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Anti-hemostatic Effects of a Serpin from the Saliva of the Tick Ixodes ricinus

2006; Elsevier BV; Volume: 281; Issue: 36 Linguagem: Inglês

10.1074/jbc.m604197200

1083-351X

Pierre-Paul Prévôt, Benoît Adam, Karim Zouaoui Boudjeltia, Michel Brossard, Laurence Lins, P. Cauchie, Robert Brasseur, Michel Vanhaeverbeek, Luc Vanhamme, Edmond Godfroid,

Protein Hydrolysis and Bioactive Peptides

Serpins (serine protease inhibitors) are a large family of structurally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses revealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in the interaction between serpins and serine proteases. Only serine proteases were inhibited by purified recombinant Iris (rIris), whereas mutants L339A and A332P were found devoid of any protease inhibitory activity. The highest Ka was observed with human leukocyte-elastase, suggesting that elastase-like proteases are the natural targets of Iris. In addition, mutation M340R completely changed both Iris substrate specificity and affinity. This likely identified Met-340 as amino acid P1 in the RCL. The effects of rIris and its mutants were also tested on primary hemostasis, blood clotting, and fibrinolysis. rIris increased platelet adhesion, the contact phase-activated pathway of coagulation, and fibrinolysis times in a dose-dependent manner, whereas rIris mutant L339A affected only platelet adhesion. Taken together, these results indicate that Iris disrupts coagulation and fibrinolysis via the anti-proteolytic RCL domain. One or more other domains could be responsible for primary hemostasis inhibition. To our knowledge, this is the first ectoparasite serpin that interferes with both hemostasis and the immune response. Serpins (serine protease inhibitors) are a large family of structurally related proteins found in a wide variety of organisms, including hematophagous arthropods. Protein analyses revealed that Iris, previously described as an immunomodulator secreted in the tick saliva, is related to the leukocyte elastase inhibitor and possesses serpin motifs, including the reactive center loop (RCL), which is involved in the interaction between serpins and serine proteases. Only serine proteases were inhibited by purified recombinant Iris (rIris), whereas mutants L339A and A332P were found devoid of any protease inhibitory activity. The highest Ka was observed with human leukocyte-elastase, suggesting that elastase-like proteases are the natural targets of Iris. In addition, mutation M340R completely changed both Iris substrate specificity and affinity. This likely identified Met-340 as amino acid P1 in the RCL. The effects of rIris and its mutants were also tested on primary hemostasis, blood clotting, and fibrinolysis. rIris increased platelet adhesion, the contact phase-activated pathway of coagulation, and fibrinolysis times in a dose-dependent manner, whereas rIris mutant L339A affected only platelet adhesion. Taken together, these results indicate that Iris disrupts coagulation and fibrinolysis via the anti-proteolytic RCL domain. One or more other domains could be responsible for primary hemostasis inhibition. To our knowledge, this is the first ectoparasite serpin that interferes with both hemostasis and the immune response. Ticks are blood-sucking arthropods that infest a large variety of vertebrate hosts (mammals, birds, reptiles, and amphibians) in many parts of the world (1Sauer J.R. McSwain J.L. Bowman A.S. Essenberg R.C. Annu. Rev. Entomol. 1995; 40: 245-267Crossref PubMed Scopus (197) Google Scholar). To complete their blood meal, blood-sucking arthropods express a wide range of anti-hemostatic molecules in their saliva, including vasodilators, inhibitors of the platelet aggregation, and anti-coagulants (2Champagne D.E. Curr. Drug Targets. 2004; 4: 375-396Google Scholar). Tick saliva and salivary gland extracts are also known to modulate the host's defense mechanisms (3Ribeiro J.M. Infect Agents Dis. 1995; 4: 143-152PubMed Google Scholar, 4Kopecky J. Kuthejlova M. Parasite Immunol. 