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• W2034915097 abstract "To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the α clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig α-chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-Pro-Leu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe8 to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by α1-antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster α-chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe- and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala216 side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase. To explore guinea pigs as models of chymase biology, we cloned and expressed the guinea pig ortholog of human chymase. In contrast to rats and mice, guinea pigs appear to express just one chymase, which belongs to the α clade, like primate chymases and mouse mast cell protease-5. The guinea pig enzyme autolyzes at Leu residues in the loop where human chymase autolyzes at Phe. In addition, guinea pig α-chymase selects P1 Leu in a combinatorial peptide library and cleaves Ala-Ala-Pro-Leu-4-nitroanilide but has negligible activity toward substrates with P1 Phe and does not cleave angiotensin I. This contrasts with human chymase, which cleaves after Phe or Tyr, prefers P1 Phe in peptidyl 4-nitroanilides, and avidly hydrolyzes angiotensin I at Phe8 to generate bioactive angiotensin II. The guinea pig enzyme also is inactivated more effectively by α1-antichymotrypsin, which features P1 Leu in the reactive loop. Unlike mouse, rat, and hamster α-chymases, guinea pig chymase lacks elastase-like preference for P1 Val or Ala. Partially humanized A216G guinea pig chymase acquires human-like P1 Phe- and angiotensin-cleaving capacity. Molecular models suggest that the wild type active site is crowded by the Ala216 side chain, which potentially blocks access by bulky P1 aromatic residues. On the other hand, the guinea pig pocket is deeper than in Val-selective chymases, explaining the preference for the longer aliphatic side chain of Leu. These findings are evidence that chymase-like peptidase specificity is sensitive to small changes in structure and provide the first example of a vertebrate Leu-selective peptidase. Chymases are serine peptidases expressed and secreted mainly by mast cells. They are proposed to play a variety of roles in host defense, homeostasis and disease, including anti-parasite defense (1Knight P.A. Wright S.H. Lawrence C.E. Paterson Y.Y. Miller H.R. J. Exp. Med. 2000; 192: 1849-1856Crossref PubMed Scopus (247) Google Scholar), blood pressure regulation (2Ju H. Gros R. You X. Tsang S. Husain M. Rabinovitch M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 7469-7474Crossref PubMed Scopus (88) Google Scholar, 3Li M. Liu K. Michalicek J. Angus J.A. Hunt J.E. Dell'Italia L.J. Feneley M.P. Graham R.M. Husain A. J. Clin. Invest. 2004; 114: 112-120Crossref PubMed Scopus (91) Google Scholar), connective tissue turnover (4Tchougounova E. Lundequist A. Fajardo I. Winberg J.O. Abrink M. Pejler G. J. Biol. Chem. 2005; 280: 9291-9296Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), angiogenesis (5Coussens L.M. Raymond W.W. Bergers G. Laig-Webster M. Behrendtsen O. Werb Z. Caughey G.H. Hanahan D. Genes Dev. 1999; 13: 1382-1397Crossref PubMed Scopus (791) Google Scholar), cardiovascular remodeling (6Nishimoto M. Takai S. Kim S. Jin D. Yuda A. Sakaguchi M. Yamada M. Sawada Y. Kondo K. Asada K. Iwao H. Sasaki S. Miyazaki M. Circulation. 2001; 104: 1274-1279Crossref PubMed Scopus (70) Google Scholar), ischemia-reperfusion injury (7Abonia J.P. Friend D.S. Austen Jr., W.G. Moore Jr., F.D. Carroll M.C. Chan R. Afnan J. Humbles A. Gerard C. Knight P. Kanaoka Y. Yasuda S. Morokawa N. Austen K.F. Stevens R.L. Gurish M.F. J. Immunol. 2005; 174: 7285-7291Crossref PubMed Scopus (72) Google Scholar), lung fibrosis (8Sakaguchi M. Takai S. Jin D. Okamoto Y. Muramatsu M. Kim S. Miyazaki M. Eur. J. Pharmacol. 