TMPRSS13, a type II transmembrane serine protease, is
inhibited by hepatocyte growth factor activator inhibitor
type 1 and activates pro-hepatocyte growth factor
Tomio Hashimoto
1
, Minoru Kato
2
, Takeshi Shimomura
2
and Naomi Kitamura
1
1 Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta,
Midori-ku, Yokohama, Japan
2 Advanced Medical Research Laboratory, Mitsubishi Tanabe Pharma Corporation, Kamoshida-cho, Aoba-ku, Yokohama, Japan
Introduction
Type II transmembrane serine proteases (TTSPs) are
structurally defined by the presence of a short N-termi-
nal cytoplasmic domain, a transmembrane domain
located near the N-terminus, and a C-terminal extra-
cellular serine protease domain. In addition, TTSPs
possess a stem region that may contain a diverse array
Keywords
activation of pro-hepatocyte growth factor;
hepatocyte growth factor activator inhibitor
type 1 (HAI-1); Kunitz-type inhibitor;
TMPRSS13; type II transmembrane serine
protease (TTSP)
Correspondence
N. Kitamura, Department of Biological
Sciences, Graduate School of Bioscience
and Biotechnology, Tokyo Institute of

functions as an HGF-converting protease, the activity of which may be
regulated by HAI-1.
Abbreviations
BSA, bovine serum albumin; ERK, extracellular signal-regulated kinase; HA, haemagglutinin; HAI-1, hepatocyte growth factor activator
inhibitor type 1; HAI-2, hepatocyte growth factor activator inhibitor type 2; HGF, hepatocyte growth factor; HGFA, hepatocyte growth factor
activator; HPAI, highly pathogenic avian influenza; IC
50,
the concentration of inhibitor that inhibited the enzymatic activity by 50% compared
with the uninhibited control; LDL, low-density lipoprotein; MSPL, mosaic serine protease large form; PBS, phosphate-buffered saline;
TTSP, type II transmembrane serine protease.
4888 FEBS Journal 277 (2010) 4888–4900 ª 2010 The Authors Journal compilation ª 2010 FEBS
of protein domains [1,2]. The human TTSP family
consists of 17 members, which are classified into four
subfamilies [2]. TTSPs are synthesized as inactive sin-
gle-chain pro-enzymes, the proteolytic cleavage of
which is required for the enzymes to exert their activity
[2]. Several members of the TTSP family have been
shown to have pivotal functions in development and
homeostasis [1,2]. Moreover, recent studies revealed
that some members are involved in tumorigenesis
and viral infections [3]. However, the physiological and
pathological functions of most members of the TTSP
family remain to be investigated.
The activities of some members of the TTSP family
are regulated by endogenous protease inhibitors, which
include Kunitz-type inhibitors and serpins [2]. Hepato-
cyte growth factor activator inhibitor type 1 (HAI-1),
a Kunitz-type serine protease inhibitor, is implicated in
the inhibition of two members of the TTSP family,
matriptase and hepsin. HAI-1 was originally identified

ing from a human lung cDNA library using degenerate
primers designed on the basis of the conserved cata-
lytic motif of known trypsin-type serine proteases
[11,12]. The amino acid sequence of TMPRSS13 is
identical to that of MSPL except for an insertion of
five amino acids in the N-terminal cytoplasmic region
and the C-terminal end following the protease domain,
in which TMPRSS13 has eight amino acids and MSPL
has a different 27 amino acids [12]. MSPL and
TMPRSS13 preferentially recognize cleavage sites con-
sisting of paired basic amino acid residues [12].
Recently, MSPL and TMPRSS13 have been shown to
be candidates for haemagglutinin (HA)-processing pro-
teases of highly pathogenic avian influenza (HPAI)
viruses. Namely, a full-length recombinant HA of an
HPAI virus was efficiently converted to mature HA
subunits with membrane-fused giant cell formation in
MSPL- or TMPRSS13-transfected cells, but not in
untransfected cells. Furthermore, infection and multi-
plication of the HPAI virus were detected in the trans-
fected cells [13]. MSPL and TMPRSS13 are expressed
in a variety of tissues, and predominantly in lung,
placenta, pancreas and prostate [12]. Therefore, in
addition to the function in HA processing, MSPL and
TMPRSS13 may have physiological functions in these
tissues that remain to be explored.
Here, we characterize in detail the inhibitory effect
of HAI-1 on TMPRSS13. Moreover, we demonstrate
a possible physiological function of TMPRSS13, that
is its HGF-converting activity.

