Purification and Cloning of Hepatocyte Growth Factor Activator Inhibitor Type 2, a Kunitz-type Serine Protease Inhibitor

Abstract

Hepatocyte growth factor (HGF) activator is a serine protease responsible for proteolytic activation of HGF in response to tissue injury and thus plays an important role in the regulation of biological functions of HGF in regenerating tissue. We previously purified an inhibitor of HGF activator (HGF activator inhibitor type 1, HAI-1) from the conditioned medium of a human stomach carcinoma cell line MKN45 and cloned its cDNA. HAI-1 is a novel member of the Kunitz family of serine protease inhibitors. In the present study, we purified a second type of HGF activator inhibitor (HAI-2) from the conditioned medium of MKN45 cells and molecularly cloned its cDNA. The cDNA sequence revealed that HAI-2 is derived from a precursor protein of 252 amino acids and contains two Kunitz domains, indicating that HAI-2 is also a member of the Kunitz family of serine protease inhibitors. The primary translation product of HAI-2 has a hydrophobic sequence in the COOH-terminal region, suggesting that, like HAI-1, HAI-2 is produced in a membrane-associated form and secreted in a proteolytically truncated form. Because HAI-2 and HAI-1 are potent inhibitors specific for HGF activator, they may be involved in regulation of proteolytic activation of HGF in injured tissues. Hepatocyte growth factor (HGF) activator is a serine protease responsible for proteolytic activation of HGF in response to tissue injury and thus plays an important role in the regulation of biological functions of HGF in regenerating tissue. We previously purified an inhibitor of HGF activator (HGF activator inhibitor type 1, HAI-1) from the conditioned medium of a human stomach carcinoma cell line MKN45 and cloned its cDNA. HAI-1 is a novel member of the Kunitz family of serine protease inhibitors. In the present study, we purified a second type of HGF activator inhibitor (HAI-2) from the conditioned medium of MKN45 cells and molecularly cloned its cDNA. The cDNA sequence revealed that HAI-2 is derived from a precursor protein of 252 amino acids and contains two Kunitz domains, indicating that HAI-2 is also a member of the Kunitz family of serine protease inhibitors. The primary translation product of HAI-2 has a hydrophobic sequence in the COOH-terminal region, suggesting that, like HAI-1, HAI-2 is produced in a membrane-associated form and secreted in a proteolytically truncated form. Because HAI-2 and HAI-1 are potent inhibitors specific for HGF activator, they may be involved in regulation of proteolytic activation of HGF in injured tissues. Hepatocyte growth factor (HGF) 1The abbreviations used are: HGF, hepatocyte growth factor; HAI-1 and -2, hepatocyte growth factor activator inhibitor type 1 and type 2, respectively; APP, β-amyloid precursor protein; TFPI, tissue factor pathway inhibitor; UTI, urinary trypsin inhibitor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid; PAGE, polyacrylamide gel electrophoresis; PCR polymerase chain reaction; ConA, concanavalin A; bp, base pair(s). 1The abbreviations used are: HGF, hepatocyte growth factor; HAI-1 and -2, hepatocyte growth factor activator inhibitor type 1 and type 2, respectively; APP, β-amyloid precursor protein; TFPI, tissue factor pathway inhibitor; UTI, urinary trypsin inhibitor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PBS, phosphate-buffered saline; TFA, trifluoroacetic acid; PAGE, polyacrylamide gel electrophoresis; PCR polymerase chain reaction; ConA, concanavalin A; bp, base pair(s). activator is a blood coagulation factor XII-like serine protease that converts the inactive single chain form of HGF to the active heterodimeric form in injured tissues (1Miyazawa K. Shimomura T. Kitamura A. Kondo J. Morimoto Y. Kitamura N. J. Biol. Chem. 1993; 268: 10024-10028Abstract Full Text PDF PubMed Google Scholar, 2Miyazawa K. Shimomura T. Kitamura N. J. Biol. Chem. 1996; 271: 3615-3618Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). HGF activator is produced and secreted by parenchymal liver cells (3Okajima A. Miyazawa K. Naitoh Y. Inoue K. Kitamura N. Hepatology. 