inhibition of membrane-type serine protease 1/matriptase
TRANSCRIPT
J Nutr Sci Vitaminol, 49, 27-32, 2003
Inhibition of Membrane-Type Serine Protease 1/Matriptase by
Natural and Synthetic Protease Inhibitors
Yoshie YAMASAKI, Shigeki SATOMI, Nobuhito MURAI, Satoshi TSUZUKI and Tohru FUsHIKI*
Laboratory of Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
(Received August 26, 2002)
Summary Membrane-type serine protease 1 (MT-SP1), identical to matriptase, is a recently identified type II transmembrane serine protease. MT-SP1/matriptase is of considerable interest for the development, homeostasis, and cancer invasion and metastasis of epithelial tissues. The administration of inhibitors for MT-SP1/matriptase may be effective to suppress the development of tumors where the enzyme may be involved. In the present study, we produced a secreted form of recombinant MT-SP1/matriptase (ekMT-SP1s) that can be activated by enterokinase in vitro and investigated the inhibitory ability of various protease inhibitors toward the recombinant enzyme. The enterokinase-treated ekMT-SP 1s (active ekMT-SP1s) cleaved various peptidyl-4-methylcoumaryl-7-amide (MCA) substrates with arginine (or lysine) residue at position P1, and the best substrate was t-butyloxycarbonyl (Boc)-Gln-Ala-Arg-MCA. The specificity for the synthetic and natural substrates of the active ekMT-SP1s was in good agreement with that of the natural enzyme. Endogenous protease inhibitors tested, except for antithrombin III, showed no or little inhibition on the cleavage of Boc-Gln-Ala-Arg-MCA by the active ekMT-SP1s. Aprotinin showed strong inhibitory activity toward the cleavage. Food-derived inhibitors, such as soybean trypsin inhibitor, Bowman-Birk inhibitor, and lima bean trypsin inhibitor inhibited it, while chicken ovomucoid did not. Synthetic inhibitors tested inhibited it, and among them, the inhibitory effect of FOY 305 was strongest. The present findings provide important information for the suppression of cancer invasion and metastasis for which MT-SP1/matriptase is responsible.Key Words membrane-type serine protease 1 (MT-SP1)/matriptase, protease inhibitors, suppression of tumor development
It is known that protease inhibitors suppress the development of tumors in several different tissues such as the colon, liver, lungs, esophagus, and cheek pouch
(1-4). Although the mechanism of action of protease inhibitors as anticarcinogenic agents is poorly understood, it is likely that they are reversing the events in tumor development, presumably by stopping the ongoing processes. In fact, the effects of protease inhibitors that are thought to be related to anticarcinogenic activity include the ability to affect the levels of certain types of proteolytic activities (1). It has, however, remained unclear what types of proteases are responsible for tumor development that can be suppressed by the administration of protease inhibitors.
* To whom correspondence should be addressed .E-mail: [email protected]: BBI, Bowman-Birk inhibitor; Boc-, t-butyl
oxycarbonyl-; HAI-1, hepatocyte growth factor activator inhibitor-1; HGF, hepatocyte growth factor; LBTI, lima bean trypsin inhibitor; MCA, 4-methylcoumaryl-7-amide; MT-SP1, membrane-type serine protease 1; PAGE, polyacrylamide gel electrophoresis; PAR-2, protease-activated receptor-2; PCR, polymerise chain reaction; PMSF, phenylmethylsulfonyl fluoride; PSTI, pancreatic secretory trypsin inhibitors; SBTI, soybean trypsin inhibitor; SDS, sodium dodecylsulfate; uPA, urokinase-type plasminogen activator.
Membrane-type serine protease 1(MT-SP1), identical to matriptase, is a recently identified type II transmembrane serine protease (Fig. 1) (5, 6). In vitro, MT-SP1/matriptase has been shown to cleave and activate single chain-urokinase type plasminogen activator (uPA) (7-9), activate protease-activated receptor-2 (PAR-2) (7), cleave the precursor form of hepatocyte growth factor (HGF) to produce its active form (8), and digest extracellular matrix proteins directly (9). The identification of these molecules as putative in vivo substrates suggests that MT-SP1/matriptase physiologically regulates the functions mediated by these molecules, such as cell adhesion and/or migration. It has also been shown that the activity of MT-SP1/matriptase is inhibited by hepatocyte growth factor activator inhibitor-1
(HAI-1) (10). HAI-1 is found predominantly in the columnar epithelium of many tissue types and is upregulated in injured or regenerative tissues (11). HAI-1 has been considered to suppress the growth and motility of carcinoma cells by inhibiting the generation of active uPA by MT-SP1/matriptase, active HGF by MT-SP1/matriptase or hepatocyte growth factor activator, or by inhibiting the activity of additional unidentified serine proteases. Recently, HAI-1 immunoreactivity in human primary colorectal carcinomas was found to be decreased significantly in cells within colon carcinomas
27
28 YAMASAKI Y et al.
relative to adjacent normal mucosa or adenomas (12).
