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J. Biol. Chem., Vol. 279, Issue 13, 12734-12743, March 26, 2004
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From the
Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, **Department of Molecular Virology and Oncology, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, and Department of Pathology, School of Medicine, Keio University, Shinjuku-ku, Tokyo 160-0016,-Japan
Received for publication, September 8, 2003 , and in revised form, December 28, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As a member of the MMP family, MT1-MMP has a propeptide, a catalytic domain, hinge, and a hemopexin-like (HPX) domain starting from the N terminus, and this extracellular portion is linked to the membrane through the transmembrane domain, which follows a short cytoplasmic tail composed of 20 amino acids (1, 2, 5). As an invasion-promoting enzyme, the activity, localization, and turnover of MT1-MMP are tightly regulated during cell locomotion (3). For example, MT1-MMP localizes at the leading edge of migrating cells, and this localization is regulated through interaction between CD44 and the HPX domain (6). CD44 is a cell adhesion molecule that acts as a receptor for hyaluronan and mediates flexible adhesion to the provisional hyaluronan-rich matrix in the dynamic ruffling membrane area. Such adhesion is expected to be followed by a firmer adhesion through integrins to generate force for migration (3). At the migration edge MT1-MMP forms oligomers through the HPX domain with possible participation of the cytoplasmic tail, the hinge region, and extracellular matrix that binds to the molecule (7–10). Activation of proMMP-2 by MT1-MMP is expected to be carried out efficiently within the oligomeric complex at the leading edge (7). MT1-MMP exposed on the cell surface is regulated negatively by TIMPs (5), auto-degradation (11), and internalization (12, 13). Internalization of MT1-MMP depends on the cytoplasmic tail, and an LLY motif in the region was found to act as a binding site for AP-2 complex that mediates incorporation of target proteins into clathrin-coated pits (12). Deletion of the cytoplasmic tail inhibits not only the internalization of MT1-MMP but also the invasion-promoting activity against the reconstituted basement membrane (Matrigel) mediated by MT1-MMP (12, 14). Thus, the proteolytic activity of MT1-MMP alone is not enough for the invasion-promoting activity, and it has to be regulated further by the function of the cytoplasmic tail.
Although the cytoplasmic tail is short, containing 20 amino acids, it is reported to affect functions of MT1-MMP such as the formation of oligomers (8, 9), localization of the enzyme to the proteolytically active protrusions (invadopodia) (15), internalization (12, 13), and cell migration and invasion (12, 14). However, it has not been elucidated how the cytoplasmic tail affects these functions except for the mechanism of internalization. To obtain clues about the mechanism by which the cytoplasmic tail regulates MT1-MMP, we attempted to isolate genes whose products interact with this portion using the yeast two-hybrid screening system. Using a cDNA library established from human fibroblast WI-38 cells we isolated a new gene whose product shows homology to members of the Cupin superfamily, a new family composed of proteins with diverse functions and which has a conserved three-dimensional structure (16). We named this protein MT1-MMP cytoplasmic tail-binding protein-1 (MTCBP-1). MTCBP-1 is expressed as a 19-kDa protein and is co-localized with MT1-MMP at the adherent membrane edge. It was also found in the cytoplasm and nucleus. The formation of a complex between MTCBP-1 and MT1-MMP was confirmed by co-immunoprecipitation. Characteristics of MTCBP-1 and its possible role as an invasion suppressor are discussed.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Northern Blot Analysis—Northern blot analysis was performed using The Human Multiple Tissue Northern (MTNTM) blot membrane (Clontech). Fragments of the cDNAs for human MT1-MMP, MTCBP-1, and glyceraldehyde-3-phosphate dehydrogenase were labeled with [
-32P]dCTP (3000 Ci/mmol, Amersham Biosciences) and used as probes.
Reverse Transcription (RT)-PCR—First-strand cDNA was synthesized from 3 µg of total RNA using 0.3 µg of each random primer (Invitrogen) and 200 units of Superscript II RNase H reverse transcriptase (RTase) (Invitrogen). After removal of the random primers, 1 µl of the RT product was used as a template for PCR (25 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min). Primer sequences are indicated in the figure legends. Glyceraldehyde-3-phosphate dehydrogenase mRNA was amplified similarly as an internal control.