1998; 20: 169-174PubMed Google Scholar, 5Wikel S.K. Annu. Rev. Parasitol. 1996; 84: 304-309Google Scholar, 6Brossard M. Wikel S.K. Parasitology. 2004; 129: 161-176Crossref PubMed Scopus (200) Google Scholar). Both anti-hemostatic and immunosuppressive compounds were identified, isolated, and characterized from soft and hard ticks. These compounds include histamine-binding proteins, tissue factor pathway inhibitor-like proteins, anti-thrombin-like proteins, and anti-complement factors (7Hoffmann A. 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Finally, Ribeiro and co-workers explored the sialome of the tick I. scapularis (19Valenzuela J.G. Francischetti I.M. Pham V.M. Garfield M.K. Mather T.N. Ribeiro J.M. J. Exp. Biol. 2002; 205: 2843-2864Crossref PubMed Google Scholar, 20Ribeiro J.M. Alarcon-Chaidez F. Francischetti I.M. Mans B.J. Mather T.N. Valenzuela J.G. Wikel S.K. Insect Biochem. Mol. Biol. 2006; 36: 111-129Crossref PubMed Scopus (307) Google Scholar) and uncovered a large variety of putative bioactive agents. These studies all identified some serine protease inhibitors, containing serpin, kunitz, kazal, or α-macroglobulin motifs (21Kanost M.R. Dev. Comp. Immunol. 1999; 23: 291-301Crossref PubMed Scopus (366) Google Scholar). To date, ∼500 serpins have been identified in a large variety of species, including animals, viruses, and plants. On average, serpins are 350-400 amino acids long. In human plasma, they make up ∼2% of the total protein amount, of which 70% is α-1-antitrypsin. Both extracellular and intracellular serpins have been identified (22Van Gent D. Sharp P. Morgan K. Kalsheker N. J. Biochem. Cell Biol. 2003; 35: 1536-1547Crossref PubMed Scopus (168) Google Scholar). Members of the superfamily of serine protease inhibitors are expressed in a cell-specific manner and are involved in a number of fundamental biological processes such as blood coagulation, complement activation, fibrinolysis, angiogenesis, inflammation, and tumor suppression (23Davie E.W. Fujikawa K. Kisiel W. Biochemistry. 1991; 30: 10363-10370Crossref PubMed Scopus (1618) Google Scholar, 24Hekman C.M. Loskutoff D.J. Semin. Thromb. Hemost. 1987; 13: 514-527Crossref PubMed Scopus (89) Google Scholar, 25Rubin H. Nat. Med. 1996; 2: 632-633Crossref PubMed Scopus (48) Google Scholar). The protein structure of serpins is usually characterized by three β-sheets (A, B, and C) and eight or nine α-helices (26Silverman G.A. Bird P.I. Carrell R.W. Church F.C. Coughlin P.B. Gettins P.G. Irving J.A. Lomas D.A. Luke C.J. Moyer R.W. Pemberton P.A. Remold-O'Donnell E. Salvesen G.S. Travis J. Whisstock J.C. J. Biol. Chem. 2001; 276: 33293-33296Abstract Full Text Full Text PDF PubMed Scopus (1060) Google Scholar). A typical feature of serpins is the reactive center loop (RCL), 3The abbreviations used are: RCL, reactive center loop; HLE, human leukocyte elastase; Iris, I. ricinus immunosuppressor; PAI-1, plasminogen activator inhibitor, type 1; PDB, Protein Data Bank; PPE, pancreatic porcine elastase; RBD, rIris, recombinant Iris; r.m.s.d., root mean square deviation; t-PA, tissue plasminogen activator; HBSS, Hanks' balanced salt solution; ECLT, euglobulin clot lysis time; ANOVA, analysis of variance; MMP, metalloprotease; PFA, platelet function analyzer.3The abbreviations used are: RCL, reactive center loop; HLE, human leukocyte elastase; Iris, I. ricinus immunosuppressor; PAI-1, plasminogen activator inhibitor, type 1; PDB, Protein Data Bank; PPE, pancreatic porcine elastase; RBD, rIris, recombinant Iris; r.m.s.d., root mean square deviation; t-PA, tissue plasminogen activator; HBSS, Hanks' balanced salt solution; ECLT, euglobulin clot lysis time; ANOVA, analysis of variance; MMP, metalloprotease; PFA, platelet function analyzer. a protein motif composed of ∼20 amino acids, located near the C terminus of the protein. This motif contains a scissile bond between residues