Proteolysis of the Membrane Type-1 Matrix Metalloproteinase Prodomain
2007; Elsevier BV; Volume: 282; Issue: 50 Linguagem: Inglês
10.1074/jbc.m706290200
ISSN1083-351X
AutoresVladislav S. Golubkov, Alexei V. Chekanov, Sergey A. Shiryaev, Alexander E. Aleshin, Boris I. Ratnikov, Katarzyna Gawlik, Ilian Radichev, Khatereh Motamedchaboki, Jeffrey W. Smith, Alex Y. Strongin,
Tópico(s)Signaling Pathways in Disease
ResumoMembrane type-1 matrix metalloproteinase (MT1-MMP) exerts its enhanced activity in multiple cancer types. Understanding the activation process of MT1-MMP is essential for designing novel and effective cancer therapies. Like all of the other MMPs, MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its inhibitory prodomain. Proteolytic processing of the prodomain transforms the zymogen into a catalytically active enzyme. A sequential, two-step activation process is normally required for MMPs. Our in silico modeling suggests that the prodomain of MT1-MMP exhibits a conserved three helix-bundled structure and a "bait" loop region linking helixes 1 and 2. We hypothesized and then confirmed that in addition to furin cleavage there is also a cleavage at the bait region in the activation process of MT1-MMP. A two-step sequential activation of MT1-MMP is likely to include the MMP-dependent cleavage at either P47GD↓L50 or P58QS↓L61 or at both sites of the bait region. This event results in the activation intermediate. The activation process is then completed by a proprotein convertase cleaving the inhibitory prodomain at the R108RKR111↓Y112 site, where Tyr112 is the N-terminal residue of the mature MT1-MMP enzyme. Our findings suggest that the most efficient activation results from a two-step mechanism that eventually is required for the degradation of the inhibitory prodomain and the release of the activated, mature MT1-MMP enzyme. These findings shed more light on the functional role of the inhibitory prodomain and on the proteolytic control of MT1-MMP activation, a crucial process that may be differentially regulated in normal and cancer cells. Membrane type-1 matrix metalloproteinase (MT1-MMP) exerts its enhanced activity in multiple cancer types. Understanding the activation process of MT1-MMP is essential for designing novel and effective cancer therapies. Like all of the other MMPs, MT1-MMP is synthesized as a zymogen, the latency of which is maintained by its inhibitory prodomain. Proteolytic processing of the prodomain transforms the zymogen into a catalytically active enzyme. A sequential, two-step activation process is normally required for MMPs. Our in silico modeling suggests that the prodomain of MT1-MMP exhibits a conserved three helix-bundled structure and a "bait" loop region linking helixes 1 and 2. We hypothesized and then confirmed that in addition to furin cleavage there is also a cleavage at the bait region in the activation process of MT1-MMP. A two-step sequential activation of MT1-MMP is likely to include the MMP-dependent cleavage at either P47GD↓L50 or P58QS↓L61 or at both sites of the bait region. This event results in the activation intermediate. The activation process is then completed by a proprotein convertase cleaving the inhibitory prodomain at the R108RKR111↓Y112 site, where Tyr112 is the N-terminal residue of the mature MT1-MMP enzyme. Our findings suggest that the most efficient activation results from a two-step mechanism that eventually is required for the degradation of the inhibitory prodomain and the release of the activated, mature MT1-MMP enzyme. These findings shed more light on the functional role of the inhibitory prodomain and on the proteolytic control of MT1-MMP activation, a crucial process that may be differentially