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Departamento de Bioquimica y Biologia Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain [E. L., A. M. P., J. P. F., C. L-O.], Department of Neurosurgery, Hyogo College of Medicine, Hyogo 663, Japan [A. N.] and School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom [V. K., G. M.]
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Introduction |
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The MT-MMP subfamily is the most recently described subclass of MMPs and is composed of four members that have been identified using the reverse transcriptase-PCR technique and degenerate primers corresponding to conserved regions of MMP genes (5, 6, 7, 8) . MT-MMPs are type I membrane proteins with a single membrane-spanning domain and a short cytoplasmic tail located after the hemopexin domain, which are characteristic of most MMPs. In addition, they contain a conserved sequence of basic amino acids between the propeptide and catalytic domains that has been implicated in the intracellular activation of these membrane proteases by furin or furin-like enzymes (9) , although extracellular mechanisms for MT-MMPs activation have been also proposed (10) . MT-MMPs have raised additional interest for their role as cell surface activators of progelatinase A, which has a propeptide that is not generally susceptible to the serine proteinase-mediated process of activation occurring in other pro-MMPs. Because gelatinase A is an important enzyme for basement membrane invasion due to its ability to degrade type IV collagen, its activation, mediated by MT-MMPs on the tumor cell surface, is thought to play a critical role in the invasive phenotype of tumor cells (11 , 12) . Furthermore, although MT-MMPs were first characterized by virtue of their ability to activate progelatinase A, we have recently provided evidence that their activating role may be also extended to other MMP family members, such as procollagenase-3, which is efficiently activated by MT1-MMP (13) . This has led us to propose that these three enzymes could form a proteolytic cascade operating in those physiological and pathological conditions, including tumor processes, in which MT1-MMP, gelatinase A, and collagenase-3 are coexpressed (14) . In addition to this role of MT-MMPs as membrane-bound activators of other MMPs, several studies have shown that these membrane proteases can also degrade a number of extracellular matrix proteins, such as gelatin, fibronectin, vitronectin, fibrillar collagens, or aggrecan (15) . Furthermore, Hiraoka et al. (16) have recently provided evidence that MT1-MMP has the ability to regulate neovascularization processes by acting as a pericellular fibrinolysin. These enzymatic activities extend the number of biological functions in which MT-MMPs could be involved and suggest that their proposed ability to favor the invasive potential of tumor cells may not necessarily be the result of progelatinase A activation at the cell surface.
Identification of new members of the MT subclass of MMPs would be important for a better understanding of the properties of these membrane proteinases, the biological functions of which appear to be distinct from previously described family members. In this work, we report the molecular cloning of a novel human MT-MMP that has been called MT5-MMP and show that it is localized in the plasma membrane. We also report the chromosomal location of the MT5-MMP gene (MMP-24) and analyze its expression in normal and tumor tissues. Finally, we describe the expression of the gene in Escherichia coli and perform an analysis of the enzymatic activity of the recombinant MT5-MMP, including its characterization as a progelatinase A activator.
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Materials and Methods |
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gt11 and two Northern blots containing polyadenylated RNAs from different human tissues were from Clontech (Palo Alto, CA). A human PAC was provided by the Human Genome Mapping Resource Center (Cambridgeshire, United Kingdom). A human BAC library was provided by Dr. P. J. de Jong (Roswell Park Cancer Institute, Buffalo, NY). Restriction endonucleases and other reagents used for molecular cloning were from Boehringer Mannheim (Mannheim, Germany). Synthetic oligonucleotides were prepared in an Applied Biosystems (Foster City, CA) model 392A DNA synthesizer. Double-stranded DNA probes were radiolabeled with [32P] dCTP (3000 Ci/mmol) purchased from Amersham International (Buckinghamshire, United Kingdom) using a commercial random-priming kit from the same company.
Probe Preparation and Screening of a Human Brain cDNA Library.