called P1 and P1′, which is cleaved by the target protease. Once cleaved, the RCL domain traps the protease and moves to the opposite pole of the serpin through the β-sheet A. This tight association results in an irreversible loss of structure and distortion of both the protease and the serpin. Inside the RCL domain the hinge region (amino acids P15-P9) is implicated in the stabilization of the interaction with the protease and provides mobility for the integration of the RCL in β-sheet A. Some serpin family members, while structurally similar to inhibitor serpins, have no inhibitory functions. These non-inhibitory serpins include corticosteroid binding globulin and thyroxine binding globulin, which are involved in hormone transport, angiotensinogen, which is a peptide hormone precursor, and ovalbumin. These serpins retain the mobility of the RCL domain. However, there is no evidence for a conformational change following cleavage at their putative reactive centers. A few serpins have been recently isolated from the hard ticks Amblyomma hebraeum, Amblyomma variegatum, Boophilus microplus, Hemaphysalis longicornis, I. scapularis, and Rhipicephalus appendiculatus (27Imamura S. da Silva Vaz Junior I. Sugino M. Ohashi K. Onuma M. Vaccine. 2005; 23: 1301-1311Crossref PubMed Scopus (123) Google Scholar, 28Kazimirova M. Jancinova V. Petrikova M. Takac P. Labuda M. Nosal R. Exp. Appl. Acarol. 2002; 28: 97-105Crossref PubMed Scopus (23) Google Scholar, 29Mulenga A. Tsuda A. Onuma M. Sugimoto C. Insect. Biochem. Mol. Biol. 2003; 33: 267-276Crossref PubMed Scopus (57) Google Scholar, 30Sugino M. Imamura S. Mulenga A. Nakajima M. Tsuda A. Ohashi K. Onuma M. Vaccine. 2003; 21: 2844-2851Crossref PubMed Scopus (78) Google Scholar). These proteins are mainly expressed in tick salivary glands and are secreted in the saliva. Most of them have been proposed as targets in an anti-tick mixture vaccine (30Sugino M. Imamura S. Mulenga A. Nakajima M. Tsuda A. Ohashi K. Onuma M. Vaccine. 2003; 21: 2844-2851Crossref PubMed Scopus (78) Google Scholar, 31Mulenga A. Sugino M. Nakajim M. Sugimoto C. Onuma M. J. Vet. Med. Sci. 2001; 63: 1063-1069Crossref PubMed Scopus (59) Google Scholar, 32Mulenga A. Sugimoto C. Onuma M. Microbes Infect. 2000; 2: 1353-1361Crossref PubMed Scopus (63) Google Scholar, 34Narasimhan S. Montgomery R.R. DePonte K. Tschudi C. Marcantonio N. Anderson J.F. Sauer J.R. Cappello M. Kantor F.S. Fikrig E. Proc. Natl. Acad. Sci. U. S. A. 2004; 3: 1141-1146Crossref Scopus (107) Google Scholar). Recently, we have characterized a protein, which is up-regulated during the blood meal and modulates both the innate and acquired immunity of the host (35Leboulle G. Crippa M. Decrem Y. Mejri N. Brossard M. Bollen A. Godfroid E. J. Biol. Chem. 2002; 277: 10083-10089Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Accordingly, we named the protein Iris for "I. ricinus immunosuppressor." Iris was also found to be related to the pig leukocyte elastase inhibitor (35Leboulle G. Crippa M. Decrem Y. Mejri N. Brossard M. Bollen A. Godfroid E. J. Biol. Chem. 2002; 277: 10083-10089Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In this report, we present the cloning and the expression of Iris as well as single point mutants in insect cells. Structural analysis and directed mutagenesis confirmed that Iris is a serpin with Met-340 as the P1 residue. We also show that Iris is a specific elastase inhibitor that interferes with the coagulation pathways and the fibrinolysis process via the RCL domain. Finally, we show that Iris inhibits platelet adhesion via another functional domain. To our knowledge, this is the first ectoparasite serpin that alters both hemostasis and the immune response. Plasmid Construction, Protein Expression, and Purification—The coding sequence for Iris (formerly Seq24; accession number AJ269658) was subcloned from vector pCDNA3.1-V5-His/Iris (35Leboulle G. Crippa M. Decrem Y. Mejri N. Brossard M. Bollen A. Godfroid E. J. Biol. Chem. 