Cell Culture and Transfection—Human fetus lung normal diploid fibroblasts (WI-38 and TIG-20), human fibrosarcoma (HT1080), and green monkey kidney (COS-1) cells were cultured at 37 °C in Dulbecco's modified Eagle's medium (Sigma). Human fibrosarcoma (SW684) cells were cultured at 37 °C in Leibovitz's L-15 medium (Sigma). All media were supplemented with 10% fetal bovine serum. Cells were seeded in 6-well plates at 1.0–1.5 x 105 cells/well, and transfection was carried out after 16 h using FuGENE 6TM (Roche Applied Science) according to the manufacturer's instructions.
Immunoprecipitation—Transfected cells (1.5 x 105 cells) cultured in 6-well dishes were lysed in a lysis buffer (1% Brij-99, 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% deoxycholic acid, and 0.1% SDS) in the presence of a protease inhibitor mixture (Roche Applied Science). The cell lysate was clarified by centrifugation at 15,000 rpm for 15 min, and the supernatant was incubated with anti-FLAG M2 antibody-conjugated agarose beads (Sigma) for 2 h at 4 °C. After the beads were collected and washed three times with lysis buffer, precipitates were eluted using a FLAG peptide and then analyzed by Western blotting using the same antibody.
Subcellular Fractionation—After washing, cells in a culture dish were collected with a cell scraper on ice and suspended in 1 ml of 10 mM Tris-buffer, pH 7.4, containing 250 mM sucrose and proteinase inhibitor mixture (Roche Applied Science). Then the cells were homogenized using a Dounce homogenizer (20 strokes) and centrifuged in a microtube at 1500 rpm for 10 min at 4 °C to remove the nucleus and undisrupted cells. The supernatant fraction was collected and centrifuged further at 65,000 rpm for 1 h at 4 °C using a Beckman rotor (NTV-90). Plasma membrane fraction was recovered in the pellet, and cytoplasmic proteins were in the supernatant.
Western Blot Analysis—Human tissue samples (Protein MedleyTM) (Clontech) and cells in culture were lysed in a SDS sample buffer containing 2-mercaptoethanol. Proteins were separated on SDS-PAGE and transferred to a nitrocellulose membrane (Hybond-ECL, Amersham Biosciences). After the blocking of the membrane with 10% fat-free dry milk in Tris-buffered saline (20 mM Tris-HCl, pH 7.5, 150 mM NaCl), the membrane was probed with antibodies for detection. The membrane was further probed with alkaline phosphatase-conjugated anti-mouse IgG or anti-rabbit IgG (Sigma) to visualize the reacted antibody.
Purification of Recombinant MTCBP-1 and Preparation of a Polyclonal Antibody—Recombinant human MTCBP-1 was expressed in the Escherichia coli strain BL21 (DE3) pLysS (Stratagene) by transfecting MTCBP-1/pRSET B vector plasmid (Invitrogen), with which expression of the gene can be induced by adding 0.4 mM isopropyl-1-thio-
-D-galactopyranoside to the culture medium. Cells were collected and sonicated in a TNC buffer (50 mM Tris-HCl, 150 mM NaCl, 10 mM CaCl2, and 0.02% NaN3) containing 2 mM phenylmethylsulfonyl fluoride. Supernatant was collected, and the His6-tagged protein was purified on a chelating Sepharose column. The bound protein was eluted with TNC buffer containing 500 mM imidazole and separated further with a gel filtration column of Sephacryl S-200 (Amersham Biosciences). To remove the His tag, EnterokinaseMaxTM (Invitrogen) was used under the reaction conditions recommended by the manufacturer.
To obtain polyclonal antibody for MTCBP-1 two female rabbits were immunized with the purified recombinant protein conjugate emulsified with an equal volume of Freund's complete adjuvant. A week after the last injection serum was obtained from the animals and subjected to a 40% saturated ammonium sulfate fractionation. Antibody reactive to MTCBP-1 was further purified using an affinity column of Sepharose 4B conjugated with recombinant MTCBP-1 (rMTCBP-1).
