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J. Biol. Chem., Vol. 277, Issue 16, 14053-14059, April 19, 2002
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From the
Received for publication, December 21, 2001
Hepatocyte growth factor activator inhibitor type
1 (HAI-1) is a Kunitz-type serine protease inhibitor identified as a
strong inhibitor of hepatocyte growth factor (HGF) activator and
matriptase. HAI-1 is first produced in a membrane-integrated form with
two Kunitz domains in its extracellular region, and subsequent
ectodomain shedding releases two major secreted forms, one with a
single Kunitz domain and one with two Kunitz domains. To determine the roles of the Kunitz domains in the inhibitory activity of HAI-1 against
serine proteases, we constructed various HAI-1 mutant proteins and
examined their inhibitory activity against HGF activator and trypsin.
The N-terminal Kunitz domain (Kunitz I) had potent inhibitory activity
against both HGF activator and trypsin, whereas the C-terminal Kunitz
domain (Kunitz II) had only very weak inhibitory activity against HGF
activator, although its potency against trypsin was equivalent to that
of Kunitz I. These results indicate that Kunitz I is the
functional domain of HAI-1 for inhibiting the HGF-converting activity
of HGF activator. Furthermore, the presence of two Kunitz domains
affected the inhibitory activity of HAI-1 against HGF activator, and it
showed a similar, but not additive, level of inhibitory activity
against trypsin when compared with that of the individual Kunitz
domains. These results suggest that serine protease binding
sites of Kunitz I and Kunitz II are located close to each other and
that proteolytic processing to generate HAI-1 with only one Kunitz
domain regulates the activity of HAI-1.
Hepatocyte growth factor activator inhibitor type 1 (HAI-1)1 is a Kunitz-type
serine protease inhibitor (1) initially identified as a strong
inhibitor of hepatocyte growth factor (HGF) activator. HGF activator is
a blood coagulation factor XII-like serine protease that catalyzes
proteolytic conversion of the inactive single-chain precursor of HGF to
the active two-chain form in response to tissue injury (2, 3). The
activated HGF is involved in cell growth during the repair of injured
tissues (4-7). Furthermore, HAI-1 has been shown to be up-regulated
along with repair of injured tissues (8, 9). Thus, HAI-1 may play a
crucial role in tissue repair by regulating the activity of a growth
factor-activating enzyme. HAI-1 was also identified from human breast
milk in a complex with another serine protease, named matriptase (10). Matriptase is an epithelial cell-derived serine protease that has extracellular matrix-degrading activity and has been proposed to
play a role in breast cancer invasion (11, 12). Thus, HAI-1 may also
function as a regulator of tumor progression by controlling the
activity of an extracellular matrix-degrading enzyme.
The primary translation product of HAI-1, which was predicted from the
cDNA sequence, consists of characteristic structural domains,
including two Kunitz domains, a low density lipoprotein receptor
(LDLR)-like domain between the Kunitz domains, and a transmembrane
domain (1). Immunoblotting analysis revealed that HAI-1 is first
produced in a 66-kDa membrane-integrated form, and subsequent
ectodomain shedding releases two major secreted forms from the cell
surface into the extracellular space, and the sizes of the two secreted
forms are 40/39 and 58 kDa (13). The 40/39- and 58-kDa HAI-1 have the
same N-terminal sequence; thus, they are produced from the
membrane-integrated form by different C-terminal processing. The
40/39-kDa HAI-1 has one Kunitz domain and shows strong inhibitory
activity against the HGF-converting activity of HGF activator. The
58-kDa HAI-1 has two Kunitz domains and shows markedly weak inhibitory
activity against HGF activator (13). Thus, the presence of the
C-terminal region including the C-terminal Kunitz domain (Kunitz II) in
the 58-kDa HAI-1 may interfere with the binding of HGF activator to the
reactive site of the N-terminal Kunitz domain (Kunitz I), whereas
elimination of this region by proteolytic processing could lead to
strong binding of HGF activator to HAI-1.