2002; 277: 10083-10089Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) between the KpnI/AgeI restriction sites of vector pBlueBac4.5-V5-His (Invitrogen) in-frame with the coding sequence of the V5 and His epitopes at the C terminus. Recombinant baculoviruses were made by recombination between pBlueBac/Iris and Bac-N-Blue linear DNA virus (Invitrogen). Recombinant viruses were selected and amplified according to the manufacturer's instruction. SF9 cells were infected with a high titer stock of recombinant baculovirus and were incubated for 55 h at 27 °C. Cells were then lysed using the French press, and spun at 106 × g for 40 min. The recombinant protein was further purified in one step from the cleared supernatant by chelating chromatography over Ni2+-Sepharose (Qiagen). Recombinant Iris was eluted in 50 mm Tris-HCl (pH 7.5) containing 500 mm NaCl and 150 mm imidazole. All purification steps were carried out at 4 °C. Typically, 50 mg of purified rIris was obtained per liter of suspension culture. Site-directed Mutagenesis of Iris in pBlueBac4.5 V5 His—Mutants were produce by using the QuikChange PCR mutagenesis kit (Stratagene). The following PCR primers were used to generate P9, P2, and P1 mutants, respectively: 5′-GCA CAG AGG CTG CAG CTC CCA CTG CCA TAC CC-3′ (forward A332P), 5′-GGG TAT GGC AGT GGG AGC TGC AGC CTC TGT GC-3′ (reverse A332P); 5′-GCC ATA CCC ATT ATG GCG ATG TGC GCG AGA TTT CC-3′ (forward L339A), 5′-GGA AAT CTC GCG CAC ATC GCC ATA ATG GGT ATG GC-3′ (reverse L339A); 5′-GCC ATA CCC ATT ATG TTG CGG TGC GCG AGA TTT CC-3′ (forward M340R), 5′-GGA AAT CTC GCG CAC CGC AAC ATA ATG GGT ATG GC-3′ (reverse M340R); where the underlined nucleotides generate the mutation. Supercompetent XL1-Blue cells were transformed according to the manufacturer's instructions, and the plasmids were purified to confirm the sequence modifications by sequencing. Molecular Modeling—Three-dimensional models of Iris were generated using the homology modeling software Modeler 6.2 (36Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10456) Google Scholar). To construct a model, the sequence of Iris was aligned to a homologous sequence whose three-dimensional structure had been experimentally determined. For that purpose, the Protein Data Bank (PDB) was screened using the Blast algorithm. α-Antitrypsin (PDB code: 1ATU) was selected as the template for the native model and horse leukocyte elastase inhibitor (PDB code: 1HLE) for the cleaved model. The percentages of identity with Iris are 32 and 42%, respectively, and the percentages of similarity 51 and 59%, respectively. To further examine the homology, the hydrophobic cluster analysis plots of both sequences were compared. Briefly, the hydrophobic cluster analysis method is based on a two-dimensional helical plot of the sequence and allows detection of hydrophobic clusters in proteins (37Gaboriaud C. Bissery V. Benchetrit T. Mornon J.P. FEBS Lett. 1987; 224: 149-155Crossref PubMed Scopus (542) Google Scholar). The stereochemical quality of the minimized three-dimensional model was assessed using Procheck (38Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Both models had no residues located in the disallowed regions of the Φ, Ψ angle pairs of the Ramachandran plot, indicating correct stereochemistry. Molecular views were drawn with WinMGM (39Rahman M. Brasseur R. J. Mol. Graph. 1994; 12: 212-218Crossref PubMed Scopus (32) Google Scholar). A model of the interaction between Iris and a protease was built using Swissmodel (40Schwede T. Kopp J. Guex N. Peitsch M.C. Nucleic Acids Res. 2003; 31: 3391-3395Crossref Scopus (4448) Google Scholar). The template was the complex between trypsin and alaserpin A353K (PDB code: 1K9O). The Pex algorithm was used to analyze the residues in this interaction. This method extracts numeric and string descriptions of protein structures from PDB files and lists parameters such as the dihedral angles, the N-H bond lengths, the secondary structure, the closest residues in the interaction, and the minimal distance of the interaction (41Thomas A. Bouffioux O. Geeurickx D. Brasseur R. Proteins Struct. Funct. Genet. 