Title: Tissue Expression, Protease Specificity, and Kunitz Domain Functions of Hepatocyte Growth Factor Activator Inhibitor-1B (HAI-1B), a New Splice Variant of HAI-1
Abstract: Hepatocyte growth factor activator inhibitor-1 (HAI-1) is an integral membrane protein expressed on epithelial cells and contains two extracellular Kunitz domains (N-terminal KD1 and C-terminal KD2) known to inhibit trypsin-like serine proteases. In tumorigenesis and tissue regeneration, HAI-1 regulates the hepatocyte growth factor (HGF)/c-Met pathway by inhibiting the activity of HGF activator (HGFA) and matriptase, two serine proteases that convert pro-HGF into its biologically active form. By screening a placental cDNA library, we discovered a new splice variant of HAI-1 designated HAI-1B that contains an extra 16 amino acids adjacent to the C terminus of KD1. To investigate possible consequences on Kunitz domain function, a soluble form of HAI-1B (sHAI-1B) comprising the entire extracellular domain was produced. First, we found that sHAI-1B displayed remarkable enzyme specificity by potently inhibiting only HGFA (IC50 = 30.5 nm), matriptase (IC50 = 16.5 nm), and trypsin (IC50 = 2.4 nm) among 16 serine proteases examined, including plasminogen activators (urokinase- and tissue-type plasminogen activators), coagulation enzymes thrombin, factors VIIa, Xa, XIa, and XIIa, and activated protein C. Relatively weak inhibition was found for plasmin (IC50 = 399 nm) and plasma kallikrein (IC50 = 686 nm). Second, the functions of the KD1 and KD2 domains in sHAI-1B were investigated using P1 residue-directed mutagenesis to show that inhibition of HGFA, matriptase, trypsin, and plasmin was due to KD1 and not KD2. Furthermore, analysis by reverse transcription-PCR demonstrated that HAI-1B and HAI-1 were co-expressed in normal tissues and various epithelial-derived cancer cell lines. Both isoforms were up-regulated in eight examined ovarian carcinoma specimens, three of which had higher levels of HAI-1B RNA than of HAI-1 RNA. Therefore, previously demonstrated roles of HAI-1 in various physiological and pathological processes likely involve both HAI-1B and HAI-1. Hepatocyte growth factor activator inhibitor-1 (HAI-1) is an integral membrane protein expressed on epithelial cells and contains two extracellular Kunitz domains (N-terminal KD1 and C-terminal KD2) known to inhibit trypsin-like serine proteases. In tumorigenesis and tissue regeneration, HAI-1 regulates the hepatocyte growth factor (HGF)/c-Met pathway by inhibiting the activity of HGF activator (HGFA) and matriptase, two serine proteases that convert pro-HGF into its biologically active form. By screening a placental cDNA library, we discovered a new splice variant of HAI-1 designated HAI-1B that contains an extra 16 amino acids adjacent to the C terminus of KD1. To investigate possible consequences on Kunitz domain function, a soluble form of HAI-1B (sHAI-1B) comprising the entire extracellular domain was produced. First, we found that sHAI-1B displayed remarkable enzyme specificity by potently inhibiting only HGFA (IC50 = 30.5 nm), matriptase (IC50 = 16.5 nm), and trypsin (IC50 = 2.4 nm) among 16 serine proteases examined, including plasminogen activators (urokinase- and tissue-type plasminogen activators), coagulation enzymes thrombin, factors VIIa, Xa, XIa, and XIIa, and activated protein C. Relatively weak inhibition was found for plasmin (IC50 = 399 nm) and plasma kallikrein (IC50 = 686 nm). Second, the functions of the KD1 and KD2 domains in sHAI-1B were investigated using P1 residue-directed mutagenesis to show that inhibition of HGFA, matriptase, trypsin, and plasmin was due to KD1 and not