In addition, ittt has very recently been reported that the
increased expression of MT-SP1/matriptase occurs in
advanced colorectal cancers possibly because the en
zyme is stabilized by glycosylation in which ƒÀ1-6-N-
acetylglucosaminyltransferase is involved (13). These
findings indicate that an imbalance in the MT-SP1/ma
triptase: HAI-1 ratio could be critical in the develop
ment of tumors. At least, it is believed that tumor devel
opment responsible for such an imbalance may be sup
pressed by the administration of inhibitors for MT-
SP1/matriptase. Thus, the investigation of MT-SP1/ma
triptase inhibitors would be of importance.
In the present study, we investigated natural and syn
thetic inhibitors for MT-SP1/matriptase. Inhibition of
the enzyme by protease inhibitors was determined
using a secreted form of recombinant MT-SP1/matrip
tase. Cleavage of a peptidyl-4-methylcoumaryl-7-amide
(MCA) substrate by recombinant MT-SP1/matriptase
was inhibited by plant-derived natural inhibitors but
not by an animal-derived inhibitor, ovomucoid, or en
dogenous protease inhibitors such as pancreatic secre
tory trypsin inhibitor. A synthetic trypsin inhibitor,
FOY-305 strongly inhibited it. The administration of
plant-derived inhibitors or some synthetic inhibitors
may be effective for the suppression of cancer invasion
and metastasis, at least in which MT-SP1/matriptase
may be involved.
MATERIALS AND METHODS
Materials. COS-1 monkey kidney cells were ob
tained from the Health Science Research Resources
Bank (Osaka, Japan). Soybean trypsin inhibitor (SBTI,
type I-S), Bowman-Birk inhibitor (BBI), lima bean
trypsin inhibitor (LBTI), chicken ovomucoid, ƒ¿1-antit
rypsin, ƒ¿2-macroglobulin, and 9-aminoacridine were
purchased from Sigma (St. Louis, MO, USA). Aprotinin
and a recombinant enterokinase were purchased from
Takara Shuzo Co. (Kyoto, Japan). Antithrombin III and
phenylmethylsulfonyl fluoride were purchased from
Wako Pure Chemical Industries, Ltd. (Tokyo, Japan).
FOY-305 and human recombinant pancreatic secretory
trypsin inhibitor (PSTI) were gifts from Mochida
Pharmaceutical Co. (Tokyo, Japan). Various peptidyl-
MCA substrates and leupeptin were purchased from
Peptide Institute Co. (Osaka, Japan). The synthetic
oligonucleotides used were purchased from ESPEC
Oligoservice Inc. (Ibaraki, Japan). Restriction endonu
cleases and protein size marker (broad range) were pur
chased from New England Biolabs Inc. (Beverly, MA,
USA). KODdash_??_ used for polymerase chain reaction
(PCR) amplification, and other enzymes used for vector
construction were purchased from Toyobo (Osaka,
Japan). Other reagents used were of analytical grade.
Production of recombinant MT-SP1/matriptase (ekMT-
SPls). A secreted form of recombinant rat MT
- SP1/matriptase named ekMT-SP1s, in which the acti
vation cleavage motif, T610KQAR614, of the wild-type
MT-SP1/matriptase was substituted to DDDDK, the en
terokinase cleavage motif, was prepared in the present
study (Fig. 1). The vector construction was performed
as follows. PCR amplification was carried out using
plasmid pT7-MT-SP1s (9) as the template and two sets
of primers 5•Œ-acgacaagGTGGTTGGTGGCACG-3•Œ, 5•Œ-
GTAACTATCGTCTTGAGTCC-3•Œ and 5•Œ-TAGTTAGGC
CACCACTTCA-3•Œ, 5•Œ-cgtcatcAAAGGATCGCAGCCCAC-
3•Œ. The two PCR products were blunt-ended with T4
DNA polymerase, digested with AIWNI, and then ligated
to each other to generate plasmid pT7-ekMT-SP1s. A
BamHI digested 2.0kb fragment of pT7-ekMT-SP1s was
ligated into pSec-MT-SP1s digested by BamHI to gener
ate pSec-ekMT-SP1s. The procedure for the production
and purification of ekMT-SP1s was essentially the same
as done on a secreted form of recombinant MT-SP1/ma
triptase named MT-SP1s (9) except for the final prepa
ration of the active form of ekMT-SP1s. The purified la
tent ekMT-SP1s was incubated with a recombinant en
terokinase for 16h at room temperature, and then the
enterokinase was removed by Enterokinase Capture
AgaroseTM (Novagen Inc., Madison, WI, USA).