2004; 493: 173-176Crossref PubMed Scopus (37) Google Scholar), airway inflammation (9Greco M.N. Hawkins M.J. Powell E.T. Almond Jr., H.R. de Garavilla L. Hall J. Minor L.K. Wang Y. Corcoran T.W. Di Cera E. Cantwell A.M. Savvides S.N. Damiano B.P. Maryanoff B.E. J. Med. Chem. 2007; 50: 1727-1730Crossref PubMed Scopus (52) Google Scholar, 10de Garavilla L. Greco M.N. Sukumar N. Chen Z.W. Pineda A.O. Mathews F.S. Di Cera E. Giardino E.C. Wells G.I. Haertlein B.J. Kauffman J.A. Corcoran T.W. Derian C.K. Eckardt A.J. Damiano B.P. Andrade-Gordon P. Maryanoff B.E. J. Biol. Chem. 2005; 280: 18001-18007Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and venom detoxification (11Metz M. Piliponsky A.M. Chen C.C. Lammel V. Abrink M. Pejler G. Tsai M. Galli S.J. Science. 2006; 313: 526-530Crossref PubMed Scopus (281) Google Scholar). Most chymases can cleave a variety of extracellular peptides and proteins, including endogenous targets like angiotensin I and exogenous pathogen or allergen-derived targets like profilin (12Mellon M.B. Frank B.T. Fang K.C. J. Immunol. 2002; 168: 290-297Crossref PubMed Scopus (28) Google Scholar). Because the pool of cleavable host and pathogen targets is large, chymases have the potential to influence a broad range of events associated with mast cell activation and secretion. The closest relatives of chymases are cathepsin G and granzyme B-like proteins, which are expressed in myelomonocytes and cytolytic lymphocytes, respectively, in addition to mast cells. Genes encoding these enzymes are tightly clustered in mammalian genomes. In humans, four genes in this group are expressed: chymase (CMA1), cathepsin G (CTSG), granzyme B (GZMB), and granzyme H (GZMH). In mice and rats, however, chymase and granzyme B-like genes markedly expanded and diversified by gene duplication, conversion, and point mutation (13Caughey G.H. Curr. Resp. Med. Rev. 2006; 2: 263-277Crossref PubMed Scopus (13) Google Scholar, 14Gallwitz M. Reimer J.M. Hellman L. Immunogenetics. 2006; 58: 655-669Crossref PubMed Scopus (53) Google Scholar). The rat locus has an estimated 66 genes, of which 28 may be expressed (15Puente X.S. Lopez-Otin C. Genome Res. 2004; 14: 609-622Crossref PubMed Scopus (158) Google Scholar). Although products of chymase, cathepsin G, and granzyme genes are structurally and phylogenetically related, they have a range of roles and target specificities. Granzyme B, for example, is an Aspase that cleaves specific host proteins and kills tumor and other target cells by triggering apoptosis, although humans and mice differ in this regard (16Kaiserman D. Bird C.H. Sun J. Matthews A. Ung K. Whisstock J.C. Thompson P.E. Trapani J.A. Bird P.I. J. Cell Biol. 2006; 175: 619-630Crossref PubMed Scopus (158) Google Scholar). Cathepsin G, on the other hand, has broad activity (tryptic, chymotryptic, Met-ase) (17Polanowska J. Krokoszynska I. Czapinska H. Watorek W. Dadlez M. Otlewski J. Biochim. Biophys. Acta. 1998; 1386: 189-198Crossref PubMed Scopus (66) Google Scholar) and regulates adhesion-dependent function of neutrophils (18Raptis S.Z. Shapiro S.D. Simmons P.M. Cheng A.M. Pham C.T. Immunity. 2005; 22: 679-691Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Within the chymase group, expressed proteins examined to date are either primarily chymotryptic (hydrolyzing peptides after residues with aromatic side chains) or elastolytic (hydrolyzing after residues with small, aliphatic side chains) (19Kunori Y. Koizumi M. Masegi T. Kasai H. Kawabata H. Yamazaki Y. Fukamizu A. Eur. J. Biochem. 