A computer search of the GenBank database of human ESTs for entries with similarity to previously described MMPs led us to identify a sequence (accession no. AA324134) derived from a cerebellum cDNA clone, showing significant similarity with the hemopexin domain of MMPs. To obtain this DNA fragment, we performed PCR amplification of a panel of cDNAs (Quick Screen; Clontech) with two specific primers, 5'-TTCAACACAGTGGCCCTCTTC-3' and 5'-CCCCCAGGCTGTGGGGGTA-3', derived from the AA324134 sequence. The PCR was carried out in a GeneAmp 2400 PCR system from Perkin-Elmer/Cetus (Norwalk, CT) for 40 cycles of denaturation (94°C, 15 s), annealing (57°C, 15 s), and extension (72°C, 30 s). The 245-bp PCR product, amplified from human brain cDNA was sequenced and found to be virtually identical (99% identities) to the EST sequence. This cDNA fragment was then radiolabeled and used to screen a human brain cDNA library according to standard procedures.
5'-Extension of Isolated cDNAs.
The 5' ends of cloned cDNAs were extended by successive cycles of RACE using RNA from human brain and kidney and the Marathon cDNA amplification kit (Clontech), essentially as described by the manufacturer. Each cycle of RACE allowed the extension of 60–100 bp of cDNA toward the 5' end. After cloning and sequencing the amplified products, we synthesized new specific oligonucleotides and used them for the next RACE experiment. Finally, the complete cDNA was obtained by PCR amplification using the Expand Long PCR kit (Boehringer Mannheim). The PCRs were performed for 35 cycles of denaturation (94°C, 15 s), annealing (64°C, 15 s), and extension (68°C, 2 min) with primers 5'-ATGGCTATCTGCTTCCCTATGACC-3' and 5'-GCACCCATTCCTGGACTGGCCGC-3'. Following gel purification, the amplification product was cloned and sequenced by the dideoxy chain termination method, using the Sequenase Version 2.0 kit (United States Biochemical, Cleveland, OH). All nucleotides were identified in both strands. Computer analysis of DNA and protein sequences was performed with the GCG software package of the University of Wisconsin Genetics Computer Group.
Chromosomal Mapping.
Fluorescent in situ hybridization mapping of genomic DNA clones for MT5-MMP was performed as described previously (4)
. Briefly, DNA from isolated PAC and BAC clones were obtained with the standard alkaline lysis method using Qiagen columns (Qiagen, Chatsworth, CA) and nick-translated with biotin-16-dUTP. Then, labeled probes were hybridized to normal male metaphase chromosomes obtained from phytohemagglutinin-stimulated cultured lymphocytes and detected using two avidin-fluorescein layers. Chromosomes were DAPI-banded and images were captured in a Zeiss axiophot fluorescent microscope equipped with a charged coupled device camera (Photometrics).
Northern Blot Analysis.
Nylon filters containing 2 µg of poly(A)+ RNA of different normal human tissues or 10 µg of total RNA from human tumors were prehybridized at 42°C for 3 h in 50% formamide, 5x SSPE [1 x = 150 mM NaCl, 10 mM NaH2PO4, and 1 mM EDTA (pH 7.4)], 10x Denhardt’s solution, 2% SDS, and 100 µg/ml denatured herring sperm DNA and then hybridized with radiolabeled MT5-MMP full-length cDNA for 20 h under the same conditions. Filters were washed with 0.1x SSC-0.1% SDS for 2 h at 50°C and exposed to autoradiography. RNA integrity and equal loading was assessed by hybridization with an actin probe.
Construction of Expression Vectors for MT5-MMP and Expression in Escherichia coli.
