Identification of membrane-bound serine proteinase
matriptase as processing enzyme of insulin-like growth
factor binding protein-related protein-1
(IGFBP-rP1/angiomodulin/mac25)
Sanjida Ahmed
1,2
, Xinlian Jin
1
, Motoki Yagi
1,2
, Chie Yasuda
1
, Yuichiro Sato
1,2
, Shouichi Higashi
1
,
Chen-Yong Lin
3
, Robert B. Dickson
3
and Kaoru Miyazaki
1,2
1 Division of Cell Biology, Kihara Institute for Biological Research, Yokohama City University, Japan
2 Graduate School of Integrated Sciences, Yokohama City University, Japan
3 Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA
Insulin-like growth factor (IGF) binding proteins
(IGFBPs) regulate cellular proliferation by modulating
the actions of insulin and IGFs [1,2]. Recent studies
have revealed a group of IGFBP-related proteins
(IGFBP-rPs), which have low affinity for IGFs ⁄

their membrane fraction had an IGFBP-rP1-cleaving activity. The mem-
brane fraction contained an 80-kDa gelatinolytic enzyme, which was
identified as the membrane-type serine proteinase matriptase (MT-SP1)
by immunoblotting. When the membrane fraction was separated by
SDS ⁄ PAGE, the IGFBP-rP1-cleaving activity comigrated with matriptase.
A soluble form of matriptase purified in an inhibitor-free form efficiently
cleaved IGFBP-rP1 at the same site as that found in a naturally cleaved
IGFBP-rP1. Furthermore, small interfering RNAs for matriptase efficiently
blocked both the matriptase expression and the cleavage of
IGBP-rP1 in OVISE cells. These results demonstrate that IGFBP-rP1 is
processed to the two-chain form by matriptase on the cell surface.
Abbreviations
AEBSF, 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride; AGM, angiomodulin; FBS, fetal bovine serum; HAI-1, hepatocyte growth
factor activator inhibitor-1; HGF, hepatocyte growth factor; HLE, hepatocellular carcinoma; IGF, insulin-like growth factor; IGFBP, IGF-binding
protein; IGFPB-rP1, IGFBP-related protein-1; MMP, matrix metalloproteinase; MT-SP1, membrane-type serine proteinase matriptase; PSF,
prostacyclin-stimulating factor; TAF, tumor-derived cell adhesion factor; siRNA, small interfering RNA; tPA, tissue plasminogen activator uPA,
urokinase-type plasminogen activator.
FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS 615
Angiomodulin (AGM) was initially purified as a
tumor-derived cell adhesion factor (TAF) from human
bladder carcinoma cells [4,5]. Its cDNA was cloned
from human leptomeningial cells as mac25 [6] and from
human fibroblasts as prostacyclin-stimulating factor
(PSF) [7]. Since AGM has a relatively low structural
homology to IGFBPs, the name of IGFBP-related pro-
tein-1 (IGFBP-rP1) has recently been proposed for
AGM ⁄ mac25 ⁄ PSF. AGM ⁄ IGFBP-rP1 exerts a weak
cell adhesion activity through heparan sulfate proteog-
lycans on the cell surface [4,5,8] and stimulates cell
growth in culture medium containing insulin or IGFs

prostate specific antigen [28] and MMP-3 [29], have
been reported to cleave IGFBPs. The proteolysis of
IGFBPs is thought to modulate the actions of IGFs
towards cells [29].
We recently found that IGFBP-rP1 is converted
from a single-chain form to a two-chain form by the
action of a trypsin-like serine proteinase [10]. The pro-
teolytic processing of IGFBP-rP1 greatly reduced its
insulin ⁄ IGF-dependent growth promoting activity but
enhanced its syndecan-1-mediated cell adhesion activity
[10]. We report here that the membrane-bound serine
proteinase matriptase is responsible for the processing
of IGFBP-rP1.
Results
Expression and processing of IGFBP-rP1
in various cell lines
We have reported that IGFBP-rP1 is proteolytically
converted to a two-chain form during purification [10].
The cleavage of IGFBP-rP1 leads to complete loss
of insulin ⁄ IGF-1-dependent cell growth-stimulatory
activity due to its loss of insulin ⁄ IGF-binding ability.
To examine whether the specific cleavage of IGFBP-
rP1 also occurs in cultured cell systems, we tested
expression and processing of IGFBP-rP1 in eight
human cancer cell lines and one immortalized epithe-
lial cell line (HEK293). When the conditioned media
were analyzed by immunoblotting after reducing
SDS ⁄ PAGE, seven of the nine cell lines tested were
found to secrete IGFBP-rP1 protein (Fig. 1). Among
them, OVISE ovarian adenocarcinoma cells and