2002; 269: 5921-5930Crossref PubMed Scopus (60) Google Scholar, 20Karlson U. Pejler G. Tomasini-Johansson B. Hellman L. J. Biol. Chem. 2003; 278: 39625-39631Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Even among chymotryptic chymases, there is a range of catalytic competence and target preference. For example, mammalian chymotryptic chymases vary in their tendency to hydrolyze after one or the other of two aromatic residues in angiotensin I (21Caughey G.H. Raymond W.W. Wolters P.J. Biochim. Biophys. Acta. 2000; 1480: 245-257Crossref PubMed Scopus (173) Google Scholar, 22Muilenburg D.J. Raymond W.W. Wolters P.J. Caughey G.H. Biochim. Biophys. Acta. 2002; 1596: 346-356Crossref PubMed Scopus (21) Google Scholar). Cleavage at one site activates angiotensin I to angiotensin II, whereas cleavage at the other destroys activity. These preferences are influenced by sometimes subtle variations of amino acids in the vicinity of the substrate-binding site (22Muilenburg D.J. Raymond W.W. Wolters P.J. Caughey G.H. Biochim. Biophys. Acta. 2002; 1596: 346-356Crossref PubMed Scopus (21) Google Scholar). In the case of elastolytic chymases, notably the mouse chymase MCP-5 and its rat ortholog, the switch from chymotryptic to elastolytic specificity is attributable to natural mutation of a single amino acid (19Kunori Y. Koizumi M. Masegi T. Kasai H. Kawabata H. Yamazaki Y. Fukamizu A. Eur. J. Biochem. 2002; 269: 5921-5930Crossref PubMed Scopus (60) Google Scholar, 20Karlson U. Pejler G. Tomasini-Johansson B. Hellman L. J. Biol. Chem. 2003; 278: 39625-39631Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Similar elastolytic properties have recently been described for hamster α chymase (23Kervinen J. Abad M. Crysler C. Kolpak M. Mahan A.D. Masucci J.A. Bayoumy S. Cummings M.D. Yao X. Olson M. de Garavilla L. Kuo L. Deckman I. Spurlino J. J. Biol. Chem. 2008; 283: 427-436Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar). Thus, small changes in structure, including alteration of just one amino acid in the vicinity of the substrate-binding site, can cause large changes in function. Guinea pigs are used to model allergic events and a variety of other phenomena. Although the properties of trypsin-like mast cell tryptases have been explored in guinea pigs, the enzymatic properties of their chymases have been an enigma, especially because unlike in primates, dogs, and hamsters, there is little evidence that chymase-like enzymes in guinea pigs contribute to angiotensin I-induced vascular contraction (24Miyazaki M. Takai S. Jin D. Muramatsu M. Pharmacol. Ther. 2006; 112: 668-676Crossref PubMed Scopus (99) Google Scholar). However, guinea pigs can respond to exogenous chymotryptic peptidase, because human chymase injected into guinea pig skin increases microvascular permeability and provokes neutrophilic and eosinophilic inflammation (25He S. Walls A.F. Br. J. Pharmacol. 1998; 125: 1491-1500Crossref PubMed Scopus (131) Google Scholar). The present work shows that guinea pigs express an α-chymase-like peptidase with novel specificity for Leu in peptide and protein targets. Cloning and Sequencing of Guinea Pig Chymase cDNA— Fresh guinea pig (Cavia porcellus) heart muscle was harvested into RNAlater (Qiagen). Total RNA was extracted from homogenates following the RNeasy Mini procedure (Qiagen). Single-stranded cDNA was synthesized using random hexamers. To amplify a portion of guinea pig chymase by homology PCR, rodent mast cell protease consensus sequence was used to design the following primers: 5′-GAGTCAAAGCCACACTCCCGCCCTTACATGG and 5′-KYRCACASDARDGGNCCNCCDGAGTCYCC. Amplimers were cloned into pCR2.1-TOPO-TA (Invitrogen) and sequenced. Primers for rapid amplification of cDNA ends were designed from the obtained sequence. Amplimers obtained by 3′-rapid amplification of cDNA ends using primer 5′-GAGAAAGACTGTAGTGGTTTCTTGATAC were cloned and sequenced. To obtain complete cDNA sequence, nested reverse transcriptase-PCR was performed with a 5′-rapid amplification of cDNA ends protocol (Invitrogen), using 5′-ACCCCTAGGTTGACTGTTAG and 5′-GTATCAAGAAACCACTACAGTCTTTCTC as first and second stage primer, respectively. The resulting amplimer was cloned and sequenced. To assess for potential polymorphisms, protein-coding sequence was verified by PCR cloning and DNA sequencing of a transcript