Indirect Immunofluorescence Staining—Transfected HT-1080 cells were seeded on fibronectin/gelatin-coated coverslips. After 24 h the cells were washed with phosphate-buffered saline three times and fixed with 3% paraformaldehyde in phosphate-buffered saline. To detect the MTCBP-1 signal, cells were permeabilized with 0.1% Triton X-100 in phosphate-buffered saline for 10 min at room temperature. After blocking treatment with 5% goat serum and 3% bovine serum albumin in phosphate-buffered saline for 1 h at room temperature, cells were reacted with rabbit anti-MTCBP-1 antibody or mouse anti-FLAG M2 antibody at room temperature for 2 h. To visualize MTCBP-1 and FLAG-tagged MT1-MMP, cells were further incubated with AlexaTM488-conjugated goat anti-rabbit IgG (Molecular Probes, Inc., OR) and Cy3-conjugated goat anti-mouse IgG (Jackson ImmunoResearch laboratories, Inc.). Fluorescence signals were detected using a Bio-Rad MRC-1024 confocal laser microscope.
Gelatin Zymography—Gelatin zymography was conducted with an SDS-polyacrylamide gel containing gelatin (0.8 mg/ml) as described previously (4). The samples were mixed with SDS/PAGE loading buffer without a reducing agent and subjected to electrophoretic analysis at room temperature. Enzyme activity was visualized as negative staining with Coomassie Brilliant Blue R-250.
Phagokinetic Track Motility Assay—The phagokinetic track motility assay was performed as described previously (12). Colloidal gold-coated coverslips were placed in a 12-well plate, and transfected cells were seeded at 3 x 103 cells/well. After 12 h of incubation at 37 °C, the phagokinetic tracks were visualized under bright-field illumination using a CoolSNAP-fx monochrome CCD camera (Roper Scientific). The track area was measured using NIH Image software Version 1.62.
Matrigel Invasion Assay—The Matrigel invasion assay was performed as described previously (7) according to the manufacturer's instructions (BD Biosciences). MTCBP-1 plasmid was introduced into HT-1080 cells with a plasmid for the expression of green fluorescent protein. The transfected cells were suspended in Dulbecco's modified Eagle's medium without serum and seeded onto a Matrigel-coated filter (8-µm pore) in the Transwell chamber. Fetal bovine serum (10%) was added to the medium in the lower chambers and incubated for 12 h at 37 °C. Non-invading cells remaining on the upper surface of the filter were removed and fixed with 3% paraformaldehyde. The green fluorescent protein-positive cells on the lower surface of the filter were enumerated under microscope at a magnification of x400. Each assay was performed in triplicate, and five microscopic fields from each of the three filters were counted.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ARD has a -barrel hold that is also known as a double stranded helix (DSBH) domain (20) and is characteristically conserved in members of the Cupin superfamily that was proposed recently (16). Despite this structural conservation, the members of this family vary widely in function and include metabolic enzymes, transcription factors, scaffold proteins etc.
Interaction between MTCBP-1 and MT1-MMP in Cells— rMTCBP-1 was expressed in E. coli and purified as described under "Materials and Methods." Rabbit polyclonal antibody was also prepared using rMTCBP-1 as an immunogen. To examine whether MTCBP-1 binds to the cytoplasmic tail of MT1-MMP directly, a cytoplasmic tail peptide with His6 tag (CP-WT) and a control peptide with a randomized sequence (CP-RD) were prepared. The peptides were immobilized on a nitrocellulose membrane filter and then blotted with rMTCBP-1. The protein bound to the filter was visualized using anti-MTCBP-1 antibody conjugated with alkaline phosphatase (Fig. 3A). MTCBP-1 was bound to CP-WT but not CP-RD. The binding to CP-WT was specific because it was blocked almost completely with an excess amount of the CP-WT peptide but not with CP-RD.
