(Received for publication, August 6, 1996, and in revised form, November 7, 1996)
From the Hepatocyte growth factor (HGF) activator is a
serine protease that is produced and secreted by the liver and
circulates in the blood as an inactive zymogen. In response to tissue
injury, the HGF activator zymogen is converted to the active form by
limited proteolysis. The activated HGF activator converts an inactive single chain precursor of HGF to a biologically active heterodimer in
injured tissue. The activated HGF may be involved in the regeneration of the injured tissue. In this study, we purified an inhibitor of HGF
activator from the conditioned medium of a human MKN45 stomach
carcinoma cell line and molecularly cloned its cDNA. The sequence
of the cDNA revealed that the inhibitor has two well defined Kunitz
domains, suggesting that the inhibitor is a member of the Kunitz family
of serine protease inhibitors. The sequence also showed that the
primary translation product of the inhibitor has a hydrophobic sequence
at the COOH-terminal region. Inhibitory activity toward HGF activator
was detected in the membrane fraction as well as in the conditioned
medium of MKN45 cells. These results suggest that the inhibitor may be
produced as a membrane-associated form and secreted by the producing
cells as a proteolytically truncated form.
Hepatocyte growth factor (HGF),1 also
known as scatter factor, is a mesenchymally derived heparin-binding
glycoprotein that is secreted as an inactive single chain precursor
from producing cells, and normally it remains in this form (1),
probably associated with the extracellular matrix in the producing
tissues (2). In response to tissue damage such as hepatic and renal
injury, the inactive single chain form is converted to an active
heterodimeric molecule exclusively in the injured tissue by limited
proteolysis at a single site (1). The proteolytically activated HGF may be involved in regeneration of the injured tissue, because HGF is a
potent mitogen for a variety of cells such as hepatocytes and renal
tubular epithelial cells (3-5). Thus, the biological effects of HGF in
the injured tissues are regulated through proteolytic processing. This
processing is mediated by an enzymic activity that is induced in the
injured tissue (1). Four proteases are reported to activate HGF
in vitro. These are HGF activator, urokinase, tissue-type
plasminogen activator, and blood coagulation factor XIIa (2, 6-9).
Recently, we identified HGF activator as a processing enzyme in the
injured liver (10).
HGF activator, a novel serine protease, was isolated from fetal bovine
serum (6) and from human serum (7). The sequence of the HGF activator
cDNA revealed that HGF activator purified from serum is derived
from the COOH-terminal half-region of a precursor protein and that the
precursor has a similar overall domain organization to blood
coagulation factor XII (7). The precursor protein circulates in the
blood as an inactive zymogen. The zymogen is activated by limited
proteolysis by thrombin, and the NH2-terminal half region
of the zymogen is removed by plasma kallikrein in vitro
(11). Tissue injury often leads to local blood coagulation. When
coagulation is initiated, thrombin and kallikrein are generated from
pro-proteins. Activated thrombin and kallikrein may participate in the
activation of the HGF activator zymogen and in the removal of the
NH2-terminal half of the zymogen, respectively.
The activation potential of HGF activator may be neutralized by the
activity of an inhibitor(s). The activity of HGF activator is not
inhibited by serum protease inhibitors such as antithrombin III,
C1-inhibitor, The following cell lines were
obtained from the indicated sources: human melanoma cell lines C32 and
A375, human lung carcinoma cell line A549, human lung fibroblast cell
line HLF, human hepatoma cell lines Hep G2 and PLC/PRF/5, and human
colon carcinoma cell line CCL229 from American Type Culture Collection
(Rockville, MD); human stomach carcinoma cell lines HSC-3 and MKN45 and
human lung carcinoma cell lines PC-9 and PC-3 from IBL (Gunma, Japan); human lung carcinoma cell line HLC-1 and human stomach carcinoma cell
line IG-1 from Department of Physiology, Keio University (Tokyo,
Japan); immortalized cell line of human fetal liver NuE from Dr. N. Ishida (Tohoku University, Sendai, Japan). All cells were grown in eRDF
medium containing 5% fetal bovine serum. At confluence, the cells were
washed twice with serum-free medium and further cultured in fresh
serum-free medium for 4 days.