TMPRSS13 expressed in E. coli showed weak protease
activity, probably because of incorrect protein folding.
We therefore expressed pro-TMPRSS13 in mammalian
cells. To obtain pro-TMPRSS13 from conditioned
medium of mammalian cells, we constructed an expres-
sion vector encoding a secreted form of this protein
that lacked the cytoplasmic and transmembrane
domains. In addition, the putative activation cleavage
sequence (AMTGR325) was replaced with the entero-
kinase recognition sequence (DDDDK) for activation
in vitro, and the protein was tagged at the C-terminus
with myc-His for purification and immunoblot analysis
(Fig. 1A). COS-7 cells were transiently transfected with
the expression vector. The protein was purified from
the conditioned medium of the transfected cells. The
immunoblot analysis of the purified protein using
an anti-c-Myc IgG showed a band of 63 kDa under
reducing and nonreducing conditions (Fig. 1B,C), indi-
cating that pro-TMPRSS13 was highly expressed in
this system.
To activate pro-TMPRSS13, we treated the protein
with enterokinase. The immunoblot analysis of the
reaction product using the anti-c-Myc IgG showed a
band of 37 kDa under reducing conditions (Fig. 1B),
and that of 67 kDa under nonreducing conditions
(Fig. 1C). The 37 kDa band probably corresponded to
the protease domain of TMPRSS13, suggesting the
proteolytic activation of the pro-protein. Detection of
the 67 kDa band suggests that the pro-protein was
cleaved at a single site, and the cleaved protein is a

C
SS
Enterokinase
(kDa)
100
50
37
25
IB: anti-c-Myc
63
250
12
IB: anti-c-Myc
Reduced
Nonreduced
1 2
150
75
(kDa)
100
50
37
25
63
250
150
75
A
BC
Fig. 1. Production and activation of the recombinant pro-TMPRSS13. (A) Schematic representation of the structure of pro-TMPRSS13 (wild-

consists of the N-terminal region (N), two Kunitz
domains (K1 and K2), and the low-density lipoprotein
(LDL) receptor class A domain (L) between the
Kunitz domains (Fig. 2A). Inhibition by aprotinin was
compared with that by HAI-1, because aprotinin has
been shown to efficiently inhibit the protease activity
of TMPRSS13 [12]. TMPRSS13 (100 pm) was incu-
bated with various concentrations of HAI-1–NK1,
HAI-1–NK1LK2 and aprotinin, and protease activity
was measured using the synthetic substrate. Figure 2C
shows the dose dependence of the inhibitory activities.
HAI-1–NK1 had the most potent inhibitory effect
(IC
50
= 2.18 ± 0.18 nm). HAI-1–NK1LK2 and apro-
tinin showed much weaker inhibitory activity than
HAI-1–NK1.
Hepatocyte growth factor activator inhibitor type 2
(HAI-2), also known as placental bikunin, is also a
transmembrane Kunitz-type serine protease inhibitor
[15,16]. HAI-2 has been shown to inhibit matriptase
and hepsin [9,10,17]. Thus, we examined the effect of a
soluble form of HAI-2 (Fig. 2B) on the protease activ-
ity of TMPRSS13. HAI-2 inhibited TMPRSS13
(IC
50
= 1.54 ± 0.01 nm) (Fig. 2C), and the IC
50
was
similar to that of HAI-1–NK1. However, the inhibi-

