Glycobiology Advance Access originally published online on October 9, 2003
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Glycobiology vol 14 no 2 pp. 139-146, 2004
© Oxford University Press 2004; all rights reserved.
Addition of ß1-6 GlcNAc branching to the oligosaccharide attached to Asn 772 in the serine protease domain of matriptase plays a pivotal role in its stability and resistance against trypsin
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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ß1-6 GlcNAc branching, a product of N-acetylglucosaminyltransferase V (GnT-V), is a key structure that is associated with malignant transformations and cancer metastasis. Although a number of reports concerning tumor metastasis–related glycoproteins that contain ß1-6 GlcNAc branching have appeared, the precise function of ß1-6 GlcNAc branching on glycoproteins remains to be elucidated. We previously reported on the importance of ß1-6 GlcNAc branching on matriptase in terms of proteolytic degradation in tumor metastasis. We report here that matriptase purified from GnT-V transfectant (ß1-6 GlcNAc matriptase) binds strongly to L4-PHA, which preferentially recognizes ß1-6 GlcNAc branches of tri- or tetraantennary sugar chains, indicating that the isolated matriptase contains ß1-6 GlcNAc branching. The ß1-6 GlcNAc matriptase was resistant to autodegradation, as well as trypsin digestion, compared with matriptase purified from mock-transfected cells. Furthermore, N-glycosidase-F treatment of ß1-6 GlcNAc matriptase greatly reduced its resistance to degradation. An analysis of matriptase mutants that do not contain potential N-glycosylation sites clearly shows that the ß1-6 GlcNAc branching on N-glycans attached to Asn 772 in the serine protease domain plays a major role in trypsin resistance. This is the first example of a demonstration of a direct relationship between ß1-6 GlcNAc branching and a biological function at the protein level.
Key words: ß1-6 GlcNAc branching / GnT-V / matriptase / protease
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Introduction |
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Changes in the oligosaccharide structure of glycoproteins are linked to malignant transformation and previous studies have revealed that ß1-6 GlcNAc branching, a product of UDP-GlcNAc:
-mannoside ß1,6-N-acetylglucosaminyltransferase (GnT-V), is a key factor in tumor metastasis (Dennis et al., 1987
; Rademacher et al., 1988; Hakomori, 1989). We and another group independently succeeded in the purification and cDNA cloning of GnT-V from rat kidney (Shoreibah et al., 1992, 1993) and human lung cancer cells (Gu et al., 1993; Saito et al., 1994), respectively. An involvement of GnT-V in malignant transformations has been demonstrated in animal models (Miyoshi et al., 1993; Demetriou et al., 1995; Granovsky et al., 2000) as well as clinical studies (Seelentag et al., 1998; Murata et al., 2000; Ito et al., 2001). However, a detailed explanation of how GnT-V promotes tumor metastasis is not currently available.
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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Analysis of oligosaccharide structures on matriptase purified from GnT-V transfectants
Human matriptase contains four potential N-linked glycosylation sites (Lin et al., 1999a
). Although matriptase is known to contain sugar chains, the biological effects of these chains, especially ß1-6 GlcNAc branching on matriptase, has not been fully investigated. To determine the biological effects of ß1-6 GlcNAc branching, we purified matriptase from GnT-V transfectants of MKN45 cells, which secrete a large amount of glycoproteins containing ß1-6 GlcNAc branching. Under nonreducing conditions, several bands were observed at approximately 70–80 kDa by silver staining and western blot (Figure 1A and B). To examine how the sugar chains were modified by GnT-V, lectin blotting with leukoagglutinating phytohemagglutinin (L4-PHA) was performed. As expected, ß1-6 GlcNAc matriptase was strongly stained with L4-PHA compared with control matriptase (Figure 1C). These results indicate that the GnT-V activity led to an increase in the amount of ß1-6 GlcNAc branching of N-glycans on matriptase. On deglycosylation treatment (N-glycosidase-F) of control and ß1-6 GlcNAc matriptase, all bands moved to lower molecular sizes (Figure 2), suggesting that the several forms of matriptase in the conditioned medium all contained sugar chains.
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ß1-6 GlcNAc branching inhibits autodegradation
To evaluate the effect of ß1-6 GlcNAc branching on matriptase, we investigated the autodegradation of the ß1-6 GlcNAc modified enzyme. It is most likely that matriptase was degraded by itself in the reaction solution because a secreted type of matriptase from cancer cells has enzymatic activity as a protease (Lin et al., 1997
; Ihara et al., 2002). After 12–24 h of incubation, nearly all of matriptase was degraded. This autodegradation process was markedly delayed in the case of ß1-6 GlcNAc matriptase (Figure 3A and B).