The conditioned medium was harvested,
clarified by centrifugation, and concentrated 20-fold by
ultrafiltration using a YM30 membrane (Amicon). The concentrates were
assayed for inhibitory activity toward HGF activator. To prepare
membrane extract, the cells (1 × 107 cells) were
washed three times with Dulbecco's sodium phosphate-buffered saline
(PBS) and harvested by a cell scraper. The collected cells were
suspended in 10 ml of 20 mM Tris-HCl (pH 8.0), disrupted by
sonication at 4 °C, and centrifuged at 1,000 × g at
4 °C for 10 min. The supernatant was again centrifuged at
100,000 × g at 4 °C for 1 h. The pellet was
suspended in 1.5 ml of PBS containing 1% Brij 58, sonicated, and
centrifuged at 100,000 × g at 4 °C for 1 h.
The resultant supernatant (membrane extract) was assayed for inhibitory
activity toward HGF activator. Protein concentration in the conditioned
medium and membrane extract was measured using a bicinchoninic acid
protein assay kit (Pierce) with bovine serum albumin as a standard. The
single chain precursor of HGF and HGF activator was prepared as
described (6, 7). After 10 µl of 900 ng/ml HGF activator and 40 µl
of concentrated conditioned medium or membrane extract were incubated
at 37 °C for 30 min, 10 µl of 1.5 mg/ml single chain HGF was added
as a substrate, and the mixture was further incubated at 37 °C for
2 h. The mixture was then analyzed by SDS-polyacrylamide gel
electrophoresis (PAGE) under reducing conditions. The gel was stained
with Coomassie Brilliant Blue, and the bands were scanned using a
Flying-Spot Scanner CS-9000 (Shimadzu).
To purify HAI, MKN45 cells were cultured in roller bottles
(850, Falcon). At confluence, the cells were washed twice with serum-free medium and further cultured in serum-free medium. After 3-6
days, the conditioned medium was harvested. Ten liters of medium was
concentrated to about 300 ml by 30 K OMEGA membrane (FILTRON) and
applied to a heparin-Sepharose CL-6B (Pharmacia Biotech Inc.) column
(2.5 × 10 cm) pre-equilibrated with PBS. The pass-through
fractions were collected and then applied to a ConA-Sepharose
(Pharmacia) column (1 × 5 cm) pre-equilibrated with PBS. Proteins
were eluted with PBS containing 200 mM
The concentration of HAI in the sample was determined by the
phenylthiocarbamyl method (12). The final preparation of the protein
was hydrolyzed with hydrochloric acid, and the resulting free amino
acids were converted to phenylthiocarbamyl derivatives (phenylthiocarbamyl-amino acid) by phenyl isothiocyanate.
Phenylthiocarbamylamino acids were separated by a YMC pack ODS-A
column (0.46 × 15 cm), and the quantity of HAI in the sample was
calculated.
To determine the
NH2-terminal amino acid sequence of the purified HAI, the
protein that was eluted from the C4 column and dried was reduced with
2-mercaptoethanol in 1 M Tris-HCl (pH 8.6) containing 6 M guanidine hydrochloride and 2 mM EDTA at
40 °C for 2 h. The reduced protein was then carboxymethylated
with monoiodoacetic acid at room temperature for 1 h, separated by
reverse-phase HPLC on a C4 column, and sequenced using an Applied
Biosystems 470A Protein Sequencer. To determine the internal amino acid
sequence of HAI, the protein was digested with Achromobacter
protease-I. The digested peptides were separated by reverse-phase HPLC
on a YMC pack C8 column (0.46 × 15 cm) and sequenced.
HGF activator (18 ng)
or factor XIIa (38 ng) was mixed with various concentrations of HAI in
40 µl of PBS containing 0.05% CHAPS and incubated at 37 °C for 30 min. Five microliters of 1.5 mg/ml single chain HGF in PBS containing
0.05% CHAPS and 5 µl of 100 µg/ml dextran sulfate
(Mr cut-off, 500,000, Sigma) were added to the
mixture and further incubated (2 h for HGF activator and 24 h for
factor XIIa). The mixture was analyzed by SDS-PAGE under reducing
conditions. The amounts of single chain HGF and the heterodimeric form
were measured by Flying-Spot Scanner. The inhibitory activity of HAI
against each protease was estimated by calculating the ratio of the
remaining single chain form to total HGF.