cleavage.
Processing of IGFBP-rP1 by matriptase S. Ahmed et al.
616 FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS
Analysis of serine proteinases secreted
by various cell lines
Trypsin-type serine proteinases present in the condi-
tioned media of the nine cell lines were analyzed by
gelatin zymography (Fig. 3A). To eliminate the activit-
ies of metalloproteinases, the renatured gel was first
treated with 1 mm EDTA and then incubated in the
presence of 10 mm CaCl
2
, as described in Experimental
procedures. Of the nine cell lines tested, OVISE,
Fig. 1. Analysis of noncleaved and cleaved
forms of IGFBP-rP1 in conditioned media of
eight cancer cell lines and one immortalized
cell line (HEK293). Concentrated conditioned
media were prepared from the cultures of
the nine indicated human cell lines, and
each sample containing the same amount of
protein (5 lg) was subjected to SDS ⁄ PAGE
under reducing conditions on a 14% gel,
followed by immunoblotting with the anti-
TAF ⁄ IGFBP-rP1 antibody (upper panel). Bars
indicate the noncleaved form (33 kDa) and
the cleaved form (25 kDa). The cleaved form
is detected highly in OVISE and MKN-45 cell
lines and slightly in HLE and HEK293 cell
lines. As a loading control, the same vol-

conditioned media. Bars indicate the
molecular size in kDa.
S. Ahmed et al. Processing of IGFBP-rP1 by matriptase
FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS 617
MKN-45 and DLD-1 cells commonly expressed a
major gelatinolytic activity at approximately 75 kDa
and additional weak activities at 95 kDa, 105 kDa and
lower molecular weight positions. The 75 kDa activity
was faintly detected in HSC-4 and HEK293 cells.
To date, several families of type II transmembrane
serine proteinases have been identified [30]. Among
them, MT-SP1 is known to be expressed in many types
of epithelial cell lines and carcinoma cell lines, and to
be proteolytically released from the cell membranes
[21,31,32]. The molecular size of a major form of sol-
uble matriptase is approximately 75 kDa. To deter-
mine whether or not the 75 kDa proteinase in Fig. 3A
was matriptase, the conditioned media of the nine cell
lines were subjected to immunoblotting with an anti-
matriptase antibody (M32). As shown in Fig. 3B, the
conditioned media of OVISE, MKN-45 and DLD-1
cells clearly showed doublet bands at approximately
75 kDa and two minor bands at 95 and 110 kDa. The
conditioned media of HSC-4 and HEK293 cells also
showed weak immunoreactive bands at the same posi-
tions. These results attributed the gelatinolytic activit-
ies at 75, 95 and 105 kDa in Fig. 3A to matriptase.
The 75 kDa doublet has been reported to result from
glycosylation [33]. The glycosylation is thought to
increase the stability of this protein against trypsin and

75
EJ-1
ECV-304
HT1080
OVISE
MKN-45
HLE
HSC-4
DLD-1
HEK293
A
B
Fig. 3. Analysis of serine proteinases secre-
ted by nine human cell lines. Conditioned
media were prepared from the cultures of
the indicated, nine human cell lines. The
concentrated conditioned media containing
the same amount of protein (5 lg) were
subjected to gelatin zymography (A) and
immunoblotting with the antimatriptase
antibody M32 (B) as described in ‘Experi-
mental procedures’. In the gelatin zymogra-
phy, metalloproteinase activities were
eliminated by incubating the renatured gel
with 1 m
M EDTA for 30 min. (A) Gelatin
zymograms of the conditioned media from
the nine cell lines. The conditioned media of
OVISE and MKN-45, which showed high
IGFBP-rP1 cleavage in Fig. 1, showed a

