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

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) 1 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 (1,2). HGF activator is produced and secreted by parenchymal liver cells (3) and circulates in the blood as an inactive zymogen (4). 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 (2,5). The activated HGF may be involved in tissue repair after the injury because HGF is a potent mitogen for a variety of cells (6 -8). 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 (9). 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 (9). Because the inhibitor efficiently neutralizes the activity of HGF activator in vitro and the mRNA of the inhibitor is expressed in various tissues (9), 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.

EXPERIMENTAL PROCEDURES
Materials-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 * This work was supported in part by research grants from the Ministry of Education, Science, Sports and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EBI Data Bank with accession number(s) AB006534.
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 ϫ 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 ϫ 15 cm) chromatography. TFPI was eluted with a linear gradient of 10 -70% acetonitrile/isopropanol (3/7) containing 0.1% trifluoroacetic acid (TFA). The ␤-amyloid precursor protein (APP) was purified from the concentrated serum-free conditioned medium of RCM-1 cells (10, 11) using the same procedure as for TFPI.
Assay for Inhibitory Activity against HGF Activator-Five l of the fractions for assay were incubated with 10 l of 900 ng/ml HGF activator in 35 l of PBS containing 0.05% CHAPS at 37°C for 30 min. The mixtures were added to 10 l 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).
Purification of HAI-2 from the Conditioned Medium of MKN45 Cells-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 ϫ 10 cm) pre-equilibrated with PBS. The unadsorbed fractions were collected and then applied to a ConA-Sepharose column (1 ϫ 5 cm) pre-equilibrated with PBS. The unadsorbed fractions were concentrated and dialyzed against 10 mM sodium phosphate buffer (pH 6.8) containing 1 M ammonium sulfate by a YM 30 membrane. The concentrate was applied to a phenyl-5PW column (0.75 ϫ 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 ϫ 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 ϫ 7.5 cm) preequilibrated with the dialysis buffer. The unadsorbed fractions were collected and then applied to a gel-filtration Ashahipack GS520 column (0.76 ϫ 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 ϫ 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.
Deglycosylation-Fifty l of 20 g/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 l of 10% octylglucopyranoside, 0.5 l of 2-mercaptoethanol and 1 l of 200 units/ml N-glycosidase F were added to the mixture. The mixture was incubated at 37°C for 20 h, and the deglycosylated product was analyzed by SDS-PAGE under reducing conditions.
Amino Acid Sequence Analysis-To determine the NH 2 -terminal amino acid sequence of the purified HAI-2, the final preparation from the C8 reverse-phase chromatography was sequenced using an Applied Biosystems 470A Protein Sequencer. The internal amino acid sequences of HAI-2 were determined as described (9).
Dose Response of the Inhibitory Activity of HAI-2 against HGF Activator-Two g/ml of HGF activator was mixed with various concentrations of HAI-2 (0 -2.4 g/ml) in 40 l of PBS containing 0.05% CHAPS. After incubation at 37°C for 30 min, 5 l of 2.4 mg/ml single-chain HGF in PBS containing 0.05% CHAPS and 5 l of 100 g/ml dextran sulfate (molecular mass: 500 kDa) were added to the mixture and further incubated at 37°C for 10 min. The mixture was 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. The inhibitory activities of HAI-2 were estimated by calculating the ratio of the single-chain to heterodimeric form of HGF.