1997; 25: 97-102Crossref PubMed Scopus (55) Google Scholar) and circulates in the blood as an inactive zymogen (4Shimomura 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). In response to tissue injury, the zymogen is converted to an active serine protease. The activated HGF activator acquires strong heparin binding affinity and is localized to the injured tissue. The localized HGF activator catalyzes the activation of the inactive single chain HGF that resides in the tissue (2Miyazawa K. Shimomura T. Kitamura N. J. Biol. Chem. 1996; 271: 3615-3618Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 5Miyazawa K. Shimomura T. Naka D. Kitamura N. J. Biol. Chem. 1994; 269: 8966-8970Abstract Full Text PDF PubMed Google Scholar). The activated HGF may be involved in tissue repair after the injury because HGF is a potent mitogen for a variety of cells (6Gohda E. Tsubouchi H. Nakayama H. Hirono S. Sakiyama O. Takahashi K. Miyazaki H. Hashimoto S. Daikuhara Y. J. Clin. Invest. 1988; 81: 414-419Crossref PubMed Scopus (633) Google Scholar, 7Igawa T. Kanda S. Kanetake H. Saitoh Y. Ichihara A. Tomita Y. Nakamura T. Biochem. Biophys. Res. Commun. 1991; 174: 831-838Crossref PubMed Scopus (261) Google Scholar, 8Rubin J.S. Chan A.M.-L. Bottaro D.P. Burgess W.H. Taylor W.G. Cech A.C. Hirschfield D.W. Wong J. Miki T. Finch P.W. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 415-419Crossref PubMed Scopus (467) Google Scholar). Thus, HGF activator is a specific enzyme that localizes the activity of HGF to the injured tissue. The activity of HGF activator may be regulated by one or more inhibitors. We previously purified an inhibitor of HGF activator from the conditioned medium of a human stomach carcinoma cell line MKN45 and molecularly cloned its cDNA. The nucleotide sequence of the cDNA revealed that the inhibitor has two well defined Kunitz domains, indicating that it is a new member of the Kunitz family of serine protease inhibitors (9Shimomura 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 (236) Google Scholar). The sequence also showed that the primary translation product of the inhibitor has a hydrophobic sequence in the COOH-terminal region, suggesting that the inhibitor is produced in a membrane-associated form and secreted by the producing cells in a proteolytically truncated form (9Shimomura 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 (236) Google Scholar). Because the inhibitor efficiently neutralizes the activity of HGF activator in vitro and the mRNA of the inhibitor is expressed in various tissues (9Shimomura 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 (236) Google Scholar), it may be responsible for suppressing the activity of HGF activator after tissue repair. In the present study, we found a second inhibitor of HGF activator in the conditioned medium of MKN45 cells. We purified it from the conditioned medium and cloned its cDNA. The nucleotide sequence of the cDNA revealed that this newly identified inhibitor is also a Kunitz-type serine protease inhibitor. We designated this protein as HGF activator inhibitor type 2 (HAI-2). To distinguish the previously identified inhibitor from this new protein, we redesignated it as HGF activator inhibitor type 1 (HAI-1). Both HAI-1 and HAI-2 are potent inhibitors of HGF activator and have similar structural domains. However, there are some differences in structure and properties between these inhibitors. We compare here the structures and properties of HAI-1 and HAI-2. Cell lines and reagents were obtained as follows: human stomach carcinoma cell line MKN45 from IBL (Gunma, Japan); human lung carcinoma cell line HLC-1 from the Department of Physiology, Keio University (Tokyo, Japan); human rectal adenocarcinoma cell line RCM-1 from Dr. H. Kataoka (Department of Pathology, Miyazaki Medical College, Miyazaki, Japan); heparin-Sepharose