2001; 43: 28-36Crossref PubMed Scopus (27) Google Scholar). Enzymatic Assays—To assay the inhibition of proteases, Iris was mixed in a microcuvette with 5-20 μl of a protease at an equimolecular ratio in 50 mm Tris-HCl (pH 7.5) containing 100 mm NaCl. Residual activity was measured by adding 1 ml of 0.5 mm of an appropriate chromogenic substrate in 50 mm Tris-HCl, pH 7.5, 100 mm NaCl. The increase in absorbance at 405 nm over time was measured. Two variants of the assay were performed: (i) in a first, residual activity was measured after incubation for 1 h at 37 °C, (ii) in a second, activity was measured as function of the incubation time. In the former case, residual activity in the presence of ovalbumin, which is a serpin with no inhibitory activity, was taken as the 100% reference point. rIris was tested in the presence of the following subset of enzyme-substrate systems: human thrombin and N-benzoyl-Phe-Val-Arg-p-Na; human leukocyte elastase (HLE) and N-(methoxysuccinyl)-Ala-Ala-Pro-Val-p-Na; porcine pancreatic elastase (PPE, Roche Applied Science), and N-methoxysuccinyl-Ala-Ala-Pro-Val-p-Na; human plasmin and N-p-tosyl-Gly-Pro-Lys-p-Na; human factor Xa and factor Xa chromogenic substrate; recombinant tissue plasminogen activator (t-PA, Hyphen Biomed, France) and t-PA chromogenic substrate; and human trypsin and N-benzoyl-Phe-Val-Arg-p-Na. We also tested two cysteine proteases (papain with pGlu-Phe-Leu-p-Na and caspase 3 with N-acetyl-Asp-Glu-Val-Asp-p-Na) and one human metalloelastase (MMP12 with MMP chromogenic substrate, Biomol). All products were purchased from Sigma-Aldrich, unless otherwise indicated. Measurement of Association Rate Constants—The association rate constant (ka) describes the inhibition kinetics of free serine proteases by serpins. It was determined by mixing proteases and rIris for variable periods of time before addition of the substrate (0.5 mm), which slows down the association process enough to allow measurement of residual enzyme activity (42Frommherz K.J. Faller B. Bieth J.G. J. Biol. Chem. 1991; 266: 15356-15362Abstract Full Text PDF PubMed Google Scholar). Considering that the dissociation reaction is slow with respect to the interval of time needed to follow association, the rate association, -d[E]/dt is given by Equation 1 (42Frommherz K.J. Faller B. Bieth J.G. J. Biol. Chem. 1991; 266: 15356-15362Abstract Full Text PDF PubMed Google Scholar).-d[E]dt=ka·[E][I](Eq. 1) The rate of association was either under second order conditions ([E]0 = [I]0) or pseudo-first order conditions ([I]0 > 10[E]0). In the former case, non-linear regression analysis was used to fit the data to the following second-order equation,[E]=[E]0(1+[E]0·ka·t) where E is the concentration of free enzyme at any time, t, and E0 is the concentration at t = 0. E0 and E are proportional to the rate of substrate hydrolysis at t = 0 and at any time, respectively. When pseudo-first order rate constants were used, the data were fitted to the following exponential equation,[E]=[E]0·e-ka·[I]0·t where I0 is the I concentration at t = 0. Clotting Assays—The tests were performed on a pool of citrated platelet-poor human plasma from six healthy, fully informed, and consenting adult volunteers. Aliquots were stored at -80 °C until analysis. Recombinant Iris was dialyzed in phosphate-buffered saline devoid of Ca2+ and Mg2+. The effects of 1.5-6 μm rIris on coagulation time were evaluated on BCT machine (Dade, Behring). Phospholipids and activators (Innovin 1/200 for tissue factor, PTT 1/4 for contact phase activation) were added to 50 μl of spiked plasma. Clotting time was recorded after the addition of 50 μl of 25 mm CalCl2. Cell-based clotting time assays were performed by adding 6 μm rIris to a mixture of human plasma and the human monocytes THP1 (106 cells/ml final). The mix was then incubated at 37 °C for 5 min. Clotting times were measured after addition of 100 μl of 25 mm CaCl2. Euglobulin Clot Lysis Time—The euglobulin fraction was prepared as described by Zouaoui Boudjeltia et al. (43Zouaoui Boudjeltia K. Cauchie P. Remacle C. Guillaume M. Brohee D. Hubert J.L. Vanhaeverbeek M. BMC Biotech. 