K1
LDLA
N
K2 TM
(2)
myc-His-
N
N
C
(3)
myc-His-
C
myc-His-
C
SP
K1
N
TMPRSS13 activity (%)
Inhibitor concentration (nM).
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LDLA
N
K2
N
C
HAI-2
NK1
Aprotinin
NK1LK2
(2)
N
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SP
K1 K2
(1)
C
N
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TM
0
10
20
30
40
50
60
70
80
90
100
0.01 0.1 1 10 100 1000
Fig. 2. Dose dependence of the inhibitory activity of soluble
forms of HAI-1 and HAI-2 against the protease activity of
TMPRSS13. (A) Schematic representation of the structure of the
full-length HAI-1 (1) and soluble forms of HAI-1, HAI-1–NK1LK2
(2) and HAI-1–NK1 (3), tagged at the C-terminus with myc-His.
SP, signal peptide; N, N-terminal region; K1, Kunitz domain 1;
LDLA, LDL receptor class A domain; K2, Kunitz domain 2; TM,
transmembrane domain. (B) Schematic representation of the
structure of the full-length HAI-2 (1) and a soluble form of HAI-2
tagged at the C-terminals with myc-His (2). (C) Dose dependence
of the inhibitory activity of soluble forms of HAI-1 and HAI-2
against the protease activity of TMPRSS13. TMPRSS13 was
incubated with various concentrations of HAI-1–NK1 (
•
), HAI-1–
NK1LK2 (j), aprotinin (m) or HAI-2 (r). Then, Pyr-RTKR-MCA
was added, and after further incubation, the fluorescence of the

NK1 (5 lm). The pretreatment of TMPRSS13 with
HAI-1–NK1 did not generate the 60 and 32 kDa bands
(Fig. 4C), indicating that HAI-1–NK1 inhibits the
pro-HGF converting activity of TMPRSS13.
A
TMPRSS13 (nM) TMPRSS13 (nM)
50
37
75
(kDa)
50
37
75
(kDa)
50
75
100
(kDa)
50
75
100
(kDa)
NK1 (n
M)
Not boiled
Boiled Boiled
NK1LK2 (nM)
Not boiled
2 20 200
20

TMPRSS13 (n
M) TMPRSS13 (nM)
50
37
75
(kDa)
50
37
75
100
NK1 (n
M)
Boiled
NK1LK2 (nM)
Boiled
2 20 200
20
2 20 200
20 20 20 20 20 20 20
(kDa)
IB: HAI-1
IB: TMPRSS13
*
**
*
Fig. 3. TMPRSS13 forms complexes with HAI-1–NK1. TMPRSS13
at the indicated concentrations was incubated with 20 n
M HAI-1–
NK1 and HAI-1–NK1LK2 at 37 °C for 2 h. After the addition of SDS
sample buffer with 100 m

HGF processing (%)
Protease concentration (nM)
0
20
40
60
80
100
3.4 6.8 13.5 27 54
C
HAI-1-NK1 (µM)
TMPRSS13 (n
M)
54 54
5
0
00
Fig. 4. Proteolytic conversion of pro-HGF by TMPRSS13 and its inhi-
bition by HAI-1–NK1. (A) Pro-HGF (2 l
M) was incubated with various
concentrations of TMPRSS13. The reaction mixtures were separated
by SDS ⁄ PAGE under reducing conditions. The gel was stained with
Coomassie Brilliant Blue. (B) The intensity of the band of pro-HGF
was quantified with NIH
IMAGEJ software and the percentage of HGF
processed was calculated. (C) Pro-HGF (2 l
M) was incubated with
TMPRSS13 (54 n
M) pretreated with or without HAI-1–NK1 (5 lM).
The reaction mixtures were analysed as described in (A).

treated with the TMPRSS13-cleaved pro-HGF than in
HepG2 cells treated with the uncleaved pro-HGF or
with TMPRSS13 (Fig. 5B). Finally, we analysed the
biological response of HepG2 cells by observing their
scattering phenotype. Treatment with the TMPRSS13-
cleaved pro-HGF induced a scattering of cell colonies,
whereas no scattering was observed in the cells treated
with the uncleaved pro-HGF or with TMPRSS13
(Fig. 5C). These results indicate that TMPRSS13 con-
verts the inactive pro-HGF into the active two-chain
form of HGF.
Co-expression of TMPRSS13 and HAI-1 mRNA in
cultured cell lines
Because TMPRSS13 and HAI-1 are both transmem-
brane proteins, HAI-1 is probably co-expressed with
TMPRSS13 in the same cells to function as a physio-
logical inhibitor of the protease. We examined the
co-expression of TMPRSS13 and HAI-1 mRNA in
cultured cell lines by RT-PCR. We analysed five
human carcinoma cell lines: a lung carcinoma cell line
A549, a colon carcinoma cell line LoVo, stomach car-
cinoma cell lines MKN45 and MKN74, and HepG2.
A549 and LoVo cells have been shown to express
TMPRSS13 mRNA [13]. MKN45 cells were used for
identification of HAI-1 proteins [4]. MKN74 and
HepG2 cells have been shown to respond to HGF [20].
TMPRSS13 mRNA was detected in MKN45 and
MKN74 cells, but not in A549, LoVo and HepG2
cells. On the other hand, HAI-1 mRNA was detected
in LoVo, MKN45, MKN74 and HepG2 cells (Fig. 6).