CL-6B and ConA-Sepharose from Pharmacia Biotech Inc.; phenyl-5PW column from TOSOH; PL-SAX column from Polymer Laboratories, Ltd.; HCA A-4007 column from Mitsui-touatsu Chemical; Ashahipack GS520 column from Asahi Chemical Industries, Ltd.; YMC C4 and C8 columns from YMC; dextran sulfate, CHAPS and porcine pancreas trypsin from Sigma; YM 30 membrane from Amicon; Affi-Gel 10 from Bio-Rad; N-glycosidase F from Boehringer Mannheim; octylglucopyranoside from Nacalai tesque Inc.; human urinary trypsin inhibitor (UTI) from Mochida Pharmaceutical Co., Ltd; and bicinchoninic acid protein assay kit from Pierce Chemical Co. Trypsin and HGF activator immobilized affinity columns were prepared by coupling 13 mg of trypsin and 1.6 mg of HGF activator with 10 and 5 ml of Affi-Gel 10, respectively, by the method described in the instruction manual. To obtain tissue factor pathway inhibitor (TFPI), the concentrated serum-free conditioned medium of HLC-1 cells was applied to a trypsin immobilized column (1 × 13 cm) equilibrated with phosphate-buffered saline (PBS). The adsorbed fractions were eluted from the column with 10 mm HCl, neutralized immediately by 1 m Tris-HCl buffer (pH 8.0), and then were subjected to a reverse-phase C4 column (0.46 × 15 cm) chromatography. TFPI was eluted with a linear gradient of 10–70% acetonitrile/isopropanol (3/7) containing 0.1% trifluoroacetic acid (TFA). The β-amyloid precursor protein (APP) was purified from the concentrated serum-free conditioned medium of RCM-1 cells (10Kataoka H. Nabeshima K. Komada N. Koono M. Virchows Arch. B Cell Pathol. 1989; 57: 157-165Crossref Scopus (43) Google Scholar, 11Murayama T. Kataoka H. Koita H. Nabeshima K. Koono M. Virchows Arch. B Cell Pathol. 1991; 60: 263-270Crossref Scopus (15) Google Scholar) using the same procedure as for TFPI. Five μl of the fractions for assay were incubated with 10 μl of 900 ng/ml HGF activator in 35 μl of PBS containing 0.05% CHAPS at 37 °C for 30 min. The mixtures were added to 10 μl of 1.5 mg/ml single-chain HGF and further incubated at 37 °C for 2 h. The mixtures were then analyzed by SDS-PAGE under reducing conditions. The gel was stained with Coomassie Brilliant Blue and scanned by a Flying-Spot Scanner CS-9000 (Shimadzu). Ten liters of the serum-free conditioned medium of MKN45 cells was concentrated to about 300 ml. The concentrate was applied to a heparin-Sepharose CL-6B column (2.5 × 10 cm) pre-equilibrated with PBS. The unadsorbed fractions were collected and then applied to a ConA-Sepharose column (1 × 5 cm) pre-equilibrated with PBS. The unadsorbed fractions were concentrated and dialyzed against 10 mm sodium phosphate buffer (pH 6.8) containing 1m ammonium sulfate by a YM 30 membrane. The concentrate was applied to a phenyl-5PW column (0.75 × 7.5 cm) pre-equilibrated with the dialysis buffer. The unadsorbed fractions containing the inhibitory activity were collected and dialyzed against 20 mm Tris-HCl buffer (pH 8.0) containing 0.05% CHAPS and then applied to an anion exchange PL-SAX column (0.46 × 5 cm) pre-equilibrated with the dialysis buffer. Elution was performed with a linear gradient of 0–500 mm NaCl. The HAI-2 protein was eluted at 0–150 mm NaCl. The fractions were dialyzed against 5 mm sodium phosphate buffer (pH 6.8) containing 0.05% CHAPS. The dialysate was applied to a hydroxyapatite HCA A-4007 column (0.4 × 7.5 cm) pre-equilibrated with the dialysis buffer. The unadsorbed fractions were collected and then applied to a gel-filtration Ashahipack GS520 column (0.76 × 50 cm) pre-equilibrated with PBS containing 0.05% CHAPS. The HAI-2 protein was collected at 40–20 kDa fractions and then finally purified by a reverse-phase YMC C8 column (0.46 × 15 cm). Elution was performed with a linear gradient of 10–50% of acetonitrile/isopropanol (3/7) containing 0.1% TFA. The fractions containing HAI-2 protein were neutralized with 1 m Tris-HCl buffer (pH 8.0) and then dried under vacuum conditions. The dried samples were dissolved in PBS containing 0.05% CHAPS and