The cell surface HAI-1 integrated in epithelial cell membranes has a
high affinity for HGF activator and forms a complex with the serine
protease. Treatment of the cells with phorbol 12-myristate 13-acetate
or interleukin 1 Whereas regulated proteolytic processing of HAI-1 has been well
characterized (13, 14), the roles of the characteristic structural
domains in the inhibitory activity against serine proteases remain to
be elucidated. Therefore, in this study, we constructed various HAI-1
mutant proteins and examined their inhibitory activity against HGF
activator and trypsin. We found that Kunitz I had potent inhibitory
activity against both HGF activator and trypsin, whereas Kunitz II had
very weak inhibitory activity against HGF activator, although its
potency against trypsin was equivalent to that of Kunitz I. These
results indicate that Kunitz I is the functional domain of HAI-1 for
inhibition of HGF activator. We also found that the presence of two
Kunitz domains affected the inhibitory activity of HAI-1 against HGF
activator and showed a similar, but not additive, level of the
inhibitory activity against trypsin when compared with that of the
individual Kunitz domains. These results suggest that serine protease
binding sites of Kunitz I and Kunitz II are located close to each other
and that proteolytic processing to generate HAI-1 with only one Kunitz domain regulates the activity of HAI-1.
Materials--
Reagents were obtained as follows: CHAPS and
bovine serum albumin (BSA) fraction V were obtained from Sigma, bovine
pancreas trypsin was obtained from Roche Diagnostics GmbH, nonspecific mouse IgG was obtained from Organo Teknika Corp., Alexa 488-conjugated sheep IgG anti-mouse IgG was obtained from Molecular Probe, and enhanced chemiluminescence Western blotting detection reagents were
obtained from Amersham Biosciences, Inc. Monoclonal antibodies (mAbs)
against HAI-1, C76-18 and 1N7, and mAb against HGF activator, A23, were
prepared as described previously (3, 8, 13). Single-chain HGF and
HGF activator were prepared as described previously (2, 4).
Construction and Transfection of Expression Plasmids for HAI-1
Mutant cDNAs--
We used the pME18S vector (15) to express
various mutant proteins of HAI-1. The cDNA encoding full-length
HAI-1 was inserted into the NotI site of the pME18S vector
(pME18S-HAI-1). To prepare an expression plasmid encoding a secreted
form of HAI-1 (HAI-1-NK1LK2), a cDNA fragment (0.6 kb) was
amplified by PCR using full-length HAI-1 cDNA as a template and two
oligonucleotide primers, 5'-GCCGCGGCCAACGTCACAG-3' and
5'-GCGCGGCCGCGACAAGA CT-3'. The PCR product was digested
with BglII and NotI, generating a 0.5-kb
fragment. The 0.7-kb BglII-NotI fragment of
pME18S-HAI-1 was replaced with the 0.5-kb fragment (pME18S-HAI-1-NK1LK2). To prepare expression plasmids encoding HAI-1
deletion mutants, cDNAs encoding the deletion mutants were amplified by PCR using full-length HAI-1 cDNA as a template and a
set of individual oligonucleotide primers. After the PCR products were
digested with EcoRI and NotI, they were inserted
into the EcoRI and NotI sites of the pME18S
vector. These HAI-1 mutant constructs were transiently transfected into
COS-7 cells by the DEAE-dextran method (16). To prepare expression
plasmids encoding HAI-1 with point mutations in the P1 positions of the
Kunitz domains (R260L, K385L, and R260L/K385L), the pME18S-HAI-1-NK1LK2
plasmid was mutated using a set of individual oligonucleotide primers and the QuikChange Site-Directed Mutagenesis Kit (Stratagene). These
mutant constructs and pSV2 neo DNA were co-transfected into CHO cells
by the LipofectAMINE method (17). After transfection, the cells were
cultured in medium containing 0.4 mg/ml G418 and 10% fetal bovine
serum. G418-resistant colonies were selected and screened for
expression of the mutant HAI-1 proteins by an enzyme immunoassay.