regulated in normal and cancer cells. Matrix metalloproteinases (MMPs), 2The abbreviations used are:MMPmatrix metalloproteinaseAATα1-antitrypsinMSmass spectrometryMT1-MMPmembrane type-1 MMPMT1-CAT, MT1-PEX and MT1-PROthe catalytic domain, the hemopexin domain, and the prodomain of MT1-MMP, respectivelyPDXα1-antitrypsin variant PortlandPBSphosphate-buffered salineTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineMES4-morpholineethanesulfonic acid. a family comprised of 25 individual zinc-dependent proteolytic enzymes, are classified as either soluble or membrane-tethered proteinases. The latter exhibit the presence of either a transmembrane domain or a glycosylphosphatidylinositol anchor (1Visse R. Nagase H. Circ. Res. 2003; 92: 827-839Crossref PubMed Scopus (3702) Google Scholar) and because of their association with cell surfaces play key roles in pericellular proteolysis (2Wolf K. Wu Y.I. Liu Y. Geiger J. Tam E. Overall C. Stack M.S. Friedl P. Nat. Cell Biol. 2007; 9: 893-904Crossref PubMed Scopus (774) Google Scholar). MMPs are especially important to the aberrant proteolysis associated with pathologies including cancer, arthritis, and cardiovascular diseases (3Mott J.D. Werb Z. Curr. Opin. Cell Biol. 2004; 16: 558-564Crossref PubMed Scopus (880) Google Scholar). All individual members of the MMP family are synthesized as latent zymogens. The active site zinc of the MMP catalytic domain is coordinated with the three histidines of the active site and with the cysteine of the "cysteine switch" motif of the N-terminal prodomain (4Van Wart H.E. Birkedal-Hansen H. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 5578-5582Crossref PubMed Scopus (1211) Google Scholar). matrix metalloproteinase α1-antitrypsin mass spectrometry membrane type-1 MMP the catalytic domain, the hemopexin domain, and the prodomain of MT1-MMP, respectively α1-antitrypsin variant Portland phosphate-buffered saline N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 4-morpholineethanesulfonic acid To date, the crystal structure of only a few individual MMP zymogens, including MMP-1, MMP-2, MMP-3, and MMP-9, has been solved. The common characteristic of the prodomain structure of these four MMPs is the presence of the three helixes that are perpendicular to each other (5Jozic D. Bourenkov G. Lim N.H. Visse R. Nagase H. Bode W. Maskos K. J. Biol. Chem. 2005; 280: 9578-9585Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 6Elkins P.A. Ho Y.S. Smith W.W. Janson C.A. D'Alessio K.J. McQueney M.S. Cummings M.D. Romanic A.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1182-1192Crossref PubMed Scopus (128) Google Scholar, 7Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Crossref PubMed Scopus (485) Google Scholar, 8Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M. Cameron P.M. Esser C.K. Hagmann W.K. Hermes J.D. Springer J.P. Protein Sci. 1995; 4: 1966-1976Crossref PubMed Scopus (272) Google Scholar). Hydrophobic interactions between the helixes maintain the stability of the three-helix bundle of the prodomain. In the course of the two-step activation, the external active protease initially cleaves the susceptible bond at the "bait" region of the prodomain of MMP-1, MMP-2, and MMP-9. This event destroys the helix bundle and leads to the generation of the activation intermediate. The activation intermediate is processed further by either an external proteinase or autocatalytically resulting in the complete removal of the residual prodomain sequence (9Maskos K. Biochimie (Paris). 2005; 87: 249-263Crossref PubMed Scopus (154) Google Scholar). The activation process involves proprotein convertases, autocatalysis. and other activated MMPs and it may occur either intracellularly or extracellularly (10Murphy G. Stanton H. Cowell S. Butler G. Knauper V. Atkinson S. Gavrilovic J. APMIS. 