. Chem. 1989; 264: 8126-8134Abstract Full Text PDF PubMed Google Scholar, 7Ross R. Lancet. 1989; : 1179-1182Abstract PubMed Scopus (268) Google Scholar). The activities of neutrophil elastase and cathepsin G are primarily regulated by the serine proteinase inhibitors (serpins), 1The abbreviations used are: α1PI, α-1 proteinase inhibitor; PAI-1, plasminogen activator inhibitor type-1; α1ACT, α-1-antichymotrypsin; LRP, low-density lipoprotein receptor-related protein; PAI-1AV, PAI-1V343A A346V; PAI-1F, PAI-1R346F; PAI-1A, PAI-1R346A; fMLF, formyl-Met-Leu-Phe; RAP, receptor-related protein; PMSF, phenylmethylsulfonyl-fluoride; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; LDL, low density lipoprotein; NE, neutrophil elastase; serpin; RCL, reactive center loop. α1-proteinase inhibitor (α1PI, also called α1antitrypsin) and α1-antichymotrypsin (α1ACT), respectively. These inhibitors are abundant plasma proteins, present in the circulation at high concentrations (8Forsyth S. Horvath A. Coughlin P. Genomics. 2003; 81: 336-345Crossref PubMed Scopus (70) Google Scholar). The importance of these inhibitors in regulating neutrophil proteinase activity in extravascular tissues has been demonstrated in individuals with α1PI deficiency who are more likely to develop early onset emphysema because of degradation of lung elastin (9Birrer P. Respiration. 1995; 62: 25-28Crossref PubMed Scopus (54) Google Scholar, 10Coakley R.J. Taggart C. O'Neill S. McElvaney N.G. Am. J. Med. Sci. 2001; 321: 33-41Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar). Neutrophil elastase and cathepsin G are very basic proteins (pI ≥8.5) that bind negatively charged molecules like DNA and heparin with high affinity. Their binding to these anionic surfaces limits the accessibility of their active site to both small substrates and serpins. α1PI and α1ACT are very effective inhibitors of neutrophil elastase and cathepsin G in solution phase, but efficiency is greatly reduced in the presence of these surfaces. The reason for this is thought to be because of the phase separation of neutrophil elastase and cathepsin G from α1PI and α1ACT due to the inability of the inhibitors to bind these surfaces. Partitioning of these proteinases from their endogenous inhibitors could explain the tissue damage associated with many chronic inflammatory disorders because they are protected from inhibitors by anionic macromolecules present in cell debris (11Ermolieff J. Boudier C. Laine A. Meyer B. Bieth J.G. J. Biol. Chem. 1994; 269: 29502-29508Abstract Full Text PDF PubMed Google Scholar, 12Duranton J. Boudier C. Belorgey D. Mellet P. Bieth J.G. J. Biol. Chem. 2000; 275: 3787-3792Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 13Duranton J. Belorgey D. Carrere J. Donato L. Moritz T. Bieth J.G. FEBS Lett. 2000; 473: 154-156Crossref PubMed Scopus (28) Google Scholar). Unlike α1PI and α1ACT, which are efficient inhibitors of proteinases in solution phase, other serpins are efficient inhibitors of their target proteinases when they are bound to a surface. A classic example is antithrombin-III, whose rate of inhibition is enhanced by heparin (14Jordan R.E. Oosta G.M. Gardner W.T. Rosenberg R.D. J. Biol. Chem. 1980; 255: 10081-10090Abstract Full Text PDF PubMed Google Scholar). Another serpin that is able to inhibit proteinases bound to surfaces is plasminogen activator inhibitor type-1 (PAI-1), which is the principal inhibitor of the plasminogen activators (PAs), urokinase-type plasminogen activator and tissue-type plasminogen activator in vivo (15Fay W.P. Shapiro A.D. Shih J.L. Schleef R.R. Ginsburg D. N. Engl. J. Med. 1992; 327: 1729-1733Crossref PubMed Scopus (233) Google Scholar, 16Carmeliet P. Kieckens L. Schoonjans L. Ream B. Van Nuffelen A. Prendergast G. Cole M. Bronson R. Collen D. Mulligan R.C. J. Clin. Investig. 1993; 92: 2746-2755Crossref PubMed Scopus (302) Google Scholar). Both PAs bind to a variety of surfaces, including cellular receptors, fibrin, and heparin. Surface binding, however, does not protect them from inhibition by PAI-1 (17Keijer J. Linders M. Wegman J.J. Ehrlich H.J. Mertens K. Pannekoek H. Blood. 