Total RNA was prepared from MKN45 cells by
acid guanidinium thiocyanate/phenol/chloroform extraction (13), and
poly(A) RNA was purified by oligo(dT) affinity
chromatography. The primers, 5 Total RNA (10 µg) from MKN45 cells was
denatured, electrophoresed (14), 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 32P-labeled probe as
described (15). The membranes were washed with 1 × SSC containing
1% SDS at 50 °C. The hybridization probe was the 480-bp PCR
fragment.
We screened
serum-free conditioned media from a variety of human cell lines for HGF
activator-inhibitory activity. Six out of 14 conditioned medium samples
contained significant inhibitory activity (Table I).
Among them, five cell lines (three lung and two stomach carcinoma cell
lines) produced high levels of the inhibitory activity. Thus, one
(MKN45) of these cell lines was used for purification of a molecule
with the inhibitory activity. An inhibitor (HAI) was purified from the
serum-free conditioned medium of MKN45 by a seven-step procedure
described under "Experimental Procedures". Analysis of the purified
protein by SDS-PAGE revealed two bands of 39 and 40 kDa under reducing
conditions (Fig. 1). When the NH2-terminal
amino acid sequence of the purified protein was analyzed, only one
sequence was obtained (Table II). Thus, the apparent
heterogeneity of the purified protein on the gel may be caused by a
difference in the COOH-terminal sequence or in glycosylation.
Inhibition of HGF activator by conditioned medium from human cell
lines
Amino acid sequences of HAI-derived peptides
Research Center,
2-antiplasmin, and
1-proteinase inhibitor. Furthermore, HGF activator is
active in serum (9). Thus, serum protease inhibitors are not
responsible for inhibiting the activity of HGF activator. It is likely
that cells of tissues produce the inhibitor of HGF activator. We
therefore searched human cell lines for the inhibitor and found that
conditioned media from various cell lines contained the inhibitory
activity for HGF activator. To characterize the molecule for the
inhibitory activity, we purified the protein and cloned its cDNA.
The nucleotide sequence of the cDNA revealed that the inhibitor is
a Kunitz-type of serine protease inhibitor. We designated this newly
identified serine protease inhibitor as HGF activator inhibitor
(HAI).
Cell Lines and Cell Culture
-methyl-D-mannoside. The eluates were concentrated, and
the buffer was exchanged for 10 mM sodium phosphate (pH
6.8) containing 1 M ammonium sulfate, using a YM30
membrane. The concentrates were applied to a phenyl-5PW (TOSOH) column
(0.75 × 7.5 cm) pre-equilibrated with the same buffer. Proteins
were eluted with a decreasing linear gradient of ammonium sulfate from
1 to 0 M. The HAI protein was eluted at 500-250
mM ammonium sulfate. The fraction was dialyzed against 20 mM Tris-HCl (pH 8.0) containing 0.05% CHAPS and then
applied to an anion exchange PL-SAX (Polymer Laboratories Ltd.) 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 protein was eluted with 50-200 mM NaCl and then
dialyzed against 5 mM sodium phosphate (pH 6.8) containing
0.05% CHAPS. The dialysate was applied to a hydroxyapatite HCA A-4007
(Mitsui-touatu Chemical) column (0.4 × 7.5 cm) pre-equilibrated
with the dialysis buffer. The pass-through fraction was collected and
then gel-filtrated on an Ashahipak GS520 (Asahi Chemical Industries,
Ltd.) column (0.76 × 50 cm) pre-equilibrated with PBS containing
0.05% CHAPS. The HAI protein was collected at 50-30 kDa and finally
purified by reverse-phase high performance liquid chromatography (HPLC) on a YMC pack C4 (YMC) column (0.46 × 15 cm). Elution was
performed with a linear gradient of 10-50% acetonitrile/isopropyl
alcohol (3:7) containing 0.07% trifluoroacetic acid at a flow rate of 1 ml/min for 30 min. The HAI fractions were neutralized with 1 M Tris-HCl (pH 8.0), dried under a vacuum, and then
dissolved in PBS containing 0.05% CHAPS. The preparation was analyzed
by SDS-PAGE and stained with silver.