)1
pro-HGF. Cells
were also treated with purified active HGF at 50 ngÆmL
)1
(Active
HGF). (A) Cells were cultured for 5 min. Lysate of the cells was
immunoblotted with the anti-phospho-c-Met IgG (upper panel) and
anti-c-Met IgG (lower panel). (B) Cells were cultured for 5 min.
Lysate of the cells was immunoblotted with the anti-phospho-
ERK1 ⁄ 2 IgG (upper panel) and anti-ERK1 ⁄ 2 IgG (lower panel).
(C) Cells were cultured for 4 days. The morphology of the cells
was analysed by light microscopy.
T. Hashimoto et al. Protease TMPRSS13 is inhibited by HAI-1
FEBS Journal 277 (2010) 4888–4900 ª 2010 The Authors Journal compilation ª 2010 FEBS 4893
Discussion
In this study, we tested the inhibitory effect of HAI-1
on the protease activity of several members of the
TTSP family using enzymes expressed in E. coli.We
found that the protease activity of TMPRSS13 was
inhibited by HAI-1, but that of TMPRSS11A, HAT-
like 4 and HAT-like 5 was not. TMPRSS11A, HAT-
like 4, and HAT-like 5 belong to the HAT ⁄ DESC
subfamily [2]. Mouse DESC1, also of the HAT ⁄ DESC
subfamily, forms stable inhibitory complexes with plas-
minogen activator inhibitor-1 and protein C inhibitor
[21]. Thus, these serpins might be endogenous inhibi-
tors of TMPRSS11A, HAT-like 4 and HAT-like 5.
The protease activity of TMPRSS13 expressed in
E. coli was weak, probably because of incorrect pro-
tein folding. Thus, we expressed the enzyme in mam-

mRNA is also highly expressed in placenta, pancreas
and prostate [4]. Thus, the 40 kDa form of HAI-1
could function as an endogenous regulator of
TMPRSS13 in these tissues.
HAI-1–NK1LK2 had a much weaker inhibitory
effect against TMPRSS13 than HAI-1–NK1 (Fig. 2).
Moreover, no complex of HAI-1–NK1LK2 and
TMPRSS13 was detected in the in vitro binding assays
(Fig. 3). These results indicate that HAI-1–NK1LK2
only weakly associates with TMPRSS13. HAI-1–
NK1LK2 consists of the N-terminal region, the first
Kunitz domain, the LDL receptor class A domain,
and the second Kunitz domain, and corresponds to the
58 kDa form of HAI-1 identified in the conditioned
medium of cultured carcinoma cells [14]. Weaker
inhibitory activity of HAI-1–NK1LK2 against HGFA
and matriptase was also observed, and an idea that the
second Kunitz domain may obstruct the protease-bind-
ing site of the first Kunitz domain was proposed
[22,23]. The present results indicate that this idea may
also apply to TMPRSS13. The weaker inhibitory activ-
ity of HAI-1–NK1LK2 was prominent against
TMPRSS13, compared with that against HGFA and
matriptase. Thus, the presence of the second Kunitz
domain may more strongly affect the binding of the
first Kunitz domain to TMPRSS13.
A soluble form of HAI-2, another Kunitz-type
inhibitor, also inhibited the protease activity of
TMPRSS13, with an IC
50