1999; 107: 38-44Crossref PubMed Scopus (391) Google Scholar, 11Kotra L.P. Cross J.B. Shimura Y. Fridman R. Schlegel H.B. Mobashery S. J. Am. Chem. Soc. 2001; 123: 3108-3113Crossref PubMed Scopus (26) Google Scholar). The two-step proteolytic removal of the prodomain results in the activation of the latent MMP zymogen and the generation of a mature enzyme with full proteolytic activity. Invasion-promoting MT1-MMP (MMP-14) is expressed in many cancer types in which the protease functions as one of the main mediators of proteolytic events on the cell surface (12Itoh Y. Seiki M. J. Cell Physiol. 2006; 206: 1-8Crossref PubMed Scopus (420) Google Scholar, 13Zucker S. Pei D. Cao J. Lopez-Otin C. Curr. Top. Dev. Biol. 2003; 54: 1-74Crossref PubMed Google Scholar, 14Yana I. Seiki M. Clin. Exp. Metastasis. 2002; 19: 209-215Crossref PubMed Scopus (81) Google Scholar, 15Seiki M. Cancer Lett. 2003; 194: 1-11Crossref PubMed Scopus (363) Google Scholar). Multiple cleavage targets of MT1-MMP include the extracellular matrix components (types 1, 2, and 3 collagens, laminin 1 and 5, fibrin, fibronectin, and vitronectin) and cell-surface receptors including CD44, integrin αvβ3, transglutaminase, and low density lipoprotein receptor-related protein (15Seiki M. Cancer Lett. 2003; 194: 1-11Crossref PubMed Scopus (363) Google Scholar, 16Seiki M. Yana I. Cancer Sci. 2003; 94: 569-574Crossref PubMed Scopus (127) Google Scholar, 17Itoh Y. IUBMB Life. 2006; 58: 589-596Crossref PubMed Scopus (145) Google Scholar, 18Holmbeck K. Bianco P. Yamada S. Birkedal-Hansen H. J. Cell Physiol. 2004; 200: 11-19Crossref PubMed Scopus (150) Google Scholar). MT1-MMP is also a physiological activator of soluble MMP-2 (19Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2379) Google Scholar, 20Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar, 21Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar). MT1-MMP acts as a growth factor in malignant cells and usurps tumor growth control (22Hotary K.B. Allen E.D. Brooks P.C. Datta N.S. Long M.W. Weiss S.J. Cell. 2003; 114: 33-45Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). Like all of the other members of the MMP family, MT1-MMP is synthesized as a proteolytically inert zymogen. Evidence suggests that the processing of the zymogen into the catalytically active MT1-MMP enzyme involves proprotein convertases including furin, PACE4, PC6, and PC7 (23Remacle A.G. Rozanov D.V. Fugere M. Day R. Strongin A.Y. Oncogene. 2006; 25: 5648-5655Crossref PubMed Scopus (60) Google Scholar, 24Osenkowski P. Toth M. Fridman R. J. Cell Physiol. 2004; 200: 2-10Crossref PubMed Scopus (166) Google Scholar, 25Yana I. Weiss S.J. Mol. Biol. Cell. 2000; 11: 2387-2401Crossref PubMed Scopus (271) Google Scholar). The requirement for furin activity in the activation of the cellular MT1-MMP and MT3-MMP zymogens was convincingly demonstrated (26Kang T. Nagase H. Pei D. Cancer Res. 2002; 62: 675-681PubMed Google Scholar, 27Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (534) Google Scholar). We, however, hypothesized that similar to the other MMPs, the activation pathway of MT1-MMP, in addition to the known proprotein convertase cleavage, involves a previously uncharacterized cleavage step that leads to the activation intermediate. The intermediate is then processed by proprotein convertases with the generation of the mature and fully active enzyme of MT1-MMP. To determine whether this hypothesis is correct, we performed an extensive biochemical analysis of the cleavage events that target the prodomain of MT1-MMP. As a result of these studies, we suggest