Enzyme and inhibitor assays. The substrate speci
ficity of the ekMT-SP1s was determined as described
previously (9). For the inhibitor assay, t-butyloxycar
Fig. 1. Predicted domain structure of rat MT-SP1
(wild-type) and diagram of expression construct for recombinant MT-SP1/matriptase. A schematic representation of the structure of rat MT-SP1/matriptase is presented. MT-SP1/matriptase consists of 855 amino acids. TM, transmembrane domain; CUB, complement factor 1R-urichin embryonic growth factor-bone mor
phogenetic protein domain; L, low-density lipoprotein receptor module; SP, serine protease (catalytic) domain. The predicted disulfide linkages are shown labeled as SS. The activation cleavage site (indicated by arrow) and its surrounding sequence are shown in MT-SP1/matriptase (wild-type). The amino acid sequence is shown in the single-letter code, and the amino acid numbering starts from the putative N-terminal of the protein. The enterokinase recognition site (indicated by arrow) was shown in ekMT-SP1s. The recombinant is a secreted form in which the signal anchor and cytosolic domain are replaced by the human immunoglobulin k-chain signal peptide and S-tag sequence (S-tag).
MT-SP1/Matriptase Inhibitors 29
bonyl (Boc)-Gln-Ala-Arg-MCA substrate was used.
Aliquots (1ng) of the enterokinase-treated ekMT-SP1s
were preincubated with each inhibitor in a buffer
(25mm HEPES, pH 7.5, 145mm NaCl, and 0.1% Triton
X-100) for 10min at 37•Ž in the final volume of 80ƒÊL.
The reaction was initiated by adding the substrate to a
final concentration of 125ƒÊM. After incubation for
20min, the reaction was terminated by adding 350ƒÊL
of 0.1M sodium acetate buffer, pH 4.0, containing
0.05M monochloroacetic acid, and absorbance at
370nm was measured.
RESULTS
Production and characterization of recombinant MT-
SP1/matriptase, ekMT-SP1s
In the present study, we produced a secreted form of
recombinant MT-SP1/matripatse named ekMT-SPIs
that can be activated by enterokinase in vitro. As
shown in Fig. 1, 15 nucleotides corresponding to amino
acid residues T610KQAR614 (N-terminal side at the acti
vation cleavage site) of wild-type MT-SP1/matriptase
were substituted to those corresponding to DDDDK, the
enterokinase recognition sequence, to create pSec-
ekMT-SPls. Transiently transfected COS-1 cells with
pSec-ekMT-SPls secreted ekMT-SP1s to a final concen
tration of about 10ƒÊg/mL in conditioned media. The
purified ekMT-SP1s gave signals of about 95 and
90kDa on sodium dodecylsulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under reducing and non-re
ducing conditions, respectively (Fig. 2). After enteroki
nase treatment, the ekMT-SP1s gave signals of about
15, 24, and 30kDa under reducing conditions and
about 16, 24, and 50kDa under non-reducing condi
tions (Fig. 2), showing that this form of recombinant
was cleaved to a disulfide-linked form. We previously
produced a secreted form of recombinant MT-SPI/ma
triptase named MT-SP1s in which the activation cleav
age site remains natural (9). The purified latent MT-
SP1s was cleaved at the site by trypsin to be activated
(9). The sizes of the latent ekMT-SP1s and fragment
pattern of enterokinase-treated ekMT-SP1s (active
ekMT-SP1s) are compatible with that of the latent MT-
SP1s and those of trypsin-treated MT-SP1s (9), respec
tively.
Fig. 2. SDS-PAGE and silver staining of purified recombinant ekMT-SP1s. Purified ekMT-SP1s was treated without (-) and with (+) a recombinant enterokinase
(rEK) as described in Materials and Methods, and samples were analyzed by SDS-PAGE and silver staining under reducing (Red) and nonreducing (NR) condi
tions. Lane M shows the positions of protein size markers. The molecular masses of the standards are indicated on the left in kilodaltons (kDa).