1991; 78: 1254-1261Crossref PubMed Google Scholar, 18Cubellis M.V. Andreasen P. Ragno P. Mayer M. Dano K. Blasi F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4828-4832Crossref PubMed Scopus (160) Google Scholar, 19Keijer J. Linders M. van Zonneveld A.-J. Ehrlich H.J. de Boer J.-P. Pannekoek H. Blood. 1991; 78: 401-409Crossref PubMed Google Scholar, 20Edelberg J.M. Reilly C.F. Pizzo S.V. J. Biol. Chem. 1991; 266: 7488-7493Abstract Full Text PDF PubMed Google Scholar). Besides the ability of PAI-1 to inhibit PAs bound to receptors and surfaces, PAI-1 is able to mediate rapid cellular clearance of the target proteinase. Upon complex formation with a proteinase, PAI-1 undergoes a rapid conformational change that increases its affinity for the clearance receptors of the low density lipoprotein (LDL) receptor family. These include the LDL receptor-related protein (LRP), the very low density lipoprotein receptor, and Megalin (21Stefansson S. Kounnas M.Z. Henkin J. Mallampalli R.K. Chappell D.A. Strickland D.K. Argraves W.S. J. Cell Sci. 1995; 108: 2361-2368Crossref PubMed Google Scholar, 22Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 23Argraves K.M. Battey F.D. MacCalman C.D. McCrae K.R. Gafvels M. Kozarsky K.F. Chappell D.A. Strauss III, J.F. Strickland D.K. J. Biol. Chem. 1995; 270: 26550-26557Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). We previously showed that the high affinity binding of PAI-1-proteinase complexes to LRP was independent of the proteinase but was mediated through a cryptic site on PAI-1 that is exposed when it is in a covalent complex with a proteinase. Furthermore, this enhanced clearance was significantly more efficient compared with clearance of proteinases, such as thrombin, in complex with other serpins, including antithrombin-III, α1PI, and heparin cofactor-II (22Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). Thus, PAI-1 not only inhibits proteinases but also specifically promotes their clearance. In this study we have taken advantage of these innate properties of PAI-1, which normally is cleaved by these proteinases, and designed mutations in the reactive center loop (RCL) of PAI-1, which converts it to an efficient inhibitor of either pancreatic elastase or chymotrypsin or of the neutrophil proteinases, neutrophil elastase and cathepsin G. The efficiency of these PAI-1 mutants is not adversely affected by surfaces such as heparin or DNA, and they are markedly more efficient at promoting the cellular clearance and degradation of neutrophil proteinases compared with their natural serpin inhibitors. Together, these properties make the PAI-1 mutants more effective in vivo than their endogenous inhibitors at reducing the neutrophil proteinase concentrations in a model of lung inflammation. Proteins and Reagents—Active human neutrophil elastase, cathepsin G, and α1-antichymotrypsin were purchased from Athens Biochemicals (Athens, GA). Chromogenic substrates N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (for cathepsin G and chymotrypsin), N-succinyl-Ala-Ala-Ala-Pro-Val-p-nitroanilide (for neutrophil elastase), N-succinyl-Ala-Ala-Ala-p-nitroanilide (for pancreatic elastase), bovine serum albumin, bovine chymotrypsin, hexadecyl-trimethyl ammonium bromide, endotoxin, formyl-Met-Leu-Phe (fMLF), and heparin were purchased from Sigma. Human pancreatic elastase was a generous gift from Dr. S. Olson (University of Illinois at Chicago). Salmon sperm DNA was purchased from Invitrogen. Human α1 proteinase inhibitor was a generous gift from Dr. A. M. Ralston (J. H. Holland Laboratory.). The human 39-kDa receptor-associated protein (RAP) and the human LRP were generous gifts from Dr. D. K. Strickland (J. H. Holland Laboratory.). Heparin-Sepharose, phenyl-Sepharose, and PD-10 prepacked gel filtration columns were purchased from Amersham Biosciences. Na125I and 125I Bolton-Hunter reagent were purchased from Amersham Biosciences and ICN (Irvine, CA), respectively. Preparation of PAI-1 Mutant Inhibitors