cDNA Cloning-Total RNA was prepared from MKN45 cells by acid guanidinium thiocyanate/phenol/chloroform extraction (12), and poly(A) RNA was purified by oligo(dT) affinity chromatography. The primers 5Ј-AAGGT(G/T)GT(G/T)GG(G/T)(A/C)G(G/T)TG(T/C)(A/C)G-3Ј and 5Ј-C(G/T)CCGTA(G/T)ACGAA(G/T)A(G/A)(T/C)TG(G/A)C-3Ј were chemically synthesized. Using the primers and poly(A) RNA as a template, DNA fragments were amplified by reverse transcription-polymerase chain reaction (PCR), and an 85-bp fragment was generated. The DNA fragment was subcloned and sequenced. The fragment was used as a probe for screening a cDNA library. To construct the cDNA library, cDNA was synthesized from poly(A) RNA of MKN45 cells using a cDNA synthesis kit with oligo(dT) primer (Pharmacia). The cDNA with EcoRI adaptors at both ends was ligated to EcoRI-digested ZAPII vector (Stratagene) and packaged in vitro using Gigapack Gold (Stratagene). Hybridization to nylon replica membranes (Hybond-N ϩ , Amersham) was performed at 42°C for 16 h with 32 P-labeled probe in a solution containing 50% formamide, 5 ϫ Denhardt's solution (0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, and 0.1% Ficoll), 0.75 M NaCl, 50 mM sodium phosphate (pH 7.4), 6 mM EDTA, 0.5% SDS, and 100 g/ml salmon testis DNA. The probe was labeled using multiprime DNA labeling system (Amersham Life Science Inc.). The membranes were washed twice with 0.1 ϫ SSC containing 1% SDS at 42°C for 30 min.
Northern Blotting-Total RNA (10 g) from MKN45 cells was denatured, electrophoresed (13), and transferred to a nylon membrane (Biodyne). Human adult and fetal multiple tissue Northern blot membranes were purchased from CLONTECH. The membranes were hybridized at 42°C for 16 h with the 32 P-labeled probe as described (14). The membranes were washed twice with 1 ϫ SSC containing 1% SDS at 42°C for 30 min. The hybridization probe was the 648-bp NcoI fragment.
Measurement of Relative Amounts of HAI-1 and HAI-2 in the Conditioned Medium of MKN45 Cells-The serum-free conditioned medium of MKN 45 cells was applied to a trypsin immobilized column (1 ϫ 13 cm) equilibrated with PBS. The adsorbed proteins were eluted from the column with 10 mM HCl. The eluted fractions were neutralized immediately by 1 M Tris-HCl buffer (pH 8.0) and directly applied to an HGF activator immobilized column (1 ϫ 6 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 ϫ 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. Assay for Inhibitory Activities of Kunitz-type Serine Protease Inhibitors against HGF Activator-HAI-1, HAI-2, APP, TFPI, or UTI was mixed with 100 ng/ml of HGF activator in 45 l of PBS containing 0.05% CHAPS and incubated at 37°C for 30 min. The mixtures were supplemented with 5 l 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 CS-9000.

RESULTS
Purification of HAI-2 from the Conditioned Medium of MKN45 Cells-HAI-1 was previously purified from serum-free conditioned medium of MKN45 cells by a seven-step procedure (9). The conditioned medium was concentrated and applied to a heparin-Sepharose column. The unadsorbed fractions were applied to a ConA-Sepharose column, and the inhibitory activities were detected both in the unadsorbed and adsorbed fractions. The HAI-1 protein was recovered from the adsorbed fraction (9). In this study, we purified a protein for the activity (HAI-2) in the unadsorbed fraction by an additional five-step procedure using the columns used for the purification of HAI-1. Table I shows elution characteristics of HAI-2 and HAI-1. The elution characteristics of HAI-2 on hydrophobic, gel filtration, and reverse-phase chromatographies were distinctly different from those of HAI-1. The final preparation of HAI-2 on reversephase chromatography revealed a broad band of 23 to 31 kDa on SDS-PAGE (15.0% acrylamide) under reducing conditions (Fig. 1, lane 1).
The broad migration of HAI-2 on SDS-PAGE may be caused by N-glycans bound to HAI-2. We therefore deglycosylated HAI-2 using an N-glycosidase and analyzed it by SDS-PAGE. The deglycosylated HAI-2 protein migrated as two bands of 14.0 and 13.5 kDa (Fig. 1, lane 2), indicating that large Nlinked sugar chains were bound to the HAI-2 protein. When the NH 2 -terminal amino acid sequence of the purified protein was analyzed before deglycosylation, only one sequence was obtained ( Fig. 2A). Thus, the apparent heterogeneity of the deglycosylated protein on the gel might be caused by a difference in the COOH-terminal sequence. In addition to the NH 2 -terminal sequence, the two internal amino acid sequences were obtained from peptide fragments produced by digestion of the purified protein with Achromobacter protease-I ( Fig. 2A). The sequence of peptide 1 showed extensive similarity (53.6% identity) to that of a part of the first Kunitz domain of HAI-1 (Fig.  2B), suggesting that HAI-2 is also a Kunitz-type serine protease inhibitor.
