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• W2045491115 abstract "Members of the membrane-type matrix metalloproteinase (MT-MMPs) family are dual regulators of extracellular matrix remodeling through direct degradation of extracellular matrix components and activation of other latent MMPs. However, the structural basis of this functional diversity remains poorly understood. In an attempt to dissect the structural determinants for MT-MMP function, we performed domain exchange experiments between MT1-MMP and its close relative MT3-MMP and analyzed the exchange chimeras for pro-MMP-2 activation and collagen degradation at the cellular level. Our results indicate that catalytic domains determine the pattern of pro-MMP-2 activation, whereas pexin-like domains modulate the level of activation. On the other hand, both the catalytic and pexin-like domains of MT1-MMP are required for strong collagenolysis because exchanging either domain with that of MT3-MMP yielded significantly lower activity, and the introduction of the MT1-MMP catalytic or pexin-like domain into MT3-MMP failed to generate any significant enhancement of collagenolytic activity compared with wild-type MT3-MMP. Interestingly, the cytoplasmic domain of MT1-MMP behaves as a negative regulator not only for MT1-MMP itself, but also for MT3-MMP in both pro-MMP-2 activation and collagenolysis, consistent with and extending our recent findings (Jiang, A., Lehti, K., Wang, X., Weiss, S. J., Keski-Oja, J., and Pei, D. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13693-13698). Taken together, these results demonstrate that domains in MT-MMPs function differently toward a given substrate and thus should be targeted differentially for future therapeutic development. Members of the membrane-type matrix metalloproteinase (MT-MMPs) family are dual regulators of extracellular matrix remodeling through direct degradation of extracellular matrix components and activation of other latent MMPs. However, the structural basis of this functional diversity remains poorly understood. In an attempt to dissect the structural determinants for MT-MMP function, we performed domain exchange experiments between MT1-MMP and its close relative MT3-MMP and analyzed the exchange chimeras for pro-MMP-2 activation and collagen degradation at the cellular level. Our results indicate that catalytic domains determine the pattern of pro-MMP-2 activation, whereas pexin-like domains modulate the level of activation. On the other hand, both the catalytic and pexin-like domains of MT1-MMP are required for strong collagenolysis because exchanging either domain with that of MT3-MMP yielded significantly lower activity, and the introduction of the MT1-MMP catalytic or pexin-like domain into MT3-MMP failed to generate any significant enhancement of collagenolytic activity compared with wild-type MT3-MMP. Interestingly, the cytoplasmic domain of MT1-MMP behaves as a negative regulator not only for MT1-MMP itself, but also for MT3-MMP in both pro-MMP-2 activation and collagenolysis, consistent with and extending our recent findings (Jiang, A., Lehti, K., Wang, X., Weiss, S. J., Keski-Oja, J., and Pei, D. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 13693-13698). Taken together, these results demonstrate that domains in MT-MMPs function differently toward a given substrate and thus should be targeted differentially for future therapeutic development. Remodeling of the extracellular matrix (ECM), 1The abbreviations used are: ECM, extracellular matrix; MMP, matrix metalloproteinase; MT-MMP, membrane-type matrix metalloproteinase; MDCK, Madin-Darby canine kidney; DMEM, Dulbecco's modified Eagle's medium. such as the abundant type I collagen, is believed to be mediated primarily by members of the matrix metalloproteinase (MMP) family, especially the collagenases and MMP-1, -8, and -13 (1Woessner Jr., J.F. FASEB J. 1991; 5: 2145-2154Crossref PubMed Scopus (3091) Google Scholar, 2Overall C.M. Lopez-Otin C. Nat. Rev. Cancer. 