of Neutrophil Proteinases—A preferred method for producing PAI-1 mutants utilizes the commercially available Transformer site-directed mutagenesis kit (Clontech, Palo Alto, CA) (22Stefansson S. Muhammad S. Cheng X.F. Battey F.D. Strickland D.K. Lawrence D.A. J. Biol. Chem. 1998; 273: 6358-6366Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). This kit uses a mismatched primer form of mutagenesis and is compatible with double stranded plasmid DNA. Thus the mutants can be constructed directly onto existing expression plasmids. The PAI-1A346V-V343A and PAI-1R346F mutants were made using the oligonucleotides (5′-CATAGCCTCAGCCGTCATGGCCCCCGAG-3′ and 5′-CATAGTCTCAGCCTTCATGGCCCCCGAGGAG-3′, respectively). These PAI-1 mutants were expressed and purified as described (24Lawrence D. Strandberg L. Grundström T. Ny T. Eur. J. Biochem. 1989; 186: 523-533Crossref PubMed Scopus (118) Google Scholar) and isolated and purified as described by Kvassman and Shore (25Kvassman J.-O. Shore J.D. Fibrinolysis. 1995; 9: 215-221Crossref Scopus (51) Google Scholar). Preparations with significant endotoxin levels as determined by coagulase kit (Bio-Whittacker, Walkersville, MD) following the phenyl-Sepharose step were further purified by either additional passes through phenyl-Sepharose or Detoxi-Gel (Pierce) until endotoxin levels were at or below 10 units/mg. Biacore Analysis of the Binding of the Proteinases, Inhibitors, and Their Complexes to LRP—Affinities of pancreatic elastase, neutrophil elastase, chymotrypsin, and cathepsin G for LRP either in complex with α1PI, α1ACT, PMSF, or PAI-1 mutants were measured by surface plasmon resonance using a BIA 3000 optical biosensor (Biacore AB, Uppsala, Sweden). Purified human LRP was immobilized at the level of 3000 response units. Remaining binding sites were blocked by 1 m ethanolamine, pH 8.5, and unbound proteins were washed out with 0.5% SDS. A flow cell with immobilized ovalbumin at the level of 500 response units was used as a control for nonspecific protein binding. All binding reactions were performed in standard HBS-P buffer, pH 7.4, containing 10 mm HEPES, 150 mm NaCl, and 0.005% Tween 20. Binding of proteases, inhibitors, and their complexes to LRP was measured at 25 °C at a flow rate of 30 μl/min for 4 min, followed by 4 min of dissociation. Chip surfaces were regenerated with subsequent 1-min pulses of 1 m NaCl, pH 4.0, and 1 m NaCl containing 10 mm NaOH, followed by 2 min of washing with HBS-P. Binding of proteinases, inhibitors, and proteinase-inhibitor complexes was measured using a range of concentrations (10–0.15-nm). Collected data were analyzed with BIA evaluation 3.0 software (Biacore) using global analysis to fit a 1:1 Langmuire binding model with mass transfer limitation. Cellular Endocytosis and Degradation Assays of Proteinases—A rat pretype-II pneumocyte cell line (T-II) was a generous gift from Dr. R. K. Mallampalli (University of Iowa College of Medicine, Iowa City, IA) (26Mallampalli R.K. Floerchinger C.S. Hunninghake G.W. In Vitro Cell Dev. Biol. 1992; 28A: 181-187Crossref PubMed Scopus (19) Google Scholar). These cells were grown in Dulbecco's modified Eagle's medium (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) containing penicillin and streptomycin. For endocytosis and degradation studies, T-II cells were seeded onto 12-well plates, (1–3 × 105 cells/well) coated with 0.1% gelatin and allowed to adhere for 18 h at 37 °C, 5% CO2 in Dulbecco's modified Eagle's medium containing 10% bovine calf serum. Before addition of radiolabeled proteinases, cells were washed twice using serum-free Dulbecco's modified Eagle's medium and incubated for 30 min in serum-free Dulbecco's modified Eagle's medium containing 1% bovine serum albumin before addition of either preformed 125I-labeled proteinase-inhibitor complexes or 125I-labeled proteinases. Where indicated, RAP (1 μm) was added prior to the 125I proteinase-inhibitor complexes. Quantitation of the endocytosed and degraded ligand was done as described (27Stefansson S. Lawrence D.A. Argraves W.S. J. Biol. Chem. 