-GGNGCNGAYTGYTTRAA-3
and
5
-GGNGCNGAYTGYCTNAA-3
(primer 1), 5
-GTRTCYAANACRAANCC-3
and
5
-GTRTCNAGNACRAANCC-3
(primer 2), 5
-CCNCCRTANACRAANGA-3
and
5
-CCNCCRTANACRAARCT-3
(primer 3), and 5
-CCCCAYAAYTCNACYTG-3
and
5
-CCCCANAGYTCNACYTG-3
(primer 4) (N = A, G, C, or T,
Y = C or T, and R = A or G) were chemically synthesized.
Using primers 1 and 2, and poly(A) RNA as a template, DNA fragments
were amplified by reverse transcription-polymerase chain reaction, and
a 56-bp fragment was generated. The DNA fragment was subcloned and
sequenced. The primer, 5
-AACAGCTTTACCG-3
(primer 5), which is part of
the new sequence, was chemically synthesized. Using primers 3 and 5 and
poly(A) RNA as a template, DNA fragments were amplified by reverse
transcription-polymerase chain reaction. The products were further
amplified using primers 4 and 5, and a 480-bp fragment was generated.
Using the fragment as a probe, a human placenta cDNA library
(Clontech) was screened to obtain full-length cDNA.
Purification of an Inhibitor of HGF Activator (HAI) from the
Conditioned Medium of a Stomach Carcinoma Cell Line
Cell
line
Origin
Inhibitory activitya
C32
Human
melanoma
A375
Human melanoma
PC-9
Human lung carcinoma
++
PC-3
Human lung
carcinoma
+
A549
Human lung carcinoma
++
HLC-1
Human lung carcinoma
++
HLF
Human lung
fibroblast
±
HSC-3
Human stomach carcinoma
++
MKN45
Human stomach carcinoma
++
IG-1
Human stomach
carcinoma
±
PLC/PRF5
Human hepatoma
±
HepG2
Human
hepatoma
±
NuE
Immortalized cells from human fetal
liver
±
CCL229
Human colon carcinoma
±
a
±, less than 10%; +, 10-50%, ++, more than 50%.
Fig. 1.
SDS-PAGE of purified human HAI. The
final preparation of the C4 reverse phase chromatography was analyzed
by SDS-PAGE (12.5% acrylamide) under reducing conditions and stained
with silver. Molecular mass markers are shown in kilodaltons on the right.
[View Larger Version of this Image (34K GIF file)]
Peptide no.
Amino acid sequencea
N-terminal
GPPPAPPGLPAGADCLNSFTAGVPGFVLDTXASVSNGATF
1
VQPQXPLVLK
2
SFVYGGXLGNK
3
DVENTDWRLLRGDTDVRVERK
4
AWAGIDLK
5
DPNQVELWGLK
6
XGTYLFQLTV
a
Amino acids not determined are denoted by
X.
To determine the internal amino acid sequences, the purified protein was carboxymethylated and digested with Achromobacter protease-I, and the resulting peptide fragments were separated by reverse-phase HPLC. Six partial sequences were determined (Table II). None of the peptide sequences nor the NH2-terminal sequence matched those in the Swiss Prot or NBRF protein sequence data bases, indicating that HAI is a novel protein.
Properties of the Purified HAIFig. 2 shows
the dose-response curve of the inhibitory activity of HAI. In these
reactions, HGF activator (450 ng/ml) was mixed with various
concentrations of purified HAI and incubated for 30 min to form an
enzyme-inhibitor complex. Then remaining HGF-converting activity in the
mixture was measured. The concentration of HAI for 50% inhibition was
about 250 ng/ml. Considering the molecular masses of HAI and HGF
activator, HAI forms about an equimolar complex with HGF activator
within 30 min.