analyzed by SDS-PAGE. The gel was stained with silver. The concentrations of proteins were determined using a bicinchoninic acid protein assay kit, with bovine serum albumin as a standard. Fifty μl of 20 μg/ml HAI-2 protein in PBS containing 5 mm EDTA was first denatured by heating at 100 °C in the presence of 0.2% SDS. After denaturation, 5 μl of 10% octylglucopyranoside, 0.5 μl of and 1 μl of N-glycosidase F were added to the The was incubated at 37 °C for 20 and the product was analyzed by SDS-PAGE under reducing conditions. To the amino acid sequence of the purified the from the C8 reverse-phase was using an The amino acid of HAI-2 were determined as described (9Shimomura 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 (236) Google Scholar). μg/ml of HGF activator was with various concentrations of HAI-2 in μl of PBS containing 0.05% CHAPS. After at 37 °C for 30 5 μl of mg/ml single-chain HGF in PBS containing 0.05% CHAPS and 5 μl of 100 μg/ml dextran sulfate were added to the and further incubated at 37 °C for 10 min. The was then analyzed by SDS-PAGE under reducing conditions. The gel was stained with Coomassie Brilliant Blue and scanned by a Flying-Spot Scanner The inhibitory of HAI-2 were by the of the single-chain to heterodimeric form of was prepared from MKN45 cells by acid N. Biochem. PubMed Scopus Google Scholar), and was purified by affinity chromatography. The and were the and as a were by chain and an was The was and The was used as a for a cDNA To the cDNA cDNA was from of MKN45 cells using a cDNA kit with The cDNA with at was and vitro using to was performed at °C for with in a containing 5 × bovine serum 0.1% and 0.1% m 50 mm sodium phosphate (pH mm and 100 μg/ml The was using The were with × containing at °C for 30 min. from MKN45 cells was H. D. H. PubMed Scopus Google Scholar), and to a membrane and tissue were from The were at °C for with as described K. Kitamura A. Naka D. Kitamura N. J. Biochem. 1991; PubMed Scopus Google Scholar). The were with 1 × containing at °C for 30 min. The was the The serum-free conditioned medium of cells was applied to a trypsin immobilized column (1 × 13 cm) equilibrated with PBS. The adsorbed proteins were eluted from the column with 10 mm The eluted fractions were neutralized immediately by 1 m Tris-HCl buffer (pH 8.0) and applied to an HGF activator immobilized column (1 × cm) equilibrated with PBS. The adsorbed proteins were eluted with 10 mm HCl, and the eluted fractions were collected and neutralized with 1 m Tris-HCl buffer (pH 8.0) and then subjected to reverse-phase YMC C4 column (0.46 × 15 cm) chromatography. Elution was with a linear gradient of 10–70% acetonitrile/isopropanol (3/7) containing 0.1% TFA. The concentrations of proteins were determined using a bicinchoninic acid protein assay kit, with bovine serum albumin as a standard. HAI-1, APP, TFPI, or was with 100 ng/ml of HGF activator in μl of PBS containing 0.05% CHAPS and incubated at 37 °C for 30 min. The mixtures were with 5 μl of 1 mg/ml single-chain HGF in PBS containing 0.05% CHAPS and further incubated at 37 °C for 2 h. The mixtures were then analyzed by SDS-PAGE under reducing conditions. The gel was stained with Coomassie Brilliant Blue and scanned by a Flying-Spot Scanner HAI-1 was previously purified from serum-free conditioned medium of MKN45 cells by a procedure (9Shimomura 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 (236) Google Scholar). The conditioned medium was concentrated and applied to a heparin-Sepharose The unadsorbed fractions were applied to a ConA-Sepharose and the inhibitory were in the unadsorbed and adsorbed The HAI-1 protein was from the adsorbed (9Shimomura 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 (236) Google Scholar). In this study, we purified a protein for the activity (HAI-2) in the unadsorbed by an procedure using the columns used for the of of HAI-2 and The of HAI-2 gel and reverse-phase were from of The of HAI-2 reverse-phase revealed a of to kDa SDS-PAGE under reducing of HAI-2 