KD2. Furthermore, analysis by reverse transcription-PCR demonstrated that HAI-1B and HAI-1 were co-expressed in normal tissues and various epithelial-derived cancer cell lines. Both isoforms were up-regulated in eight examined ovarian carcinoma specimens, three of which had higher levels of HAI-1B RNA than of HAI-1 RNA. Therefore, previously demonstrated roles of HAI-1 in various physiological and pathological processes likely involve both HAI-1B and HAI-1. Hepatocyte growth factor activator inhibitor-1 (HAI-1) 1The abbreviations used are: HAI-1, hepatocyte growth factor activator inhibitor-1; HGF, hepatocyte growth factor; HGFA, hepatocyte growth factor activator; pro-HGF, single-chain hepatocyte growth factor; u-PA, urokinase-type plasminogen activator; CHO, Chinese hamster ovary; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline; KD1 and KD2, N- and C-terminal Kunitz domain of HAI-1B; sHAI-1B, soluble form of HAI-1B encompassing the extracellular domain. is an integral cell surface protein of 66 kDa expressed on epithelial cells (1Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 2Kataoka H. Shimomura T. Kawaguchi T. Hamasuna R. Itoh H. Kitamura N. Miyazawa K. Koono M. J. Biol. Chem. 2000; 275: 40453-40462Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar, 3Kataoka H. Suganuma T. Shimomura T. Itoh H. Kitamura N. Nabeshima K. Koono M. J. Histochem. Cytochem. 1999; 47: 673-682Crossref PubMed Scopus (104) Google Scholar). HAI-1 is known to inhibit the enzymatic activity of HGF activator (HGFA) (1Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar, 4Miyazawa K. Shimomura T. Kitamura A. Kondo J. Morimoto Y. Kitamura N. J. Biol. Chem. 1993; 268: 10024-10028Abstract Full Text PDF PubMed Google Scholar) and matriptase (5Takeuchi T. Shuman M.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11054-11061Crossref PubMed Scopus (232) Google Scholar, 6Lin C.-Y. Anders J. Johnson M. Sang Q.A. Dickson R.B. J. Biol. Chem. 1999; 274: 18231-18236Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar, 7Lin C.-Y. Anders J. Johnson M. Dickson R.B. J. Biol. Chem. 1999; 274: 18237-18242Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 8Benaud C. Dickson R.B. Lin C.-Y. Eur. J. Biochem. 2001; 268: 1439-1447Crossref PubMed Scopus (120) Google Scholar, 9Lee S.-L. Dickson R.B. Lin C.-Y. J. Biol. Chem. 2000; 275: 36720-36725Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar), two trypsin-like serine proteases capable of converting the inactive single-chain form of hepatocyte growth factor (pro-HGF) (10Gak E. Taylor W.G. Chan A.M.-L. Rubin J.S. FEBS Lett. 1992; 311: 17-21Crossref PubMed Scopus (82) Google Scholar, 11Naka D. Ishii T. Yoshiyama Y. Miyazawa K. Hara H. Hishida T. Kitamura N. J. Biol. Chem. 1992; 267: 20114-20119Abstract Full Text PDF PubMed Google Scholar, 12Naldini L. Tamagnone L. Vigna E. Sachs M. Hartmann G. Birchmeier W. Daikuhara Y. Tsubouchi H. Blasi F. Comoglio P.M. EMBO J. 1992; 11: 4825-4833Crossref PubMed Scopus (523) Google Scholar, 13Hartmann G. Naldini L. Weidner K.M. Sachs M. Vigna E. Comoglio P.M. Birchmeier W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 11574-11578Crossref PubMed Scopus (192) Google Scholar, 14Lokker N.A. Mark M.R. Luis E.A. Bennett G.L. Robbins K.A. Baker J.B. Godowski P.J. EMBO J. 1992; 11: 2503-2510Crossref PubMed Scopus (239) Google Scholar) into its biologically active two-chain form (HGF). When activated HGF binds to its receptor c-Met, it promotes phospho-transfer activity of the intracellular tyrosine kinase domain leading to activation of multiple intracellular signaling pathways. Therefore, as an inhibitor of HGFA and matriptase, HAI-1 may control the local generation of HGF and thus modulate the activity of the HGF/c-Met receptor system, which is involved in such biological processes as tissue regeneration, morphogenesis, and tumorigenesis (reviewed in Refs. 15Trusolino L. Comoglio P.M. Nat. Rev. Cancer. 