A 815-bp fragment of the MT5-MMP cDNA encoding the prodomain and catalytic domain of this protein, was generated by PCR amplification with primers 5'-AGTCCTATGGCTATCTGCTTCCC-3' and 5'-CCGGAAGAGGGCCACTGTGTTG-3'. The PCR amplification was performed for 30 cycles of denaturation (95°C, 15 s), annealing (54°C, 15 s), and extension (68°C, 1 min), using the Expand Long High Fidelity PCR kit and the GeneAmp 9700 PCR system. The PCR-amplified product was then ligated in the Sma1 site of the pGEX 3X expression vector. The expression vector was transformed into BL21(DE3)pLysS competent E. coli cells and grown on agar plates containing chloramphenicol and ampicillin. Single colonies were used to inoculated 2-ml cultures in 2YT medium supplemented with 33 µg/ml chloramphenicol and 50 µg/ml ampicillin. Five hundred µl of the corresponding culture was used to inoculate 200 ml of 2YT medium containing the above antibiotics. After culture reached an A600 of 0.6, expression was induced by addition of isopropyl-1-thio-ß-D-galactopyranoside (0.5 mM final concentration) followed by further incubation for 3–20 h at 30°C. Cells were collected by centrifugation, washed, and resuspended in 0.05 volumes of PBS. Finally, cells were lysed by using a French press and centrifuged at 20,000 x g for 20 min at 4°C. The soluble extract was treated with glutathione-Sepharose 4B (Pharmacia) and eluted with glutathione elution buffer [10 mM reduced glutathione in 50 mM Tris-HCl, (pH 8.0)] following the manufacturer’s instructions. Determination of the TIMP inhibition profile and substrate specificity of recombinant MT5-MMP versus synthetic MMP substrates was determined as described previously (4
, 13)
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Assay of Progelatinase A (MMP-2) and Progelatinase B (MMP-9) Activation.
Human progelatinase A and progelatinase B were obtained from the supernatant of the fibrosarcoma cell line HT1080. Glutathione-Sepharose beads absorbed to GST-MT5-MMP fusion protein were incubated for 3 h with HT1080 conditioned medium in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM CaCl2, and progelatinase activation analyzed by means of gelatin zymography.
Construction of Eukaryotic Expression Vectors for MT5-MMP-HA and Immunolocalization.
An MT5-MMP cDNA fragment encoding amino acids G89-V645 was PCR-amplified and cloned in the blunt-ended NheI site of a modified pCEP-pU secretion vector (kindly provided by Dr. E. Kohfeld, Max-Planck-Institut, Martinsried, Germany). In addition, a 24-bp linker coding for the HA epitope of human influenza virus was ligated in frame to the 3' end of the MT5-MMP insert. Thus, the resulting MT5-MMP protein was HA-tagged at the COOH terminus. COS-7 cells were transfected with 1 µg of plasmid DNA, using Fugene 6 reagent (Boehringer Mannheim) according to the manufacturer’s instructions. Forty-eight h after transfection, cells were fixed for 10 min in cold 4% paraformaldehyde in PBS for 10 min, washed in PBS, and incubated for 10 min in 0.2% Triton X-100 in PBS. Fluorescent detection was performed by incubating the slides with monoclonal antibody 12CA5 (Boehringer Mannheim) against HA (diluted 1:2500), followed by another incubation with goat antimouse fluoresceinated antibody (diluted 1:50). After washing in PBS, slides were mounted with Vectashield (Vector Laboratories, Burlingame, CA) and observed in a BioRad confocal laser microscope. COS-7 extracts were also obtained for Western blot analysis of the MT5-MMP-HA protein.
Preparation of Cell Membrane Fractions and Western Blot Analysis.
COS-7 cells were transiently transfected with the pCEP-MT5-MMP-HA plasmid as described previously. Cells were rinsed in PBS and scraped from the plates. Membrane fractions were prepared essentially following the procedure described by Strongin et al. (11)
. Extracts were separated by SDS-PAGE and analyzed by Western blotting with an anti-HA monoclonal antibody and detected with an ECL chemiluminiscent kit (Amersham).
In Vitro Transcription and Translation.
cDNA insert from plasmid pCEP-MT5-MMP-HA was released by HindIII/XhoI and cloned in pcDNA3 (Invitrogen). One µg of this plasmid was then transcribed and translated using the coupled reticulocyte TNT T7 Kit (Promega) in the presence of [35S]methionine (Amersham), following the manufacturer’s instructions. Protein translation products were analyzed by SDS-PAGE, followed by overnight autoradiography.