HGF activator is homologous to blood coagulation factor XIIa. Factor XIIa can activate single chain HGF in vitro, although the specific activity of factor XIIa is lower than that of HGF activator (9). We therefore examined whether or not HAI inhibits the HGF-converting activity of factor XIIa and found that it did not, even when a 5-fold molar excess of HAI was incubated with factor XIIa (Fig. 2). Thus, HAI is specific for HGF activator in HGF-converting activity.
Isolation of cDNA Clone and DNA Sequence AnalysisTwo
hexapeptide sequences, Gly-Ala-Asp-Cys-Leu-Asn and
Gly-Phe-Val-Leu-Asp-Thr in the NH2-terminal sequence (Table
II), were used to design degenerate oligonucleotide primers for PCR
amplification of the sequence for the NH2-terminal region.
PCR amplification of MKN45 RNA resulted in a cDNA fragment with the
expected size of about 56 bp. The cDNA fragment was subcloned and
sequenced. The cDNA clone encoded 19 amino acids, including 7 amino
acids in the NH2-terminal amino acid sequence between the
two hexapeptide sequences used for the primer design. Thirteen
nucleotides from the obtained sequence were used as a 5 primer for
further PCR amplification together with a sequence corresponding to the
hexapeptide Ser-Phe-Val-Tyr-Gly-Gly in peptide 2 (Table II) as a 3
primer. The PCR amplification products were further amplified using the 5
primer and the sequence corresponding to the hexapeptide
Gln-Val-Glu-Leu-Trp-Gly in peptide 5 (Table II) as a 3
primer. The PCR
amplification resulted in a cDNA fragment of about 480 bp. The
cDNA fragment was subcloned and sequenced. The cDNA clone
encoded 160 amino acids, including the sequences of peptides 1, 3, and
4 in Table II. Northern blotting using the PCR clone as a probe
revealed that the human placenta produced significant amounts of
mRNA of HAI. Thus, a cDNA library from the human placenta
(Clontech) was screened using the PCR clone as a probe. Eighty-four
hybridization-positive clones were obtained from about 4 × 105 phage. The largest clone was sequenced to determine the
primary structure of human HAI. The determined nucleotide sequence of the cDNA is shown in Fig. 3.
Predicted Amino Acid Sequence of HAI
The amino acid sequence
of HAI deduced from the cDNA sequence is also shown in Fig. 3. The
translation initiation site was assigned to the first methionine codon
because the sequence GCGG matches a favorable Kozak
consensus sequence (16). This methionine is followed by a hydrophobic
region (Fig. 4), and the NH2-terminal amino
acid of the purified protein is located at 36th residue downstream of
the methionine. Thus, the hydrophobic region may represent a signal
peptide sequence. Because the reading frame is open upstream of the
first methionine codon, it is possible that translation is initiated
further upstream beyond the boundary of the cDNA sequence. The open
reading frame that starts from the putative ATG codon consists of 513 amino acids, and the protein product has a calculated molecular mass of
56,893. Excluding the putative signal peptide, the mature form of the
protein consists of 478 amino acids and has a calculated molecular mass
of 53,319. The apparent molecular mass of HAI purified from the
conditioned medium of MKN45 cells was about 40 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 region of 23 amino acids is present in the COOH-terminal region (Fig. 4), suggesting that the primary translation product is a
membrane-associated protein. There are three potential
N-glycosylation sites with the canonical Asn-X-
(Ser/Thr). A comparison of the predicted protein sequence of HAI with
sequences in the Swiss Prot and NBRF protein data base revealed three
regions with characteristic structural features. The two regions
(residues 250-300 and 375-425) showed extensive similarity to the
Kunitz-type sequence of serine protease inhibitors (Fig.
5A). Thus, HAI appears to be a Kunitz-type serine protease inhibitor. The other region (residues 319-353) located
between the two Kunitz domains showed similarity to the ligand binding
domain of the low density lipoprotein (LDL) receptor and related
proteins (Fig. 5B). Other regions did not show clear-cut similarity to any protein sequences within the data base entries.