2002; 2: 289-300Crossref PubMed Scopus (631) Google Scholar, 16Maulik G. Shrikhande A. Kijima T. Ma P.C. Morrison P.T. Salgia R. Cytokine Growth Factor Rev. 2002; 13: 41-59Crossref PubMed Scopus (366) Google Scholar, 17van der Voort R. Taher T.E.I. Derksen P.W.B. Spaargaren M. van der Neut R. Pals S.T. Adv. Cancer Res. 2000; 79: 39-90Crossref PubMed Google Scholar, 18Trusolino L. Pugliese L. Comoglio P.M. FASEB J. 1998; 12: 1267-1280Crossref PubMed Scopus (73) Google Scholar). The activation of the HGF-converting enzymes represents yet another level of HGF/c-Met pathway regulation. Similar to the coagulation factors, HGFA is mainly produced in the liver and circulates in blood as zymogen (19Shimomura T. Kondo J. Ochiai M. Naka D. Miyazawa K. Morimoto Y. Kitamura N. J. Biol. Chem. 1993; 268: 22927-22932Abstract Full Text PDF PubMed Google Scholar), but it can also be produced by cancer cells (20Parr C. Jiang W.G. Int. J. Oncol. 2001; 19: 857-863PubMed Google Scholar). During blood coagulation HGFA is converted into its active two chain form by thrombin (19Shimomura T. Kondo J. Ochiai M. Naka D. Miyazawa K. Morimoto Y. Kitamura N. J. Biol. Chem. 1993; 268: 22927-22932Abstract Full Text PDF PubMed Google Scholar). Matriptase is a type II transmembrane serine protease (21Hooper J.D. Clemente J.A. Quigley J.P. Antalis T.M. J. Biol. Chem. 2001; 276: 857-860Abstract Full Text Full Text PDF PubMed Scopus (323) Google Scholar) expressed as a single-chain form on epithelial cell types (5Takeuchi T. Shuman M.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11054-11061Crossref PubMed Scopus (232) Google Scholar, 22Oberst M. Anders J. Xie B. Singh B. Ossandon M. Johnson M. Dickson R.B. Lin C.-Y. Am. J. Pathol. 2001; 158: 1301-1311Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). It has been suggested that sphingosine 1-phosphate, a serum-derived lipoprotein, is able to convert matriptase zymogen into its enzymatically active two chain form (23Benaud C. Oberst M. Hobson J.P. Spiegel S. Dickson R.B. Lin C.-Y. J. Biol. Chem. 2002; 277: 10539-10546Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In addition to HGFA and matriptase, there are a number of other serine proteases that, at least in vitro, convert pro-HGF into its active form. One of them, coagulation factor XIIa (24Shimomura T. Miyazawa K. Komiyama Y. Hiraoka H. Naka D. Morimoto Y. Kitamura N. Eur. J. Biochem. 1995; 229: 257-261Crossref PubMed Scopus (167) Google Scholar), is not inhibited by HAI-1 (1Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar). The ability of HAI-1 to inhibit urokinase-type plasminogen activator (u-PA) (12Naldini L. Tamagnone L. Vigna E. Sachs M. Hartmann G. Birchmeier W. Daikuhara Y. Tsubouchi H. Blasi F. Comoglio P.M. EMBO J. 1992; 11: 4825-4833Crossref PubMed Scopus (523) Google Scholar, 25Mars W.M. Zarnegar R. Michalopoulos G.K. Am. J. Pathol. 1993; 143: 949-958PubMed Google Scholar, 26Naldini L. Vigna E. Bardelli A. Follenzi A. Galimi F. Comoglio P.M. J. Biol. Chem. 1995; 270: 603-611Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar) and two newly identified pro-HGF activators, plasma kallikrein and coagulation factor XIa (27Peek M. Moran P. Mendoza N. Wickramasinghe D. Kirchhofer D. J. Biol. Chem. 2002; 277: 47804-47809Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), is unknown. HAI-1 is expressed in many organs and specifically localizes to the surface of epithelial cells, particularly of the columnar epithelium (3Kataoka H. Suganuma T. Shimomura T. Itoh H. Kitamura N. Nabeshima K. Koono M. J. Histochem. Cytochem. 