Dose Dependence of the Inhibitory Activity of the Purified HAI-2- Fig. 3 shows the dose-response curve of the inhibitory activity of HAI-2. In these reactions, HGF activator (2 g/ml) was mixed with various concentrations of HAI-2 and incubated for 30 min to form an enzyme-inhibitor complex. Then remaining HGF-converting activity was measured. The concentration of HAI-2 for 50% inhibition was about 300 ng/ml. Considering the molecular masses of the protein portions of HAI-2 (14 kDa) and HGF activator (34 kDa), HAI-2 forms about an equimolar complex with HGF activator.
Isolation of cDNA Clone and DNA Sequence Analysis-The NH 2 -terminal sequence, Lys-Val-Val-Gly-Arg-Cys-Arg, and the COOH-terminal sequence, Cys-Gln-Leu-Phe-Val-Tyr-Gly-Gly, in peptide 1 ( Fig. 2A) were used to design degenerate oligonucleotide primers for PCR amplification of the sequence for peptide 1. A lysine residue preceding the NH 2 -terminal sequence was predicted because the peptide fragments were obtained by digestion with a lysylendopeptidase. The cysteine residues were not determined by the amino acid sequence analysis ( Fig. 2A), but they were predicted because the sequence of peptide 1 is similar to the first Kunitz domain of HAI-1. PCR amplification of MKN45 RNA resulted in a cDNA fragment with the expected size of about 85 bp. The cDNA fragment was subcloned and sequenced. The amino acid sequence predicted from the cDNA sequence completely matched that of peptide 1. The PCR clone was thus used as a probe to screen a cDNA library constructed from MKN45 cells. Twenty- two hybridization-positive clones were obtained from about 1.5 ϫ 10 5 phage. The largest clone was sequenced to determine the primary structure of human HAI-2 (Fig. 4).
Predicted Amino Acid Sequence of HAI-2-The amino acid sequence of HAI-2 predicted from the cDNA sequence is shown in Fig. 4. The translation initiation site was assigned to the first methionine codon because in-frame stop codons are present upstream of the methionine codon. The open reading frame that starts from the ATG codon consists of 252 amino acids, and the protein product has a calculated molecular mass of 28,169. The first methionine is followed by a hydrophobic sequence, and the NH 2 -terminal amino acid of the purified protein is located at the 28th residue downstream of the methionine, suggesting that the hydrophobic region represents a signal peptide sequence. Excluding the signal peptide, the mature form of the protein consists of 225 amino acids and has a calculated molecular mass of 25,415. The apparent molecular mass of the protein portion of HAI-2 purified from the conditioned medium of MKN45 cells was about 14 kDa, as determined by SDS-PAGE. Thus, the protein purified from the conditioned medium appears to be a processing product cleaved at the COOH-terminal region. A hydrophobic sequence of 24 amino acids is present in the COOH-terminal region, suggesting that the primary translation product is a membrane-asso-ciated protein. There are two potential N-glycosylation sites with the canonical Asn-Xaa-(Ser/Thr). A comparison of the protein sequence of HAI-2 with sequences in the SwissProt and the National Biomedical Research Foundation protein data base revealed that two regions (residues 38 -88 and 133-183) showed extensive similarity to the Kunitz-type sequence of serine protease inhibitors (Fig. 5). Thus, HAI-2 appears to be a Kunitz-type serine protease inhibitor.
Tissue Distribution of HAI-2 mRNA-The size and tissue distribution of HAI-2 mRNA was determined by Northern blotting with poly(A) RNAs from various human tissues (Fig. 6). A transcript of 1.6 kilobases was detected in MKN45 cells from which we purified the HAI-2 protein. The transcript was detected in a variety of human adult and fetal tissues. Among them, the expression level of HAI-2 mRNA was relatively high in the adult placenta, kidney, pancreas, prostate, testis, thyroid, and trachea.