Immunoblotting Analysis--
COS-7 cells transfected with the
expression plasmids encoding HAI-1 deletion mutants were cultured in a
10-cm culture dish with Dulbecco's modified Eagle's medium (DMEM)
containing 10% fetal bovine serum. At 24 h after transfection,
the cells were washed twice with serum-free medium and further cultured
in fresh serum-free medium. After 2 days, the conditioned medium was
harvested and then clarified by centrifugation before being
concentrated 20-fold by ultrafiltration using a YM30 membrane (Amicon)
and analyzed by immunoblotting. CHO cell clones expressing HAI-1 mutant proteins were cultured in a 10-cm culture dish with enriched
RPMI1640/DMEM/F-12 (eRDF) medium containing 5% fetal bovine
serum. At confluence, the cells were washed twice with serum-free
medium and then further cultured in fresh serum-free medium. After 2-6
days, the conditioned medium for immunoblotting analysis was prepared
as described above for COS-7 cells. To prepare cell extracts, the cells
were washed with cold 0.9% NaCl and treated with 1 ml of 10%
trichloroacetic acid for 15 min on ice. The trichloroacetic
acid-insoluble fraction was pelleted by centrifugation. The pellet was
then dissolved in 150 µl of 7 M urea containing 2%
Triton X-100 and 5% 2-mercaptoethanol. After an additional
centrifugation, the supernatant was recovered and analyzed for
immunoblotting. The samples for immunoblotting were separated by
SDS-PAGE under reducing conditions. The proteins were transferred to a
nitrocellulose membrane (Hybond ECL; Amersham Biosciences, Inc.) and
incubated with the anti-HAI-1 mAb for 2 h followed by an
incubation with horseradish peroxidase-conjugated secondary antibodies
for 1 h. Immunoreactive proteins were visualized with an enhanced
chemiluminescence Western blotting detection system.
Assay for Inhibitory Activity of HAI-1 Mutant Proteins against
HGF Activator and Trypsin--
Concentrated conditioned medium
containing each HAI-1 mutant was mixed with 1.5 ng of HGF activator or
1.1 ng of trypsin in 40 µl of phosphate-buffered saline with 0.05%
CHAPS and then incubated for 30 min at 37 °C. After the incubation,
5 µl of 1.5 mg/ml single-chain HGF (for HGF activator) or 6 µl of
1.0 mg/ml BSA (for trypsin) in phosphate-buffered saline containing
CHAPS was added to the respective mixture, and the samples were
incubated for an additional 2 h. The reaction products were then
separated by SDS-PAGE under reducing conditions. The gel was stained
with Coomassie Brilliant Blue, and the bands were scanned using a
Flying-Spot Scanner CS-900 (Shimadzu). The inhibitory activity against
HGF activator was estimated by calculating the ratio of the remaining
single-chain HGF to two-chain HGF. The inhibitory activity against
trypsin was estimated by calculating the remaining BSA.
Immunocytostaining--
CHO cell clones expressing full-length
HAI-1 and HAI-1-LK2M as well as MKN45 cells were cultured in 8-well
glass chamber slides with eRDF medium containing 5% fetal bovine
serum. At semiconfluence, the cells were washed with cold
phosphate-buffered saline containing 0.1% gelatin and 0.1%
NaN3 (washing buffer) and incubated with 200 µl of 50 µg/ml anti-HAI-1 mAb (C76-18) or 500 µg/ml control mouse IgG for 60 min on ice. After the incubation, the cells were washed with washing
buffer and incubated with Alexa 488-conjugated anti-mouse IgG. The
cells were then washed with washing buffer and visualized by a confocal
immunofluorescence microscope. For detection of bound HGF activator,
the cells were incubated with 5 µg/ml HGF activator for 30 min on
ice. The cells were then washed with washing buffer and incubated with
200 µl of 50 µg/ml anti-HGF activator mAb (A23) before being
visualized as described above.
Construction and Expression of HAI-1 Deletion Mutant
cDNAs--
The extracellular region of HAI-1 (Fig.
1) consists of a long N-terminal region
(N), Kunitz I (K1), an LDLR-like domain
(L), and Kunitz II (K2). To determine the role of
each domain within the extracellular region of HAI-1 in the inhibitory
activity against serine proteases, we constructed five deletion mutant
cDNAs (Fig. 1). To obtain sufficient amounts of secreted proteins
in conditioned medium, we deleted the transmembrane region in all
constructs. HAI-1-NK1LK2 contained almost the entire extracellular
region including Kunitz I and Kunitz II. HAI-1-NK1 contained Kunitz I but lacked the LDLR-like domain and Kunitz II, whereas HAI-1-NK1L lacked only Kunitz II. HAI-1-LK2 contained Kunitz II but lacked the
N-terminal region and Kunitz I. HAI-1-N consisted of only the
N-terminal region. The constructs were inserted into expression vector
pME18S and transiently transfected into COS-7 cells. Proteins secreted
into the conditioned medium were analyzed by immunoblotting. Two
monoclonal antibodies, C76-18 and 1N7, were used for the
immunoblotting. C76-18, which was directed against an epitope located
in the N-terminal region, recognized HAI-1-NK1LK2, HAI-1-NK1,
HAI-1-NK1L and HAI-1-N, whereas 1N7, which was directed against an
epitope located in Kunitz II, recognized HAI-1-NK1LK2 and HAI-1-LK2.