the SDS treatment. In addition, immunoblotting under
reducing conditions suggested that the majority of the
75-kDa immunoreactive band was a single-chain latent
matriptase (data not shown) [32].
The correlation between the IGFBP-rP1-cleaving
activity (Fig. 1) and the expression of matriptase in
MKN-45 and OVISE cell lines suggested matriptase as
a candidate for the IGFBP-rP1-processing enzyme.
Moreover, it has been reported that matriptase prefers
basic P1, P3 and P4 residues to cleave substrates [33].
This consensus sequence is found in the cleavage site
sequence of IGFBP-rP1, where Lys97, Lys95 and
Arg94 residues are located at P4, P3 and P1 sites,
respectively [10]. These facts prompted us to examine
whether or not matriptase is the IGFBP-rP1-processing
enzyme. However, when purified IGFBP-rP1 was
incubated with the conditioned medium of OVISE or
DLD-1 cells, the latter of which contained the highest
amount of matriptase, its processing to a two-chain
form was not observed (data not shown).
IGFBP-rP1-cleaving activity of membrane fraction
from OVISE cells
As IGFBP-rP1 is cleaved in cultured OVISE cells, we
next examined the possibility that cell-associated serine
proteinase(s) might cleave IGFBP-rP1. A membrane
fraction of OVISE cells was prepared as described
under ‘Experimental procedures’ and incubated with
purified IGFBP-rP1. Figure 5A (lanes 1 and 2) shows
that the cleaved form of IGFBP-rP1 significantly
increased during the incubation. This cleavage was

buffer after the SDS ⁄ PAGE and subsequent renaturation (lanes 3
and 4). –, No addition, +, addition. Ordinate indicates the molecular
size in kDa. Arrowheads show the matriptase bands. Other experi-
mental conditions are described in ‘Experimental procedures’ and
in the legends to Fig. 3A.
S. Ahmed et al. Processing of IGFBP-rP1 by matriptase
FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS 619
4–6 had the IGFBP-rP1-cleaving activity and fraction
6 showed the highest activity. When the proteins in
the gel pieces were analyzed by immunoblotting, an
80 kDa matriptase was also detected in fraction 6
(Fig. 6B), indicating that this membrane-bound serine
proteinase is the prime candidate for the IGFBP-rP1-
cleaving proteinase in OVISE cells.
IGFBP-rP1-cleaving activity of purified matriptase
The results of the SDS ⁄ PAGE separation of the mem-
brane-bound enzyme suggested that the soluble form
of matriptase might cleave IGFBP-rP1 if separated
from inhibitors. To test this possibility, the condi-
tioned medium of DLD-1 cells, which contained the
highest activity of matriptase, was separated by
SDS ⁄ PAGE and assayed for the IGFBP-rP1-cleaving
80
B
80
1 2
A
123
33
25

lowed by 37 °C for 24 h. The incubated samples were subjected to
immunoblotting under reducing conditions with the anti-TAF ⁄
IGFBP-rP1 antibody. (None), IGFBP-rP1 incubated without gel frac-
tion. Bars indicate the noncleaved form (33 kDa) and the cleaved
form (25 kDa). Fraction 6 shows the highest activity. (B) Fraction-
ation of matriptase. Gel fractions from another lane were used to
detect matriptase. Each gel fraction was extracted with the SDS
sample buffer and applied to SDS ⁄ PAGE under nonreducing condi-
tions on a 10% gel and immunoblotted with the antimatriptase anti-
body M32. An arrow indicates an immunoreactive band of
matriptase. MF, the membrane fraction before separation. Other
experimental conditions are described in ‘Experimental procedures’.
Processing of IGFBP-rP1 by matriptase S. Ahmed et al.
620 FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS
activity. The IGFBP-rP1-cleaving activity was detected
at fractions corresponding to approximately 75 and
95 kDa, which also showed immunoreactive bands to
the antimatriptase antibody in immunoblotting (data
not shown). A similar result was also obtained when
the conditioned medium of OVISE cells was separated
by SDS ⁄ PAGE (data not shown). These results
strongly suggested that soluble forms of matriptase are
able to cleave IGFBP-rP1 in the absence of HAI-1 or
other inhibitors.
To further confirm this possibility, we purified an
inhibitor-free, soluble form of matriptase from the
conditioned medium of DLD-1 cells, as described in
‘Experimental procedures’. The purified matriptase
preparation contained the 75-kDa matriptase and a
few minor proteins, as analyzed by SDS ⁄ PAGE