Detection of Inhibitory Activity toward HGF Activator in Membrane Fraction of MKN45 Cells
The cDNA sequence of HAI suggests
that the primary translation product is a membrane-associated protein.
We therefore examined whether inhibitory activity toward HGF activator
was detected in membrane fraction. Membrane extract and conditioned
medium of MKN45 cells were prepared and assayed for the inhibitory
activity. Protein concentration in the membrane extract and conditioned medium was 290 and 620 µg/ml, respectively. Significant activity was
detected in the membrane extract (Fig. 6). Considering
the protein concentration in each fraction, the activity in the
membrane fraction was about 80% that in the conditioned medium. These
results suggest that HAI may be produced as a membrane-associated form and is secreted as a proteolytically truncated form.
Tissue Distribution of HAI mRNA
We determined the size
and tissue distribution of HAI mRNA by Northern blotting with
poly(A) RNAs from various human tissues (Fig. 7). A
major transcript of 2.5 kb was detected in MKN45 cells where we
purified the HAI protein. In addition, a minor transcript of 5.6 kb was
also detected in MKN45 cells. A transcript of 2.5 kb was detected in a
variety of human adult and fetal tissues. Among them, the expression
level of HAI mRNA was relatively high in the adult placenta,
kidney, pancreas, prostate, and small intestine. It was also high in
the fetal kidney. However, although the level was low in the adult
lung, it was relatively high in the fetal lung.
In this study, we found an inhibitory activity toward HGF
activator in serum-free conditioned media of various human cell lines.
We purified the inhibitor protein, HAI, from the conditioned medium of
MKN45 stomach carcinoma cells. The purified HAI has a molecular mass of
about 40 kDa. The primary structure of the protein was predicted from
the sequence of the cDNA for human HAI. The structure of human HAI
is schematically summarized in Fig. 8. The primary
translation product consists of 513 amino acid residues. The
NH2-terminal 35 residues may serve as a signal peptide. The
mature protein appears to be membrane-bound, because a hydrophobic
region of about 20 amino acids is present at the COOH-terminal region.
HAI has two well defined Kunitz domains. The Kunitz domain is typically
about 60 amino acids in length and contains three disulfide bonds. It
was first recognized as the functional domain of bovine pancreatic
trypsin inhibitor (17). Thereafter, the domain was found in several
mammalian serine protease inhibitors. Thus, one or both of the Kunitz
domains found in HAI appear to be responsible for the inhibitory
activity of the protein.
The first and second Kunitz domains of human HAI show the highest
homology (47% identity) to those of human -amyloid precursor protein (APP) and human APP homolog protein, respectively. APP is the
precursor protein of amyloid
-protein which is present in neutritic
plaque and cerebrovascular deposits in individuals with Alzheimer's
disease and Down's syndrome (18). The Kunitz domain is located in the
middle of APP (19-21), and it efficiently functions as an inhibitor of
several serine proteases (22). The primary translation product of APP
has a hydrophobic sequence at the COOH-terminal region, and thus it
appears to be a membrane-bound protein. Oltersdorf et al.
(23) and Van Nostrand et al. (24) reported that protease
nexin II (PNII) is a secreted form of APP. PNII is a protease inhibitor
that forms SDS-resistant inhibitory complexes with epidermal growth
factor-binding protein, the
-subunit of nerve growth factor, and
trypsin. Smith et al. (25) reported that an inhibitor of
coagulation factor XIa purified from the serum-free conditioned medium
of HepG2 liver cells is also a secreted form of APP. Truncated forms of
APP are derived from their cognate membrane-associated forms by
proteolysis and have apparently lost the cytoplasmic and the
transmembrane domains (26). Thus, PNII and factor XIa inhibitor are
proteolytically truncated forms of the transmembrane form of APP. HAI
purified from the serum-free conditioned medium of MKN45 cells has a
molecular mass of about 40 kDa, which is smaller than the predicted
molecular mass (53,319) of the primary translation product, and thus it
may lack the putative membrane-associated and cytoplasmic domains.