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MT1-MMP Recruits MTCBP-1 to the Plasma Membrane Fraction—If MTCBP-1 bound MT1-MMP, the proteins would co-localize in the cells. MT1-F and MTCBP-1 were co-expressed in HT1080 cells, and localization of the products was examined by immunostaining (Fig. 3C). MT1F was detected on the cell surface especially at the ruffling edge when the cells were examined without permeabilization (no treatment with Triton X-100). Under the same conditions MTCBP-1 was not detected as it is a cytoplasmic protein. After the cells were treated with Triton X-100, co-localization of MTCBP-1 and MT1F was detected at the periphery of the cells (Fig. 3C). Some MT1F signals within the cells presumably represent the translation products in vesicles and the Golgi apparatus. Although MTCBP-1 was also detected in the cytoplasm, its localization did not coincide with that of MT1F. Some MTCBP-1 was detected in the nucleus as well, indicating the possibility that MTCBP-1 shuttles between the three compartments, membrane, cytoplasm, and nucleus.
To confirm the interaction of both proteins further, plasma membrane-enriched fraction was prepared and examined by Western blotting (Fig. 3D). Accumulation of MTCBP-1 in the membrane fraction was not much when it is expressed without MT1F. However, co-expression increased the amount of MTCBP-1 in the membrane fraction significantly. This accumulation was dependent on the cytoplasmic tail of MT1F because co-expression of dCPF failed to increase this amount. Appropriate fractionation was monitored using marker proteins such as transferring receptor (TfnR) for membrane proteins and actin for cytoplasmic proteins. Expression of MT1F and dCPF did not affect the expression levels of MTCBP-1 and the fractionation (Fig. 3D). Thus, the results strongly suggest that MT1-MMP forms a complex with MTCBP-1 within the cells.
Expression and Tissue Distribution of MTCBP-1—The expression of MTCBP-1 mRNA in different human tissues was examined by Northern blotting and compared with that of MT1-MMP (Fig. 4A). Three MTCBP-1 transcripts (1.2, 1.5, and 2.0 kb) were detected in tissue samples. The three presumably represent transcripts terminating at different sites. Corresponding to the three transcripts, three potential poly(A) signals can be seen in the 3'-non-coding region of the 1.6-kb cDNA sequence as indicated in Fig. 1. The expression levels were higher in heart and lower in brain and placenta (Fig. 4A). On the other hand, MT1-MMP mRNA was predominantly expressed in lung and placenta, where MTCBP-1 expression is low. Thus, MT1-MMP may not require MTCBP-1 to function, at least in these tissues.
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Inverse Correlation of MTCBP-1 and MT1-MMP Expression in the Transformed and Non-transformed Human Cell Lines— MT1-MMP is frequently expressed in human tumors and tumor-derived cell lines. Thus, expression of MTCBP-1 and MT1-MMP was examined further using cell lines to confirm the relationship between the two (Fig. 5, A and B). As tumor cell lines, four gastric carcinoma (MKN-7, MKN-28, NUGU-3, and NUGC-4) and two fibrosarcoma (HT1080 and SW684) cell lines were analyzed by RT-PCR using specific primers (Fig. 5A). Non-transformed fibroblasts WI-38 and TIG-20 were also examined. MT1-MMP mRNA was detected in the tumor cell lines, reported at higher levels than in the non-transformed cells (Fig. 5B). In contrast, although MTCBP-1 mRNA was detected in all the cells, the expression was weak in the transformed cells compared with the non-transformed cells. Although we analyzed more than 20 different types of human tumor cell lines, none of them expressed a level of MTCBP-1 mRNA comparable with that in the non-transformed fibroblasts (data not shown). The amount of the transcripts measured by real-time PCR after reverse transcription revealed that the expression of MTCBP-1 mRNA in the fibrosarcomas was less than 5–10% that in the non-transformed fibroblasts (data not shown). Expression of the protein products was also confirmed by Western blotting (Fig. 5C). Thus, the expression of MTCBP-1 appears to be down-regulated in the tumor-derived cell lines compared with the non-transformed cell lines and inversely correlated with that of MT1-MMP. These results raise the possibility that MTCBP-1 is a negative regulator of MT1-MMP function.