that the activation of MT1-MMP involves a two-step mechanism in which the processing of the prodomain sequence at the P47GD↓L50 and P58QS↓L61 cleavage sites generates the intermediate form. The activation intermediate is then processed by a proprotein convertase cleaving at the R108RKR↓Y112 site and generating the fully activated enzyme of MT1-MMP. Antibodies, Reagents, and Cells—Rabbit polyclonal antibody (AB815) against the hinge region of MT1-MMP and the murine monoclonal antibody (clone 3G4) against the catalytic domain of MT1-MMP and the hydroxamate inhibitor GM6001 were from Chemicon (Temecula, CA). The mouse monoclonal antibody against a V5 epitope was from Invitrogen. Sulfosuccinimidyl-6-(biotinamido)hexanoate (EZ-Link sulfo-NHS-Long Chain(LC)-biotin) was from Pierce. Human glioma U251 cells were originally from ATCC (Manassas, VA). Cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. As a control, we used U251 cells stably transfected with the two original pcDNA3.1-zeo and-neo plasmids (mock cells). For MT1-MMP overexpression, U251 cells were transfected with the wild-type MT1-MMP (MT1-WT cells) and the MT1-MMP mutant constructs including the R89A mutant with the inactivated R89RPR92 putative furin cleavage motif (R89A cells), the ARAA mutant with the inactivated furin R108RKR111 cleavage site (ARAA cells), and the R89A/ARAA double mutant (R89A/ARAA cells). In addition, we used WT cells that stably co-expressed MT1-MMP with the α1-antitrypsin Portland furin inhibitor (PDX) (MT1/PDX cells). The MT1/PDX cells were initially transfected with MT1-MMP and then with PDX. All these cell lines were constructed and have been extensively characterized in our earlier works (23Remacle A.G. Rozanov D.V. Fugere M. Day R. Strongin A.Y. Oncogene. 2006; 25: 5648-5655Crossref PubMed Scopus (60) Google Scholar, 28Deryugina E.I. Bourdon M.A. Luo G.X. Reisfeld R.A. Strongin A. J. Cell Sci. 1997; 110: 2473-2482Crossref PubMed Google Scholar, 29Rozanov D.V. Deryugina E.I. Ratnikov B.I. Monosov E.Z. Marchenko G.N. Quigley J.P. Strongin A.Y. J. Biol. Chem. 2001; 276: 25705-25714Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar, 30Golubkov V.S. Boyd S. Savinov A.Y. Chekanov A.V. Osterman A.L. Remacle A. Rozanov D.V. Doxsey S.J. Strongin A.Y. J. Biol. Chem. 2005; 280: 25079-25086Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). MT1-MMP Constructs Expressed in Breast Carcinoma MCF-7 Cells—The double L50D/L61D MT1-MMP mutant (MT1-L50D/L61D) was generated by PCR mutagenesis using the wild-type MT1-MMP template. The following oligonucleotide primers were used for mutagenesis: 5′-TGGCTACCTGCCTCCCGGGGACGATCGTACCCACACACAGCGCTC-3′ (MT1-L50D-forward) and 5′-GAGCGCTGTGTGTGGGTACGATCGTCCCCGGGAGGCAGGTAGCCA-3′ (MT1-L50D-reverse), and 5′-AGCGCTCACCCCAGTCAGACTCAGCTGCCATCGCTGCCATG-3′ (MT1-L61D-forward) and 5′-CATGGCAGCGATGGCAGCTGAGTCTGACTGGGGTGAGCGCT-3′ (MT1-L61D-reverse) (mutant positions are underlined). The mutant sequence was confirmed by DNA sequencing. The wild-type and the constructed L50D/L61D MT1-MMP mutant were cloned into the pLenti6/V5-GW/lacZ lentiviral plasmid vector. The lentiviral constructs were used to transfect breast carcinoma MCF-7 cells using Lipofectamine 2000. The stable transfectants were selected with 10 μg/ml blasticidin. Blasticidin-resistant clones expressing the wild-type MT1-MMP (MT1-V5 cells) and the L50D/L61D mutant construct (MT1-L50D/L61D cells) were selected by Western blotting with the V5 antibody. Several independent clones of MT1-L50D/L61D were randomly selected (numbers 5, 7, and 8) and then used for further analysis. MCF-7 cells stably expressing the wild-type MT1-MMP (MT1-WT cells) and the catalytically inert E240A mutant as well as the mock