and with the inhibitory mm of exchange mm of mm of (3/7) containing 0.1% (3/7) containing 0.1% (3/7) containing 0.1% TFA. in a new The of HAI-2 SDS-PAGE may be to HAI-2. We HAI-2 using an N-glycosidase and analyzed it by SDS-PAGE. The HAI-2 protein as two of and kDa 1, indicating that were to the HAI-2 the amino acid sequence of the purified protein was analyzed one sequence was obtained 2 Thus, the of the protein the gel be by a in the COOH-terminal In to the the two amino acid were obtained from produced by of the purified protein with The sequence of 1 showed to that of a of the first Kunitz of HAI-1 2 suggesting that HAI-2 is also a Kunitz-type serine protease inhibitor. the of the inhibitory activity of HAI-2. In these HGF activator was with various concentrations of HAI-2 and incubated for 30 to form an activity was The of HAI-2 for was about 300 the of the protein of HAI-2 and HGF activator HAI-2 about an with HGF The and the COOH-terminal in 1 2 were used to for PCR of the sequence for the sequence was because the were obtained by with a The were determined by the amino acid sequence 2 they were because the sequence of 1 is similar to the first Kunitz of PCR of MKN45 in a cDNA with the of about The cDNA was and The amino acid sequence from the cDNA sequence that of The PCR was thus used as a to a cDNA from MKN45 cells. were obtained from about 1.5 × The was to the primary structure of human HAI-2 The amino acid sequence of HAI-2 from the cDNA sequence is in The translation was to the first because are present of the The that from the of 252 amino and the protein product has a of The first is by a hydrophobic and the amino acid of the purified protein is at the of the suggesting that the hydrophobic a the the form of the protein of amino acids and has a of The of the protein of HAI-2 purified from the conditioned medium of MKN45 cells was about as determined by SDS-PAGE. Thus, the protein purified from the conditioned medium to be a product at the COOH-terminal hydrophobic sequence of amino acids is present in the COOH-terminal region, suggesting that the primary translation product is a membrane-associated are two with the of the protein sequence of HAI-2 with in the and the protein base revealed that two and showed to the Kunitz-type sequence of serine protease inhibitors Thus, HAI-2 to be a Kunitz-type serine protease inhibitor. The and tissue of HAI-2 mRNA was determined by with from various human tissues of 1.6 was in MKN45 cells from we purified the HAI-2 The was in a variety of human and tissues. the of HAI-2 mRNA was in the and a of trypsin and HGF activator immobilized affinity column of HAI-2 and HAI-1 in the conditioned medium of MKN45 were The serum-free conditioned medium of MKN45 was applied to a trypsin immobilized affinity and the adsorbed fractions were applied to an HGF activator immobilized affinity The adsorbed fractions were analyzed by C4 reverse-phase chromatography. The inhibitory activity was in the adsorbed fractions in affinity and in the unadsorbed the C4 reverse-phase were amino acid sequence revealed that 1 and to HAI-2 and HAI-1, the amino acid sequence of 2 was two and were to of and in HAI-1, indicating that the proteins in 2 are at the of truncated HAI-1 proteins inhibitory activity HGF activator inhibitor HAI-2 and HAI-1 was by this affinity Thus, the inhibitory activity HGF activity in the conditioned medium of MKN45 cells is derived from HAI-1 and HAI-2. The proteins in were and were by a protein assay The of HAI-2 was to that of HAI-1 in the conditioned medium of MKN45 cells. APP, TFPI, and have Kunitz that are responsible for the inhibitory of these proteins against serine S. M. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar, Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar). we these inhibitors an inhibitory the activity of HGF activator The inhibitory of and TFPI against HGF activator were at and that of was these proteins the proteolytic activity of trypsin We previously identified and cloned an HGF activator inhibitor from MKN45 human stomach carcinoma cells (9Shimomura 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 (236) Google Scholar). In the present study, we identified a second type of HGF activator inhibitor (HAI-2) from the same cells. The inhibitor protein was purified from the conditioned medium of the cells. The primary structure of the protein was from the sequence of the cDNA for human HAI-2. The structure of human HAI-2 is in with the structure of human The primary translation product of HAI-2 of 252 amino acid The may as a HAI-2 has two Kunitz domains. Kunitz is about amino acids in and contains is as the of serine protease inhibitors M. Biochem. PubMed Scopus Google Scholar). Thus, one or of the Kunitz in HAI-2 to be responsible for the inhibitory The protein also has a hydrophobic sequence of about 20 amino acids in the COOH-terminal region, suggesting that it may be The protein of HAI-2 purified from the conditioned medium of MKN45 cells has a of about is the of the primary translation Thus, HAI-2 purified from the conditioned medium to be a proteolytically truncated form of the membrane-associated form. HAI-1 also has two Kunitz and the COOH-terminal hydrophobic region, indicating that the structures of the are similar between HAI-1 and HAI-2. However, HAI-1 has structures that are found in a the first Kunitz and a structure similar to the binding of the between the two Kunitz (9Shimomura 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 (236) Google Scholar). The of the protein of HAI-2 purified from the conditioned medium of MKN45 cells is about to amino suggesting that the truncated form of HAI-2 is produced by the proteolytic that the second Kunitz was for the of the truncated form of HAI-1 (9Shimomura 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 (236) Google Scholar). the of the HAI-2 activity showed that HAI-2 purified from the conditioned medium an with HGF was for HAI-1 and HGF activator (9Shimomura 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 (236) Google Scholar). Thus, the first Kunitz in HAI-2 as well as HAI-1 may in the inhibitory activity HGF The first Kunitz of HAI-2 the to the first Kunitz of HAI-1 Kunitz of human serine protease suggesting that the amino acid between the an important role in the of between the inhibitors and HGF The of HAI-2 by an N-glycosidase its indicating that are to the protein of HAI-2. The of HAI-1 was also by N-glycosidase Shimomura and T. the to was that for HAI-2. Thus, HAI-2 and HAI-1 are HAI-1 to ConA-Sepharose (9Shimomura 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 (236) Google Scholar), HAI-2 is a that the present in J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). The are and In and are to and structures are In more or to the M. J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar). Thus, the to HAI-2 are to the structure are that the of in the regulation of protease and A. T. K. Biochem. Biophys. Res. Commun. PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, S. J.S. J. Biol. Chem. 1988; Full Text PDF PubMed Google Scholar). The to HAI-2 have similar in Both HAI-2 and HAI-1 are potent inhibitors of HGF activator, the Kunitz type serine protease APP, TFPI, and UTI, or inhibitory activity HGF serum serine protease inhibitors as and the activity of HGF activator T. Miyazawa K. Y. H. Naka D. Morimoto Y. Kitamura N. J. Biochem. PubMed Scopus Google Scholar). that HAI-2 and HAI-1 as specific inhibitors of HGF of HAI-2 and HAI-1 mRNA are in some tissues as the In these the of the inhibitors may the of HGF tissue of mRNA of HAI-2 and HAI-1 were in various human and tissues in of HAI-2 mRNA is that of HAI-1 about of inhibitors were in the conditioned medium of MKN45 cells. that HAI-2 and HAI-1 HGF However, these inhibitors have some are to the protein of HAI-2 and HAI-2 was adsorbed by the hydrophobic column HAI-1 indicating that HAI-2 is more properties may in inhibitory HGF Thus, further of HAI-2 and HAI-1 is to these inhibitors against HGF