The immunoblotting analysis revealed that sufficient amounts of HAI-1
mutant proteins were secreted into the conditioned medium (Fig.
2). It has been shown that MKN45 human
gastric carcinoma cells produce two major secreted forms of HAI-1,
40/39- and 58-kDa HAI-1 (13). However, the 40/39-kDa form was barely
detected in the conditioned medium of COS-7 cells transfected with
full-length HAI-1 or HAI-1-NK1LK2 (Fig. 2A, lanes 3 and
4), indicating that its contribution to the inhibitory
activity was negligible. Thus, the inhibitory activity present in the
conditioned medium depended mostly on HAI-1 proteins with two Kunitz
domains. The mutant proteins were quantified by scanning densitometry
of the immunoblots using purified HAI-1 as a standard and assayed for
their inhibitory activity against serine proteases.
Assay for the Inhibitory Activity of HAI-1 Mutant Proteins against
HGF Activator and Trypsin--
We examined the inhibitory activity of
the HAI-1 mutant proteins against two serine proteases, HGF activator
and trypsin. To assess the inhibitory activity against HGF activator,
the protease was mixed with each mutant protein and incubated for 30 min to allow formation of the enzyme-inhibitor complex. Next,
single-chain HGF was added as a substrate, and the mixture was
incubated for an additional 2 h. The samples were then analyzed by
SDS-PAGE. To assess the inhibitory activity against trypsin, the enzyme was mixed with each mutant protein and incubated for 30 min. Next, BSA
was added as a substrate, and the mixture was incubated for an
additional 2 h before being analyzed by SDS-PAGE. Fig.
3 shows representative patterns of the
SDS-PAGE analysis. Single-chain HGF was converted to two chains (a
heavy chain and a light chain) by HGF activator in the absence of HAI-1
(Fig. 3A, lanes 2 and 4). This conversion was
inhibited in the presence of the conditioned medium of MKN45 cells
(Fig. 3A, lane 3), wild-type HAI-1 (Fig. 3A, lane
5), or mutant proteins that contained Kunitz I (Fig. 3A,
lanes 6-8), but not in the presence of mutant proteins that contained only the N-terminal region (Fig. 3A, lane 9) or
Kunitz II (Fig. 3A, lane 10). These results suggest that
Kunitz I, and not Kunitz II, is the functional domain for inhibition of
the HGF-converting activity of HGF activator. BSA was degraded by trypsin in the absence of HAI-1 (Fig. 3B, lanes 2 and
4). This degradation was inhibited in the presence of the
conditioned medium of MKN45 cells (Fig. 3A, lane 3),
wild-type HAI-1 (Fig. 3B, lane 5), or mutant proteins that
contained Kunitz I and/or Kunitz II (Fig. 3B, lanes 6-8 and
10), but not in the presence of a mutant protein that
consisted of only the N-terminal region (Fig. 3B, lane 9).
These results indicate that both Kunitz I and Kunitz II have inhibitory
activity against trypsin.
Dose Dependence of the Inhibitory Activity of HAI-1 Mutant Proteins
against HGF Activator and Trypsin--
To quantitate the inhibitory
activity of the Kunitz domains in HAI-1, the dose dependence of the
inhibitory activity of mutant proteins was examined. Fig.