from a guinea pig from an alternate supplier. Phylogenetic Analysis—Guinea pig chymase amino acid sequence was compared with that of cathepsin G and 27 other chymase-like mammalian peptidases using Geneious software (Biomatters). Rooted dendrograms were prepared from aligned sequences using the unweighted pair group method with arithmetic mean (UPGMA) 2The abbreviations used are: UPGMA, unweighted pair group with arithmetic mean; MES, 2-(N-morpholino)ethanesulfonic acid; NA, nitroanilide; MCP, mast cell protease; Fmoc, N-(9-fluorenyl)methoxycarbonyl; contig, group of overlapping clones. or neighbor-joining techniques with bootstrap resampling. Generation of Humanized Mutant Guinea Pig Chymase cDNA—Guinea pig chymase was partially humanized using a QuikChange multisite-directed mutagenesis kit (Stratagene) with primer 5′-GCCCAGGGCATTGTATCCTATGGTCATCGGAATG. This changes the codon encoding specificity triad residue Ala216 into a codon for Gly. The resulting A216G mutant cDNA was used to express recombinant mutant enzyme, as described below. Expression and Mutagenesis of Recombinant Chymases—For expression of guinea pig chymases in BL21(DE3) E. coli, primers 5′-CATATGGATGACGACGACAAGCTCCCATTACCGGCCGTG and 5′-GCGGCCGCTTAATTTGCTTTCAAGATCTTGTTGATC were used to PCR-clone mature peptide-coding sequence into NdeI and NotI sites of vector pET28a (Stratagene). Primers were designed so that enteropeptidase cleavage of expressed product, which has a 26-residue prosequence containing hexahistidine and an enteropeptidase cleavage recognition sequence, yields a peptide matching that of predicted wild type mature protein. Inclusion bodies containing recombinant guinea pig chymase were isolated and purified from E. coli extracts. Insoluble protein from lysed cells was dissolved in 0.1 m Tris-HCl (pH 8.0), 1 mm β-mercaptoethanol, and 6 m guanidine HCl and chromatographed in the same buffer over an Ni2+-nitrilotriacetic acid column. Purified, denatured protein then was incubated with a mixture of oxidized glutathione (150 mm) and reduced glutathione (1.5 mm) and then dialyzed overnight against H2O (pH 4–4.5) at 4 °C. Precipitated protein was recovered by centrifugation and dissolved in 6 m guanidine HCl, 20 mm EDTA (pH 4.5). Refolding was accomplished by dilution into a large volume of refolding buffer: 50 mm Tris-HCl (pH 8.0), 0.5 m arginine, 1 mm EDTA, and 0.5 mm cysteine. After incubation for 2 days at 4 °C, the preparation was concentrated and dialyzed in 50 mm Tris-HCl (pH 7.0) containing 0.3 m NaCl and 1 mm β-mercaptoethanol, purified by Ni2+-nitrilotriacetic acid chromatography, and activated by incubation with enteropeptidase. The activated preparation was further purified by gel filtration in Superdex 75 using running buffer containing 50 mm Tris-HCl (pH 8.0), 0.3 m NaCl, 1 mm tris(2-carboxyethyl) phosphine, and 10% glycerol, followed by rechromatography in 50 mm MES (pH 5.5), 150 mm NaCl, 1 mm tris(2-carboxyethyl) phosphine, and 10% glycerol. The purified protein was monomeric by gel filtration and analytical ultracentrifugation. For kinetic and inhibitor studies, recombinant human chymase expressed in insect cells was prepared, activated, and purified as described (21Caughey G.H. Raymond W.W. Wolters P.J. Biochim. Biophys. Acta. 2000; 1480: 245-257Crossref PubMed Scopus (173) Google Scholar). Active chymase concentration in samples used in kinetic studies was estimated using the specific activity of highly purified recombinant human chymase assayed under standard conditions (21Caughey G.H. Raymond W.W. Wolters P.J. Biochim. Biophys. Acta. 2000; 1480: 245-257Crossref PubMed Scopus (173) Google Scholar). Protein Sequencing—Purified, self-incubated, recombinant wild type guinea pig chymase (∼100 pmol) was desalted by application to and washing of a polyvinylidene difluoride membrane. Membrane-bound protein was subjected directly to N-terminal amino acid sequencing on a Procise 494 HT sequencer (PerkinElmer