2 Department of Biochemistry, Osaka University Graduate School of Medicine, B1, 2-2, Yamadaoka, Suita, Osaka 565-0871, Japan; 3 Department of Molecular Biochemistry and Clinical Investigations, Osaka University University Graduate School of Medicine, 1-7 Yamadaoka, Suita, Osaka 565-0871, Japan; and 4 Department of Oncology, Lombardi Cancer Center, Georgetown University, Medical Center, Washington, DC 20007
Received on June 25, 2003; revised on September 26, 2003; accepted on September 29, 2003
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Abstract |
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Key words: ß1-6 GlcNAc branching / GnT-V / matriptase / protease
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Introduction |
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Recently we reported that matriptase levels are dramatically increased in a gastric cancer cell line transfected with GnT-V (Ihara et al., 2002). Matriptase was originally isolated as a type II transmembrane serine protease expressed in human breast carcinoma (Shi et al., 1993
) and breast milk (Lin et al., 1999b
) and has subsequently been cloned (Lin et al., 1999a
; Takeuchi et al., 1999
). An alternative name for this enzyme is membrane type serine protease-1 (MT-SP1). Matriptase/MT-SP1 plays important roles in cell migration, extracellular matrix degradation, and the activation of single-chain urokinase-plasminogen activator and hepatocyte growth factor, all of which are well-known prometastatic factors in cancer (Lee et al., 2000
; Takeuchi et al., 2000
; Satomi et al., 2001
).
Our previous studies report a dramatic increase in the expression of matriptase protein in GnT-V transfectants that was not due to an enhanced expression of mRNA but rather to the stabilization of the proteins. When cell lysates prepared from GnT-V transfectants were incubated in a buffer without protease inhibitors at 37°C, the degradation of matriptase was delayed to a considerable extent, compared with those from mock transfectants (Ihara et al., 2002). The next question to be raised is why proteins that contain ß1-6 GlcNAc branching (such as matriptase) are resistant to degradation. Three scenarios are possible: (1) Matriptase with ß1-6 GlcNAc branching could be resistant to degradation by proteases. (2) Certain inhibitors that serve to reduce the degradation of matriptase could be induced/activated in GnT-V transfectants. (3) Certain proteases that degrade matriptase are inhibited in GnT-V transfectants.
In the current study, we report on the purification and characterization of matriptase from mock and GnT-V transfectants (control matriptase and ß1-6 GlcNAc matriptase, respectively), in attempt to elucidate the role played by ß1-6 GlcNAc branching in the sugar chain of matriptase. The findings show that matriptase that had been glycosylated by the addition of ß1-6 GlcNAc branching, hereafter referred to as ß1-6 GlcNAc matriptase, has an increased stability and is resistant to trypsin, despite the fact that no changes in specific protease activity are evident. Furthermore, site-directed mutagenesis studies revealed that Asn 772 located in the serine protease domain is essential for the ß1-6 GlcNAc branching mediated stabilization of matriptase.
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Results |
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Enzymatic activities of control, ß1-6 GlcNAc matriptase, and deglycosylated ß1-6 GlcNAc matriptase
The specific activities of control and ß16 GlcNAc matriptases were evaluated by measurement of their gelatinolytic activities on gelatin zymography (Figure 5). The initial rate of degradation of gelatin cannot affect by the modification of the sugar chains (Figure 5A). Because the incubation time for gelatin zymography was approximately 6 h, the gelatinolytic activities of control and ß1-6 GlcNAc matriptases were forward to be almost the same level. On the other hand, Figure 5B shows that ß1-6 GlcNAc matriptase had a little higher gelatinolytic activity as compared to control matriptase. Because the incubation time used for the zymography was approximately 20 h, it might be sufficient to destroy matriptase itself; the difference in gelatinolytic activities seemed to reflect the difference of the autodegradation of two kinds of matriptases, which was blocked in the case of ß1-6 GlcNAc matriptase. When the protease activity was assayed using various synthetic fluorescent peptide substrates, the enzymatic activities of control matriptase, ß1-6 GlcNAc matriptase, and deglycosylated matriptase were almost indistinguishable, as shown in Table I. These results indicate that ß1-6 GlcNAc branching affected the stabilization of matriptase and its resistance to degradation but not its specific enzymatic activity.