1996; 271: 8215-8220Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar) with minor modifications. Briefly, cellular degradation of the 125I-labeled proteinases was determined by removing the medium and precipitating it using 10% trichloroacetic acid and 5% phosphotungstic acid (final concentration). Acid-soluble radioactivity was taken to represent degraded ligands. For quantitation of endocytosis, cell layers were washed twice with serum-free medium and incubated in serum-free medium containing trypsin and proteinase K (0.5 mg/ml) and 0.5 mm EDTA for 2–5 min at 4 °C. The cells were then centrifuged at 6000 × g for 2 min, and the radioactivity in the cell pellet was taken to represent the amount of endocytosed ligand. To evaluate the effects of α1PI and PAI-1AV on the endocytosis and degradation of exogenously added 125I elastase, cells were grown in medium containing 10% serum as described above. Washed monolayers were then incubated with 125I elastase (10 nm) for 30 min, after which increasing concentrations of either α1PI or PAI-1AV were added and incubated for 4 h at 37 °C to determine endocytosis or 18 h for to determine degradation. Kinetics of Proteinase Inhibition by α1PI, α1ACT, and PAI-1 Mutants—Before measuring the second order rate constant for the inhibition of the neutrophil proteinases, the stoichiometry of the inhibitor as a proteinase substrate to a proteinase inhibitor (SI value) was determined. Briefly, a constant concentration of the neutrophil proteinases was incubated with an increasing concentration of the proteinase inhibitors in the presence or absence of heparin or DNA. After 30 min the appropriate chromogenic proteinase substrate was added to the reaction mixture, and the proteinase activity was measured. The results were plotted as activity of the proteinase versus the inhibitor concentration divided by the enzyme concentration. The intercept on the x-axis is therefore an indicator of whether the inhibitor inhibits in a 1:1 molar ratio or whether higher concentrations of the inhibitors are required. Two methods were used to obtain kinetic parameters for the inhibition of the proteinases. For slow reactions (ki < 105), the PAI-1 mutants will be assumed to inhibit target proteinases by the two-step inhibitory mechanism first described for irreversible inhibitors by Kitz and Wilson (28Kitz R. Wilson I.B. J. Biol. Chem. 1962; 237: 3245-3249Abstract Full Text PDF PubMed Google Scholar) and Hastings et al. (29Hastings G.A. Coleman T.A. Haudenschild C.C. Stefansson S. Smith E.P. Barthlow R. Cherry S. Sandkvist M. Lawrence D.A. J. Biol. Chem. 1997; 272: 33062-33067Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). For fast reactions between the proteinases and the inhibitors (ki >105), the scheme described in Lawrence et al. (24Lawrence D. Strandberg L. Grundström T. Ny T. Eur. J. Biochem. 1989; 186: 523-533Crossref PubMed Scopus (118) Google Scholar) was used. The effects of heparin and DNA on the kinetics of inhibition were determined in the presence of either 0.1 mg/ml heparin or 0.1 mg/ml DNA. In Vivo Lung Inflammation Assay—In vivo lung inflammation assays were performed essentially as described (31Eyles J.E. Williamson E.D. Alpar H.O. Int. J. Pharm. 1999; 189: 75-79Crossref PubMed Scopus (52) Google Scholar), with some modifications. Briefly, male C57BL/6J mice (age 8–12 weeks) under anesthesia were intranasally instilled with either PBS or a mixture of endotoxin and fMLF, 100 and 50 μg, respectively, in a total volume of 150 μl in PBS. PAI-1AV and PAI-1F mutants, 50 μg/25 μg, respectively, or α1AP/α1ACT, 50 μg/25 μg, respectively, in a total volume of 150 μl in PBS were administered along with the endotoxin/fMLF instillation mixture. Additionally, PAI-1AV/PAI-1F, 100 μg/25 μg, respectively, or α1AP/α1ACT, 100 μg/25 μg, respectively, in a total volume of 150 μl or PBS were injected intraperitonally to both PBS- and endotoxin/fMLF-treated mice immediately following nasal instillation and again 16–17 h postinstillation. At 24–26 h postinstillation, animals were anesthetized and