1999; 47: 673-682Crossref PubMed Scopus (104) Google Scholar, 22Oberst M. Anders J. Xie B. Singh B. Ossandon M. Johnson M. Dickson R.B. Lin C.-Y. Am. J. Pathol. 2001; 158: 1301-1311Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar, 28Kataoka H. Meng J.-Y. Itoh H. Hamasuna R. Shimomura T. Suganuma T. Koono M. Histochem. Cell Biol. 2000; 114: 469-475Crossref PubMed Scopus (27) Google Scholar). In addition, the expression of HAI-1 is enhanced or induced during tissue regeneration and inflammation and may regulate the HGFA-mediated activation of pro-HGF (3Kataoka H. Suganuma T. Shimomura T. Itoh H. Kitamura N. Nabeshima K. Koono M. J. Histochem. Cytochem. 1999; 47: 673-682Crossref PubMed Scopus (104) Google Scholar, 29Itoh H. Kataoka H. Tomita M. Hamasuna R. Nawa Y. Kitamura N. Koono M. Am. J. Physiol. 2000; 278: G635-G643Crossref PubMed Google Scholar). Moreover, HAI-1 and its target proteases HGFA and matriptase are implicated in the progression of breast cancer (20Parr C. Jiang W.G. Int. J. Oncol. 2001; 19: 857-863PubMed Google Scholar, 22Oberst M. Anders J. Xie B. Singh B. Ossandon M. Johnson M. Dickson R.B. Lin C.-Y. Am. J. Pathol. 2001; 158: 1301-1311Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar), hepatocellular carcinoma (30Nagata K. Hirono S. Ido A. Kataoka H. Moriuchi A. Shimomura T. Hori T. Hayashi K. Koono M. Kitamura N. Tsubouchi H. Biochem. Biophys. Res. Commun. 2001; 289: 205-211Crossref PubMed Scopus (28) Google Scholar), and ovarian cancer (31Oberst M.D. Johnson M.D. Dickson R.B. Lin C.-Y. Singh B. Stewart M. Williams A. al-Nafussi A. Smyth J.F. Gabra H. Sellar G.C. Clin. Cancer Res. 2002; 8: 1101-1107PubMed Google Scholar). In colorectal cancer, HAI-1 expression diminishes during the adenoma to adenocarcinoma transition, resulting in an imbalance between HGFA and its inhibitor, which was interpreted as contributing to the invasive tumor phenotype (32Kataoka H. Uchino H. Denda K. Kitamura N. Itoh H. Tsubouchi H. Nabeshima K. Koono M. Cancer Lett. 1998; 128: 219-227Crossref PubMed Scopus (25) Google Scholar, 33Kataoka H. Hamasuna R. Itoh H. Kitamura N. Koono M. Cancer Res. 2000; 60: 6148-6159PubMed Google Scholar). A similar enzyme/inhibitor imbalance was observed in ovarian cancer, in which cancer progression was associated with a marked reduction in HAI-1 antigen, whereas matriptase was only moderately diminished (31Oberst M.D. Johnson M.D. Dickson R.B. Lin C.-Y. Singh B. Stewart M. Williams A. al-Nafussi A. Smyth J.F. Gabra H. Sellar G.C. Clin. Cancer Res. 2002; 8: 1101-1107PubMed Google Scholar). Elevated levels of matriptase mRNA have also been observed in a wide variety of transformed cell lines (34Bhatt A.S. Takeuchi T. Yistra B. Ginzinger D. Albertson D. Shuman M.A. Craik C.S. Biol. Chem. 2003; 384: 257-266Crossref PubMed Scopus (27) Google Scholar). Additional studies using tissue microarrays have also implicated matriptase and HAI-1 in the progression of node-negative breast cancer (35Kang J.Y. Dolled-Filhart M. Ocal I.T. Singh B. Lin C.-Y. Dickson R.B. Rimm D.L. Camp R.L. Cancer Res. 2003; 63: 1101-1105PubMed Google Scholar). Therefore, HAI-1 may regulate the local generation of active HGF by HGFA or matriptase. In addition, HAI-1 may have a role in regulating matriptase-specific activities that could contribute to tumorigenicity and inflammation, such as the activation of u-PA and of G protein-coupled protease activated receptor-2 (9Lee S.-L. Dickson R.B. Lin C.