Relative Amounts of HAI-2 and HAI-1 in the Conditioned Medium of MKN45 Cells-Using a combination of trypsin and HGF activator immobilized affinity column chromatography, relative amounts of HAI-2 and HAI-1 in the conditioned medium of MKN45 were measured. The serum-free conditioned medium of MKN45 was directly applied to a trypsin immobilized affinity column, and the adsorbed fractions were applied to an HGF activator immobilized affinity column. The adsorbed fractions were analyzed by C4 reverse-phase chromatography. The inhibitory activity was quantitatively recovered in the adsorbed fractions in each affinity chromatography and did not remain in the unadsorbed fractions. On the C4 reverse-phase chromatograph, three peaks were detected (Fig. 7A). NH 2 -terminal amino acid sequence analysis revealed that peaks 1 (at 15.0 min) and 3 (at 24.2 min) corresponded to HAI-2 and HAI-1, respectively. When the NH 2 -terminal amino acid sequence of peak 2 (at 22.9 min) was analyzed, two sequences (Arg-Gln-Leu-Arg and Thr-Gln-Gly-Phe) were obtained. These sequences correspond to those of residues 150 -153 and 154 -157 in HAI-1, indicating that the proteins in peak 2 are processing products cleaved at the NH 2 -terminal region of HAI-1. These truncated HAI-1 proteins exhibited inhibitory activity toward HGF activator (data not shown). No inhibitor other than HAI-2 and HAI-1 was detected by this affinity purification procedure. Thus, the inhibitory activity toward HGF activity in the conditioned medium of MKN45 cells is derived from HAI-1 and HAI-2. The proteins in each peak were fractionated and their contents were quantified by a protein assay (Fig. 7B). The amount of HAI-2 was almost equal to that of HAI-1 in the conditioned medium of MKN45 cells.
Examination of the Inhibitory Activity of Kunitz-type Inhibitors against HGF Activator-APP, TFPI, and UTI have Kunitz domains that are responsible for the inhibitory activities of these proteins against serine proteases (15)(16)(17). Therefore, we examined whether these inhibitors exhibited an inhibitory effect on the HGF-converting activity of HGF activator (Fig. 8). The inhibitory activities of APP and TFPI against HGF activator were very weak even at high concentrations, and that of UTI was not detected although these proteins strongly inhibited the proteolytic activity of trypsin (data not shown). DISCUSSION We previously identified and cloned an HGF activator inhibitor (HAI-1) from MKN45 human stomach carcinoma cells (9). 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 predicted from the sequence of the cDNA for human HAI-2. The structure of human HAI-2 is schematically summarized in Fig. 9 together with the structure of human HAI-1. The primary translation product of HAI-2 consists of 252 amino acid residues. The NH 2 -terminal 27 residues may serve as a signal peptide. HAI-2 has two Kunitz domains. A Kunitz domain is typically about 60 amino acids in length and contains three disulfide bonds. It is recognized as the functional domain of serine protease inhibitors (18). Thus, one or both of the Kunitz domains in HAI-2 appears to be responsible for the inhibitory activity. The protein also has a hydrophobic sequence of about 20 amino acids in the COOH-terminal region, suggesting that it may be membrane-associated. The protein portion of HAI-2 purified from the conditioned medium of MKN45 cells has a molecular mass of about 14 kDa, which is smaller than the predicted molecular mass (25,415) of the primary translation product. Thus, HAI-2 purified from the conditioned medium appears to be a proteolytically truncated form of the membrane-associated form. HAI-1 also has two Kunitz domains and the COOH-terminal  reverse-phase chromatography. The samples containing HAI-1 and HAI-2 were prepared from the conditioned medium of MKN45 cells by two-step affinity chromatography on Affi-Gel 10 with immobilized trypsin and HGF activator. The preparation was analyzed using C4 reversephase chromatography. B, the protein contents of each peak were calculated by a protein assay. hydrophobic region, indicating that the overall structures of the characteristic domains are similar between HAI-1 and HAI-2. However, HAI-1 has additional structures that are not found in HAI-2: a long NH 2 -terminal region preceding the first Kunitz domain and a structure similar to the ligand binding domain of the low density lipoprotein receptor between the two Kunitz domains (9).