perfused with PBS. Lungs were then excised, washed briefly with 0.3% hexadecyl-trimethyl ammonium bromide in Tris-buffered saline, and stored on dry ice, followed by homogenization in Tris-buffered saline containing 1% bovine serum albumin and 0.3% hexadecyl-trimethyl ammonium bromide, followed by centrifugation to remove particulates. Neutrophil elastase and cathepsin G activities were measured in the lung homogenates using the chromogenic substrates described above. The effectiveness of the inhibitors as assessed by the neutrophil proteinase load in the lungs was determined by subtracting the activities of animals treated intranasally with PBS from the endotoxin/fMLF-treated animals, with or without added proteinase inhibitors. (Approved Animal Welfare Assurance A3379–01 registered with the Public Health Services, Office of Laboratory Animal Welfare). Generation and Characterization of PAI-1 Mutants That Inhibit Pancreatic and Neutrophil Proteinases—Serpins act as “suicide inhibitors” because when a proteinase cleaves a serpin at a site within the RCL, termed the scissile bond (P1-P1′), it traps the proteinase in a non-reversible covalent complex (32Huntington J.A. Pannu N.S. Hazes B. Read R.J. Lomas D.A. Carrell R.W. J. Mol. Biol. 1999; 293: 449-455Crossref PubMed Scopus (118) Google Scholar, 33Lawrence D.A. Nat. Struct. Biol. 1997; 4: 339-341Crossref PubMed Scopus (50) Google Scholar). In many cases the amino acid composition of the serpin scissile bond reflects the substrate specificity of the target proteinase. The scissile bond of wild-type PAI-1 is Arg-346-Met-347, which enables it to inhibit many serine proteinases with trypsin-like specificities, but kinetic analysis showed that it is the most effective inhibitor of both PAs (34Hekman C.M. Loskutoff D.J. Arch. Biochem. Biophys. 1988; 262: 199-210Crossref PubMed Scopus (91) Google Scholar). Because PAI-1 is an efficient inhibitor of its target proteinases both in solution and in solid phase (17Keijer J. Linders M. Wegman J.J. Ehrlich H.J. Mertens K. Pannekoek H. Blood. 1991; 78: 1254-1261Crossref PubMed Google Scholar, 18Cubellis M.V. Andreasen P. Ragno P. Mayer M. Dano K. Blasi F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 4828-4832Crossref PubMed Scopus (160) Google Scholar, 19Keijer J. Linders M. van Zonneveld A.-J. Ehrlich H.J. de Boer J.-P. Pannekoek H. Blood. 1991; 78: 401-409Crossref PubMed Google Scholar, 20Edelberg J.M. Reilly C.F. Pizzo S.V. J. Biol. Chem. 1991; 266: 7488-7493Abstract Full Text PDF PubMed Google Scholar) and because it is very efficient at promoting their cellular clearance, (27Stefansson S. Lawrence D.A. Argraves W.S. J. Biol. Chem. 1996; 271: 8215-8220Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar), we wanted to determine whether we could redirect PAI-1 specificity toward neutrophil elastase and cathepsin G, which wt PAI-1 does not inhibit, (35Wu K. Urano T. Ihara H. Takada Y. Fujie M. Shikimori M. Hashimoto K. Takada A. Blood. 1995; 86: 1056-1061Crossref PubMed Google Scholar, 36Iacoviello L. Kolpakov V. Salvatore L. Amore C. Pintucci G. de Gaetano G. Donati M.B. Arterioscler. Thromb. Vasc. Biol. 1995; 15: 2037-2046Crossref PubMed Scopus (27) Google Scholar), and still retain these properties. Mutations in a serpin scissile bond have been shown to redirect it to target another proteinase (37Owen M.C. Brennan S.O. Lewis J.H. Carrell R.W. N. Engl. J. Med. 1983; 309: 694-698Crossref PubMed Scopus (323) Google Scholar). Therefore we tested whether a PAI-1 mutant with the P1 Arg-346 residue changed to an alanine (PAI-1R346A, referred herein as PAI-1A) (38Sherman P.M. Lawrence D.A. Yang A.Y. Vandenberg E.T. Paielli D. Olson S.T. Shore J.D. Ginsburg D. J. Biol. Chem. 1992; 267: 7588-7595Abstract Full Text PDF PubMed Google Scholar), could inhibit pancreatic elastase, which prefers Ala as the P1 residue. The PAI-1A has no inhibitory activity against urokinase-type plasminogen activator (data not shown) but retained the ability to bind vitronectin with affinity, equal to wild-type PAI-1 (39Stefansson S. Lawrence D.A. Nature. 