4A shows the dose-dependence
curves for the inhibitory activity of deletion mutants against HGF
activator. HAI-1-NK1 showed a dose-dependence curve similar to that of
HAI-1-NK1L, and the concentration for 50% inhibition was about 2 nM. HAI-1-NK1LK2 showed a curve similar to that of purified
58-kDa HAI-1, and the concentration for 50% inhibition was about 6 nM. The concentration of HAI-1-LK2 for 50% inhibition was
about 19 nM. These results indicate that HAI-1 with only
Kunitz I shows the most potent inhibitory activity against HGF
activator and that HAI-1 with two Kunitz domains is about 3-fold
less potent, whereas HAI-1 with only Kunitz II is about 10-fold less
potent. Fig. 4B shows the dose-dependence curves of the
inhibitory activity against trypsin. All HAI-1 mutant proteins showed
similar dose-dependence curves, and the concentration for 50%
inhibition was about 1-2 nM.
Immunocytostaining Analysis for the Binding of a
Membrane-integrated Form of HAI-1 with Only Kunitz II to HGF
Activator--
The membrane-integrated form of full-length HAI-1
showed strong binding to HGF activator, whereas the secreted form of
HAI-1 containing almost the entire extracellular region (the 58-kDa HAI-1) showed rather weak binding (14). Thus, it is possible that the
membrane-integrated form of HAI-1-LK2 may be capable of binding to HGF
activator. To examine this possibility, cDNA encoding the
HAI-1-LK2M protein that contained the transmembrane region and
C-terminal cytoplasmic tail of HAI-1 (Fig.
5A) was constructed and stably
transfected into CHO cells. The ability of the expressed proteins to
bind HGF activator was tested by immunocytostaining. For positive
controls, CHO cells expressing full-length HAI-1 and MKN45 cells
expressing a high level of endogenous HAI-1 were also analyzed.
Immunoblotting analysis of the cell extracts suggested that the
membrane-integrated form of HAI-1-LK2M was expressed in the transfected
CHO cells (Fig. 5B, lane 6). Immunocytostaining with a mAb
against HAI-1 confirmed the presence of HAI-1-LK2M on the cell surface
(Fig. 5C, g). However, no binding of HGF activator was
observed on the surface of the cells expressing HAI-1-LK2M (Fig.
5C, k). On the other hand, obvious binding of HGF activator
was observed on the cell surface of the positive control cells (Fig.
5C, j and l). These results indicate that Kunitz
II of HAI-1 has a low affinity for HGF activator both in the secreted
form and in the membrane-integrated form.
Analysis of the Inhibitory Activity of HAI-1 Mutant Proteins with a
Point Mutation in the P1 Positions--
The amino acid residues in the
P1 positions of the reactive sites of various Kunitz domains were
assigned as essential for their inhibitory activity (18, 19). To
further characterize the roles of the Kunitz domains in the inhibitory
activity of HAI-1-NK1LK2, we constructed cDNAs encoding
HAI-1-NK1LK2 mutants in which an arginine residue (Arg260)
of the P1 position in Kunitz I or a lysine residue (Lys385)
of the P1 position in Kunitz II was individually changed to a leucine
residue (R260L and K385L, respectively). In addition, cDNA encoding
a HAI-1-NK1LK2 mutant in which both Arg260 and
Lys385 were changed to leucine was also constructed
(R260L/K385L). These cDNAs as well as cDNA encoding
HAI-1-NK1LK2 were stably transfected into CHO cells because transient
transfection of these mutant cDNAs into COS-7 cells did not produce
sufficient amounts of mutant proteins. Cell lines that stably produced
and secreted proteins of each mutant were obtained. Immunoblotting
analysis showed that sufficient mutant proteins with molecular mass of
58 kDa were obtained in the conditioned medium (Fig.
6). These proteins were assayed for their
inhibitory activity against HGF activator and trypsin.
Fig. 7A shows the
dose-dependence curves of the inhibitory activity against HGF
activator. HAI-1-NK1LK2 with a mutation in Kunitz II (K385L) showed
significantly weaker inhibitory activity than intact HAI-1-NK1LK2, and
the concentration for 50% inhibition was 13 nM. A mutation
in Kunitz I (R260L) led to a further reduction in the inhibitory
activity, and the concentration for 50% inhibition was 42 nM. Almost no activity was observed in the protein with point mutations in both Kunitz domains (R260L/K385L). Fig.
7B shows the dose-dependence curves of the inhibitory
activity against trypsin. A mutation in Kunitz I (R260L) or Kunitz II
(K385L) did not significantly affect the inhibitory activity. Almost no
activity was observed in the protein with point mutations in both
Kunitz domains (R260L/K385L).
