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Identification and Characterization of the Fifth Membrane-type Matrix Metalloproteinase MT5-MMP

1999; Elsevier BV; Volume: 274; Issue: 13 Linguagem: Inglês

10.1074/jbc.274.13.8925

ISSN

1083-351X

Autores

Duanqing Pei,

Tópico(s)

Peptidase Inhibition and Analysis

Resumo

A new member of the membrane-type matrix metalloproteinase (MT-MMP) subfamily tentatively named MT5-MMP was isolated from mouse brain cDNA library. It is predicted to contain (i) a candidate signal sequence, (ii) a propeptide region with the highly conserved PRCGVPD sequence, (iii) a potential furin recognition motif RRRRNKR, (iv) a zinc-binding catalytic domain, (v) a hemopexin-like domain, (vi) a 24-residue hydrophobic domain as a potential transmembrane domain, and (vii) a short cytosolic domain. Reverse transcriptase-polymerase chain reaction analysis of its transcripts indicates that MT5-MMP is expressed in a brain-specific manner consistent with the origin of its EST clone from cerebellum. It is also highly expressed during embryonic development at stages day 11 and 15. Like other MT-MMPs, MT5-MMP specifically activates progelatinase A when co-expressed in Madin-Darby canine kidney cells. Its ability to activate progelatinase A is dependent on its proteolytic activity since a mutation converting Glu to Ala in the zinc binding motif HE255LGH renders MT5-MMP inactive against progelatinase A. In contrast to other MT-MMPs, MT5-MMP tends to shed from cell surface as soluble proteinases, thus offering flexibility as both a cell bound and soluble proteinase for extracellular matrix remodeling processes. Taken together, these properties serve to distinguish MT5-MMP as a versatile MT-MMP playing an important role in extracellular matrix remodeling events in the brain and during embryonic development. A new member of the membrane-type matrix metalloproteinase (MT-MMP) subfamily tentatively named MT5-MMP was isolated from mouse brain cDNA library. It is predicted to contain (i) a candidate signal sequence, (ii) a propeptide region with the highly conserved PRCGVPD sequence, (iii) a potential furin recognition motif RRRRNKR, (iv) a zinc-binding catalytic domain, (v) a hemopexin-like domain, (vi) a 24-residue hydrophobic domain as a potential transmembrane domain, and (vii) a short cytosolic domain. Reverse transcriptase-polymerase chain reaction analysis of its transcripts indicates that MT5-MMP is expressed in a brain-specific manner consistent with the origin of its EST clone from cerebellum. It is also highly expressed during embryonic development at stages day 11 and 15. Like other MT-MMPs, MT5-MMP specifically activates progelatinase A when co-expressed in Madin-Darby canine kidney cells. Its ability to activate progelatinase A is dependent on its proteolytic activity since a mutation converting Glu to Ala in the zinc binding motif HE255LGH renders MT5-MMP inactive against progelatinase A. In contrast to other MT-MMPs, MT5-MMP tends to shed from cell surface as soluble proteinases, thus offering flexibility as both a cell bound and soluble proteinase for extracellular matrix remodeling processes. Taken together, these properties serve to distinguish MT5-MMP as a versatile MT-MMP playing an important role in extracellular matrix remodeling events in the brain and during embryonic development. Members of the matrix metalloproteinase (MMP) 1The abbreviations used are:MMP, matrix metalloproteinase; MT1-, MT2-, MT3-, MT4-, and MT5-MMP, membrane-type matrix metalloproteinase-1, -2, -3, -4, -5; ECM, extracellular matrix; RT-PCR, reverse transcription-polymerase chain reaction; MDCK, Madin-Darby Canine Kidney; EST, expressed sequence tag; GST, glutathione S-transferase.1The abbreviations used are:MMP, matrix metalloproteinase; MT1-, MT2-, MT3-, MT4-, and MT5-MMP, membrane-type matrix metalloproteinase-1, -2, -3, -4, -5; ECM, extracellular matrix; RT-PCR, reverse transcription-polymerase chain reaction; MDCK, Madin-Darby Canine Kidney; EST, expressed sequence tag; GST, glutathione S-transferase. family have been well documented as critical players in the breakdown of extracellular matrix (ECM) under both physiological as well as diseased conditions ranging from embryo implantation to cancer progression (1Woessner Jr., J.F. 