The active ekMT-SP1s cleaved various MCA substrates with Arg or Lys residue at position P1 in the following order: Boc-Gln-Ala-Arg-MCA>Boc-benzyl-Glu-Gly-Arg-MCA>Boc-Leu-Gly-Arg-MCA>Boc-Phe-Ser-Arg-MCA>Boc-Val-Pro-Arg-MCA>Boc-Gly-Lys-Arg-MCA>Boc-Leu-Arg-Arg-MCA>Boc-Gly-Lys-Arg-MCA>Boc-Glu-Lys-Lys-MCA. The active ekMT-SP1s was also shown to cleave single-chain uPA to produce its active form (Table 1). Purified human matriptase was shown to cleave Boc-Gln-Ala-Arg-MCA most efficiently and to activate uPA (6, 8) (Table 1), suggesting that the current ekMT-SP1s exhibits a natural enzymatic property. The substrate specificity for the active
Table 1. Comparison of substrate specificity for wild-type MT-SP1/matriptase, MT-SP1s, and ekMT-SP1s.
a Data were obtained from Refs, 6 and 8.b Data were obtained from Ref , 9.
c ND, not determined.
Boc-: t-butyloxycarbonyl-. MCA: 4-methylcoumaryl-7-amide. sc-uPA: single-chain urokinase-type plasminogen activator.
30 YAMASAKI Y et al.
ekMT-SP1s is in agreement with that for the active MT-
SP1s (9) (Table 1).
Inhibition of MT-SP1/matriptase by protease inhibitors
The inhibition of MT-SP1/matriptase by protease in
hibitors was examined using the active ekMT-SP1s and
Boc-Gln-Ala-Arg-MCA. We first tested the inhibitory
activity of typical endogenous protease inhibitors (Table
2). al-Antitrypsin at the normal plasma level of
1mg/mL (15) showed 11% inhibition toward the sub
strate cleavage of the active ekMT SPls. ƒ¿2-
Macroglobulin at the concentration of 0.01mg/mL
showed no inhibitory action. Antithrombin III at the
normal plasma level of 0.3mg/mL (15) showed 40% in
hibition. PSTI at the normal plasma level of 1ng/mL
(16) showed no inhibition. PSTI at a super-physiologi
cal dose (0.1mg/mL) also showed no inhibitory activity.
Aprotinin (basic pancreatic trypsin inhibitor), a serine
Table 2. Effect of endogenous protease inhibitors and
aprotinin on recombinant MT-SP1/matriptase (ekMT-
SP1s) activity. The active ekMT-SP1s (1ng) was prein
cubated with each inhibitor in 25mM HEPES, pH 7.5,
145mM NaCl, and 0.1% Triton X-100 for 10min at
37•Ž. The remaining activity was assayed by incuba
tion with Boc-Gln-Ala-Arg-MCA for 20min.
Boc-: t-butyloxycarbonyl-. MCA: 4-methylcoumaryl-7-
amide.
protease inhibitor produced in bovine pancreas or lung,
strongly inhibited it (IC50_??_10nM). As shown in Fig. 3,
all of the plant-derived protease inhibitors tested inhib
ited the active ekMT-SP1s in the following order:
LBTI>BBI>SBTI. An animal-derived protease inhibitor,
chicken ovomucoid, showed no inhibitory activity.
Synthetic protease inhibitors tested showed inhibitory
activity in the following order: FOY-305>leupeptin>9-
aminoacridine>phenylmethylsulfonyl fluoride (PMSF)
(Fig. 4).
DISCUSSION
In the present study, we investigated the effects of MT-
SP1/matriptase inhibitors using a secreted form of the
recombinant enzyme. We previously produced and
characterized a secreted form of recombinant MT-
SP1/matriptase named MT-SP1s (9). The MT-SP1s that
was purified as single-chain proteins showed no prote
olytic activity, but incubation of the latent MT-SP1s
with pancreatic trypsin resulted in activation accompa
nied by limited cleavage at the activation cleavage site
between Arg614 and Val615 (Fig. 1). The active MT-SP1s
as well as the natural enzyme purified from human milk
(8) cleaved various MCA substrates with Arg (or Lys)
residue at position P1, indicating that MT-SP1/matrip
tase is an enzyme that has trypsin-like specificity. The
most preferable substrate was Boc-Gln-Ala-Arg-MCA.
However, the productivity of MT-SP1s was very low; the
final concentration was approx. 0.2ƒÊg/mL in a condi
tioned medium of the transiently transfected COS-1
cells. In addition, the activation step by trypsin in vitro
was not easy to be controlled. In the present study,
therefore, we produced ekMT-SP1s that can be acti
vated by enterokinase, a highly specific serine protease,
and found that (i) the productivity of ekMT-SP1s in
COS-1 cells was more than 50 times higher than that of
MT-SP1s, and (ii) the activation step of the latent ekMT
- Fig. 3. Inhibition of Boc-Gln-Ala-Arg-MCA cleavage of recombinant MT-SP1/matriptase (ekMT-SP1s) by plant-derived
protease inhibitors. BBI, LBTI, and SBTI were preincubated at various concentrations with 1ng of enterokinase-treated
ekMT-SP1s for 10min at 37•Ž, and then the reaction was initiated by adding the substrate. After 20min incubation at
37•Ž, the reaction was terminated and the remaining activity (absorbance 370nm) was measured. Activity without any
inhibitors was taken as 100%. Assays were done in triplicate.