and its splice variant (HAI-1B) [35]. GAPDH mRNA was used as an
internal control.
Protease TMPRSS13 is inhibited by HAI-1 T. Hashimoto et al.
4894 FEBS Journal 277 (2010) 4888–4900 ª 2010 The Authors Journal compilation ª 2010 FEBS
TMPRSS13 formed complexes (Fig. 3). The weak
complex formation may be related to the characteristic
association of the protease–inhibitor pair.
In the present study we have shown that
TMPRSS13 converted the single-chain pro-HGF to a
two-chain form in vitro (Fig. 4). We proved that the
two-chain form of HGF is biologically active, by three
assessments. Its treatment of HepG2 cells induced the
tyrosine phosphorylation of c-Met, enhanced the phos-
phorylation of ERK, and induced the scattering
phenotype (Fig. 5). Thus, the proteolytic cleavage of
pro-HGF by TMPRSS13 generates a biologically
active HGF. The concentration for half-maximal activ-
ity of TMPRSS13 was 15 nm (Fig. 4B). This value was
0.17 nm for HGFA under similar reaction conditions
[24]. Thus, the specific activity of TMPRSS13 is
approximately 90-fold lower than that of HGFA.
TMPRSS13 preferentially recognizes cleavage sites
consisting of paired basic amino acid residues (RR or
KR at positions P2 and P1). In addition, the presence
of a basic amino acid residue (R or K) at position P4
enhances the efficiency of cleavage [13]. The HA protein
of an HPAI virus strain with the KKKR motif at the
cleavage site was efficiently converted to mature HA
subunits in TMPRSS13-transfected cells [13], suppor-
ting the preference for the cleavage sequences in sub-

responsible for the activation of HGF in the injured
intestinal mucosa, but not in other injured tissues [32].
Thus, other serine proteases are probably involved in
the activation of HGF in these tissues.
Several serine proteases have been shown to convert
pro-HGF to the active form in vitro. They include serine
proteases involved in blood coagulation, such as plasma
kallikrein, and coagulation factors XIa and XIIa
[24,33]. These serine proteases might be responsible for
the activation of HGF in injured tissues. Matriptase
and hepsin, members of the TTSP family, also convert
pro-HGF to the active form [9,10,15]. Thus, it is possi-
ble that these TTSPs function as HGF-converting pro-
teases in injured tissue. A two-step model for the
activation of HGF in injured tissues has been proposed.
When tissue injury occurs, circulating plasma serine
proteases, such as HGFA, are activated in response to
the activation of the coagulation cascade and inflamma-
tion. The activated proteases convert pro-HGF to the
active form (the first step). Subsequently, the activated
HGF functions as a mitogen for the epithelial cells. The
proliferating epithelial cells produce TTSPs, such as
matriptase. The TTSPs convert pro-HGF to the active
form (the second step). The activated HGF is involved
in further proliferation of the epithelial cells [32].
TMPRSS13 might also function as an HGF-converting
protease in the second step, because it appears to be
expressed in epithelial cells [13]. The specific activity of
the HGF conversion of TMPRSS13 is much lower than
that of HGFA as described above. However,

primer containing an XbaI site, which also had a point
mutation replacing the stop codon with a Leu codon. The
PCR product was subcloned into a mammalian expression
vector, p3xFLAG-CMV14 (Sigma, St. Louis, MO, USA).
To construct an expression vector encoding pro-
TMPRSS13 lacking the cytoplasmic and transmembrane
domains, a cDNA sequence encoding amino acid residues
187–567 was amplified by PCR using p3xFLAG-CMV14-
TMPRSS13 as a template. The PCR product was
subcloned into the EcoRI and PstI sites of a mammalian
expression vector, pSecTag2C (Invitrogen, Carlsbad, CA,
USA). The activation cleavage site (A321MTGR325) was
replaced with the enterokinase recognition sequence as
described above.
To construct an E. coli expression vector encoding pro-
TMPRSS13, the cDNA sequence was excised by digestion
with HindIII and XbaI from p3xFLAG-CMV14-
TMPRSS13, and subcloned into an expression vector,
pcDNA3.1 ⁄ myc-His-A (Invitrogen). The activation cleavage
site was replaced with the enterokinase recognition
sequence as described above. A cDNA sequence encoding
amino acid residues 315–567 with the C-terminally tagged
myc-His sequence was amplified by PCR, and subcloned
into the EcoRI and PstI sites of an E. coli expression
vector, pMAL-c2X.
To construct an E. coli expression vector encoding HAI-
1–K1, the cDNA sequence encoding amino acid residues
241–305 was amplified by PCR using cDNA of HAI-1 [4]
as a template. The PCR product was subcloned into the
BamHI and XbaI sites of the vector, pcDNA3.1 ⁄ myc-His-