1996; 383: 441-443Crossref PubMed Scopus (607) Google Scholar). The rate of inhibition (ki) of pancreatic elastase by PAIA is 4.6 × 105m–1 s–1, which is comparable with the rate obtained by Laurent and Bieth (40Laurent P. Bieth J.G. Biochim. Biophys. Acta. 1989; 994: 285-288Crossref PubMed Scopus (11) Google Scholar) for α1PI (4.7 × 105m–1 s–1). These results demonstrate that the specificity of PAI-1 can be redirected toward pancreatic elastase with a rate of inhibition similar to α1PI. To target serine proteinases with chymotrypsin-like specificities, a second PAI-1 mutant was generated with a phenylalanine at the P1 position (PAI-1R346F, referred herein as PAI-1F). This mutant was tested against chymotrypsin, and the rate of inhibition of PAI-1F was 6.1 × 104m–1 s–1 compared with 8.1 × 105m–1 s–1 for α1ACT reported by Rubin et al. (41Rubin H. Wang Z.M. Nickbarg E.B. McLarney S. Naidoo N. Schoenberger O.L. Johnson J.L. Cooperman B.S. J. Biol. Chem. 1990; 265: 1199-1207Abstract Full Text PDF PubMed Google Scholar). The results reported above demonstrate that the proteinase specificity of PAI-1 can be easily redirected by changing the amino acid composition of the P1 residue without a substantial loss in the rate of inhibition in solution phase. Therefore, these PAI-1 mutants were tested for their ability to inhibit other serine proteinases with similar specificity, specifically neutrophil elastase and cathepsin G. Kinetic analysis of PAI-1A inhibition of neutrophil elastase yielded a ki of 1.4 × 103m–1 s–1, which is about 300-fold less than the rate of inhibition of pancreatic elastase by PAI-1A (ki = 4.6 × 105m–1 s–1) and is ∼10,000 fold less than α1PI inhibition of neutrophil elastase (ki = 1.3 × 107m–1 s–1). The decrease in the ability of PAI-1A to inhibit neutrophil elastase was surprising because it efficiently inhibited pancreatic elastase with a rate equal to that of α1PI. This discrepancy could be because of the preference of neutrophil elastase for valine over alanine as the P1 residue or because the proteinase was cleaving the RCL at another site than the P1-P1′, rendering it inactive as was demonstrated for wtPAI-1 (35Wu K. Urano T. Ihara H. Takada Y. Fujie M. Shikimori M. Hashimoto K. Takada A. Blood. 1995; 86: 1056-1061Crossref PubMed Google Scholar). Sequence analysis of the reaction mixture of PAI-1A with neutrophil elastase showed that PAI-1A was being cleaved at both the Val-343-Ser-344 position (P3-P4) and at Ala-346-Met-347 (P1-P1′, data not shown), indicating that the inefficiency of PAI-1A might be due in part to this non-productive cleavage at Val-343-Ser-344, coupled with the P1 alanine being a suboptimal residue for neutrophil elastase. Therefore, to improve PAI-1A as an inhibitor of neutrophil elastase, the Val-343 residue was replaced with alanine and the Ala-346 was replaced with valine. The resulting mutant, PAI-1V343A A346V (referred herein as PAI-1AV), has the non-productive Val-343 cleavage site changed to alanine, which is a suboptimal cleavage site for neutrophil elastase, and the alanine at the P1 position changed to valine, which is preferred by neutrophil elastase as demonstrated by small peptide substrates (43Koehl C. Knight C.G. Bieth J.G. J. Biol. Chem. 2003; 278: 12609-12612Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Valine was also chosen as the P1 residue because studies by Shubeita et al. (44Shubeita H.E. Cottey T.L. Franke A.E. Gerard R.D. J. Biol. Chem. 1990; 265: 18379-18385Abstract Full Text PDF PubMed Google Scholar) demonstrated that replacing the P1-P1′ residues of wt-PAI-1 with those of α1PI (Met-Ser) did not convert PAI-1 into an inhibitor of elastase or trypsin. Additionally, the PAI-1AV mutant does not alter the length of the PAI-1 RCL, whic" @default.
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• W2078969730 title "Mutants of Plasminogen Activator Inhibitor-1 Designed to Inhibit Neutrophil Elastase and Cathepsin G Are More Effective in Vivo than Their Endogenous Inhibitors" @default.
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