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Med. 1993; 4: 197-250Crossref PubMed Scopus (2633) Google Scholar). The addition of a hemopexin-like domain to this basic design eventually led to the evolution of the rest of the MMP family and confers them with specificity in substrate as well as inhibitor bindings (5Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (699) Google Scholar, 6Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Crit. Rev. Oral Biol. Med. 1993; 4: 197-250Crossref PubMed Scopus (2633) Google Scholar). While the majority of the MMPs are secreted as soluble enzymes into extracellular milieu, a subset of MMPs have been identified in recent years to contain additional sequences downstream of the hemopexin-like domain capable of anchoring the MMPs on plasma membrane (7Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2367) Google Scholar, 8Will H. Hinzmann B. Eur. J. Biochem. 1995; 231: 602-608Crossref PubMed Scopus (317) Google Scholar, 9Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Crossref PubMed Scopus (447) Google Scholar, 10Puente X.S. Pendás A.M. Llano E. Velasco G. López-Otı́n C. Cancer Res. 1996; 56: 944-949PubMed Google Scholar). Named after the putative transmembrane domains as membrane-type matrix metalloproteinase 1 to 4 (MT1-, MT2-, MT3-, and MT4-MMPs), these enzymes have been proposed to be the master switches of ECM turnover based on the purported ability of MT-MMPs to activate other MMPs such as progelatinase A and collagenase 3: two degradative enzymes widely implicated in tumor invasion and metastasis (7Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2367) Google Scholar, 11Vassalli J.-D. Pepper M.S. Nature. 1994; 370: 14-15Crossref PubMed Scopus (177) Google Scholar, 12Knauper V. Will H. Lopez-Otin C. Smith B. Atkinson S.J. Stanton H. Hembry R.M. Murphy G. J. Biol. Chem. 1996; 271: 17124-17131Abstract Full Text Full Text PDF PubMed Scopus (619) Google Scholar). However, MT-MMPs themselves are synthesized in latent forms and activation is required for them to exert any proteolytic function (13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 14d'Ortho M.P. Will H. Atkinson S. Butler G. Messent A. Gavrilovic J. Smith B. Timpl R. Zardi L. Murphy G. Eur J. Biochem. 1997; 250: 751-757Crossref PubMed Scopus (383) Google Scholar, 15Sato H. Kinoshita T. Takino T. Nakayama K. Seiki M. FEBS Lett. 1996; 393: 101-104Crossref PubMed Scopus (300) Google Scholar, 16Ohuchi E. Imai K. Fujii Y. Sato H. Seiki M. Okada Y. J. Biol. Chem. 1997; 272: 2446-2451Crossref PubMed Scopus (829) Google Scholar). The mechanism responsible for MT-MMP activation appears to be mediated by members of the proprotein convertase family which can specifically cleave off the prodomain at the carboxyl side of the conserved RXRXKR motif sandwiched between the pro- and catalytic domains of all MT-MMPs, a mechanism first demonstrated in stromelysin-3 (13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 14d'Ortho M.P. Will H. Atkinson S. Butler G. Messent A. Gavrilovic J. Smith B. Timpl R. Zardi L. Murphy G. Eur J. Biochem. 1997; 250: 751-757Crossref PubMed Scopus (383) Google Scholar, 15Sato H. Kinoshita T. Takino T. Nakayama K. Seiki M. FEBS Lett. 1996; 393: 101-104Crossref PubMed Scopus (300) Google Scholar, 16Ohuchi E. Imai K. Fujii Y. Sato H. Seiki M. Okada Y. J. Biol. Chem. 1997; 272: 2446-2451Crossref PubMed Scopus (829) Google Scholar, 17Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (533) Google Scholar, 18Steiner D.F. Smeekens S.P. Ohagi S. Chan S.J. J. Biol. Chem. 1992; 267: 23435-23438Abstract Full Text PDF PubMed Google Scholar). Thus, a proprotein convertase/MT-MMP/MMP cascade could be potentially responsible for the regulation of ECM turnover at the level of zymogen activation. Despite the extensive sequence homology and functional overlap among MT-MMPs, little is known about any functional cooperation among themselves in executing ECM remodeling. Their patterns of expression suggest a complex picture with overlapping expression in both normal and tumor tissues (7Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2367) Google Scholar, 8Will H. Hinzmann B. Eur. J. Biochem. 