-Y. J. Biol. Chem. 2000; 275: 36720-36725Abstract Full Text Full Text PDF PubMed Scopus (356) Google Scholar, 36Takeuchi T. Harris J.L. Huang W. Yan K.W. Coughlin S.R. Craik C.S. J. Biol. Chem. 2000; 275: 26333-26342Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Enzyme inhibition by HAI-1 is mediated by two Kunitz domains (N-terminal KD1 and C-terminal KD2) located in the extracellular domain. Both KD1 and KD2 can engage in protease inhibition (37Denda K. Shimomura T. Kawaguchi T. Miyazawa K. Kitamura N. J. Biol. Chem. 2002; 277: 14053-14059Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar), similar to the structurally related but more promiscuous HAI-2 (38Delaria K.A. Muller D.K. Marlor C.W. Brown J.E. Das R.C. Roczniak S.O. Tamburini P.P. J. Biol. Chem. 1997; 272: 12209-12214Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 39Kawaguchi T. Qin L. Shimomura T. Kondo J. Matsumoto K. Denda K. Kitamura N. J. Biol. Chem. 1997; 272: 27558-27564Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) (also referred to as placental bikunin (40Marlor C.W. Delaria K.A. Davis G. Muller D.K. Greve J.M. Tamburini P.P. J. Biol. Chem. 1997; 272: 12202-12208Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar) or kop (41Muller-Pillasch F. Wallrapp C. Bartels K. Varga G. Friess H. Buchler M. Adler G. Gress T.M. Biochim. Biophys. Acta. 1998; 1395: 88-95Crossref PubMed Scopus (50) Google Scholar)). Three splice variants were reported for HAI-2, designated as HAI-2A, HAI-2B, and HAI-2C (42Itoh H. Yamauchi M. Kataoka H. Hamasuna R. Kitamura N. Koono M. Eur. J. Biochem. 2000; 267: 3351-3359Crossref PubMed Scopus (26) Google Scholar). They differ in RNA expression levels, tissue distribution, and the number of Kunitz domains (one or two). In the present study we describe a splice variant of HAI-1, designated HAI-1B, which contains two Kunitz domains. The generation of soluble HAI-1B allowed us to study enzyme specificity as well as the contribution of each Kunitz domain to enzyme inhibition. Moreover, the expression of HAI-1B relative to HAI-1 in tissues and cells has been investigated, and the implications on our current understanding of the biology of HAI-1 and HAI-1B are discussed. Reagents—Pro-HGF, expressed in Chinese hamster ovary (CHO) cells in the absence of serum and purified by HiTrap Sepharose SP chromatography, was obtained from David Kahn (Genentech, Inc., South San Francisco, CA). The following synthetic substrates were used to measure enzyme activities: Cholinesterase PTC (propionylthiocholine and DTNB) (Sigma) for acetylcholinesterase, Spectrozyme® fVIIa (American Diagnostica, Greenwich, CT) for HGFA, Chromozym-tPA (Roche Applied Science) for tissue factor/factor VIIa. The following substrates were from Diapharma (Westchester, OH): S2765 for matriptase, S2222 for factor Xa, S2302 for plasma kallikrein, S2366 for activated protein C and plasmin, S2444 for urokinase-type plasminogen activator (u-PA), S2288 for factor XIa, factor XIIa and tissue-type plasminogen activator, S2314 for complement factor C1s, and S2586 for chymotrypsin. Except for bovine trypsin (Worthington, Lakewood, NJ), all of the enzymes used were of human origin. Factor Xa, factor XIa, thrombin, activated protein C, and plasmin were from Haematologic Technologies (Essex Junction, VT). Plasma kallikrein and factor XIIa were from American Diagnostica. u-PA, acetylcholinesterase, and chymotrypsin were from Sigma. Complement factor C1s was from Calbiochem (San Diego, CA). Tissue-type plasminogen activator was from Genentech, Inc. Soluble human tissue factor (residues 1–219) was produced in Escherichia coli as described (43Kirchhofer D. Eigenbrot C. Lipari M.T. Moran P. Peek M. Kelley R.F. Biochemistry. 