2002; 2: 8Crossref PubMed Scopus (34) Google Scholar). Briefly, 300 μl of acetic acid (0.025%) and 3.6 ml of deionized water were added to 400 μl of pooled human plasma. The sample was centrifuged at 4.0 × 103 × g for 10 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in 400 μl of Hanks' balanced salt solution (HBSS) containing increasing concentrations of rIris, with or without added neutrophils (final concentration 5 × 106 per ml). Clot formation was started by adding 100 μl (1.5 units/ml) of thrombin. ECLT was measured by a semi-automatic method using a "Lysis Timer" device (43Zouaoui Boudjeltia K. Cauchie P. Remacle C. Guillaume M. Brohee D. Hubert J.L. Vanhaeverbeek M. BMC Biotech. 2002; 2: 8Crossref PubMed Scopus (34) Google Scholar). In addition, another test was performed to exclude the effects of the interaction of Iris on the cell surface. Cells were incubated with rIris for 1 h and then washed three times in phosphate-buffered saline. Washed cells were then resuspended in HBSS before use in the ECLT test. Primary Hemostasis—Blood samples were collected from five healthy, fully informed, and consenting volunteers in 0.13 m citrate vacuum tubes. Global platelet function was measured on PFA-100® machine (Dade, Behring) with the collagen/epinephrine cartridge. This apparatus creates an artificial vessel consisting of a bioactive membrane with a microscopic aperture (44Kundu S.K. Heilmann E.J. Sio R. Garcia C. Davidson R.M. Ostgaard R.A. Semin. Thromb. Hemost. 1995; 21: 106-112Crossref PubMed Scopus (131) Google Scholar). The sample (1/10 protein in HBSS and 9/10 anti-coagulated whole blood) was aspirated through a capillary under steady high shear rates (5000-6000 s-1) within 2 h of sample collection. The presence of the platelet agonist and the high shear rates result in a platelet plug that gradually occludes the aperture. The closure time is considered to be the time required to obtain full occlusion of the aperture. Statistical Analyses—SigmaStat® software (Jandel Scientific, Erkrath, Germany) was used for the analysis. Data are represented as mean ± S.D. and were evaluated by one-way ANOVA. The effect of rIris on platelet adhesion was evaluated by a Friedman repeated measures analysis of variance on ranks test. Molecular Modeling—Iris had previously been found to be similar to the pig leukocyte elastase inhibitor (35Leboulle G. Crippa M. Decrem Y. Mejri N. Brossard M. Bollen A. Godfroid E. J. Biol. Chem. 2002; 277: 10083-10089Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). In addition, membership in the serpin superfamily was confirmed by the high conservation of consensus residues identified by Irving and colleagues (45Irving J.A. Pike R.N. Lesk A.M. Whisstock J.C. Genome Res. 2000; 10: 1845-1864Crossref PubMed Scopus (507) Google Scholar). All conserved residues among serpins are also conserved in Iris except for the replacement of Phe-106 by Tyr, Ile-134 by Val, Gly-144 by Ser, Leu-288 by Met, and Pro-377 by Leu. Alignment of hydrophobic clusters also supported homologous folds (data not shown). Therefore, we sought to build a three-dimensional model of the entire protein based on the homology (Fig. 1) using Modeler (36Sali A. Blundell T.L. J. Mol. Biol. 1993; 234: 779-815Crossref PubMed Scopus (10456) Google Scholar). To find an accurate template, PDB was searched using the Blast algorithm. Horse leukocyte elastase inhibitor and α-antitrypsin both displaying over 30% identity with Iris (Fig. 2) were chosen as templates for the models corresponding to the cleaved state and uncleaved state, respectively (Fig. 1, A and B). Models were tested with Procheck, and all the residues were in the