Dulbecco’s modified Eagle’s medium, CHO cells and Lovo
cells were cultured in Ham’s F12 medium, and MKN45
cells and MKN74 cells were cultured in RPMI1640 med-
ium, supplemented with 10% fetal bovine serum, 100
unitsÆmL
)1
penicillin and 100 lgÆmL
)1
streptomycin at
37 °C in a humidified atmosphere containing 5% CO
2
.
Preparation and activation of pro-TMPRSS13
expressed in COS-7 cells
Cells were seeded on eight 100 mm collagen-coated plates
(Iwaki, Chiba, Japan) at a density of 1·10
6
cellsÆplate
)1
.
The cells were transfected with the expression vector encod-
ing the secreted form of pro-TMPRSS13 at 6 lgÆplate
)1
using the FuGENE-6 reagent (Roche Diagnostics, India-
napolis, IN, USA). After 24 h, the medium was replaced
with serum-free medium, and cells were further cultured for
3 days. The conditioned medium was applied to a nickel
nitrilotriacetic acid resin (EMD Chemicals), and the proteins
bound to the resin were eluted with nickel nitrilotriacetic acid
buffer (EMD Chemicals). The eluted fraction was treated

HAI-1–K1 was prepared as follows. Escherichia coli cells
were transformed with the expression vector encoding HAI-
1–K1. The cells were lysed by sonication. The lysate was
centrifuged, and the pellet was dissolved in urea (6 m). To
refold proteins, glutathione (oxidized form, 5 mm), glutathi-
one (reduced form, 1 mm) and arginine (100 mm) were
added to the solution, and the final concentration of urea
was adjusted to 0.5 m. The refolded HAI-1–K1 was purified
by column chromatography using a nickel nitrilotriacetic
acid resin, followed by dialysis against PBS. The enteroki-
nase-treated pro-TTSPs were mixed with HAI-1–K1
(0.67 lm) and incubated in the assay buffer (50 mm
Tris ⁄ HCl pH 7.5, 150 mm NaCl, and 0.05% Brij 35) for
10 min at 37 °C. Then each substrate was added to the
mixture at a final concentration of 100 lm. After incuba-
tion for 3 h at 37 °C, the amount of 7-amino-4-methyl-
coumarin liberated from the substrate was determined
fluorimetrically with excitation and emission wavelengths of
355 and 460 nm, respectively, using a fluorometer (1420
ARVOsx; Perkin Elmer Life Science, Boston, MA, USA).
HAI-1–NK1, HAI-1–NK1LK2 and HAI-2 were prepared
as follows. The expression vectors encoding HAI-1–NK1,
HAI-1–NK1LK2 and HAI-2 were introduced into CHO
cells using Superfect transfection reagent (Qiagen, Hilden,
Germany). Transfected cells were cultured at 37 °C over-
night. The medium was replaced with fresh medium con-
taining Geneticin (G418). Neomycin-resistant colonies were
selected and further cultured in a roller bottle. When the
cells became confluent, the medium was replaced with
serum-free medium, and the cells were further cultured for

and 0.002% bromophenol blue) with 100 mm dithiothreitol
was added. Some of the samples were boiled for 5 min.
Twenty microlitres of each sample was analysed by
immunoblotting.
HGF-converting activity of TMPRSS13
The recombinant pro-HGF was prepared as described pre-
viously [34]. Pro-HGF (2 lm) was mixed with various con-
centrations of TMPRSS13 in 20 lLof20mm sodium
phosphate (pH 7.3) containing 100 mm NaCl and 0.01%
Chaps and incubated at 37 °C for 2 h. The reaction mixture
was separated by SDS ⁄ PAGE under reducing conditions.
Proteins in the gel were stained with Coomassie Brilliant
Blue. The intensity of the pro-HGF band was quantified by
scanning densitometry using NIH imagej software.
To examine the inhibitory effect of HAI-1–NK1 on the
HGF-converting activity of TMPRSS13, TMPRSS13
(54 nm) was incubated with HAI-1–NK1 (5 lm)in20mm
sodium phosphate (pH 7.3) containing 100 mm NaCl and
0.01% Chaps at 37 °C for 10 min. Then, pro-HGF (2 lm)
was added to the mixture. The final volume of the mixture
was 20 l L. After incubation at 37 °C for 2 h, the reaction
mixture was analysed by SDS ⁄ PAGE, as described above.
Preparation of cell lysate
HepG2 cells were seeded at 1·10
6
cellsÆ100 mmÆplate
)1
.
They were treated with reaction mixtures of the assay for
HGF-converting activity of TMPRSS13 or with purified