cells stably transfected with the original pcDNA3.1-zeo plasmid were constructed and described earlier (29Rozanov D.V. Deryugina E.I. Ratnikov B.I. Monosov E.Z. Marchenko G.N. Quigley J.P. Strongin A.Y. J. Biol. Chem. 2001; 276: 25705-25714Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). Cloning and Expression of the Recombinant MT1-MMP Catalytic Domain, the Prodomain, and the Soluble, Catalytically Inert MT1-MMP E240A Constructs—The MT1-MMP catalytic domain (MT1-CAT) was expressed and purified as described (31Kridel S.J. Sawai H. Ratnikov B.I. Chen E.I. Li W. Godzik A. Strongin A.Y. Smith J.W. J. Biol. Chem. 2002; 277: 23788-23793Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 32Ratnikov B. Deryugina E. Leng J. Marchenko G. Dembrow D. Strongin A. Anal. Biochem. 2000; 286: 149-155Crossref PubMed Scopus (67) Google Scholar). The catalytically inert E240A full-length MT1-MMP mutant cDNA (29Rozanov D.V. Deryugina E.I. Ratnikov B.I. Monosov E.Z. Marchenko G.N. Quigley J.P. Strongin A.Y. J. Biol. Chem. 2001; 276: 25705-25714Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) was used as a template to clone the MT1-MMP prodomain (MT1-PRO) and the soluble catalytically inert MT1-MMP E240A (MT1-PRO-CAT-PEX) constructs. The MT1-PRO-CAT-PEX construct included the propeptide sequence (PRO), the inert (E240) catalytic domain (CAT), and the hemopexin (PEX) domains. To facilitate the isolation and detection in the samples, MT1-PRO-CAT-PEX was tagged with a His6 tag both C- and N-terminal. In addition, a V5 epitope sequence was linked to the C-terminal His6 tag sequence. Specifically, the forward (5′-CACCATGCATCATCATCATCATCATCACGCGCTCGCCTCCCTCGGCTC-3′) and the reverse (5′-TTAGCGCTTCCTTCGAACATTGG-3′) primers were used to obtain the MT1-PRO construct (the His6 tag sequence is underlined) in the PCR. The forward (5′-CACCATGCATCATCATCATCATCATGGCTCGGCCCAAAGCAGCAGCTTC-3′) and the reverse (5′-GCTCACCGCCCCGCCGCCCTCCTCGTC-3) primers were used in the cloning of MT1-PRO-CAT-PEX. After confirming their authenticity by sequencing, the constructs were then re-cloned into the pET101 expression vector. Competent Escherichia coli BL21(DE3) Codon Plus cells (Stratagene) were transformed with the recombinant vectors. Cells were grown at 37 °C in a Luria-Bertani broth containing ampicillin (0.1 mg/ml). Culture was induced with 1 mm isopropyl β-d-thiogalactoside for 6 h at 37 °C. E. coli cells (6 g/liter of E. coli culture) were then collected by centrifugation (5,000 × g; 15 min), re-suspended in 20 ml of 10 mm Tris-HCl, pH 8.0, containing 1 m NaCl, 1 mm phenylmethylsulfonyl fluoride and lysozyme (5 mg/ml), and disrupted on ice by sonication (30 s pulse, 30-s intervals; 8 pulses total). The MT1-PRO was purified from the supernatant fraction using a 1.6 × 10-cm Co2+-chelating Sepharose Fast Flow column (Amersham Biosciences) equilibrated with PBS supplemented with 1 m NaCl. MT1-PRO was eluted with an imidazole gradient (10–100 mm; 100 ml) in PBS, 1 m NaCl. The MT1-PRO fractions were concentrated using a 5-kDa cutoff concentrator (Millipore, Billerica, MA) and dialyzed against PBS containing 0.005% Brij35. A polyclonal antibody to the purified individual MT1-PRO was then raised in rabbits. The MT1-PRO-CAT-PEX inert construct was purified from the inclusion bodies and then refolded to restore its native conformation. The inclusion bodies (10 mg of total protein) were washed in 10 mm Tris-HCl, pH 8.0, containing 1 m NaCl and 1% Triton X-100 and then dissolved in 10 mm Tris-HCl, pH 8.0, containing 6 m guanidine hydrochloride and 10 mm 2-mercaptoethanol. The soluble material was then refolded by a 50-fold dilution in 100 mm Tris-HCl, pH 8.0, supplemented with 1 mm CaCl2, 1 mm ZnCl2, 500 