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When expression vectors for matriptase were transfected into previously transfected, mock, and GnT-V COS cells, all oligosaccharide mutants of matriptase were smaller than wild-type matriptase (Figure 6A). These results indicate that sugar chains were attached to all the three putative glycosylation sites. Resistance to trypsin was observed for wild-type matriptase, when it was transfected into GnT-V transfectants, but not for mock transfectants (Figure 6B). The bands corresponding to N302Q and N485Q were attenuated slightly but showed significant resistance to degradation in the GnT-V transfectants, whereas the N772Q was completely degraded under the same conditions (Figure 6B). To compare the presence of modification by GnT-V on these oligosaccharide mutants, we performed the L4-PHA lectin precipitation as reported previously (Ihara et al., 2002) using COS cells, which express equal amounts of matriptase with oligosaccharide mutants (Figure 6C). Wild-type and all mutants showed strong binding to L4-PHA lectin in COS GnT-V compared to COS cells. These results indicated that ß1-6 GlcNAc branching structure are equally attached to three potential oligosaccharide sites on matriptase by an action of GnT-V (Figure 6D).
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Discussion |
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A recent study by Oberst et al. (2003) suggested that sugar chains attached to Asn 302 in the first CUB (complement factor C1 s/C1r, urchin embryonic growth factor, bone morphogenetic protein) domain and Asn 772 in the serine protease domain are required for the activation of matriptase. Though ß1-6 GlcNAc branching at Asn 302, Asn 485, and Asn 772 sites was involved in the stabilization as well as resistance to degradation, that at Asn 772 site plays the most important role in the stabilization of matriptase (Figure 6). The Asn 772 is located between a ß-sheet and an
-helix in a computer graphic model (PDB#1EAX) (Friedrich et al., 2002
). According to this model, the sugar chain at Asn 772 is located at the outer solvent-exposed surface of the molecule and might be a suitable size to mask selected regions of the mastriptase molecule. To identify the autolysis and/or trypsin-catalyzed sites, we have a plan to identify the site for the degradation of matriptase. The addition of ß1-6 GlcNAc branching on oligosaccharides attached to Asn772 might mask the important structure for stabilization, thus preventing its degradation by autolysis and/or trypsin.
When the catalytic domain of matriptase is compared to other serine proteases, three catalytic triad residues and several other residues are conserved (Figure 7). Interestingly, only matriptase contains the Asn 772 N-glycosylation site among the related serine proteases (Figure 7). Therefore, the role of ß1-6 GlcNAc branching in enzyme stabilization might be specific to matriptase.
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In conclusion, the present study clearly demonstrates that ß1-6 GlcNAc branching of N-glycans attached to the Asn 772 of matriptase plays a pivotal role in its stabilization and resistance to proteolysis by trypsin but has no effect on the specific activity of matriptase.
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Materials and methods |
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Purification of matriptase
An immunoaffinity chromatography column was prepared as described previously (Lin et al., 1997). Antimatriptase antibody (mAb21-9) was immobilized on protein G sepharose beads (Amersham Bioscience, Little Chalfont, UK) as described (Harlow and Lane, 1988
).
To obtain sufficient amounts of matriptase, 2 L conditioned medium from MKN45 mock and GnT-V transfectants, which had been cultured for 72 h in serum-free conditions, were centrifuged to remove the insoluble debris. Solid ammonium sulfate was added to the conditioned medium with continuous mixing to 100% saturation, at which point a precipitate formed. The protein precipitates were obtained by centrifugation at 5000 x g for 20 min. The pellets were dissolved in water and then dialyzed against 50 mM TrisHCl (pH 7.5). Insoluble debris was cleared by centrifugation, and the supernatant was loaded on an immunoaffinity column (7 x 70 mm) at a flow rate of 7 ml/h followed by washing with 1% Triton X-100 in phosphate buffered saline (PBS). The matriptase that was bound to the column was eluted with 0.1 M glycine-HCl (pH 2.8) and stored in 50% glycerol at -20°C until used.
SDSPAGE and western blot analysis
Matriptase preparations purified (100 ng) from mock and GnT-V transfectants were electrophoresed on a 10% sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDSPAGE) under nonreducing conditions according to the previous method (Laemmli, 1970). One gel was used for silver staining to visualize proteins, the other gel were used for western blot as described previously (Ihara et al., 2002
).
Lectin blot analysis
A lectin blot analysis was performed as described previously (Yoshimura et al., 1995). Briefly, purified matriptase (1 µg) was electrophoresed on a 10% SDS-polyacrylamide gel, and then transferred onto a nitrocellurose membrane (Protran, Schleicher & Schuell). After blocking overnight at 4°C with PBS containing 3% bovine serum albumin, the filter was incubated for 1 h at room temperature with 1 µg/ml biotinylated L4-PHA (Seikagaku, Tokyo) that preferentially recognizes ß1-6 GlcNAc branches of tri- and tetraantennary sugar chains (Cummings and Kornfeld, 1982
). The washing and developing procedures have been described previously (Yoshimura et al., 1995
).