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Results |
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-phage DNA prepared from a human brain cDNA library and used as a probe to screen this library. Upon screening of 
1 x 106 plaque-forming units, six positive clones, named 1.1–1.6, were identified and characterized. DNA was isolated from these positive clones, and their nucleotide sequence was determined by standard procedures. A comparative analysis of the sequence obtained for the largest clone (1.3) with those corresponding to other MMPs, suggested that it was incomplete at the 5' end. To extend this sequence, we performed 5'-RACE experiments using a specific oligonucleotide deduced from the end of the 1.3 clone and RNA from human brain as a template. Successive 5'-RACE experiments performed in similar conditions finally led us to obtain a fragment long enough to contain most of the entire coding information for the identified MMP. However, after several 5'-RACE experiments using RNA from different human tissues, we were unable to extend the 5' sequence beyond the region coding for the putative propeptide of this MMP. To overcome this problem, likely due to the extremely high GC content of this region, we performed a genomic approach to try to complete the nucleotide sequence encoding this protease. To isolate these genomic clones, two genomic libraries (PAC and BAC) were screened with the isolated cDNA cloned as described above. A total of five positive PAC clones and 13 BAC clones were identified on the basis of their positive hybridization with the probe. Southern blot analysis of DNA isolated from these clones, followed by nucleotide sequencing of selected bands, revealed that most of them contained sequences close to the 5' end of the gene. However, only a series of restriction fragments generated from BAC clones hybridized with the most 5' cDNA probe obtained by RACE experiments. Then, a 1-kb BAC EcoRI/PstI genomic fragment positive for the 5'-RACE probe was subcloned and sequenced. Nucleotide sequence analysis of this DNA fragment revealed the presence of a region encoding a typical signal sequence as well as an in-frame ATG trinucleotide coding for the first methionine. Computer analysis of the obtained sequence (Fig. 1
; EMBL database accession no. AF131284) revealed an open reading frame coding for a protein of 645 amino acids with a predicted molecular mass of 73.2 kDa. This sequence contains two potential sites of N-glycosylation (Asn-Tyr-Thr and Asn-Lys-Thr at positions 174 and 627, respectively). Further analysis of the identified amino acid sequence revealed a significant similarity with other human MMPs, the maximum percentage of identities (63%) being with MT3-MMP. It should be also mentioned that a murine nucleotide sequence that is 94% identical to the human cDNA sequence reported herein has been recently released to the GenBank (accession no. AJ010262). According to this high percentage of identities, it is likely that the murine protein encoded by this cDNA sequence is the homologue of the human enzyme identified here.
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displays a number of features that are characteristic of MMPs. Thus, it contains a signal sequence, a prodomain with a Cys residue essential for maintaining the latency of these enzymes, a catalytic domain of 170 residues, including the consensus sequence HEXGHXXGXXHS involved in zinc binding, and a fragment of 200 amino acids with sequence similarity to hemopexin. In addition, this novel sequence contains three insertions characteristic of the MT subclass of MMPs (Fig. 1)
. The first one (IS-1, nine amino acids) is located between the propeptide and catalytic domains and ends in a RXKR furin activation consensus sequence. The second insertion (IS-2) is 8 amino acids long and is located in the catalytic domain of all MT-MMPs, with the exception of MT4-MMP. Finally, the identified sequence contains a COOH-terminal extension rich in hydrophobic residues and similar in size to the equivalent domains present in the different MT-MMPs characterized to date. On the basis of these structural characteristics, we suggest that the isolated brain cDNA codes for a novel human MT-MMP, which we propose calling MT5-MMP. Furthermore, following the nomenclature system proposed for vertebrate MMPs, we propose designating this novel MT-MMP MMP-24 because MMP-23 corresponds to the last family member first cloned by Gururajan et al. (3)
and, subsequently, by Velasco et al. (4)
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Membrane Localization of MT5-MMP.