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ß1-6 GlcNAc matriptase was resistant to trypsin but not elastase, chymotrypsin, and u-PA
We next investigated the resistance of ß1-6 GlcNAc matriptase to proteolysis by various proteases. Control and ß1-6 GlcNAc matriptases were treated with trypsin, elastase, and chymotrypsin followed by observation of the degradation profile by western blot. The ß1-6 GlcNAc matriptase showed a higher resistance to degradation by trypsin than the control (Figure 4A and C). In contrast, elastase (Figure 4B) and chymotrypsin (data not shown) led to no changes in the degradation profiles of either control or ß1-6 GlcNAc matriptase. Although urokinase plasminogen activator (u-PA) concentrations were changed from 0.001 to 1.0 mg/ml, matriptase was not degraded (data not shown). This result suggests that u-PA can not recognize matriptase as a substrate.
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These results suggest that ß1-6 GlcNAc branching masks the sites on matriptase that trypsin acts on. To investigate the issue of whether the resistance of ß1-6 GlcNAc matriptase to degradation is due to ß1-6 GlcNAc branching, the resistance of deglycosylated ß1-6 GlcNAc matriptase was determined. The resistance of ß1-6 GlcNAc matriptase to degradation was abolished by the N-glycosidase-F treatment (Figure 4D). These results suggest that the resistance to degradation and stabilization of ß1-6 GlcNAc matriptase are specifically due to the presence of ß1-6 GlcNAc branching on the sugar chains.
Enzymatic activities of control, ß1-6 GlcNAc matriptase, and deglycosylated ß1-6 GlcNAc matriptase
The specific activities of control and ß1–6 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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An important role of ß1-6 GlcNAc branching on N-glycans attached to Asn772 in matriptase
Matriptase contains four potential Asn-linked oligosaccharide linkage sites (N109, N302, N485, and N772). To determine which sites are involved in the stabilization of matriptase as the result of the added ß1-6 GlcNAc branching, we made three mutants of putative glycosylation sites (N302Q, N485Q, and N772Q). After matriptase is synthesized in a latent form, it is immediately cleaved at the Gly149 residue by proteolytic processing (Cho et al., 2001
; Oberst et al., 2003). Therefore, the Asn109 putative N-glycosylation site cannot be involved in the stabilization of active matriptase induced by the added ß1-6 GlcNAc branching.
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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The present study provides strong support for our previous finding that GnT-V promotes tumor metastasis by regulating matriptase stabilization via modulation of the carbohydrate chains of matriptase. Matriptase has been linked to tumor metastasis and progression both in an animal model (Ihara et al., 2002
) and in human cancer tissues (Benaud et al., 2002; Oberst et al., 2002; Kang et al., 2003). Matriptase is expressed in a variety of normal tissues, and specifically in epithelial tissues (Takeuchi et al., 2000), suggesting that this protease could regulate a variety of biological events. A recent study using matriptase knockout mice indicates that this protease is required for the postnatal survival of newborn mice because of its involvement in epidermal barrier function in the skin (List et al., 2002). Moreover, matriptase knockout mice showed abnormalities in hair follicle development and thymic homeostasis. These data suggest that functions of matriptase include tissue development and cancer progression at various phases. This study on the functions of carbohydrate chains of matriptase suggests an involvement of GnT-V in these matriptase-associated biological events.
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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The expression of GnT-V is linked to the prognosis of colon and breast cancers (Seelentag et al., 1998
; Murata et al., 2000), but not other cancers (Ito et al., 2001). It was thought that certain target molecule(s) of GnT-V, such as matriptase, might be involved in this discrepancy. In colon and breast cancers, ß1-6 GlcNAc matriptase might play an important role in cancer progression because of its enhanced stability. 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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Cell lines and culture conditions
A human gastric cancer cell line (MKN45) and COS cells were cultured in RPMI 1640 medium (Nikken Kagaku, Kyoto, Japan) and Dulbecco's Modified Eagle's medium, respectively, supplemented with 10% fetal bovine serum (Gibco-BRL, Rockville, MD) and antibiotics. Establishment of GnT-V transfectants using these cells were performed as described previously (Ihara et al., 2002
).
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 Tris–HCl (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.
SDS–PAGE and western blot analysis
Matriptase preparations purified (100 ng) from mock and GnT-V transfectants were electrophoresed on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) 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 Tris–HCl, 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 Tris–HCl (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 Tris–HCl (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 Tris–HCl (pH 7.5) containing 2.5% Triton X-100 and incubated overnight at 37°C in 20 mM Tris–HCl (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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This work was supported in part by a Grant-in-aid for Scientific Research (S) of 13854010 from the Japan Society for the Promotion of Science, a Grant-in-aid for Cancer Research and Scientific Research on Priority Areas number 15025238 from the Ministry of Education, Science, Sports, and Culture of Japan, and 21st Century COE program.
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Footnotes |
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1 To whom correspondence should be addressed; e-mail: seika{at}biochem.med.osaka-u.ac.jp
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Abbreviations |
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AMC, amido-4-methylcoumarin; GnT-V, UDP-GlcNAc
-mannoside ß1,6-N-acetylglucosaminyltransferase; L4-PHA, leukoagglutinating phytohemagglutinin; MT-SP1, membrane type serine protease-1; N-t-boc, N-tert-butoxycarbonyl; PBS, phosphate buffered saline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis |
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