allowed regions of the Ramachandran plot.FIGURE 2Alignment of Iris with α-antitrypsin and horse leukocyte elastase inhibitor. Amino acid sequence alignment between α-antitrypsin (PDB code: 1ATU) from residue 46 to 418, Iris from residue 1 to 377, and horse leukocyte elastase inhibitor (PDB code: 1HLE) from residue 1 to 344. This alignment was obtained using the ClustalW algorithm. Dots indicate similar amino acids, and stars indicate identical amino acids. Indents indicate gaps. Conserved domains are boxed. The open boxes indicate the position of the RCL domain and the highly conserved serpin residues (s3a domain). The shaded box stands for the serpin signature (Prosite code: PS00284) in Iris.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The root mean square deviation (r.m.s.d.) between the uncleaved model (Fig. 1A) and its template is 0.44 Å (on 363 Cα). In contrast, the calculated r.m.s.d. between ovalbumin and α-antitrypsin is 1.57 Å. We also compared the predicted RCL of Iris with the corresponding amino acid sequence of inhibitory and non-inhibitory serpins (Fig. 3). The RCL from Iris was aligned to the consensus RCL from inhibitory serpins (46Hopkins P.C. Carrell R.W. Stone S.R. Biochemistry. 1993; 32: 7650-7657Crossref PubMed Scopus (166) Google Scholar), among these Glu-324, Gly-325, Thr-326, Ala-328, and Ala-332 were perfectly conserved. All conserved amino acids essential for the inhibitory function were present in Iris (Fig. 2). This alignment suggested that Iris has an inhibitory function based on the presence of methionine 340 adjacent to the cleavage site (position P1). We further modeled and analyzed the interactions of Iris with trypsin to find the residues that make contact with the protease (Fig. 1C). The complex between trypsin and alaserpin (PDB code: 1K9O) was used as a template for this model. The r.m.s.d. between alaserpin and Iris is 0.22 Å. Using the Pex algorithm, we determined the residues involved in the interaction. Most of the important residues were located in the RCL, some specifically in the Hinge region (Fig. 3). rIris Expression and Purification—The coding sequence of Iris was cloned in the vector pBlueBac4.5-V5-His (Invitrogen) in-frame with the V5-His tag, expressed in a baculovirus system and purified to homogeneity as confirmed by SDS-PAGE (data not shown). Affinity-purified rIris migrated at an apparent mass of 48 kDa on a 10% SDS-polyacrylamide gel (data not shown). This contrasted with an expected molecular weight of 44 kDa and suggested putative posttranslational modifications. Inhibition of Protease Activity by rIris—We tested the ability of purified rIris to inhibit various serine proteases derived from human blood (leukocyte elastase, t-PA, factor Xa, plasmin, thrombin, and trypsin) or from porcine pancreas (pancreatic elastase), as well as two human cysteine proteases (caspase-3 and papain), and one human metalloelastase (MMP12). Recombinant Iris was incubated for 1 h at 37 °C with the various proteases, and the residual proteolytic activity was measured using synthetic chromogenic substrates. Most of the serine proteases were inhibited by rIris, as opposed to the two cysteine proteases and the metalloelastase, which did not show any significant drop of activity in presence of rIris (Table 1). From these data, the serine proteases could be distributed into three groups: (i) human leukocyte elastase and porcine pancreas elastase with a residual activity of ∼30%; (ii) thrombin, t-PA, and factor Xa with a residual activity near 70%; and (iii) plasmin with 100% residual activity (no inhibitory effect of rIris).TABLE 1Affinity constants and inhibition rate of proteases by rIris and its mutants Proteases were incubated at an equimolar ratio with rIris or its mutants for 1 h at 37 °C. Residual activity in the presence of ovalbumin, a serpin with no inhibitory activity, is used as the 100% reference point (see "Materials and Methods"). The mutants A332P and L339A were not tested on p