To provide further support on the subcellular distribution of MT5-MMP, we transfected COS-7 cells with pCEP-MT5-MMP-HA, a construct containing the HA epitope in the COOH terminus of MT5-MMP. Transfected cells were then analyzed by immunofluorescence with a mouse monoclonal antibody (12CA5) specific for this viral epitope. As shown in Fig. 2
, a clear fluorescent pattern surrounding the cell was visualized in a serial optical section obtained by the confocal microscope. This observation provides strong evidence that the human MT5-MMP is a membrane-bound MMP, fitting the requirement for a cell surface activator of progelatinase A. To further verify the nature of the MT5-MMP recombinant protein, we analyzed lysates from COS-7 cells transfected with the MT5-MMP-HA by SDS-PAGE, followed by Western blotting detection with anti-HA monoclonal antibody. A band of the expected molecular weight (Mr 64,000) was detected in the membrane-enriched fractions but not in the soluble fraction, reinforcing the above results proposing its membrane localization (Fig. 2)
. It is remarkable that the electrophoretical mobidlity of the protein detected in membrane extracts of COS-7-transfected cells was very similar to that obtained by SDS-PAGE analysis of the protein product generated in an in vitro transcription and translation assay of MT5-MMP cDNA (Fig. 2)
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, fluorescent signals corresponding to biotinylated MT5-MMP clones were located on chromosome 20, and no other chromosome site was labeled above background. After DAPI banding of 50 metaphases showing hybridization in both chromosomes 20, the MT5-MMP fluorescent signal was assigned to the centromeric region of the long arm of chromosome 20, in the q11.2 region. The gene encoding gelatinase B (MMP-9) has been also located in the long arm of chromosome 20 (20)
. However, this location is distinct from that reported for all MT-MMP genes, which have been mapped to chromosomes 14q11 (MT1-MMP/MMP14), 16q13 (MT2-MMP/MMP15), 8q21 (MT3-MMP/MMP16), and 12q24 (MT4-MMP/MMP17; (Refs. 21
and 22
).
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, the bacteria transformed with the recombinant plasmid contained a fusion protein of M4 64,000, which was not present in the control extracts. The fusion protein containing MT5-MMP was purified by affinity chromatography in a glutathione-Sepharose 4B column, and as shown in Fig. 4
, a single band of the expected size was detected by SDS-PAGE analysis. It is also remarkable that GST-MT5-MMP showed autoproteolytic activity, despite having a prodomain with the cysteine switch involved in maintaining the latency of the proenzyme. Similar results have also been obtained with a GST-MT1-MMP fusion protein enclosing the same structural domains as GST-MT5-MMP, and are likely due to the disturbing effect of the NH2-terminal heterologous GST sequence on the switch function of the prodomain of these enzymes (23)
. The MT5-MMP fusion protein was then used to determine its ability to activate progelatinase A (MMP-2) and progelatinase B (MMP-9; Mr 72,000 and 92,000, respectively; Fig. 4
) secreted to the medium by the human fibrosarcoma HT1080 cell line. To do that, we incubated conditioned medium from HT1080 cells with the GST-MT5-MMP fusion protein and analyzed by gelatin zymography. As can be seen in Fig. 4
, these analyses revealed that recombinant human MT5-MMP exhibits a significant proteolytic activity against progelatinase A, giving rise to a Mr 62,000 band corresponding to the active gelatinase A. In contrast, similarly to previous studies with the remaining MT-MMPs, no activation effect of MT5-MMP on progelatinase B was observed (Fig. 4)
. Finally, we examined the possibility that the proteolytic activity of recombinant MT5-MMP against progelatinase A was inhibited by EDTA, a widely used inhibitor of MMPs. In fact, as can be seen in Fig. 4
, this proteolytic activity was completely abolished by EDTA, as demonstrated by its inability to generate the Mr 62,000 band in the gelatin zymography. Furthermore, TIMP inhibition experiments revealed that TIMP-2 was an efficient inhibitor of MT5-MMP, whereas TIMP-1 was unable to inhibit its activity even when a ten-fold molar excess of inhibitor was used (Fig. 4)
. Thus, these data again confirm that MT5-MMP, is a true member of the MT-MMP family due to the fact that these proteases are not inhibited by TIMP-1. Taken together, these functional analyses suggest that the cloned cDNA encodes for a functional MT-MMP with ability to mediate the activation of progelatinase A.