1995; 231: 602-608Crossref PubMed Scopus (317) Google Scholar, 9Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Crossref PubMed Scopus (447) Google Scholar, 10Puente X.S. Pendás A.M. Llano E. Velasco G. López-Otı́n C. Cancer Res. 1996; 56: 944-949PubMed Google Scholar, 19Okada A. Bellocq J.-P. Rouyer N. Chenard M.-P. Rio M.-C. Chambon P. Basset P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2730-2734Crossref PubMed Scopus (486) Google Scholar). For example, breast cancer tissues are known to express MT1-, MT2-, MT3-, and MT4-MMPs individually or together as detected by Northern blotting and in situhybridization (19Okada A. Bellocq J.-P. Rouyer N. Chenard M.-P. Rio M.-C. Chambon P. Basset P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2730-2734Crossref PubMed Scopus (486) Google Scholar, 20Ueno H. Nakamura H. Inoue M. Imai K. Noguchi M. Sato H. Seiki M. Okada Y. Cancer Res. 1997; 57: 2055-2060PubMed Google Scholar, 21Li H. Bauzon D.E. Xu X. Tschesche H. Cao J. Sang Q.A. Mol. Carcinog. 1998; 22: 84-94Crossref PubMed Scopus (50) Google Scholar). In addition, MT1-MMP has been investigated extensively and found to be expressed in other malignant tumors such as those from human brain, colon, pancreas, liver, gastrointestinal organs, ovary, and cervix (18Steiner D.F. Smeekens S.P. Ohagi S. Chan S.J. J. Biol. Chem. 1992; 267: 23435-23438Abstract Full Text PDF PubMed Google Scholar, 22Kitagawa Y. Kunimi K. Ito H. Sato H. Uchibayashi T. Okada Y. Seiki M. Namiki M. J. Urol. 1998; 160: 1540-1545Crossref PubMed Scopus (42) Google Scholar, 23Yamamoto M. Mohanam S. Sawaya R. Fuller G.N. Seiki M. Sato H. Gokaslan Z.L. Liotta L.A. Nicolson G.L. Rao J.S. Cancer Res. 1996; 56: 384-392PubMed Google Scholar, 24Theret N. Musso O. L'Helgoualc'h A. Campion J.P. Clement B. Am. J. Pathol. 1998; 153: 945-954Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 25Imamura T. Ohshio G. Mise M. Harada T. Suwa H. Okada N. Wang Z. Yoshitomi S. Tanaka T. Sato H. Arii S. Seiki M. Imamura M. J. Cancer Res. Clin. Oncol. 1998; 124: 65-72Crossref PubMed Scopus (32) Google Scholar, 26Ohtani H. Motohashi H. Sato H. Seiki M. Nagura H. Int. J. Cancer. 1996; 68: 565-570Crossref PubMed Scopus (75) Google Scholar, 27Gilles C. Polette M. Piette J. Munaut C. Thompson E.W. Birembaut P. Foidart J.M. Int. J. Cancer. 1996; 65: 209-213Crossref PubMed Scopus (157) Google Scholar). Among tissues and cell examined, expression of MT1-, MT2-, and MT3-MMPs seems to correlate well with the activation of progelatinase A, suggesting that MT-MMPs may act cooperatively toward progelatinase A in vivo (7Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2367) Google Scholar,22Kitagawa Y. Kunimi K. Ito H. Sato H. Uchibayashi T. Okada Y. Seiki M. Namiki M. J. Urol. 1998; 160: 1540-1545Crossref PubMed Scopus (42) Google Scholar, 23Yamamoto M. Mohanam S. Sawaya R. Fuller G.N. Seiki M. Sato H. Gokaslan Z.L. Liotta L.A. Nicolson G.L. Rao J.S. Cancer Res. 1996; 56: 384-392PubMed Google Scholar, 24Theret N. Musso O. L'Helgoualc'h A. Campion J.P. Clement B. Am. J. Pathol. 1998; 153: 945-954Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 25Imamura T. Ohshio G. Mise M. Harada T. Suwa H. Okada N. Wang Z. Yoshitomi S. Tanaka T. Sato H. Arii S. Seiki M. Imamura M. J. Cancer Res. Clin. Oncol. 1998; 124: 65-72Crossref PubMed Scopus (32) Google Scholar, 26Ohtani H. Motohashi H. Sato H. Seiki M. Nagura H. Int. J. Cancer. 1996; 68: 565-570Crossref PubMed Scopus (75) Google Scholar, 27Gilles C. Polette M. Piette J. Munaut C. Thompson E.W. Birembaut P. Foidart J.M. Int. J. Cancer. 