Life Sciences). Combinatorial Peptide Library Survey of Cleavage Site Preferences—Fluorescence resonance energy transfer substrate combinatorial libraries were synthesized on PEGA1900 resin via split synthesis after standard Fmoc chemistry using 2-(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate as the condensing reagent. Fmoc-Gly-OH, Fmoc-Glu-Lucifer Yellow, and all Fmoc-protected amino acids were coupled to amino PEGA1900 resin in the presence of diisopropylethylamine in N-methylpyrrolidone. Double coupling was performed using O-(1,2-dihydro-2-oxo-1-pyridyl)-N,N,N′,N′-tetramethyluronium tetrafluoroborate in the presence of diisopropylethylamine in N-ethylpyrrolidone. Synthesis was performed on a semiautomated shaking vessel machine. For variable positions in the peptide sequences, the resin was split into 19 portions, to each of which a different Fmoc-protected amino acid (except Fmoc-Cys-OH) was coupled. The portions then were mixed together and washed. Depending on the library, splitting was repeated, or the synthesis proceeded in a single portion. The last building block was t-butoxycarbonyl-Lys-(dabsyl)-OH. Protecting groups were removed by incubation with trifluoroacetic acid/H2O/triisopropylsilane (95:3:2 by volume). For screening of the Lys(dabsyl)-QAXXXXAQQG-Glu(Gly-PEGA)-Lucifer Yellow peptide library, ∼5000 beads were washed with reaction buffer (50 mm Tris/HCl, pH 8.0, 300 mm NaCl, 1 mm tris(2-carboxyethyl)-phosphine, 10% glycerol). Purified enzyme (50 μg/ml in 1 ml) in reaction buffer was added to the resin. After shaking for 2–3 h at room temperature, beads were filtered and washed first with reaction buffer and then with 100 mm MES (pH 4.8) containing 5% glycerol and 0.05% Triton X-100. Brightly fluorescent beads identified by microscopy were isolated and submitted for N-terminal sequencing. Screening of the SSVXAXSAPG peptide library was similar. Analysis of Inhibitor Sensitivity—Enzymes were preincubated for 15 min at 25 °C with or without a potential inhibitor and then assayed for residual activity, as assessed with Suc-AAPL-4-NA (wild type guinea pig chymase) and Suc-AAPF-4-NA (A216G guinea pig mutant, human chymase, bovine chymotrypsin) substrates (Sigma). Concentrations of wild type and mutant guinea pig chymase, human chymase, and chymotrypsin (Sigma) were 110, 58, 29, and 8.4 nm, respectively. Concentrations of soybean trypsin inhibitor, phenylmethanesulfonyl fluoride, chymostatin (Sigma), α1-antitrypsin, α1-antichymotrypsin (EMD Chemicals), and E-64 (MP Biomedical) were 70 μm, 0.25 mm, 2.5 μm, 0.25 μm, 0.25 mm, and 0.5 mm, respectively. Active Site Titration—To estimate active enzyme concentration, amidolytic activity of purified preparations of wild type and A216G mutant guinea pig chymase was titrated with α1-antichymotrypsin as described by Karlson et al. (26Karlson U. Pejler G. Froman G. Hellman L. J. Biol. Chem. 2002; 277: 18579-18585Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Active site-titrated preparations were assayed under standard conditions to obtain a specific activity, which was used to determine the concentration of active enzyme in future studies. Determination of Hydrolysis Kinetics—Kinetic parameters, including catalytic constant kcat and Michaelis constant Km, were derived from double reciprocal plots of substrate concentration versus reaction velocity for wild type and A216G mutant guinea pig chymase and human chymase cleaving a panel of peptidyl 4-NA substrates and angiotensin I. Hydrolysis of 4-NA substrates was assessed spectophotometrically at 410 nm. Assay buffers contained phosphate-buffered saline with 0.01% Triton X-100 and 0.05% dimethyl sulfoxide, with concentrations of 4-NA substrates ranging from 71 to 425 μm. Cleavage of angiotensin I was monitored by reverse phase high performance liquid chromatography of fragments generated by incubation with guinea pig and human chymases using modifications of approaches