mml-arginine monohydrochloride, and 20% glycerol. The refolded MT1-PRO-CAT-PEX was next concentrated using a 30-kDa cutoff concentrator (Millipore) and purified on a 1.6 × 10-cm Co2+-chelating Sepharose Fast Flow column (Amersham Biosciences) equilibrated with PBS, 1 m NaCl. The construct was eluted with an imidazole gradient (10–500 mm gradient; 100 ml) in PBS, 1 m NaCl, concentrated using a 30-kDa cutoff concentrator and dialyzed against PBS, containing 0.005% Brij35. Cleavage of Synthetic Peptides, MT1-PRO, MT1-PRO-CAT-PEX, and Immunoprecipitated Cellular MT1-MMP—The peptides (Y44LPPGDL50RTHTQRSPQ59 and H53TQRSPQSL61 SAAIAAM68) that span the putative cleavage sites in the MT1-MMP prodomain and the corresponding mutant peptides (Y44LPPGDD50RTHTQRSPQ59 and H53TQRSPQSD61 SAAIAAM68; the mutant residues are underlined) were synthesized by GenScript (San Diego, CA). The peptides, MT1-PRO and MT1-PRO-CAT-PEX (1 μg each), were co-incubated with MT1-CAT or MMP-2 (20 nm each) for the indicated time at 37 °C in 50 mm HEPES, pH 6.8, supplemented with 10 mm CaCl2, 0.5 mm MgCl2, and 50 mm ZnCl2. MMP-2 was isolated and activated by 1 mm 4-aminophenylmercuric acetate as described earlier (20Strongin A.Y. Collier I. Bannikov G. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1995; 270: 5331-5338Abstract Full Text Full Text PDF PubMed Scopus (1438) Google Scholar, 21Strongin A.Y. Marmer B.L. Grant G.A. Goldberg G.I. J. Biol. Chem. 1993; 268: 14033-14039Abstract Full Text PDF PubMed Google Scholar). Where indicated, GM6001 (2.5 mm) was added to the reactions to inhibit MMPs. The digest samples were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) using a Bruker Daltonics Autoflex II TOF/TOF mass spectrometer to determine the mass of the cleavage products and, consequently, the location of the scissile bonds. To identify the N-terminal sequence of the cleavage fragments, the catalytically inert MT1-PRO-CAT-PEX E240A construct (5 μg) was co-incubated with MT1-CAT and furin (50 ng each) for 1 h at 37 °C in 50 mm HEPES buffer, pH 6.8. Recombinant human furin was prepared in the S2 Drosophila expression system (Invitrogen) and purified to homogeneity (33Fugere M. Limperis P.C. Beaulieu-Audy V. Gagnon F. Lavigne P. Klarskov K. Leduc R. Day R. J. Biol. Chem. 2002; 277: 7648-7656Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). The reactions were separated by SDS gel electrophoresis followed by the transfer of the protein bands on the polyvinylidene difluoride membrane and N-terminal microsequencing of the resulting bands. Microsequencing was performed at ProSeq (Boxford, MA). For the subsequent cleavage experiments, cellular MT1-MMP was immunoprecipitated, using AB815 antibody (1 μg) and Protein G-agarose beads (20 μl of a 50% slurry), for 12 h at 4 °C from the cell lysate aliquots (1 mg of total protein each) of the confluent MT1/PDX cells. The lysis buffer (20 mm Tris-HCl, 150 mm NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 1% IGEPAL, pH 7.4) was supplemented with a protease inhibitor mixture set III (Sigma) (1 mm phenylmethylsulfonyl fluoride and 10 mm EDTA). The beads were collected by centrifugation and then washed in 50 mm HEPES, pH 6.8. The samples were incubated for 30 min at 37 °C with MT1-CAT (20 ng) in 50 mm HEPES, pH 6.8, containing 10 mm CaCl2, 0.5 mm MgCl2, and 50 μm ZnCl2. The digest samples were analyzed by Western blotting with 3G4 antibody and a TMB/E substrate (Chemicon) to identify the cleavage products. Cell Surface Biotinylation—Cell surface-associated MT1-MMP was biotinylated by incubating cells (80–90% confluence) for 30 min on ice in PBS containing 0.1 mg/ml EZ-Link NHS-LC-biotin. Excess biotin was removed by washing cells in ice-cold PBS