25 kDa band of IGFBP-rP1 was determined to be
A
98
GAAAGGPG
106
, suggesting that this protein had
been cleaved between K(Lys)
97
and A(Ala)
98
. This
cleavage site was identical to that previously deter-
mined for a naturally cleaved IGFBP-rP1.
All these results demonstrate that the soluble form
of active matriptase cleaves IGFBP-rP1 but HAI-1
or some other inhibitors block its activity in culture
medium.
Effects of matriptase siRNAs on IGFBP-rP1
cleavage in OVISE cells
To show that matriptase is an endogenous IGFBP-
rP1-processing enzyme in OVISE cells, we designed
three siRNAs for matriptase and examined their effects
on the IGFBP-rP1 cleavage. As negative controls,
OVISE cells were treated with a scrambled RNA or
with the lipofectamine reagent alone. Although the
scrambled RNA had some cytotoxic effect, there was
no significant difference in the profiles of secreted pro-
teins among the control and siRNA-treated cultures
(Fig. 9A). Immunoblotting with the antimatriptase
antibody showed that all of the three siRNAs

IGFBP-rP1 was effectively reduced by the siRNA
treatment. These results confirmed that matriptase acts
as an IGFBP-rP1-processing enzyme in the culture of
OVISE cells.
Discussion
In this study, we first identified the type II membrane-
bound serine proteinase matriptase as a processing
enzyme of IGFBP-rP1 ⁄ AGM. Matriptase was initially
Fig. 8. Cleavage of IGFBP-rP1 by purified matriptase and trypsin. (A) Matriptase. IGFBP-rP1 (50 ng) was incubated with the indicated
amounts of purified matriptase at 37 °C for 5 h in 10 lL of a reaction mixture containing 20 m
M Tris ⁄ HCl (pH 7.5), 0.1 M NaCl and 10 mM
CaCl
2
. The original concentration of matriptase was determined for the total 75-kDa protein based on the band intensity relative to that of
bovine serum albumin as standard (Fig. 7A). (B) Trypsin. IGFBP-rP1 was incubated with the indicated amounts of TPCK-trypsin under the
same conditions except for the absence of CaCl
2
in the reaction mixture. The proteolytic cleavage of IGFBP-rP1 in (A) and (B) was analyzed
by immunoblotting as described in Fig. 1. Bars indicate the 33 kDa, uncleaved form and the 25 kDa, cleaved form of IGFBP-rP1.
AB C
Fig. 9. Effects of matriptase siRNAs on IGFBP-rP1 processing in culture of OVISE cells. OVISE cells at 50–60% confluence in 60 mm culture
dishes were transfected with 200 pmol of each of three siRNAs (si973, si1513 and si2578) using Lipofectamine 2000 reagent. As negative
controls, the cells were treated with a scrambled RNA (sc) or with the lipofectamine reagent alone (Cont.). These cultures were incubated in
serum-containing medium overnight, and then in serum-free medium for 2 days. The resultant conditioned media were collected and con-
centrated. The concentrated samples containing 10 lg protein were subjected to reducing SDS ⁄ PAGE followed by the Coomassie Brilliant
Blue staining (A), nonreducing immunoblotting to detect matriptase (B), and reducing immunoblotting to detect IGFBP-rP1 (C). Bars indicate
the molecular sizes of marker proteins in (A), the 110-, 95- and 75-kDa bands of matriptase in (B), and the 33-kDa, uncleaved form and the
25-kDa, cleaved form of IGFBP-rP1 in (C). The scrambled RNA-treated culture (2nd lane) was low in the total band intensity of matriptase (B)
and IGFBP-rP1 (C) as compared with the control or siRNA-treated cultures. This seemed due to the cytotoxic effect of the scrambled RNA.
Other experimental conditions are described in Experimental procedures.