MT-SP1/Matriptase Inhibitors 31
Fig. 4. Inhibition of Boc-Gln-Ala-Arg-MCA cleavage of recombinant MT-SP1/matriptase (ekMT-SP1s) by synthetic pro
tease inhibitors. FOY-305, leupeptin, 9-aminoacridine, and PMSF were preincubated at various concentrations with 1ng
of enterokinase-treated ekMT-SP1s for 10min at 37•Ž, and then the reaction was initiated by adding the substrate. After
20min incubation at 37•Ž, the reaction was terminated and the remaining activity (absorbance at 370nm) was meas
ured. Activity without any inhibitors was taken as 100%. Assays were done in triplicate.
SP1s by enterokinase was easily controlled and reproducible. As shown in Table 1, the active ekMT-SPls exhibits enzymatic properties similar to that of natural enzyme purified from human milk (6, 8). For this reason, we currently used ekMT-SP1s for investigation of the inhibitors.
MT-SP1/matriptase was initially identified as an 80kDa matrix-degrading protease from conditioned medium of human breast cancer cells (T-47D) (17), and was later purified from human breast milk as a complex with a Kunitz-type serine protease inhibitor, HAT-1 (10). Recently, HAT-1 immunoreactivity in human primary colorectal carcinomas was found to be decreased significantly in cells within colon carcinomas relative to adjacent normal mucosa or adenomas (11). In contrast, it has very recently been reported that the increased expression of MT-SP1/matriptase occurs in advanced colorectal cancers possibly because of its glycosylation (13). Thus, it has been suggested that the MT-SP1/matriptase; HAT-1 ratio could be important in the development of advanced tumors (18). In addition, any of the endogenous inhibitors currently tested showed no or little inhibitory activity toward MT-SP1/matriptase. In such a case, the exogenous administration of inhibitors may be a choice for the suppression of cancer invasion and metastasis.
It has been reported that the oral administration of BBI to min mice drastically reduces tumor development in both the small intestine and colon of the animal (4). There is a possibility that the protease inhibitor may exert these effects through its inhibitory action on MT-SP1/matriptase. In fact, the inhibitory effect of the BBI was not only previously shown using the purified enzyme (19), but also shown currently. SBTI was previously shown to inhibit the proteolytic activity of membrane-bound arginine-specific serine proteinase,
porcine orthologue of MT-SP1/matriptase (14). The inhibitory action of SBTI was confirmed by the current study (Fig. 3). LBTI showed inhibitory action on the active ekMT-SP1s, and the inhibition was strongest among the plant-derived inhibitors tested (Fig. 3). SBTI and LBTI, if they reach the colon without significant loss of activity, may be orally administered for the sup
pression of colorectal cancer invasion and metastasis.It has been reported that aprotinin (basic pancreatic
trypsin inhibitor) shows an inhibitory action on the membrane-bound arginine-specific serine proteinase
(14, 20). The inhibitory activity was also shown using active ekMT-SPls. In the present study, we found a strong inhibitory action of FOY-305 toward active ekMT-SP1s. These inhibitory capacities for the inhibitors were stronger than those of the plant-derived inhibitors tested. Synthetic inhibitors are more stable in the gastrointestinal tract and easily available. Moreover, it has been reported that camostate (FOY-305)-containing diets have little or no effect on the homeostasis of the small intestine of normal mice (21); thus, such a compound is highly applicable for the suppression of cancer growth and invasion without significant damage of other normal tissues.
In summary, we currently investigated the MT-SP1/matriptase inhibitors. None of endogenous inhibitors tested had strong inhibitory effects on the currently prepared recombinant MT-SP1/matriptase. Plant-derived and synthetic protease inhibitors inhibited proteolytic activity, indicating that the administration of some inhibitors may be effective for the prevention of tumor growth and invasion in which MT-SP1/matriptase participates.
AcknowledgmentsWe wish to thank Mochida Pharmaceutical Co. for
32 YAMASAKI Y et al.
kindly supplying a recombinant human PSTI and FOY-305. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, the Uehara Memorial Foundation, and the Asahi Glass Foundation.
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