2002; 2: 657-672Crossref PubMed Scopus (1143) Google Scholar). Based on cellular localization, MMPs can be classified into two categories: secreted or membrane-bound (3Kang T. Yi J. Guo A. Wang X. Overall C.M. Jiang W. Elde R. Borregaard N. Pei D. J. Biol. Chem. 2001; 276: 21960-21968Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). In general, the secreted MMPs are synthesized and delivered to the extracellular milieu, where they mediate ECM destruction (4Massova I. Kotra L.P. Fridman R. Mobashery S. FASEB J. 1998; 12: 1075-1095Crossref PubMed Scopus (704) Google Scholar). In contrast, members of the membrane-type MMP (MT-MMP) subgroup are anchored on the plasma membrane, presumably to mediate a more focused and dynamic attack on the ECM (5Nakahara 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 (367) Google Scholar). Among the six known MT-MMPs, MT1-, MT2-, MT3-, and MT5-MMPs form the first group with type I transmembrane domains, and MT4-MMP and MT6-MMP are members of the second group with apparent glycosylphosphatidylinositol anchors (6Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2377) Google Scholar, 7Okada 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, 8Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 9Llano E. Pendas A.M. Freije J.P. Nakano A. Knauper V. Murphy G. Lopez-Otin C. Cancer Res. 1999; 59: 2570-2576PubMed Google Scholar, 10Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 11Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (169) Google Scholar). Recent evidence from both in vitro and in vivo systems suggests that MT-MMPs may play a more important role than their secretory counterparts in mediating ECM destruction in a wide range of biological and pathological processes such as cell migration, invasion, angiogenesis, and development (5Nakahara 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 (367) Google Scholar, 12Cao J. Sato H. Takino T. Seiki M. J. Biol. Chem. 1995; 270: 801-805Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 14Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar, 15Zhou Z. Apte S.S. Soininen R. Cao R. Baaklini G.Y. Rauser R.W. Wang J. Cao Y. Tryggvason K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4052-4057Crossref PubMed Scopus (690) Google Scholar, 16Belkin A.M. Akimov S.S. Zaritskaya L.S. Ratnikov B.I. Deryugina E.I. Strongin A.Y. J. Biol. Chem. 2001; 276: 18415-18422Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 17Ellerbroek S.M. Wu Y.I. Overall C.M. Stack M.S. J. Biol. Chem. 2001; 276: 24833-24842Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 18Deryugina E.I. Soroceanu L. Strongin A.Y. Cancer Res. 2002; 62: 580-588PubMed Google Scholar). In fact, MT1-MMP has emerged as an important collagenolytic enzyme in vivo (14Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar, 15Zhou Z. Apte S.S. Soininen R. Cao R. Baaklini G.Y. Rauser R.W. Wang J. Cao Y. Tryggvason K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4052-4057Crossref PubMed Scopus (690) Google Scholar). Therefore, MT-MMPs have been dubbed as master switches for ECM proteolysis (19Sato H. Okada Y. Seiki M. Thromb. Haemostasis. 1997; 78: 497-500Crossref PubMed Scopus (103) Google Scholar), thus becoming an intensely active field of investigation in recent years. The archetypal MT1-MMP is by far the most studied and is widely considered a model system for the elucidation of MT-MMP functions (6Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2377) Google Scholar, 20Seiki M. APMIS. 1999; 107: 137-143Crossref PubMed Scopus (274) Google Scholar). The first function described for MT1-MMP is its ability to activate pro-MMP-2, an enzyme implicated in many physiological and pathological conditions such as tumor invasion and metastasis (19Sato H. Okada Y. Seiki M. Thromb. Haemostasis. 1997; 78: 497-500Crossref PubMed Scopus (103) Google Scholar). Subsequently, similar activity has been confirmed for the other type I transmembrane MT-MMPs, i.e. MT2-, MT3-, and MT5-MMPs (8Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 9Llano E. Pendas A.M. Freije J.P. Nakano A. Knauper V. Murphy G. Lopez-Otin C. Cancer Res. 1999; 59: 2570-2576PubMed Google Scholar, 10Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 21Shofuda K. Nagashima Y. Kawahara K. Yasumitsu H. Miki K. Miyazaki K. Lab. Investig. 