In this study, we found that the N-terminal Kunitz domain (Kunitz
I) of HAI-1 had strong inhibitory activity against HGF activator, whereas in contrast, the C-terminal Kunitz domain (Kunitz II) of HAI-1
had only weak inhibitory activity. From these findings, we concluded
that Kunitz I is the functional domain of HAI-1 that is responsible for
inhibition of the HGF-converting activity of HGF activator. Ectodomain
shedding of the membrane-integrated HAI-1 produces the 40/39- and
58-kDa secreted forms of HAI-1. The 40/39-kDa HAI-1 with only Kunitz I
shows much stronger inhibitory activity against HGF activator than the
58-kDa HAI-1 with Kunitz I and Kunitz II (13). Moreover, activated
matriptase forms a tight complex with HAI-1, and the molecular mass of
HAI-1 isolated from the complex was determined to be 40 kDa (20),
suggesting that HAI-1 with only Kunitz I has strong inhibitory activity
against matriptase, although functional analysis of Kunitz II against matripatse has not yet been performed. Thus, proteolytic processing to
generate HAI-1 with only Kunitz I (the 40/39-kDa HAI-1) appears to be a
crucial step to produce a form of HAI-1 that has strong affinity for
both HGF activator and matriptase.
Although Kunitz II showed weak activity against HGF activator, it had
strong inhibitory activity against trypsin, equivalent to that of
Kunitz I. Thus, Kunitz II has the potential to function as a serine
protease inhibitor but has only a weak association with HGF activator.
The amino acid residues in the P1 positions of the reactive sites in
various Kunitz domains are essential for their inhibitory activity (18,
19). The amino acid residue of the P1 position in Kunitz I is an
arginine, and that in Kunitz II is a lysine. HGF activator cleaves the
single-chain HGF precursor after the arginine residue of the P1
position (2, 21), suggesting that HGF activator has the ability to bind
an arginine residue at the P1 position, and thus it has strong affinity
for Kunitz I, but only weak affinity for Kunitz II. However, some
Kunitz-type serine protease inhibitors with an arginine residue at the
P1 position did not inhibit the HGF-converting activity of HGF
activator (22). Several amino acid residues around the P1 positions
differ between Kunitz I of HAI-1 and the Kunitz domains of these
inhibitors (1). Thus, in addition to the arginine residue of the P1
position, some of the different residues in Kunitz I may also
contribute to the strong affinity for HGF activator. Because trypsin
cleaves substrate proteins after the arginine and lysine residues of
their P1 positions (18), it can associate with both Kunitz I and Kunitz II. Furthermore, the difference in amino acid residues around the P1
positions may not affect the binding of trypsin to Kunitz I and Kunitz II.
An LDLR-like domain was assigned between Kunitz I and Kunitz II of
HAI-1 (1). It has been suggested that this domain may be involved in
formation of the inhibitor-enzyme complex because it contains many
negatively charged amino acid residues (1). In the present study,
however, we showed that the absence of this domain did not affect the
inhibitory activity of Kunitz I against both HGF activator and trypsin
under our assay conditions. Thus, the LDLR-like domain may not be
involved in association of the inhibitor with proteases. It is possible
that this domain regulates the proteolytic processing of HAI-1, as was
proposed previously (13).
Although the individual Kunitz domains of HAI-1 had similar inhibitory
activity against trypsin, the HAI-1 protein with two Kunitz domains
also showed a similar, but not additive, level of inhibitory activity
compared with that of the individual Kunitz domains. Moreover, a
mutation in the P1 position of Kunitz I or Kunitz II of HAI-1-NK1LK2
did not significantly affect the inhibitory activity against trypsin.