tion of the cleared lysate was determined with the BCA
protein assay reagent (Thermo Fisher Scientific, Rockford,
IL, USA).
Antibodies and immunoblotting
Antibodies were obtained as follows: anti-TMPRSS13 IgG
(ab59865), which recognizes the catalytic domain of
TMPRSS13, from Abcam (Cambridge, MA, USA); anti-
human HAI-1 ectodomain IgG from R&D systems (Minne-
apolis, MN, USA); anti-phospho-c-Met (Try1234 ⁄ 1235)
IgG, anti-phospho-p44 ⁄ 42 mitogen-activated protein kinase
(ERK1 ⁄ 2) (Thr202 ⁄ Tyr204) IgG and anti-p44 ⁄ 42 mitogen-
activated protein kinase (ERK1 ⁄ 2) IgG from Cell Signaling
Technology (Beverly, MA, USA); anti-c-Met IgG (c-28)
and horseradish peroxidase-conjugated anti-goat IgG
(sc-2020) from Santa Cruz Biotechnology (Santa Cruz, CA,
USA); and horseradish peroxidase-conjugated anti-rabbit
and anti-mouse IgG from GE Healthcare UK (Bucking-
hamshire, UK). The anti-c-Myc IgG was prepared as fol-
lows. An anti-c-Myc IgG hybridoma cell line (9E10) was
purchased from ATCC (Manassas, VA, USA) and cultured
in RPMI 1640 medium supplemented with 10% fetal
bovine serum. The anti-c-Myc IgG was purified from condi-
tioned medium by column chromatography using protein A
sepharose (GE Healthcare UK).
Equal amounts of protein in the cell lysate were sepa-
rated by SDS ⁄ PAGE. The proteins in the gel were trans-
ferred electrophoretically to a poly(vinylidene difluoride)
membrane (Pall Corporation, Port Washington, NY,
USA). For the detection of HAI-1 and ERK1 ⁄ 2, the
blotted membrane was treated with BSA blocking buffer

product (1 lL) was amplified by PCR using the following
gene-specific primer sets: 5¢-TCCCATCTGTAGCAGCA
ACT-3¢ and 5 ¢-GGATTTTCTGAATCGCACCT-3¢ for
TMPRSS13 (34 cycles), and 5¢-ATGGAGGCTGCTTGG
GCAACA-3¢ and 5¢-ACAGGCAGCCTCGTCGGAGG-3¢
for HAI-1 (26 cycles). The GAPDH-specific primer set,
5¢-AGGTGAAGGTCGGAGTCAAC-3¢ and 5¢-TACTCC
TTGGGAGGCCATGTG-3¢, was used for control reac-
tions (20 cycles). The PCR products were run on a 1% (for
TMPRSS13 and GAPDH) or 2.5% (for HAI-1) agarose gel
and stained with ethidium bromide.
Acknowledgement
We thank Mrs M. Kamizono for excellent technical
assistance.
References
1 Szabo R, Wu Q, Dickson RB, Netzel-Arnett S,
Antalis TM & Bugge TH (2003) Type II transmem-
brane serine proteases. Thromb Haemost 90, 185–193.
2 Bugge TH, Antalis TM & Wu Q (2009) Type II trans-
membrane serine proteases. J Biol Chem 284, 23177–
23181.
3 Choi SY, Bertram S, Glowacka I, Park YW & Pohlmann
S (2009) Type II transmembrane serine proteases in can-
cer and viral infections. Trends Mol Med 15, 303–312.
4 Shimomura T, Denda K, Kitamura A, Kawaguchi T,
Kito M, Kondo J, Kagaya S, Qin L, Takata H, Miyaz-
awa K et al. (1997) Hepatocyte growth factor activator
inhibitor, a novel Kunitz-type serine protease inhibitor.
J Biol Chem 272, 6370–6376.
5 Lin C-Y, Anders J, Johnson M & Dickson RB (1999)