1996; 65: 209-213Crossref PubMed Scopus (157) Google Scholar). With the expansion of the MT-MMP family, it becomes apparent that the function of MT-MMPs may not be only restricted to progelatinase A and collagenase 3 activation. In fact, purified MT1-MMP and MT2-MMP can degrade fibronectin, laminin, type I and III collagens, nidogen, tenascin, aggrecan, and perlecan (13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 14d'Ortho M.P. Will H. Atkinson S. Butler G. Messent A. Gavrilovic J. Smith B. Timpl R. Zardi L. Murphy G. Eur J. Biochem. 1997; 250: 751-757Crossref PubMed Scopus (383) Google Scholar, 16Ohuchi E. Imai K. Fujii Y. Sato H. Seiki M. Okada Y. J. Biol. Chem. 1997; 272: 2446-2451Crossref PubMed Scopus (829) Google Scholar). MT3-MMP appears to be able to degrade denatured type I collagen (gelatin), native type III collagen, and fibronectin based on limited studies (28Matsumoto S. Katoh M. Saito S. Watanabe T. Masuho Y. Biochim. Biophys. Acta. 1997; 1354: 159-170Crossref PubMed Scopus (84) Google Scholar, 29Shofuda K. Yasumitsu H. Nishihashi A. Miki K. Miyazaki K. J. Biol. Chem. 1997; 272: 9749-9754Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Taken together, MT-MMPs are a subgroup of versatile proteinases involved in ECM remodeling by both activating other MMPs as well as directly degrading ECM components. In contrast to secreted MMPs, MT-MMPs may express their proteolytic activities more efficiently by anchoring on cell membrane and enjoying two distinct advantageous properties, which are highly focused on ECM substrates and more resistant to proteinase inhibitors present in the extracellular milieu (11Vassalli J.-D. Pepper M.S. Nature. 1994; 370: 14-15Crossref PubMed Scopus (177) Google Scholar, 30d'Ortho M.P. Stanton H. Butler M. Atkinson S.J. Murphy G. Hembry R.M. FEBS Lett. 1998; 421: 159-164Crossref PubMed Scopus (102) Google Scholar). Recently, Nakahara and colleagues (31Nakahara H. Howard L. Thompson E.W. Sato H. Seiki M. Yeh Y. Chen W.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7959-7964Crossref PubMed Scopus (361) Google Scholar) demonstrated that MT1-MMP is localized in the invadopodia of malignant melanoma cells via the transmembrane/cytoplasmic domain, responsible for the efficient degradation of subjacent substrates and invasion into ECM in vitro. MT1-MMP can also confer mouse lung carcinoma cells metastatic phenotype upon transfection when analyzed in a tail-vein injection assay in vivo (32Tsunezuka Y. Kinoh H. Takino T. Watanabe Y. Okada Y. Shinagawa A. Sato H. Seiki M. Cancer Res. 1996; 56: 5678-5683PubMed Google Scholar). Therefore, recent attention has been shifting toward the characterization of membrane-bound MMPs and their biochemical properties (11Vassalli J.-D. Pepper M.S. Nature. 1994; 370: 14-15Crossref PubMed Scopus (177) Google Scholar). In this report, the identification and characterization of MT5-MMP, the fifth member of the MT-MMP subfamily, is described. MDCK cells and COS 7 were obtained and maintained as described previously (17Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (533) Google Scholar, 33Pei D. Yi J. Protein Expression Purif. 1998; 13: 277-281Crossref PubMed Scopus (15) Google Scholar). DNA restriction and modification enzymes were purchased from Promega (Madison, WI). Oligonucleotide primers were made by the University of Minnesota microchemical core facility. COS cells are used for transient gene expression because the pCR3.1 expression vector system is very efficient in COS cells due to the presence of SV40 T antigen, while moderately efficient in cells lacking T antigen such as MDCK cells. For progelatinase A activation, MDCK is preferred because it expresses higher levels of furin, a putative MT5-MMP activator, than COS (17Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (533) Google Scholar). The original EST clone EST27028 was obtained from American Type Cell Culture (ATCC, MD). The rest of EST27028 was sequenced by primer-walking using an ABI371 automatic sequencer. The resulting sequence was blasted against Genbank data base and aligned to 321GNFDT of MT1-MMP. The rest of EST27028 contains basically the entire hemopexin-like domain, followed by a putative transmembrane domain and cytosolic domain. Since this gene is homologous, but not identical to known MT-MMPs, it was named MT5-MMP as the fifth member of the MT-MMP subgroup. An EcoRI fragment from this clone was then isolated and used as a probe to isolate the missing part of MT5-MMP from both human and mouse brain cDNA libraries (Stratagene, CLONTECH, CA). From the human cDNA library, at least 10 clones have been isolated and sequenced to find the longest cDNA starting immediately upstream of the PRCGVPD sequence, but missing the 5′ end approximately 100-amino acid residue. Fortunately, out of 7 clones isolated from the mouse brain cDNA library, two longest clones 3 and 17 overlap to give rise to a complete open reading frame. Clone number 17 covers from the 5′-untranslated region to the seventh residue upstream of the end of MT5-MMP, thus missing the last 6 residues. Clone 3 covers the 3′ portion of the MT5-MMP cDNA. Both clones were sequenced by a combination of shotgun strategy and primer-walking from both strands to give rise to the full-length sequence of mouse MT5-MMP. Sequence alignment was performed via Internet using program Multalin version 5.3.3 at http://www.expasy.ch/www/tools.html using blosum62 with Gap weight: 12; Gap length weight: 2. The dendrogram was constructed using ClustalW program. Premade cDNA panels from mice was purchased fromCLONTECH (Palo Alto, CA) and amplified with two primers located at the 3′ portion of MT5-MMP cDNA (5′-GTGCATGCACTGGGCCATG-3′, 5′-TAGCCTTCCTGCACCCG-3′; 2 min at 94 °C for denaturation, 33 cycles of 10 s at 94 °C, 30 s at 50 °C for annealing and 30 s at 72 °C for extension, followed by 10 min extension at 72 °C). To control for the amount of cDNA used in each reaction, a parallel amplification using primers designed from the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase was performed under the same experimental conditions. The cDNA clone number 17 (missing the last 6 residues) was engineered by high fidelity PCR to give rise to MT5-MMPΔ6 which includes a FLAG tag at its COOH terminus for detection purposes using M2 monoclonal antibody (33Pei D. Yi J. Protein Expression Purif. 1998; 13: 277-281Crossref PubMed Scopus (15) Google Scholar). A chimeric primer, 5′-GTCACTTGTCATCGTCGTCCTTGTAGTCCCGCTTATAGTAGGTGAC-3′, was designed to cover part of the MT5-MMP sequence at the carboxyl end as well as the FLAG sequence. This primer was paired with T3 primer from vector to amplify MT5-MMPΔ6 from template clone number 17. The resulting fragment was cloned into pCR3.1uni and confirmed by sequencing as described (17Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (533) Google Scholar). Full-length MT5-MMP was constructed by using another primer, 5′-CTCATACCCACTCCTGGACTGGCCGCTTATAGTAGGTGAC-3′, containing the missing 6 residues at the COOH terminus to amplify the entire open reading frame with T3 primer from clone 17. The PCR fragment was cloned and characterized as described above. MT5-MMP(E252A) was made by sequential PCR as described previously using the following primers to convert Glu252 to Ala: 5′-GCCGTGCATGCACTGGGCCAT-3′ and 5′-ATGGCCCAGTGCATGCACGGC-3′. The mutant was cloned into the same pCR3.1uni vector and confirmed by DNA sequencing as described (17Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (533) Google Scholar). The antibody against human MT5-MMP was raised in rabbit using GST-hMT5-MMP (Tyr125 to Cys538) as described previously (34Pei D. Majmudar G. Weiss S.J. J. Biol. Chem. 1994; 269: 25849-25855Abstract Full Text PDF PubMed Google Scholar). DNA constructs (1.5 μg each) pCR3.1uniMT5-MMPΔ6, pCR3.1uniMT5-MMP, and pCR3.1uniMT5-MMP(E252A) were transfected into COS 7 cells and their protein products were analyzed by immunoprecipitation as described previously (13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 17Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (533) Google Scholar). To isolate cytosolic as well as membrane fractions, cells were disrupted initially by repeated freeze-thaw cycles and fractionated by centrifugation to partition into supernatants as cytosolic fraction and pellets as membrane fractions. The pellets were washed extensively and extracted with Triton X-100 (1%) in Tris-buffered saline as described (8Will H. Hinzmann B. Eur. J. Biochem. 