These results suggest that the serine protease binding sites of Kunitz
I and Kunitz II are located close to each other, and thus only one
molecule of trypsin can associate with HAI-1 even if it has two Kunitz
domains. The HAI-1 protein with two Kunitz domains showed much weaker
inhibitory activity against HGF activator than the HAI-1 protein with
only Kunitz I. Thus, the closely located Kunitz II appears to interfere
with the binding of Kunitz I to HGF activator. A substitution of the lysine residue at the P1 position of Kunitz II with a leucine residue
reduced the inhibitory activity of HAI-1-NK1LK2 against HGF activator
but not against trypsin, suggesting that a small structural change
around the P1 position affects the association of Kunitz I with HGF
activator but not with trypsin. The molecular mass of HGF activator is
34 kDa (1), whereas that of trypsin is only 23 kDa. This difference in
molecular mass may explain why trypsin has easier access than HGF
activator to the HAI-1 protein with two Kunitz domains. The
high-resolution crystallographic studies of bikunin, a serine protease
inhibitor with two Kunitz domains, revealed an interrelation of
positions of the domains (23). The two Kunitz domains of bikunin are
packed close together, but the protease binding site of the first
domain is unobstructed by the second. In contrast, the protease binding
site of the second domain is close to the first, and thus protease
binding is affected by the presence of the first domain (23, 24). In
the case of HAI-1, the protease binding of each Kunitz domain was
affected by the presence of other domain, suggesting that the
three-dimensional structure of HAI-1 with two Kunitz domains is
different from that of bikunin. The presence of the LDLR-like domain
between the two Kunitz domains of HAI-1 may contribute to a
three-dimensional structure different from that of bikunin because no
amino acid residue is present in the linking region between the two
Kunitz domains of bikunin (23). It should be noted that the
membrane-integrated form of full-length HAI-1 has a high affinity for
HGF activator, whereas the secreted 58-kDa form of HAI-1 has a weak
affinity for HGF activator (14). This observation suggests that Kunitz II in the membrane-integrated HAI-1 does not interfere with the binding
of Kunitz I to HGF activator and that proteolytic processing to
generate the secreted form of HAI-1 brings Kunitz I and Kunitz II
closer together and thus causes Kunitz II to interfere with the binding
of Kunitz I to HGF activator.
Because the proteolytic processing to generate the 40/39-kDa HAI-1
occurs between Kunitz I and Kunitz II (13), there is a possibility that
the processing may also generate a HAI-1 protein with only Kunitz II in
a secreted and/or membrane-integrated form, although no such a molecule
of HAI-1 has yet been identified. A serine protease inhibitor named
trypstatin, which consists of only the second Kunitz domain of bikunin,
was identified from mast cell (25, 26). It inhibits factor Xa,
tryptase, trypsin, and chymase. Thus, the proteolytic processing of
bikunin to eliminate the first Kunitz domain leads to an activation of
the second Kunitz domain. Kunitz II of HAI-1 has strong inhibitory
activity against trypsin, even in the presence of Kunitz I. However,
similar to the second Kunitz domain of bikunin, it may also have
inhibitory activity against other serine proteases when Kunitz I is
eliminated by proteolytic processing. Thus, identification of a HAI-1
protein with only Kunitz II and its cognate serine proteases will
reveal the novel biological functions of Kunitz II.
*
This work was supported by a grant-in-aid 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. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of
Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta, Midori-ku, Yokohama 226-8501, Japan. Tel.: 81-45-924-5701; Fax: 81-45-924-5771; E-mail: nkitamur@bio.titech.ac.jp.
Published, JBC Papers in Press, January 22, 2002, DOI 10.1074/jbc.M112263200
The abbreviations used are:
HAI-1, hepatocyte
growth factor activator inhibitor type 1;
HGF, hepatocyte growth
factor;
LDLR, low density lipoprotein receptor;
CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid;
BSA, bovine serum albumin;
mAb, monoclonal antibody;
CHO, Chinese hamster
ovary.
Functional Characterization of Kunitz Domains in
Hepatocyte Growth Factor Activator Inhibitor Type 1*
,, and¶
Department of Biological Sciences, Graduate
School of Bioscience and Biotechnology, Tokyo Institute of Technology,
Midori-ku, Yokohama 226-8501 and § Research Center,
Mitsubishi Pharma Corp., Aoba-ku, Yokohama 227-0033, Japan
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
markedly enhances release of the 58-kDa secreted
form of HAI-1, but not the 40/39-kDa secreted form of HAI-1, from the
cell surface (14). This regulated shedding appears to be mediated by a
zinc-metalloprotease. Because the 58-kDa HAI-1 has a low affinity for
HGF activator, this enzyme is released from the complex with HAI-1, and
as a result, significant HGF activator activity is recovered in
the conditioned medium (14). These observations suggest that the cell
surface HAI-1 acts not only as an inhibitor but also as a specific
acceptor of active HGF activator and that regulated ectodomain shedding ensures the concentrated pericellular activity of HGF activator under
certain cellular conditions, such as tissue injury and inflammation.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
Schematic structures of full-length HAI-1 and
constructs of the deletion mutants. The structure of full-length
HAI-1 is shown at the top; the structures of the deletion
mutants are shown below it. Amino acid numbers of the
truncated positions are shown above the structures.