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, a transcript of 4.5 kb was predominantly detected in brain, whereas transcripts of a slightly smaller size were detected in kidney, pancreas, and lung. The predominant expression of MT5-MMP in some tissues, such as pancreas and kidney, in which other members of this subfamily of MMPs are not detected at significant levels, suggests that this novel MT-MMP could participate in some of the tissue remodeling processes taking place in these tissues during physiological conditions. To ascertain the possible overexpression of human MT5-MMP in malignant tumors, we analyzed the expression of this gene in total RNAs extracted from a variety of brain tumors, including astrocytomas, anaplastic astrocytomas, glioblastomas, mixed gliomas, oligodendrogliomas, ependymomas, neurocytomas, and meningiomas. As shown in the representative experiment depicted in Fig. 5B
, MT5-MMP transcripts were significantly overexpressed in several astrocytomas, anaplastic astrocytomas, and glioblastomas, as compared with the faint band observed in the lanes corresponding to RNA from normal brain tissues. In contrast, all analyzed meiningiomas expressed very low or undetectable levels of MT5-MMP transcripts. In all cases, RNA integrity and equal loading were confirmed by hybridization with an actin probe (data not shown). Finally, preliminary results indicate that MT5-MMP is also overexpressed in other tumors from different sources, including pancreas and lung (data not shown). These results, together with the above data indicating that progelatinase A is efficiently activated by MT5-MMP, suggest that this novel membrane-bound enzyme may contribute to the generation of the gelatinolytic activity that is necessary to facilitate tumor progression.
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Discussion |
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Here, we have also provided functional evidence that MT5-MMP is an enzymatically active member of this subfamily of membrane-bound MMPs, as assessed by examining its ability to act as a progelatinase A activator. In fact, the catalytic domain of MT5-MMP produced in E. coli as a fusion with GST, is able to activate progelatinase A to its Mr 62,000 active final form. The finding that different MT-MMPs share the ability to activate this gelatinase may reflect on evolutionary adaptation to cleave similar substrates in different tissues. Consistent with this proposal, the pattern of MT5-MMP expression in human tissues is distinct from the remaining MT-MMPs. Thus, here, we have shown that this gene is abundantly expressed in brain, kidney, pancreas, and lung. None of the remaining MT-MMPs shows a similar pattern of expression (5, 6, 7, 8) . In fact, MT1-MMP is widely expressed in normal tissues but it is not detected at significantly levels in brain. MT2-MMP is also undetectable in brain, whereas MT3-MMP and MT4-MMP, which are expressed in brain, are not detected in kidney or pancreas, which are also major sources of MT5-MMP expression. On this basis, it is tempting to speculate that this novel membrane proteinase could play some specific role in any of the matrix-remodeling processes occurring in these tissues in which its levels are significantly higher when compared with those corresponding to the remaining MT-MMPs. Similarly, MT5-MMP may act as a membrane-bound progelatinase A activator in those tumors in which it is overexpressed, thus contributing to the facilitation of tumor invasion and metastasis. Interestingly, a survey of a series of brain tumors for their ability to produce MT5-MMP has revealed that this gene is significantly overexpressed in a number of astrocytomas, anaplastic astrocytomas, and glioblastomas. In contrast, all analyzed meningiomas showed very low or undetectable levels of MT5-MMP RNA transcripts. Further clinical studies, now in progress, will try to evaluate the possibility that MT5-MMP expression may have a critical role in brain tumor progression, as already shown for other MT-MMP family members overproduced in different human tumors such as breast carcinomas, lung carcinomas, or papillary thyroid carcinomas (5 , 26) .
In an attempt to provide further insights into potential associations of MT5-MMP with tumor processes, we have also established in this work the chromosomal location of the gene encoding this proteinase. According to our results, the MT5-MMP gene (MMP-24) is located at chromosome 20q11, a unique position among all MT-MMP genes mapped to date (21 , 22) . The fact that all members of the MT-MMP subfamily map at distinct chromosomes indicates that besides duplication of their putative common ancestor, transposition events to different chromosomes have played a major role in the evolutionary diversification of this gene family, as opposed to the MMPs from the 11q22 cluster that contains at least eight different family members tightly linked in a small region of the human genome (19) . Interestingly, the 20q11.2 region has been found to be a common target of genetic alterations in diverse malignancies. Thus, this region is a recurring site of breakage and translocation in multiple myeloma (27) . In addition, this region is frequently coamplified with other loci along 20q in breast carcinomas (28) . Finally, very recent studies have shown that 20q11.2 is amplified in male germ tumors with chemotherapy resistance (29) . The isolation and characterization of MT5-MMP will allow us to study the possibility that this gene could be the target of these abnormalities occurring in this region of the human genome. Finally, the availability of specific reagents for MT5-MMP generated in this work will be very helpful in examining the functional relevance of this enzyme in the context of other membrane proteases involved in tumor progression.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 This work was supported by Comisión Interministerial de Ciencia y Tecnología-Spain (Grant SAF97-0258); EU-BIOMED II (Grant BMH4-CT96-0017); the Arthritis and Rheumatism Council (to G. M.); and the Wellcome Trust (to V. K.). E. L., J. P. F., and A. M. P. are recipients of fellowships from Ministerio de Educación y Cultura (Spain) and Fuji Chemical Industries (Takaoka, Japan). 