with matriptase siRNAs efficiently blocked both mat-
riptase expression and the cleavage of IGFBP-rP1. It
is also noted that the cleavage sequence of IGFBP-rP1
is consistent with the most preferable sequence in sub-
strate proteins of matriptase [33]. Indeed, a soluble
form of matriptase purified in an inhibitor-free form
efficiently cleaved IGFBP-rP1 at the same site as that
found in a naturally cleaved IGFBP-rP1. All these
facts indicate that the membrane-bound matriptase is
a natural processing enzyme of IGFBP-rP1. On the
other hand, soluble forms of matriptase, which are
released from cell membranes by proteolysis, were
detected in the conditioned media of at least 5 cell
lines out of 9 cell lines tested. The processing of endo-
genous IGFBP-rP1 was correlated with the amount of
soluble matriptase in the conditioned media. Although
the purified soluble matriptase could cleave IGFBP-
rP1, the conditioned media of OVISE and DLD-1 cells
did not show the IGFBP-rP1-processing activity unless
they were separated by SDS ⁄ PAGE. Our recent analy-
sis has detected soluble matriptase in 19 of 24 human
carcinoma cell lines tested, which included carcinomas
of the breast, lung, stomach and colon [32]. The sol-
uble matriptase mostly existed in a single-chain, latent
form as a major component and two-chain forms
complexed with its inhibitor HAI-1, in agreement with
the past reports [18,30]. Therefore, soluble matriptase
released from cell membrane is expected to have a very
low, if any, proteolytic activity. The IGFBP-rP1-clea-
ving activities of the SDS ⁄ PAGE fractions and the

tutes three members (matriptase, matriptase 2 and
matriptase 3). It is conceivable that some of these mem-
brane-bound serine proteinases or their soluble forms
are also involved in the processing of IGFBP-rP1.
It is well known that some IGFBPs undergo proteo-
lytic cleavage [24,25]. In biological fluids, IGFBPs bind
IGFs with high affinity, protecting the growth factors
from proteolytic degradation. The proteolytic cleavage
of IGFBPs is thought to contribute to the release of
IGFs from IGF ⁄ IGFBP complexes to interact with
IGF receptors on the cell surface. This mechanism
may not be directly applicable to the case of IGFBP-
rP1, because IGFBP-rP1 has a far lower affinity for
IGFs than IGFBPs. The high affinity binding of IGFs
to IGFBPs limits the interaction of the growth factors
with the cell surface receptors. Therefore, IGFBPs gen-
erally inhibit IGF-stimulated cell growth in vitro [25].
In contrast, IGFBP-rP1 stimulates cell growth in the
S. Ahmed et al. Processing of IGFBP-rP1 by matriptase
FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS 623
presence of insulin or IGF-1 in vitro, presumably due
to its low affinity for the factors [9,10]. On the other
hand, some studies have shown that IGFBP-rP1 exhib-
its a tumor-suppressive activity when overexpressed in
cancer cells [13–15]. The apparent discrepancy between
the in vitro and in vivo studies is not clearly explained.
IGFBP-rP1 also shows IGF ⁄ insulin-independent activ-
ities. It has affinity for heparin, type IV collagen and
syndecan-1 [5,8,10]. These activities seem to be respon-
sible for the cell adhesion activity of IGFBP-rP1

raised against purified IGFBP-rP1 and purified matriptase,
respectively. All other chemicals were of analytical grade or
the highest quality commercially available.
Cell cultures and preparation of conditioned
medium
Types of human cancer cell lines used are as follows: HSC-
4, tongue squamous cell carcinoma; HT1080, fibrosarcoma;
EJ-1 and ECV-304, bladder carcinomas; DLD-1, colon
adenocarcinoma; OVISE, ovarian clear cell adenocarcino-
ma; MKN-45, adenosquamous carcinoma of the stomach;
HLE, hepatocellular carcinoma. The source and properties
of OVISE cells were described before [41]. The human
embryonic kidney cell line HEK293 (ATCC CRL-1573)
was purchased from American Type Culture Collection
(ATCC, Rockville, MD, USA). The other cell lines were
obtained from Japanese Cancer Resources Bank (JCRB) in
National Institute of Biomedical Innovation (Osaka,
Japan). To prepare conditioned medium, each cell line was
grown to semiconfluence in 90-mm culture dishes contain-
ing a 1 : 1 mixture of Dulbecco’s modified Eagles medium
and Ham’s F12 medium (Gibco; Grand Island, NY, USA),
DME ⁄ F12, supplemented with 10% fetal calf serum (FCS).
The cells were rinsed three times with serum-free
DME ⁄ F12, and the culture was further continued in the
presence or absence of various proteinase inhibitors in
serum-free DME ⁄ F12. After incubation for 2 days, the
resultant conditioned medium was collected, clarified by
centrifugation and dialyzed against distilled water at 4 °C.
The dialyzed sample was then lyophilized and dissolved in
a 100th volume of 10 mm Tris ⁄ HCl (pH 7.5) to the original