S, signal peptide; N, N-terminal
region; K1, N-terminal Kunitz domain (Kunitz I);
L, LDLR-like domain; K2, C-terminal
Kunitz domain (Kunitz II).
View larger version (15K):
[in a new window]
Fig. 2.
Immunoblotting analysis of HAI-1 deletion
mutant proteins. Proteins in the conditioned medium of
COS-7 cells transfected with full-length HAI-1 and mutant constructs
were separated by SDS-PAGE (10% polyacrylamide) and transferred to a
nitrocellulose membrane. The membrane was incubated with anti-HAI-1 mAb
C76-18 (A) or 1N7 (B) and developed with
peroxidase-conjugated secondary antibodies.
View larger version (29K):
[in a new window]
Fig. 3.
Assay for the inhibitory activity of HAI-1
deletion mutant proteins against HGF activator and trypsin.
Concentrated conditioned medium was incubated with HGF activator
(A) or trypsin (B). Single-chain HGF
(A) or BSA (B) was then added, and the mixture
was incubated further. The reaction products were separated by SDS-PAGE
(10% polyacrylamide) under reducing conditions. The gel was stained
with Coomassie Brilliant Blue. A band with high molecular mass detected
in lanes 7 and 8 in B is probably from
the conditioned medium.
View larger version (23K):
[in a new window]
Fig. 4.
Dose dependence of the inhibitory activity of
HAI-1 deletion mutants against HGF activator and trypsin. The
inhibitory activity of HAI-1 deletion mutants against HGF activator
(A) and trypsin (B) was assayed as described in
the Fig. 3 legend. The inhibitory activity against HGF activator was
determined by calculating the ratio of the remaining single-chain HGF
to two-chain HGF. The inhibitory activity against trypsin was
determined by calculating the amount of remaining BSA.
View larger version (96K):
[in a new window]
Fig. 5.
Immunocytostaining analysis for the binding
of cell surface HAI-1 proteins with only Kunitz II (HAI-1-LK2M) to HGF
activator. A, a schematic structure of HAI-1-LK2M.
B, immunoblotting analysis of HAI-1-LK2M. Proteins in
the conditioned medium (CM) and cell extracts
(CE) of CHO cells expressing full-length HAI-1
(HAI-1) or HAI-1-LK2M were separated by SDS-PAGE (10%
polyacrylamide) and analyzed as described in the Fig. 2 legend using
the anti-HAI-1 mAb 1N7. C, cytostaining analysis of
HAI-1-LK2M binding to HGF activator. Parental CHO cells, CHO cells
expressing full-length HAI-1 (HAI-1), CHO cells expressing
HAI-1-LK2M (HAI-1-LK2M), and MKN45 cells were incubated with
control mouse IgG (mIgG), anti-HAI-1 mAb (1N7),
or HGF activator plus anti-HGF activator mAb (A23). The
cells were then incubated with Alexa 488-conjugated anti-mouse IgG and
visualized by a fluorescence microscope.
View larger version (19K):
[in a new window]
Fig. 6.
Immunoblotting analysis of HAI-1 point
mutation proteins. Proteins in the conditioned medium of CHO cells
expressing HAI-1 mutants were separated by SDS-PAGE (10%
polyacrylamide) and analyzed as described in the Fig. 2 legend using
the anti-HAI-1 mAb 1N7.
View larger version (20K):
[in a new window]
Fig. 7.
Dose dependence of the inhibitory activity of
HAI-1 point mutation proteins against HGF activator and trypsin.
Inhibitory activity against HGF activator (A) and trypsin
(B) was assayed as described in the Fig. 4 legend.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
FOOTNOTES
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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