1995; 231: 602-608Crossref PubMed Scopus (317) Google Scholar). Both cytosolic as well as membrane fractions were analyzed by Western blot as described (13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). For progelatinase A activation, pCR3.1GelA (0.1 μg) was transfected either alone or with pCR3.1uniMT5-MMPΔ6, pCR3.1uniMT5-MMP, or pCR3.1uniMT5-MMP(E252A) into MDCK cells and the conditioned media were analyzed for gelatinase activity by zymography as described previously (13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Expression vector for MT5-MMP was transfected into MDCK cells and stable clones were selected and characterized as described previously (13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 17Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (533) Google Scholar). The expressed MT5-MMP products were analyzed by immunoprecipitations for both cell and secreted forms as described above. Synthetic metalloproteinase inhibitor BB-94 (5 μm, British Biotech, United Kingdom) was added to the serum-free Dulbecco's modified Eagle's medium and allowed to incubate with MT5-MMP cells. The conditioned media were analyzed for MT5-MMP activity by zymography directly, by Western blot after ∼10-fold concentration using Millipore YM10 membrane filtration as described (13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 34Pei D. Majmudar G. Weiss S.J. J. Biol. Chem. 1994; 269: 25849-25855Abstract Full Text PDF PubMed Google Scholar). A search of the public EST data base maintained in the National Center for Biotechnology Information produced a few MMP candidate genes. One such clone, EST27028 from human cerebellum with homology to stromelysin-3, actually resembles closely the hemopexin-like domain of MT1-MMP in a BLAST search of sequence data bases. To prove that this EST clone is part of a novel MT-MMP gene, the remaining portion of the open reading frame was sequenced to reveal the presence of a putative transmembrane domain and cytosolic domain. The resulting sequence shows strong homology to human MT3-MMP, thus named MT5-MMP according to the current terminology of this subgroup (7Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2367) Google Scholar, 8Will H. Hinzmann B. Eur. J. Biochem. 1995; 231: 602-608Crossref PubMed Scopus (317) Google Scholar, 9Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Crossref PubMed Scopus (447) Google Scholar, 10Puente X.S. Pendás A.M. Llano E. Velasco G. López-Otı́n C. Cancer Res. 1996; 56: 944-949PubMed Google Scholar). A 1.5-kilobase fragment from this EST clone was isolated and used as a probe to screen for full-length clones from both human and mouse brain cDNA library (from CLONTECH and Stratagene). So far, a cumulative sequence for the human MT5-MMP covers the entire predicted open reading frame except the pro-domain upstream of the conserved PRCGVPD cysteine switch. A 5′-rapid amplification of cDNA ends strategy is currently underway to recover the missing 5′ end. However, the screening for mouse MT5-MMP yielded the full-length open reading frame with both 5′- and 3′-untranslated region (Fig.1). The mouse and human MT5-MMP are over 95% homologous, thus, representing indeed a novel gene of the MT-MMP subgroup. As shown in Figs. 1 and 2, MT5-MMP possesses similar structural feature as MT1-, MT2-, and MT3-MMPs with the characteristic Pro, catalytic, hemopexin-like, stem/transmembrane/cytosolic