Processing of IGFBP-rP1 by matriptase S. Ahmed et al.
624 FEBS Journal 273 (2006) 615–627 ª 2006 The Authors Journal compilation ª 2006 FEBS
phosphatase-coupled avidin (Vector Laboratory). Immuno-
reactive signals were visualized by the enhanced chemilumi-
nescence (ECL) detection method (Amersham Biosciences,
Piscataway, NJ, USA).
Gelatin zymography
Zymography was carried out on 10% polyacrylamide gels
containing 1 mgÆmL
)1
of gelatin, as described previously
[20]. Briefly, after SDS ⁄ PAGE, the gel was incubated in
50 mm Tris ⁄ HCl (pH 7.5) buffer containing 100 mm NaCl
and 2.5% Triton X-100 at room temperature for 1.5 h to
renature the proteins on the gel. After the incubation, the
gel was treated with 50 mm Tris ⁄ HCl (pH 7.5) containing
1mm EDTA at 37 °C for 30 min to inactivate metallopro-
teinases. After the treatment, the gel was further incubated
in 50 mm Tris ⁄ HCl (pH 7.5) containing 10 mm CaCl
2
at
37 °C for 18 h. Gelatinolytic bands were visualized by
staining the gel with Coomassie Brilliant Blue R-250.
Assay of IGFBP-rP1-cleaving activity of
membrane fraction and conditioned medium
Purified IGFBP-rP1 (500 ng) was incubated with a mem-
brane fraction (20 lg protein) or concentrated conditioned
medium (20 lg protein) of OVISE or DLD-1 cells in 40 lL
of 50 mm Tris ⁄ HCl (pH 7.5) containing 0.15 m NaCl and
10 mm CaCl

concentrated SDS sample buffer and then incubated at
room temperature for 6 h. Each extract was subjected to
nonreducing SDS ⁄ PAGE on a 10% gel followed by immu-
noblotting with the antimatriptase antibody M32.
Purification of soluble matriptase
Serum-free conditioned medium of DLD-1 cells was col-
lected, concentrated by ammonium sulfate precipitation,
and subjected to molecular-sieve chromatography on a
Cellullofine GCL-2000 m column (Seikagaku Kogyo,
Tokyo, Japan) pre-equilibrated with 20 mm Tris ⁄ HCl
(pH 7.5) buffer containing 0.5 m NaCl, 0.1% CHAPS
and 0.01% Brij35. Fractions containing matriptase were
pooled, dialyzed against 20 mm Tris ⁄ HCl (pH 7.5) con-
taining 0.01% Brij35 and applied to a Reactive Red
agarose column (Sigma-Aldrich, St. Louis, MO, USA).
Proteins bound to the column were sequentially eluted
with 0.2 m and 0.4 m NaCl in the buffer. The 0.4 m
NaCl fraction was dialyzed against 20 mm Tris ⁄ HCl
(pH 8.0) and applied to a Mono Q HR5 ⁄ 5 column
(Amersham Biosciences) pre-equibrated with the same
buffer. Proteins bound to the column were eluted with a
linear gradient of 0–0.5 m NaCl. Matriptase eluted from
the column was finally applied to reverse-phase HPLC on
a TSKgel TMS-250 column (Tosoh, Tokyo, Japan) in the
presence of 0.1% trifluoroacetic acid (TFA) and eluted
with a linear gradient of 0–80% acetonitrile in 0.1%
trifluoroacetic acid. Matriptase was dissociated and separ-
ated from HAI-1 in this HPLC. The matriptase-contain-
ing fractions were concentrated by centrifugation under
vacuum and then freeze-dried. The dried materials were

work was supported by Grants-in-Aid from the Minis-
try of Education, Culture, Sports, Science and Tech-
nology and from the Ministry of Welfare and Labor
of Japan.
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