2001; 40: 675-682Crossref PubMed Scopus (50) Google Scholar). Human recombinant factor VIIa was produced in 293 cells as described (44Dennis M.S. Eigenbrot C. Skelton N.J. Ultsch M.H. Santell L. Dwyer M.A. O'Connell M.P. Lazarus R.A. Nature. 2000; 404: 465-470Crossref PubMed Scopus (203) Google Scholar). All other reagents were of the highest quality available. Cloning, Expression, and Purification of HGFA—The active enzyme form of HGFA comprises the entire B-chain (Ile408–Ser655) disulfide-linked to the C-terminal 35 residues of the A-chain (Val373–Arg407) (4Miyazawa K. Shimomura T. Kitamura A. Kondo J. Morimoto Y. Kitamura N. J. Biol. Chem. 1993; 268: 10024-10028Abstract Full Text PDF PubMed Google Scholar). Therefore, to produce recombinant active HGFA, the nucleotide sequence encoding amino acids 373–655 was cloned by PCR from an HGFA full-length clone into the baculovirus expression vector pAcGP67A (Pharmingen, San Diego, CA) immediately 3′ to the gp67 secretion signal sequence. A nucleotide sequence encoding a three-residue Ala linker and a C-terminal poly-His tag (Ala3-His8) was added to the 3′-end. Isolated plasmid DNA was transfected into Spodoptera frugiperda (Sf9) cells on plates in ESF921 medium (Expression Systems, Woodland, CA) via the Baculogold Expression System according to the manufacturer's instructions (Pharmingen, San Diego, CA). The virus was amplified three times before use in protein production. One liter of High Five™ cells (Invitrogen, San Diego, CA) growing at 5 × 105 cells/ml in suspension in ESF921 medium was infected with 8 ml of viral stock. The cultures were incubated at 27 °C for 72 h before harvesting the culture medium by centrifugation at 8,000 × g for 15 min. NiCl2, CaCl2, and Tris-HCl, pH 8.0, were added to give final concentrations of 1, 5, and 50 mm, respectively. Precipitate was removed by filtration through a 0.2-μm filter, and the medium was then applied onto a 2-ml Ni-NTA-agarose column (Qiagen). After washing with 10 column volumes of 50 mm Tris-HCl, pH 8.0, 500 mm NaCl, 5 mm imidazole, the HGFA protein was eluted with 50 mm Tris-HCl, pH 8.0, 500 mm NaCl, 250 mm imidazole. The purity of HGFA protein (∼32 kDa) was greater than 95% by SDS-PAGE analysis. N-terminal sequencing of the protein bands indicated that activation cleavage at the Arg407–Ile408 bond occurred spontaneously during the expression/purification procedures, resulting in enzymatically active two-chain HGFA. The protein concentration was determined by quantitative amino acid analysis. Cloning, Expression, and Purification of Matriptase—A full-length clone of matriptase was obtained by standard PCR protocols from a mixture of human cDNA libraries including those from brain, heart, liver, lung, and spleen using 5′ primer GGACCATGGGGAGCGATCG and 3′ primer CCTATACCCCAGTGTTCTCTTTGATCCAGT. A fragment containing the gene was excised from a 1% agarose gel, purified, and ligated into the pCR4-TOPO vector (Invitrogen) according to the manufacturer's instructions. DNA sequencing confirmed an open reading frame of 855 residues identical to that previously described (5Takeuchi T. Shuman M.A. Craik C.S. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 11054-11061Crossref PubMed Scopus (232) Google Scholar). The nucleotide sequence encoding amino acids 615–855 encoding the mature protease domain was cloned by PCR from the full-length clone ultimately into plasmid pSTII.TIR3 variant 4 (45Simmons L.C. Yansura D.G. Nat. Biotechnol. 