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v chain, tissue transglutaminase, and laminin-5 etc (3). We reported previously that the cytoplasmic tail plays some roles in the migration- and invasion-promoting activity of MT1-MMP as well (12). Thus, we examined the possibility that expression of MTCBP-1 affects the activity of MT1-MMP to promote cell migration. We used COS-1 cells that do not express endogenous MT1-MMP and are known to migrate upon expression of MT1-MMP (12). Transient expression of MT1-MMP promoted cell migration, and the effect required the cytoplasmic tail because on deletion of the tail (dCPF) the migration was not promoted as reported previously (12) (Fig. 6B). Expression of MTCBP-1 itself had no effect on cell migration. However, MTCBP-1 significantly suppressed the cell migration promoted by MT1-MMP. The effect of MTCBP-1 was specific to the migration promoted by MT1-MMP, because MTCBP-1 did not show such a suppressive effect on the 12-O-tetradecanoylphorbol-13-acetate-induced cell migration. This is consistent with the evidence that MTCBP-1 had no effect on the basal migration activity of the COS-1 cells. MTCBP-1 Reduces the Invasion-promoting Activity Mediated by MT1-MMP—To examine the effect of MTCBP-1 on invasion, we used HT1080 cells that aggressively invade a reconstituted basement membrane (Matrigel) using endogenous MT1-MMP and express low levels of MTCBP-1 (Fig. 7). In the previous study we demonstrated that specific down-regulation of MT1-MMP by RNA-mediated interference abrogated the activity to invade Matrigel (21). As expectedly from this result, invasion by the cells strictly requires MMP activity, as the synthetic MMP inhibitor BB94 or natural inhibitor TIMP-2 inhibited it efficiently (Fig. 7). TIMP-1 also inhibited the invasion although it was not comparable with that of BB94 or TIMP-2. Expression of MTCBP-1 significantly reduced the invasion (Fig. 7). Although the inhibition by MTCBP-1 was greater than by TIMP-1, it was less than that by BB94 or TIMP-2.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MTCBP-1 also showed homology to the products of prokaryotic and eukaryotic genes listed in Fig. 2. Among them, ARDs of K. pneumonia and oxytoca are the best characterized as a dioxgenase acting in the salvage pathway of methionine metabolism (22, 23). The ARD converts aci-reductone, an intermediate in the salvage pathway of methionine metabolism, into two different metabolites depending on the metal ions available (Ni2+ and Fe2+) (18). Because ARD activity is conserved in a wide array of organisms, it is possible that MTCBP-1 acts as a human ARD.
MTCBP-1 homologues in different organisms share an evolutionarily conserved domain called DSBH or -barrel hold that unites these proteins as members of the Cupin superfamily (16). A characteristic feature of this family is that the members have acquired diverse functions during evolution while retaining the DSBH scaffold. For example, they function as isomerases, epoxidases, dioxygenases, decarboxylases, transcription factors, and centromeric proteins (16). Thus, it is plausible that MTCBP-1 has acquired an ability to regulate MT1-MMP in addition to the ARD activity during the process of evolution.
MTCBP-1 Down-regulates the Cell Migration and Invasion Promoted by MT1-MMP—MTCBP-1 co-localizes with MT1-MMP at the ruffling membrane by forming a complex, and recruitment of MTCBP-1 to the membrane fraction was dependent on the cytoplasmic tail of MT1-MMP (Fig. 3D). Expression of MT1-MMP is known to promote cell migration and invasion (2, 3). Co-expression of MTCBP-1 inhibited the cell migration promoted by MT1-MMP, whereas it showed no effect on the 12-O-tetradecanoylphorbol-13-acetate-induced migration. Thus, the effect of MTCBP-1 on cell migration is specific to that promoted by MT1-MMP. MTCBP-1 also inhibited the invasion-promoting activity of MT1-MMP against Matrigel. The invasion and migration-promoting activity of MT1-MMP is mediated through proteolysis on the cell surface, because the activity was inhibited by BB94 (Figs. 6 and 7). On the other hand, MTCBP-1 did not affect proMMP-2 activation by MT1-MMP. In addition, the cytoplasmic tail of MT1-MMP has no effect on degradation of and invasion into type I collagen gel (24, 25). Thus, we speculate that the effect of the cytoplasmic tail on the proteolytic activity of MT1-MMP differs depending on its substrates. The difference may be explained by the mode of interactions of MT1-MMP with substrates. Namely, MT1-MMP binds type I collagen at the HPX domain (10) and does not require the cytoplasmic tail to degrade it (24). However, some substrates in the Matrigel may bind MT1-MMP indirectly through the membrane proteins that bind the cytoplasmic tail of MT1-MMP within the cells and bind substrates outside. In this case deletion of the tail would abolish the activity to degrade the substrates in the Matrigel and inhibit the invasion. Binding of MTCBP-1 to the cytoplasmic tail may compete this sort of interaction and eventually reduce the invasion-promoting activity of MT1-MMP.