Because the HGF activator-inhibitory activity was detected in the
membrane fraction of MKN45 cells, it is likely that the primary
translation product of HAI is a membrane-associated form. Thus, like
PNII and factor XIa inhibitor, the secreted form of HAI appears to be a
proteolytically truncated form of the membrane-associated form of
HAI.
HAI has two Kunitz domains interrupted by another domain. Two serine
protease inhibitors with two or three Kunitz domains have been
identified. Inter--trypsin inhibitor (I
TI) found in mammalian
plasma is a high molecular weight glycoprotein with two tandemly
repeated Kunitz domains in the light chain, which is one of three
polypeptide chains linked by a glycosaminoglycan (27). Tissue factor
pathway inhibitor (TFPI), which was also found in mammalian plasma, has
three tandemly repeated Kunitz domains (28). TFPI inhibits activated
factor X(Xa) directly and, in a Xa-dependent manner,
inhibits VII(a)/tissue factor activity by forming a quaternary
Xa·TFPI·VII(a)·tissue factor complex. The Kunitz domains in these
inhibitors are not interrupted by another domain. Trypsin and
chymotrypsin form equimolar complexes with I
TI. Furthermore, a
protease inhibitor, which consists of only the second Kunitz domain of
the light chain of I
TI (29), has been identified from mast cells
(30). This inhibitor, named trypstatin, markedly inhibits factor Xa and
tryptase and also inhibits trypsin and chymase. Thus, the second Kunitz
domain in the light chain of I
TI is required for the inhibition of
serine proteases, whereas the first domain does not seem to be required for this function. Site-directed mutagenesis of TFPI has revealed that
the second Kunitz domain is required for the efficient binding and
inhibition of Xa, that both Kunitz domains 1 and 2 are required for the
inhibition of VIIa/tissue factor activity, but that the third Kunitz
domain does not seem to be required for these functions (31). The
dose-response curve of the HAI activity showed that HAI purified from
the conditioned medium seems to form an equimolar complex with HGF
activator. Furthermore, the proteolytic cleavage to produce the
extracellular truncated form of HAI appears to occur within the second
Kunitz domain, because the size (about 40 kDa) of the protein implies
that it consists of about 360 amino acids. Thus, the first Kunitz
domain in HAI may be sufficient for it to exert activity.
The other characteristic structural domain in HAI is located between the two Kunitz domains. It consists of about 40 amino acid residues and bears a close resemblance to the "cysteine domain" repeat of the LDL receptor and related proteins. The LDL receptor has seven repeats of the domain (32). Each repeat has six cysteine residues, all of which are involved in disulfide bonds, and in addition it has several negatively charged amino acid residues at the COOH-terminal region. The clustering of cysteine residues in the domain of HAI is similar to that in the repeat of the LDL receptor. Furthermore, the domain of HAI has 8 negatively charged but only 2 positively charged amino acid residues. The negatively charged domain in the LDL receptor is believed to be the binding site for its positively charged apoprotein ligand (32). Although the significance of the negatively charged domain in HAI remains to be established, it may be involved in formation of the inhibitor-enzyme complex, because HGF activator shows high affinity to negatively charged substances.
HGF activator is produced mainly in the liver, and it normally circulates in the blood as an inactive zymogen. In response to tissue injury, the zymogen is activated by proteolytic processing exclusively in the injured tissue (10). The activated HGF activator acquires strong affinity for heparin, which may ensure the localization of the enzyme in injured tissue (10). This localized HGF activator activates single chain HGF that is also associated with a heparin-like molecule on the cell surface. Thus, the activity of HGF activator may be regulated by tissue-derived inhibitors. Because human HAI mRNA is expressed in various tissues, the HAI protein produced by cells of these tissues may be responsible for inhibiting HGF activator. However, HAI mRNA expression is low in some tissues, including the liver and lung. HGF is thought to play a crucial role in repair of liver and lung following injury (1, 33). In these tissues, production of HAI could be induced during tissue repair. Alternatively, another inhibitor(s) may function in these tissues. Characterizations of these inhibitors in injured tissues are needed to understand mechanisms for regulating the activation of HGF.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB000095[GenBank].