The molecular mass of the protein portion of HAI-2 purified from the conditioned medium of MKN45 cells is about 14 kDa, which corresponds to 125 amino acids, suggesting that the extracellular truncated form of HAI-2 is produced by the proteolytic cleavage that occurs within the second Kunitz domain. Similar cleavage was suggested for the generation of the extracellular truncated form of HAI-1 (9). Furthermore, the dosedependence of the HAI-2 activity showed that HAI-2 purified from the conditioned medium forms an equimolar complex with HGF activator. Similar complex formation was observed for HAI-1 and HGF activator (9). Thus, only the first Kunitz domain in HAI-2 as well as HAI-1 may function in the inhibitory activity toward HGF activator. The first Kunitz domain of HAI-2 shows the highest similarity (54% identity) to the first Kunitz domain of HAI-1 among Kunitz domains of human serine protease inhibitors, suggesting that the conserved amino acid residues between the domains play an important role in the formation of complex between the inhibitors and HGF activator.
The treatment of HAI-2 by an N-glycosidase markedly decreased its molecular mass, indicating that large N-linked sugar chains are attached to the protein portion of HAI-2. The molecular mass of HAI-1 was also decreased by N-glycosidase treatment 2 although the decrease (40 to 34 kDa) was less than that for HAI-2. Thus, both HAI-2 and HAI-1 are N-glycosylated proteins. HAI-1 binds to ConA-Sepharose (9), whereas HAI-2 does not. ConA is a legume lectin that specifically recognizes the trimannoside core present in all N-linked glycans (19). The N-linked sugar chains are generally classified into three main types: high mannose-, hybrid-and complex-type. In general, high mannose-and hybrid-type sugar chains are tightly bound to ConA and biantennary complex-type structures are weakly bound. In contrast, more extensively processed, highly branched or bisected forms do not bind to the lectin (20). Thus, the N-glycans bound to HAI-2 are likely to contain the complextype structure larger than biantennary structures. There are reports that the sugar chains of glycoproteins play roles in the regulation of folding, conformational stability, protease resistance, and intracellular trafficking (21)(22)(23). The N-glycans bound to HAI-2 might have similar roles in vivo.
Both HAI-2 and HAI-1 are potent inhibitors of HGF activator, whereas the other Kunitz type serine protease inhibitors, APP, TFPI, and UTI, show little or no inhibitory activity toward HGF activator. Furthermore, serum serine protease inhibitors such as antithrombin III, C1-inhibitor, and ␣ 2 -antiplasmin did not inhibit the HGF-converting activity of HGF activator (24). These findings suggest that HAI-2 and HAI-1 function as specific inhibitors of HGF activator in vivo. Expression levels of both HAI-2 and HAI-1 mRNA are low in some tissues such as the liver. In these tissues, the low level of the inhibitors may facilitate the action of HGF activator.
Similar tissue distributions of mRNA of HAI-2 and HAI-1 were observed in various human adult and fetal tissues except adult testis, in which expression of HAI-2 mRNA is much higher than that of HAI-1 mRNA. Furthermore, about equal amounts of both inhibitors were detected in the conditioned medium of MKN45 cells. These results suggest that HAI-2 and HAI-1 simultaneously inhibit HGF activator in vivo. However, these inhibitors have some different properties. As discussed above, different N-linked sugar chains are attached to the protein portion of HAI-2 and HAI-1. Further, HAI-2 was not adsorbed by the hydrophobic column whereas HAI-1 was, indicating that HAI-2 is more hydrophilic than HAI-1. These different properties may result in different inhibitory actions toward HGF activator. Thus, further characterization of HAI-2 2 T. Shimomura and T. Kawaguchi, unpublished results. Single-chain HGF was then added, and the mixtures were further incubated. The reaction products were subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. B, the inhibitory activity of each protease inhibitor was determined as the ratio of the remaining singlechain HGF to total HGF. ND, not detected. FIG. 9. A schematic representation of human HAI-2 together with human HAI-1. and HAI-1 is needed to elucidate how these inhibitors act against HGF activator in vivo.