and then quenched by incubating cells for 10 min in PBS containing 100 mm glycine. After washing with PBS, cells were lysed in 20 mm Tris-HCl, 150 mm NaCl, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 1% IGEPAL, pH 7.4) supplemented with a protease inhibitor mixture set III. MT1-MMP was precipitated from cell lysates using streptavidin-agarose beads and analyzed by Western blotting with the MT1-MMP antibody (3G4) followed by the goat secondary horseradish peroxidase-conjugated IgG and a TMB/M substrate (Chemicon). Gelatin Zymography—Gelatin zymography was used to determine the efficiency of MMP-2 activation by cellular MT1-MMP. Cells were plated in the wells of a 48-well plate (Costar/Corning) in serum-containing Dulbecco's modified Eagle's medium and grown to reach a 90% confluence. The medium was then replaced with serum-free Dulbecco's modified Eagle's medium supplemented with the purified MMP-2 proenzyme (100 ng/ml). After incubation for 12 h, the medium aliquots were analyzed by gelatin zymography on 10% acrylamide gels containing 0.1% gelatin (Novex) to detect the proenzyme and the activated species of MMP-2. Modeling and Multiple Sequence Alignment—The propeptide size and related annotations were obtained from the UNI-PROT data base (srs.ebi.ac.uk) (34Zdobnov E.M. Lopez R. Apweiler R. Etzold T. Bioinformatics. 2002; 18: 368-373Crossref PubMed Scopus (80) Google Scholar). The structure parameters of the propeptide were obtained from the known atomic resolution structures of the proenzymes of MMP-1 (Protein Data Bank entry 1SU3) (5Jozic D. Bourenkov G. Lim N.H. Visse R. Nagase H. Bode W. Maskos K. J. Biol. Chem. 2005; 280: 9578-9585Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), MMP-2 (PDB 1CK7) (7Morgunova E. Tuuttila A. Bergmann U. Isupov M. Lindqvist Y. Schneider G. Tryggvason K. Science. 1999; 284: 1667-1670Crossref PubMed Scopus (485) Google Scholar), MMP-3 (PDB 1SLM) (8Becker J.W. Marcy A.I. Rokosz L.L. Axel M.G. Burbaum J.J. Fitzgerald P.M. Cameron P.M. Esser C.K. Hagmann W.K. Hermes J.D. Springer J.P. Protein Sci. 1995; 4: 1966-1976Crossref PubMed Scopus (272) Google Scholar), and MMP-9 (PDB 1L6J) (6Elkins P.A. Ho Y.S. Smith W.W. Janson C.A. D'Alessio K.J. McQueney M.S. Cummings M.D. Romanic A.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2002; 58: 1182-1192Crossref PubMed Scopus (128) Google Scholar) and the catalytic. The sequence alignment was performed with CLASTALW (35Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar) and edited with Jalview (36Clamp M. Cuff J. Searle S.M. Barton G.J. Bioinformatics. 2004; 20: 426-427Crossref PubMed Scopus (1214) Google Scholar). The structure of the MT1-MMP proenzyme (residues 36–508) was modeled by the program MODPIPE (37Eswar N. John B. Mirkovic N. Fiser A. Ilyin V.A. Pieper U. Stuart A.C. Marti-Renom M.A. Madhusudhan M.S. Yerkovich B. Sali A. Nucleic Acids Res. 2003; 31: 3375-3380Crossref PubMed Scopus (388) Google Scholar) using PDB entries 1SU3 (MMP-1), 1CK7 (MMP-2), 1SLM (MMP-3) and entries 1BQQ and 1BUV (the catalytic domain of MT1-MMP) (38Fernandez-Catalan C. Bode W. Huber R. Turk D. Calvete J.J. Lichte A. Tschesche H. Maskos K. EMBO J. 1998; 17: 5238-5248Crossref PubMed Scopus (315) Google Scholar) as templates. The sequence identity of the MMP-1 template with MT1-MMP is 40% (E-value = 1e–117). The predicted structure of the MT1-MMP propeptide is in a good agreement with all structurally investigated MMP propeptides (root mean square deviation = 0.9 Å). Structural Modeling of the Three Helix-bundled MT1-MMP Prodomain—The multiple sequence alignment of the prodomain peptide sequences of several MMPs is shown in Fig. 1. There is a significant sequence homology of the helical regions and loops 2 and 3 in the peptide sequence of MMPs. In contrast, loop 1, which is the bait region in MMP-1, -2, and -9 (9Maskos K. Biochimie (Paris). 