1996; 14: 629-634Crossref PubMed Scopus (86) Google Scholar) such that Val615 immediately followed the stII signal sequence, and a His8 tag was on the C terminus. This plasmid contained a phoA promoter, the stII signal sequence, and the λto transcriptional terminator. Site-directed mutagenesis was also carried out to make the C731S mutant to avoid potential complications of an unpaired Cys in the protease domain using the oligonucleotide 5′-CGGCCCATCTCCCTGCCGGAC with the QuikChange kit (Stratagene, La Jolla, CA). As used in this paper, matriptase refers to the matriptase protease domain starting with Val615 containing C731S and a C-terminal His8 tail. E. coli strain 33D3 (W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompT Δ(nmpc-fepE) degP41 kan R) was transformed with pSTII.MTSP.PD.H8. Single colonies from a LB carbenicillin plate were inoculated into 5 ml of LB medium supplemented with carbenicillin (50 μg/ml) and grown at 30 °C on a culture wheel overnight. The 5-ml culture was diluted into 500 ml of phosphate-limiting medium (46Simmons L.C. Reilly D. Klimowski L. Raju T.S. Meng G. Sims P. Hong K. Shields R.L. Damico L.A. Rancatore P. Yansura D.G. J. Immunol. Methods. 2002; 263: 133-147Crossref PubMed Scopus (240) Google Scholar). Carbenicillin was then added to the induction culture to give a concentration of 50 μg/ml, and the culture was grown for ∼24 h at 30 °C. E. coli pastes from 500-ml shake flask cultures (6–10-g pellets) were resuspended in 10 volumes (w/v) of 20 mm Tris-HCl, pH 8.0, containing 7 m guanidine HCl. Solid sodium sulfite and sodium tetrathionate were added to make final concentrations of 0.1 and 0.02 m, respectively, and the solution was stirred overnight at 4 °C. The solution was clarified by centrifugation and loaded onto a 20-ml Qiagen Ni-NTA metal chelate column equilibrated in 20 mm Tris-HCl, pH 8.6, containing 6 m guanidine HCl. The column was washed with additional buffer containing 50 mm imidazole (Ultrol grade; Calbiochem). The protein was eluted with buffer containing 250 mm imidazole. Fractions containing the desired protein based on SDS-PAGE were pooled and diluted to 50 μg/ml with buffer containing 20 mm Tris-Cl, pH 8.6, 0.8 m arginine, 0.3 m NaCl, 20 mm glycine, 1 mm EDTA, and 1 mm cysteine. The refolding mixture was incubated overnight at 2–8 °C. The protein was subsequently concentrated 20-fold using Vivascience (Edgewood, NY) concentrator (molecular weight cut-off, 10,000) and dialyzed against 50 mm Tris-HCl, pH 8.0, and 0.15 m NaCl. The refolded protein was loaded on a Superdex 75 (Amersham Biosciences) equilibrated with the same buffer. The fractions were analyzed by SDS-PAGE (>95% purity) and enzymatic activity using a chromogenic substrate (see below) and pooled. The matriptase protease domain was also analyzed by N-terminal amino acid sequencing and electrospray mass spectrometry. Protein concentration was determined by quantitative amino acid analysis. Reverse Transcription-PCR—Human cell lines were obtained from ATCC (Manassas, VA) or BioWhittaker, Inc. (Walkersville, MD) and were cultured in recommended serum-supplemented medium. The human normal cell lines used were mammary epithelial cells, aortic smooth muscle cells, pulmonary artery smooth muscle cells, pulmonary artery endothelial cells, and umbilical artery endothelial cells. The human tumor cell lines used were: colorectal carcinoma cell lines (Colo205, HT29, HCT 116, SW480, and DLD-1), the breast carcinoma cell line BT-474, the lung carcinoma cell lines A549 and Calu-6, the pancreatic adenocarcinoma cell lines HPAC and HPAF-11, the bladder carcinoma cell line J82, the renal cell carcinoma cell line 786-0, the osteosarcoma cell line Saos-2, the rhabdomyosarcoma cell line A-673, and the prostate carcinoma cell line PC-3. For RNA isolation confluent cell layers were washed with PBS and Tri-Reagent-LS (Molecular