described previously (21Caughey G.H. Raymond W.W. Wolters P.J. Biochim. Biophys. Acta. 2000; 1480: 245-257Crossref PubMed Scopus (173) Google Scholar, 22Muilenburg D.J. Raymond W.W. Wolters P.J. Caughey G.H. Biochim. Biophys. Acta. 2002; 1596: 346-356Crossref PubMed Scopus (21) Google Scholar). Peptidases were incubated with angiotensin I (8–24 nm) in phosphate-buffered saline (pH 7.4) in aliquots of 50 μl at 37 °C. Reactions were stopped by the addition of 1 μl of 12 n HCl and then diluted with 60 μl of an aqueous solution of 10% acetonitrile, 0.1% trifluoroacetic acid. Outflow of samples chromatographed on a 2.1 × 250-mm Thermo Fisher Scientific BioBasic C-18 column with a linear gradient of 10–40% acetonitrile, 0.1% trifluoroacetic acid was monitored at 280 nm. Peak areas were determined using Unicorn 5.0 software (GE Healthcare). Molecular Modeling—Models of human and hamster 2 chymases were prepared from crystal-based coordinates from Protein Data Bank 1pjp (27Pereira P.J.B. Wang Z.M. Rubin H. Huber R. Bode W. Schechter N.M. Strobl S. J. Mol. Biol. 1999; 286: 163-173Crossref PubMed Scopus (55) Google Scholar) and 2rdl (23Kervinen J. Abad M. Crysler C. Kolpak M. Mahan A.D. Masucci J.A. Bayoumy S. Cummings M.D. Yao X. Olson M. de Garavilla L. Kuo L. Deckman I. Spurlino J. J. Biol. Chem. 2008; 283: 427-436Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar), respectively. Homology models of wild type and A216G guinea pig chymase were built by Swiss-Model (available on the World Wide Web) starting with the 1pjp scaffold. Images were produced using Chimera (available on the World Wide Web) from the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco and rendered with POV-Ray (available on the World Wide Web). Amino Acid Sequence of Guinea Pig Preprochymase—As shown in Fig. 1, the guinea pig chymase cDNA and gene sequence predict a 337-amino acid protein identical in length to human chymase and to the mouse ortholog MCP-5, both of which are α-chymases (see Fig. 2). Overall, the guinea pig sequence is slightly more similar (77% identical) to human chymase than to mouse chymase MCP-5 (75% identical), which is its closest relative among several chymase-like proteins in rodents. The guinea pig protein features a typical 19-residue signal peptide followed by an acidic dipeptide (Gly-Glu) similar or identical to the propeptide featured in other chymases and in cathepsin G, B-type granzymes, and neutrophil elastase-related peptidases, all of which are thought to be activated by dipeptidylpeptidase I (cathepsin C) (28Wolters P.J. Pham C.T. Muilenburg D.J. Ley T.J. Caughey G.H. J. Biol. Chem. 2001; 276: 18551-18556Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 29Pham C.T. Ley T.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8627-8632Crossref PubMed Scopus (338) Google Scholar, 30Adkison A.M. Raptis S.Z. Kelley D.G. Pham C.T. J. Clin. Invest. 2002; 109: 363-371Crossref PubMed Scopus (320) Google Scholar). The propeptide is followed by a 226-residue catalytic domain with an N-terminal Val, which is atypical for chymase and its relatives (which usually have an Ile at this site) but is present in some other trypsin family serine peptidases. Catalytic triad residues (His57, Asp102, and Ser195 using chymotrypsinogen numbering) essential for the amidolytic function of all serine peptidases are intact. However, “specificity triad” residues (189, 216, and 226 of chymotrypsinogen) that influence preferences for the amino acid at the site of hydrolysis in target peptides (31Wouters M.A. Liu K. Riek P. Husain A. Mol. Cell. 2003; 12: 343-354Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) are unique among enzymatically characterized serine peptidases (Table 1). The guinea pig triad differs in two residues from human chymase, which is chymotryptic, and in all triad residues in comparison with mouse MCP-5, which is elastolytic. A chymase-like peptidase we deduced from recently deposited