2005; 87: 249-263Crossref PubMed Scopus (154) Google Scholar), displays the least homology thus providing structural evidence of a unique means of the first proteolytic step of a two-step activation mechanism for each MMP. The second and the final activation step of MMPs including MT1-MMP involves cleavage at the C-terminal part of loop 3 (Fig. 1). Because the crystal structure of the MT1-MMP proenzyme is not currently available, we used in silico modeling to model the spatial structure of the MT1-MMP prodomain. The available structures of the sequence homologous MMP-1, -2, -3, and 9 were used as a template. We also built the model of MT1-MMP using the structure of the MT1-MMP catalytic domain and the structures of the prodomain and the PEX domain of MMP-1, MMP-2, and MMP-3 (Fig. 1). Computer modeling suggests that the triple helix bundle is highly conserved in MMPs including the MT1-MMP prodomain. In our model, in agreement with the solved structures for proMMP-1, proMMP-2, proMMP-3, and proMMP-9, the proMT1-MMP prodomain has a conserved 3-helix structure. Consistent with the exposure of the bait region in loop 1 in MMP-1, -2, -3, -9, and other MMPs, the loop 1 peptide sequence also appears highly accessible to the proteolytic attack in MT1-MMP. It has also been established that the MMP cleavage motifs predominantly exhibit the presence of the P3 Pro and a hydrophobic residue (especially Leu) at the P1′ position. There are two potential cleavage motifs, P47GD↓L50 and P58QS↓L61, in the loop 1 sequence of the prodomain of MT1-MMP. Because the furin cleavage of the R108RKR111 loop 3 sequence represents the final step of MT1-MMP proenzyme processing, we hypothesized that the loop 1 bait sequence is the target of either the autolytic cleavage or this processing is performed by an external protease with an MMP cleavage specificity. MT1-MMP Proteolysis of the Prodomain in Vitro—To determine whether the loop 1 sequence is susceptible to MT1-MMP autoproteolysis, we synthesized the peptides (Y44LPPGD↓ L50RTHTQRSPQ59 and H53TQRSPQS↓L61SAAIAAM68) that overlap the putative cleavage sites. We also synthesized the mutant peptides in which the cleavage sites were inactivated by the L50D and L61D mutations, respectively. The peptides were subjected to proteolysis by MT1-CAT and MMP-2. The MS analysis of the digest reactions were followed to determine the molecular mass and, consequently, the sequence of the cleavage products (Fig. 2). We determined that both MT1-CAT and MMP-2 efficiently cleaved the original peptides and generated the cleavage fragments of the expected size and the peptide sequence. Thus, the cleavage of the Y44LPPGD↓ L50RTHTQRSPQ59 resulted in peptides 44–49 (YLPPGD) and 50–59 (LRTHTQRSPQ). Similarly, peptides 53–60 (HTQR-SPQS) and 61–68 (LSAAIAAM) were detected following the cleavage of H53TQRSPQS↓L61SAAIAAM68 by MT1-CAT and MMP-2. In sharp contrast, the mutant L50D and L61D peptides exhibiting the inactivated MMP cleavage sites were completely resistant to proteolysis. GM6001, a hydroxamate inhibitor of MMPs, totally blocked the cleavage reactions (not shown). To additionally confirm these results, we isolated the recombinant MT1-PRO and then subjected the construct to MT1-CAT proteolysis and MMP-2 proteolysis (Fig. 3). The digest reactions were analyzed by SDS electrophoresis in 10–20% polyacrylamide gels in the Tris-Tricine system (Invitrogen) and also by MS. One major 6-kDa and several minor cleavage products were detected in the reactions. MS ident
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