Research Center, Cincinnati, OH) was added to the cells, and total RNA was extracted according to manufacturer's protocols. Total RNA from various human tissues was purchased from Clontech (Palo Alto, CA). The RNA samples of normal ovary and ovarian adenocarcinomas were from Clontech (see Fig. 3c, samples 1 and 5), Ambion (Austin, TX; see Fig. 3c, samples 2 and 3), the University of Michigan (see Fig. 3c, samples 4 and 12), and the Cooperative Human Tissue Network (see Fig. 3c, samples 6–11). Normal and pathological specimens were removed from patients for therapeutic procedures unrelated to this study; they were provided following appropriate Institutional Review Board review. These total RNA samples were processed by use of oligo(dT)24 and SuperScript reverse transcriptase (Invitrogen) to obtain cDNA. The cDNAs were subjected to PCR using the primer set for HAI-1B and HAI-1 or the primer set for β-actin (control). The sequences of the primers were as follows: HAI-1B and HAI-1 forward, 5′-ATGGAGGCTGCTTGGGCAACA-3′; HAI-1B and HAI-1 reverse, 5′-ACAGGCAGCCTCGTCGGAGG-3′; β-actin forward, 5′-TCACCCACACTGTGCCCATCTACGA-3′; and β-actin reverse: 5′-CAGCGGAACCGCTCATTGCCAATGG-3′. The PCR amplifications were carried out for 25 cycles of 45 s at 95 °C followed by 45 s at 55 °C and 1 min at 72 °C using Advantage-GC cDNA polymerase mix (Clontech). The PCR products were separated on a 2.5% agarose gel and then visualized by ethidium bromide staining. In some experiments the bands were excised from the gel, and the PCR products were extracted and sequenced. The obtained sequences were in full agreement with the expected sequences specific for HAI-1B (containing the 48-bp insert region) and for HAI-1 (1Shimomura T. Denda K. Kitamura A. Kawaguchi T. Kito M. Kondo J. Kagaya S. Qin L. Takata H. Miyazawa K. Kitamura N. J. Biol. Chem. 1997; 272: 6370-6376Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar), respectively. Cloning of HAI-1B—Full-length HAI-1B was obtained from a cDNA library derived from human placental RNA using oligo(dT)/NotI site as a primer and adaptor with SalI site for the second strand. The cDNA was digested with SalI and NotI; cDNAs greater than 2.8 kb were ligated to pRK5D. Single-stranded DNA of the human placental cDNA/pRK5D library was generated using standard molecular biology methods. Reverse primer (5′-ACTGGATGGCGCCTTTCCATG-3′) was annealed to the single-stranded cDNA pool and extended using T7 or T4 DNA polymerase. E. coli were transformed with the synthesized double-stranded DNA, and colonies were screened using standard filter hybridization methods. The insert size was analyzed by PCR, and the HAI-1B full-length clones were identified and confirmed by DNA sequencing. Construction, Expression, and Purification of Soluble HAI-1B—A soluble form of HAI-1B (sHAI-1B) was produced by fusing the cDNA coding for the extracellular domain (amino acids Met1–Glu465) of HAI-1B via a Met-Gly residue linker to a poly-His tag at the C terminus (Met-Gly-His8). The cDNA was then inserted into eukaryotic expression vector pSVI7.ID.LL (47Lucas B.K. Giere L.M. DeMarco R.A. Shen A. Chisholm V. Crowley C.W. Nucleic Acids Res. 1996; 24: 1774-1779Crossref PubMed Scopus (83) Google Scholar). A stable CHO cell line expressing sHAI-1B was generated using standard methods (47Lucas B.K. Giere L.M. DeMarco R.A. Shen A. Chisholm V. Crowley C.W. Nucleic Acids Res. 1996; 24: 1774-1779Crossref PubMed Scopus (83) Google Scholar). The harvested culture supernatant of the CHO stable cell line expressing sHAI-1B was filtered through a 0.2-μm filter. Sodium azide and phenylmethylsulfonyl fluoride were added to the filtered medium to give final c