domains, maintaining overall sequence identities of 53, 52.1, and 64.4%, respectively. The homology between MT5-MMP and MT1-, MT2- and MT3-MMPs is more significant among subdomains as displayed in Fig. 2 B, with identity scores 71.1, 68.8, and 86% for catalytic domains, 57.9, 56.2, and 67.4% for the hemopexin domain, as well as 23.4, 25.5, and 48.7% for the stem/transmembrane/cytosolic domain, respectively. However, MT4-MMP appears to be as distant to MT5-MMP as the other non-membrane type MMPs, e.g. stromelysin-3 and interstitial collagenase (Fig.2 B). To further establish the relatedness between MT5-MMP and other MMPs, a phylogenetic tree was constructed using alignment from the catalytic domains of 18 MMPs (not shown). As expected, both human and mouse MT5-MMP are grouped together with MT3-MMP as a sub-branch of the MT-MMP subfamily. Consistent with the homology scores, MT4-MMP is positioned between the MT-MMP subgroup and the main branch of collagenases/stromelysins/gelatinases. To gain insight into the possible role of MT5-MMP in physiological processes, normal tissues were screened for MT5-MMP expression by Northern blotting. Both human and mouse MT5-MMP appears to be expressed in minute quantity in tissues examined (data not shown). Mouse MT5-MMP appears to migrate slightly below the 28 S rRNA with an estimated size of 4.5 kilobases. A more sensitive technique, namely RT-PCR, was then employed to analyze the distribution of MT5-MMP RNA in mouse tissues. As shown in Fig.3, adult brain appears to be the major site of MT5-MMP expression, whereas testis is weakly positive. The rest of adult tissues are virtually negative for MT5-MMP RNA transcript, consistent with the Northern blot analysis (data not shown). Since MMPs have been consistently implicated during the development of embryo, MT5-MMP expression profile was also obtained among developing mouse embryos. MT5-MMP expression has an onset of day 11 and persists to day 15 before dropping around day 17 before birth (Fig. 3). Since MT5-MMP is highly homologous to other known MT-MMPs capable of activating progelatinase A (7Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2367) Google Scholar, 8Will H. Hinzmann B. Eur. J. Biochem. 1995; 231: 602-608Crossref PubMed Scopus (317) Google Scholar, 9Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Crossref PubMed Scopus (447) Google Scholar), MT5-MMP is hypothesized to be a cell membrane-associated activator of progelatinase A. To test this possibility, MT5-MMPΔ6FLAG, an expression vector derived from the clone 17 of MT5-MMP (missing the last 6 residues at the COOH terminus), was transfected into COS cells and the products were analyzed by immunoprecipitation and immunoblotting as described (17Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (533) Google Scholar). Consistent with the presence of a putative transmembrane domain at its COOH terminus, MT5-MMP is detected by immunoprecipitation as a 63-kDa major species only in the lysates, not the conditioned media, of cells transfected with MT5-MMPΔ6FLAG (Fig. 4 A, lanes 2 and4), while mock transfected cells are negative (Fig.4 A, lanes 1 and 3). As reported for MT1-MMP (7Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2367) Google Scholar,13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar), this species may represent the proenzyme of MT5-MMP. In addition, there are minor species slightly above the 63-kDa main species which may represent the minor glycosylated form. Interestingly, a visible protein species around 130 kDa was also detected from the MT5-MMPΔ6 transfected cells, which may represent the dimeric form of MT5-MMP (Fig. 4 A, lane 4). To further clarify its physical localization, transfected cells were harvested, disrupted, and separated into two main fractions: cytosolic and membrane. As shown by immunoblotting in Fig. 4 B, MT5-MMP is mainly associated with the membrane fraction, not the cytosol (Fig.

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