All the results presented support the idea that MTCBP-1 is a negative regulator of MT1-MMP through its cytoplasmic tail. Interestingly, the expression levels of MTCBP-1 in all the tumor cell lines examined was quite low compared with the non-transformed fibroblasts. Thus, down-regulation of MTCBP-1 appears to associate with tumor progression.
Subcellular Localization and Possible Physiological Functions—MTCBP-1 appears to shuttle between the three subcellular compartments, membrane, cytoplasm, and nucleus. The best characterized MTCBP-1 homologue is the ARD of K. pneumoniae (18). It is an enzyme in the salvage pathway of the methionine metabolism. As we already discussed, MTCBP-1 may be a human homologue of the bacterial ARD. Because the reactions of the metabolic pathway are to be carried out in the cytoplasm, the ARD activity of MTCBP-1 may be active when it is in the cytoplasmic fraction. In relation to the nuclear localization of MTCBP-1, the product of the yeast homologue (YMR009w) is listed as a possible interactor with a splicing factor, SNP-1, in the comprehensive yeast protein-protein interaction data base constructed by the two-hybrid methodology (26). Therefore, MTCBP-1 may regulate gene expression as a component of the splicing machinery in the nucleus. Thus, MTCBP-1 is a possible multifunctional protein carrying out different functions depending on its localization. One characteristic feature of Cupin superfamily is the diverse functions of the members, whereas they have a conserved three-dimensional structure (16). The DSBH domain conserved in the family appears flexible and able to accept such diverse functions that were necessary during evolution. Thus, it is not be surprising that MTCBP-1 has acquired multiple functions by having the DSBH domain.
In conclusion, we identified MTCBP-1 as a protein binding to the cytoplasmic tail of MT1-MMP. MTCBP-1 has a DSBH domain that is conserved in a recently identified Cupin superfamily composed of proteins with diverse functions. The expression levels of MTCBP-1 in tumor cell lines are low compared with the non-transformed fibroblasts, and enforced expression of MTCBP-1 suppressed the invasion and migration-promoting activity of MT1-MMP. Because MTCBP-1 disrupts the essential role of the cytoplasmic tail during cell migration and invasion, it may be a useful tool to analyze the regulation of MT1-MMP through the cytoplasmic tail.
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FOOTNOTES |
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* This work was supported by the Special Coordination Fund for promoting Science and Technology from the Ministry of Science and Technology of Japan and by a grant-in-aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 
Present address: Growth Factor Division, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045.
¶ Present address: Center for the Development of Molecular Target Drugs, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan.
|| Present address: Kennedy Institute of Rheumatology Division, Faculty of Medicine, Imperial College, 1 Aspenlea Rd., Hammersmith, London W6 8LH, UK.
To whom correspondence should be addressed: Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan. Tel.: 81-3-5449-5255; Fax: 81-3-5449-5414; E-mail: mseiki{at}ims.u-tokyo.ac.jp.
1 The abbreviations used are: MT-MMP, membrane-type matrix metalloproteinase; RT, reverse transcription; TIMP, tissue inhibitor of metalloproteinase; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; HPX domain, hemopexin-like domain; MTCBP-1, MT1-MMP cytoplasmic tail-binding protein-1; rMTCBP-1, recombinant MTCBP-1; ARD, aci-reductone dioxygenase; DSBH, double-stranded helix; kb, kilobases; WT, wild type.
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ACKNOWLEDGMENTS |
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REFERENCES |
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