2 To whom requests for reprints should be addressed, at Departmento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Oviedo, 33006 Oviedo, Spain. Phone: 34-985-104201; Fax: 34-985-103564; E-mail: CLO{at}DWARF1.QUIMICA.UNIOVI.ES
3 The abbreviations used are: MMP, matrix metalloproteinase; MT, membrane-type; PAC, P1 artificial chromosome; BAC, bacterial artificial chromosome; EST, expressed sequence tag; RACE, rapid amplification of cDNA ends; DAPI, diamidine-2-phenylindole hydrochloride; TIMP, tissue inhibitor of metalloproteinase; HA, hemagglutinin; GST, glutathione G-transferase.
Received 3/17/99. Accepted 4/15/99.
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P. Wang, X. Wang, and D. Pei Mint-3 Regulates the Retrieval of the Internalized Membrane-type Matrix Metalloproteinase, MT5-MMP, to the Plasma Membrane by Binding to Its Carboxyl End Motif EWV J. Biol. Chem., May 7, 2004; 279(19): 20461 - 20470. [Abstract] [Full Text] [PDF]
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M.-H. Lee, M. Rapti, V. Knauper, and G. Murphy Threonine 98, the Pivotal Residue of Tissue Inhibitor of Metalloproteinases (TIMP)-1 in Metalloproteinase Recognition J. Biol. Chem., April 23, 2004; 279(17): 17562 - 17569. [Abstract] [Full Text] [PDF]
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N. E. Sounni, C. Roghi, V. Chabottaux, M. Janssen, C. Munaut, E. Maquoi, B. G. Galvez, C. Gilles, F. Frankenne, G. Murphy, et al. Up-regulation of Vascular Endothelial Growth Factor-A by Active Membrane-type 1 Matrix Metalloproteinase through Activation of Src-Tyrosine Kinases J. Biol. Chem., April 2, 2004; 279(14): 13564 - 13574. [Abstract] [Full Text] [PDF]
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X. Wang, D. Ma, J. Keski-Oja, and D. Pei Co-recycling of MT1-MMP and MT3-MMP through the Trans-Golgi Network: IDENTIFICATION OF DKV582 AS A RECYCLING SIGNAL J. Biol. Chem., March 5, 2004; 279(10): 9331 - 9336. [Abstract] [Full Text] [PDF]
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G. Velasco-Loyden, J. Arribas, and F. Lopez-Casillas The Shedding of Betaglycan Is Regulated by Pervanadate and Mediated by Membrane Type Matrix Metalloprotease-1 J. Biol. Chem., February 27, 2004; 279(9): 7721 - 7733. [Abstract] [Full Text] [PDF]
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 A. M. Tester, M. Waltham, S.-J. Oh, S.-N. Bae, M. M. Bills, E. C. Walker, F. G. Kern, W. G. Stetler-Stevenson, M. E. Lippman, and E. W. Thompson Pro-Matrix Metalloproteinase-2 Transfection Increases Orthotopic Primary Growth and Experimental Metastasis of MDA-MB-231 Human Breast Cancer Cells in Nude Mice Cancer Res., January 15, 2004; 64(2): 652 - 658. [Abstract] [Full Text] [PDF]
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) and TIMP2 (•). Protease activity was determined using the synthetic MMP substrate McaPLGLDpaARNH2 in the presence of the indicated concentrations of TIMP-1 or TIMP-2.