Biochem J 390, 125–136.
11 Kim DR, Sharmin S, Inoue M & Kido H (2001)
Cloning and expression of novel mosaic serine proteases
with and without a transmembrane domain from
human lung. Biochim Biophys Acta 1518, 204–209.
12 Kido H & Okumura Y (2008) MSPL ⁄ TMPRSS13.
Front Biosci 13, 754–758.
13 Okumura Y, Takahashi E, Yano M, Ohuchi M,
Daidoji T, Nakaya T, Bottcher E, Garten W, Klenk
HD & Kido H (2010) Novel type II transmembrane
serine proteases, MSPL and TMPRSS13, proteolytically
activate membrane fusion activity of hemagglutinin of
highly pathogenic avian influenza viruses and induce
their multicycle replication. J Virol 84, 5089–5096.
14 Shimomura T, Denda K, Kawaguchi T, Matsumoto
K, Miyazawa K & Kitamura N (1999) Multiple sites
of proteolytic cleavage to release soluble forms of
hepatocyte growth factor activator inhibitor type 1
from a transmembrane form. J Biochem 126, 821–
828.
15 Kawaguchi T, Qin L, Shimomura T, Kondo J,
Matsumoto K, Denda K & Kitamura N (1997)
Purification and cloning of hepatocyte growth factor
activator inhibitor type 2, a Kunitz-type serine protease
inhibitor. J Biol Chem 272 , 27558–27564.
16 Marlor CM, Delaria KA, Davis G, Muller DK,
Greve JM & Tamburini PP (1997) Identification and
cloning of human placenta bikunin, a novel serine
protease inhibitor containing two Kunitz domains.
J Biol Chem 272, 12202–12208.

inhibitor type 1. J Biol Chem 277, 14053–14059.
23 Kojima K, Tsuzuki S, Fushiki T & Inouye K (2008)
Roles of functional and structural domains of
hepatocyte growth factor activator inhibitor type 1 in
the inhibition of matriptase. J Biol Chem 283, 2478–
2487.
24 Shimomura T, Miyazawa K, Komiyama Y, Hiraoka H,
Naka D, Morimoto Y & Kitamura N (1995) Activation
of hepatocyte growth factor by two homologous prote-
ases, blood coagulation factor XIIa and hepatocyte
growth factor activator. Eur J Biochem 229, 257–261.
25 Miyazawa K, Tsubouchi H, Naka D, Takahashi K,
Okigaki M, Arakaki N, Nakayama H, Hirono S, Sakiy-
ama O, Takahashi K et al. (1989) Molecular cloning
and sequence analysis of cDNA for human hepatocyte
growth factor. Biochem Biophys Res Commun 163, 967–
973.
26 Brinkmann V, Foroutan H, Sachs H, Weidner KM &
Birchmeier W (1995) Hepatocyte growth factor ⁄ scatter
factor induces a variety of tissue specific morphogenic
programs in epithelial cells. J Cell Biol 131, 1573–1586.
27 Matsumoto K & Nakamura T (1996) Emerging multi-
potent aspects of hepatocyte growth factor. J Biochem
119, 591–600.
28 Miyazawa K, Shimomura T, Naka D & Kitamura N
(1994) Proteolytic activation of hepatocyte growth
factor in response to tissue injury. J Biol Chem 269,
8966–8970.
29 Naka D, Ishii T, Yoshiyama Y, Miyazawa K, Hara H,
Hishida T & Kitamura N (1992) Activation of

hepatocyte growth factor to two chain form in serum-
free culture. Cytotechnology 8, 219–229.
35 Kirchhofer D, Peek M, Li W, Stamos J, Eigenbrot C,
Kadkhodayan S, Elliott JM, Corpuz T, Lazarus RA
& Moran P (2003) Tissue expression, protease speci-
ficity, and Kunitz domain functions of hepatocyte
growth factor activator inhibitor-1B (HAI-1B), a
new splice variant of HAI-1. J Biol Chem 278,
36341–36349.
Protease TMPRSS13 is inhibited by HAI-1 T. Hashimoto et al.
4900 FEBS Journal 277 (2010) 4888–4900 ª 2010 The Authors Journal compilation ª 2010 FEBS