N-glycosidase-F treatment
N-glycosidase-F was purchased from Roche (Indianapolis, IN). Matriptase preparations purified from GnT-V transfectant cells (100 ng, ß1-6 GlcNAc matriptase) were treated for 2 h at 37°C with 10 mU of N-glycosidase-F in a PBS solution.
Autodegradation assay
Matriptase preparations were dissolved in a reaction mixture buffer (20 mM TrisHCl, pH 8.0, and 2 mM CaCl2). The reaction mixtures were incubated for up to 24 h at 37°C. Degradation of matriptase was observed by silver staining and western blot, as already described.
Degradation of matriptase by proteases
Matriptase preparations were treated at 37°C with several types of proteases, such as trypsin, elastase, and chymotrypsin, up to 30 min in 100 mM TrisHCl (pH 8.0) and 1% Triton X-100. Degradation of matriptase was observed by western blot, as described.
The assay for protease activity
The specific activities of the control and ß1-6 GlcNAc matriptase were assayed using the fluorogenic peptides, N-tert-butoxycarbonyl (N-t-Boc)-Gln-Ala-Arg-7-amido-4-methylcoumarin (AMC), N-t-Boc--benzyl (Bz)-Glu-Ala-Arg-AMC, N-succinyl (Suc)-Ala-Phe-Lys-AMC, N-Suc-Leu-Leu-Val-Tyr-AMC, and N-Ala-Ala-Ala-Tyr-AMC (Sigma, St. Louis, MO). Forty nanograms of purified enzymes and 40 µM each substrate were added to 20 mM TrisHCl (pH 8.0) and 2 mM CaCl2 to a final volume of 100 µl and incubated for 1 h at 37°C. The reaction was stopped by adding 100 µl 30% acetic acid. After adding 800 µl water, the released AMC was measured with a fluorescence spectrophotometer (Hitachi F3500) configured with an excitation wavelength at 380 nm and an emission wavelength at 440 nm.
Gelatin zymography
Gelatin zymography was carried out as described previously with minor modifications (Ihara et al., 2002). One hundred nanograms of matriptase preparations were electrophoresed on a 10% copolymerized gel with 1 mg/ml gelatin as a substrate. The gelatin gels were washed three times with 50 mM TrisHCl (pH 7.5) containing 2.5% Triton X-100 and incubated overnight at 37°C in 20 mM TrisHCl (pH 7.5) buffer containing 5 mM CaCl2. The gel was stained with Coomassie brilliant blue.
Site-directed mutagenesis
The entire coding region of human matriptase was inserted into an expression pcDNA3.1 (Invitogen, Carlsbad, CA). Site-directed mutagenesis of the putative N-glycosylation sites was performed using the QuikChange site directed mutagenesis kit (Stratagene, La Jolla, CA). The following mutagenesis primers were used: for Asn-302-Gln, primer 1,5'-GGCACCTACCCTCCCTACCAACTGACCTTCCAC-3' and primer 2,5'-GTGGAAGGTCAGTTGGTAGGAGGGAGGGTAGGTGCC-3'; for Asn-485-Gln, primer 3,5'-CACAGCGATGAGCTCCAATGCAGTTGCGACGCCGGC-3' and primer 4,5'-GCCGGCGTCGCAACTGCATTGGAGCTCATCGCTGTG-3'; for Asn-772-Gln, primer 5,5'-GAGATCCGCGTCATCCAACAGACCACCTGCGAGAAC-3' and primer 6,5'-GTTCTCGCAGGTGGTCTGTTGGATGACGCGGATCTC.
All mutated sequences were verified by automated DNA sequencing using a Dye Terminator Cycle Sequencing Kit (Perkin-Elmer, Boston, MA) and an ABI Prism 310 Genetic Analyzer.
DNA transfection
Expression vectors (wild-type and mutated matriptase) were transfected into COS cells by an electroporation method using a Gene Pulsar (Bio-Rad, Hercules, CA), as described previously (Chu et al., 1987). The cells 1 x 107 were washed with PBS and resuspended in 800 µl PBS. Twenty micrograms of expression vectors were added to the cell suspension, followed by electroporation. The transfected cells were subjected to biochemical analyses, including measurement of the extent of matriptase degradation, at 48 h after the transfection.
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Acknowledgements |
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Footnotes |
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Abbreviations |
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References |
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