whole genome shotgun sequence of the pika contains an identical specificity triad. However, this pika chymase is not closely related to guinea pig chymase in overall structure, as demonstrated by the phylogenetic analysis in Fig. 2.FIGURE 2Tree of α/β chymases. Phylogenetic relationships between guinea pig chymase and other chymases are probed with this rooted dendrogram generated by UPGMA analysis of aligned mature chymase amino acid sequences using 1000 iterations of bootstrap resampling. The threshold for node assignment was 70%. Tines of peptidases belonging to α and β clades are black and gray, respectively, except for guinea pig chymase, which is forward-hatched. Examples of cathepsin G, the closest relative of chymases, are included for comparison. Accession numbers of sequences used in tree construction are as follows: human and mouse cathepsin G (NP_001902 and CAA55290), mouse MCP-1, -2, -4, and -5 (AAB23194, NP_032597, NP_034909, and NP_034910), rat MCP-I and -II (AAB48268 and P00700), rat MCP-4 and -5 (U67907 and NP_037224), rat vascular chymase (AAC16657), hamster chymase 1 and 2 (BAA19932 and BAA28615), gerbil chymase 1 and 2 (P50340 and P50341), opossum chymase (XP_001369716), guinea pig chymase (this work; AM851020), cattle chymase 2 (XP_593156), sheep MCP-2 (P79204), dog chymase (NP_001013442), baboon chymase (AAA91159), rhesus chymase (BAA22070), M. fascicularis chymase (crab-eating macaque; BAA22070), chimpanzee chymase (XP_001170224), and human chymase (M64269). We deduced the sequence of additional chymases from unannotated whole genome shotgun sequence as follows: American pika (Ochotona princeps, AAYZ01164217), tree shrew (AAPY01734039), mouse lemur (ABDC01231172), galago (AAQR01659070), and marmoset (contig 1790.4 from the Washington University Genome Sequencing Center, available on the World Wide Web).View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Comparison of specificity triad residuesPeptidaseSpecificity triadP1 preferenceSource/ReferenceGuinea pig chymaseSAVLeu >> Met > PheThis workGuinea pig chymase A216GSGVPhe, Tyr >> Leu > MetThis workHuman chymaseSGAPhe, Tyr >> Leu, Trp > MetRef. 43Raymond W.W. Waugh Ruggles S. Craik C.S. Caughey G.H. J. Biol. Chem. 2003; 278: 34517-34524Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar and this workDog chymaseSGAPhe, Tyr > TrpRefs. 12Mellon M.B. Frank B.T. Fang K.C. J. Immunol. 2002; 168: 290-297Crossref PubMed Scopus (28) Google Scholar, 22Muilenburg D.J. Raymond W.W. Wolters P.J. Caughey G.H. Biochim. Biophys. Acta. 2002; 1596: 346-356Crossref PubMed Scopus (21) Google Scholar, 44Powers J.C. Tanaka T. Harper J.W. Minematsu Y. Barker L. Lincoln D. Crumley K.V. Fraki J.E. Schechter N.M. Lazarus G.G. Nakajima K. Nakashino K. Neurath H. Woodbury R.G. Biochemistry. 1985; 24: 2048-2058Crossref PubMed Scopus (146) Google Scholar, and 45Caughey G.H. Leidig F. Viro N.F. Nadel J.A. J. Pharmacol. Exp. Ther. 1988; 244: 133-137PubMed Google ScholarMouse chymase 4SGAPhe > Tyr > TrpRefs. 21Caughey G.H. Raymond W.W. Wolters P.J. Biochim. Biophys. Acta. 2000; 1480: 245-257Crossref PubMed Scopus (173) Google Scholar and 35Andersson M.K. Karlson U. Hellman L. Mol. Immunol. 2008; 45: 766-775Crossref PubMed Scopus (63) Google ScholarRat chymase 1SGAPhe > Trp > Tyr >> Leu? > MetRefs. 35Andersson M.K. Karlson U. Hellman L. Mol. 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A. 2000; 97: 7754-7759Crossref PubMed Scopus (466) Google ScholarHuman PSASGSMet > Ala > Tyr > Arg > Lys > Leu > PheRef. 50Debela M. Magdolen V. Schechter N. Valachova M. Lottspeich F. Craik C.S. Choe Y. Bode W. Goettig P. J. Biol. Chem. 2006; 281: 25678-25688Abstract Full Text Full Text PDF PubMed Scopus (133) Google ScholarRat chymase 2AGAPhe > Tyr >> MetRef. 46Yoshida N. Everitt M.T. Neurath H. Woodbury R.G. Powers J.C. Biochemistry. 1980; 19: 5799-5804Crossref PubMed Scopus (56) Go" @default.
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