1998; 78: 915-923PubMed Google Scholar). Interestingly, MT1-MMP knockout mice exhibit far more severe developmental defects than MMP-2 null mice, suggesting that MT1-MMP participates in other biological functions in addition to the activation of MMP-2 (13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 14Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar, 15Zhou Z. Apte S.S. Soininen R. Cao R. Baaklini G.Y. Rauser R.W. Wang J. Cao Y. Tryggvason K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4052-4057Crossref PubMed Scopus (690) Google Scholar, 22Ohuchi E. Imai K. Fujii Y. Sato H. Seiki M. Okada Y. J. Biol. Chem. 1997; 272: 2446-2451Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar). Indeed, MT1-MMP has been previously shown to have intrinsic proteolytic activities against extracellular components, including type I collagen (13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 22Ohuchi E. Imai K. Fujii Y. Sato H. Seiki M. Okada Y. J. Biol. Chem. 1997; 272: 2446-2451Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar). The apparent failure of MT1-MMP null mice to develop normally has been linked to deficiencies in collagen metabolism in bone and the skeleton (14Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar, 15Zhou Z. Apte S.S. Soininen R. Cao R. Baaklini G.Y. Rauser R.W. Wang J. Cao Y. Tryggvason K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4052-4057Crossref PubMed Scopus (690) Google Scholar), confirming that type I collagen is an MT1-MMP substrate in vivo. In addition to collagen, fibrin has been identified as a likely substrate for MT1-MMP during angiogenesis (23Hiraoka N. Allen E. Apel I.J. Gyetko M.R. Weiss S.J. Cell. 1998; 95: 365-377Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). Although the range of substrates for MT-MMPs may continue to expand, the structural basis of MT-MMP substrate specificity remains virtually unknown. The fact that MT-MMPs are anchored on the plasma membrane represents a formidable challenge to investigate their structure/function relationship at the cellular level. Consequently, most of the characterizations for MT-MMP activity have been performed mostly with purified enzymes in well buffered reaction conditions (13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 22Ohuchi E. Imai K. Fujii Y. Sato H. Seiki M. Okada Y. J. Biol. Chem. 1997; 272: 2446-2451Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar), perhaps reflecting the function of their catalytic domains or proteolytic potentials. Recently, one cellular approach has been adapted to analyze the structure/function of MT-MMPs. By substituting the pexin-like domain of MT4-MMP into the analogous position in MT1-MMP, Itoh et al. (24Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (342) Google Scholar) concluded that the pexin-like domain of MT1-MMP is required for its ability to mediate pro-MMP-2 activation. Because MT4-MMP is not known to activate pro-MMP-2, this exchange experiment proves that the pexin-like domain of MT1-MMP plays a critical role in specifying its substrate specificity (24Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (342) Google Scholar), demonstrating the utility of domain exchanging in analyzing the structure/function relationships among closely related members of multigene families such as the MT-MMP gene family. In this study, we constructed several chimeras to define the contributions of various domains in MT1-MMP and MT3-MMP to pro-MMP-2 activation and type I collagen degradation at the cellular level. Despite the well recognized contribution of the pexin-like domains to MMP substrate specificity (24Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (342) Google Scholar), our data suggest that the catalytic domains of MT1-MMP and MT3-MMP play a dominant role in defining their activities for biological substrates. Furthermore, we demonstrate that the cytoplasmic domain of MT1-MMP behaves as a negative regulator even when substituted into MT3-MMP. These results suggest that different domains of MT-MMPs could be targeted for potential drug developments. Cell Culture and Reagents—MDCK cells and derivatives were generated and maintained as described (10Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Rabbit anti-MT3-MMP antisera were raised against a glutathione S-transferase-MT3-MMP fusion protein as described (25Kang T. Yi J. Yang W. Wang X. Jiang A. Pei D. FASEB J. 2000; 14: 2559-2568Crossref PubMed Scopus (48) Google Scholar). Affinity-purified rabbit anti-MT1-MMP antibodies 502 and 879 have been described previously (26Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (225) Google Scholar). Goat anti-rabbit secondary antibodies were purchased from Transduction Laboratories (Lexington, KY). Expression Constructs—Expression vectors carrying wild-type MT1-MMP (WT19), its cytoplasmic truncation mutant (ΔC16), and wild-type MT3-MMP have been described previously (25Kang T. Yi J. Yang W. Wang X. Jiang A. Pei D. FASEB J. 2000; 14: 2559-2568Crossref PubMed Scopus (48) Google Scholar, 26Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (225) Google Scholar). Chimeras were generated using overlapping primers designed for recombining various domains of MT1-MMP and MT3-MMP through a PCR strategy similar to the one described for MMP-11 (27Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (534) Google Scholar). The MT1-MMP/MT3-MMP exchange constructs (MT1/MT3-1, -2, -3, -4, -5, -6, and -C1) were made with two or three MT1-MMP and MT3-MMP PCR fragments as illustrated in Fig. 1. These constructs were cloned into the pCR3.1 expression vector and characterized as described previously (10Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 13Pei D. Weiss S.J. J. Biol. Chem. 1996; 271: 9135-9140Abstract Full Text Full Text PDF PubMed Scopus (366) Google Scholar, 27Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (534) Google Scholar). Cell Transfection and Generation of Stable Cell Lines—Plasmids pCR3.1-MT1WT19, pCR3.1-MT1ΔC16, and pCR3.1-MT3WT9 and MT1-MMP/MT3-MMP exchange constructs (MT1/MT3-1, -2, -3, -4, -5, -6, and -C1) were transfected into MDCK cells by LipofectAMINE (Invitrogen), and stable clones were selected in the presence of G418 as described (10Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 11Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (169) Google Scholar). The stable clones were screened by Western analysis of the cell lysates with anti-MT1-MMP antibodies 502 and 879 and anti-MT3-MMP antibody and by zymographic analysis of pro-MMP-2 activation. Ten representative clones with similar levels of protein expression were selected for further studies: MT1-19-11, MT1/MT3-1-7-8, MT1/MT3-2-9-5, MT1/MT3-3-4-1, MT1/MT3-4-14-2, MT1/MT3-5-23-10, MT1/MT3-6-17-7, MT1/MT3-C1-5-5, MT3-9(FF4-7), and MT1ΔC16-17. Zymography and Western Blotting—The basic protocols for these two assays have been described previously (10Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 11Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (169) Google Scholar). Briefly, cells were allowed to grow to confluence in 6-well plates, washed three times with phosphate-buffered saline, and allowed to incubate in the presence of Dulbecco's modified Eagle's medium (DMEM; 1 ml/well) with 5% fetal bovine serum (a source of pro-MMP-2). The synthetic MMP inhibitor BB94 (50 μm) was included in the medium as needed. After 24 h of incubation, the medium was harvested and cleared by centrifugation and analyzed on SDS-polyacrylamide gel impregnated with gelatin (1 mg/ml) as described (10Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 11Pei D. Cell Res. 1999; 9: 291-303Crossref PubMed Scopus (169) Google Scholar). The gels were incubated at 37 °C for 12 h, stained with Coomassie Blue, and destained; and images were captured by scanning. The active species of MMP-2 and its total activity were quantified by densitometry using a Stratagene Eagle-Eye system. For Western blotting, cells grown in 6-well plates were lysed in 250 μl of radioimmune precipitation assay buffer (50 mm Tris (pH 7.5), 150 mm NaCl, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 10 μm leupeptin, 0.1 μm 5-p-amidinophenylmethanesulfonyl fluoride, and 1 μm aprotinin) with 10 mm of EDTA. The lysates were centrifuged at 14,000 × g for 20 min to remove the cell debris and analyzed by Western blotting with anti-MT1-MMP antibody 502 or 879 or anti-MT3-MMP antibody and developed as described (25Kang T. Yi J. Yang W. Wang X. Jiang A. Pei D. FASEB J. 2000; 14: 2559-2568Crossref PubMed Scopus (48) Google Scholar, 26Jiang A. Lehti K. Wang X. Weiss S.J. Keski-Oja J. Pei D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13693-13698Crossref PubMed Scopus (225) Google Scholar). Invasion of Type I Collagen by MDCK Cells Transfected with MT1-MMP, MT3-MMP, and Their Derivatives—Type I collagen (0.5 ml at 2.0 mg/ml; Collaborative Research, Bedford, MA) was added to each well of a 24-well plate and incubated at 37 °C for 2 h to form a gel. Cells (2.4 × 103) were mixed with fresh medium containing 90% DMEM and 10% fetal bovine serum and added to the top of the gel with care. The medium was renewed every 2 days. BB94 (50 μm) was added as indicated. After 7 days, the cells that had invaded the collagen were photographed (magnification ×200) with a video camera attached to a Nikon microscope at the University of Minnesota Bioimaging Processing Facility as described (25Kang T. Yi J. Yang W. Wang X. Jiang A. Pei D. FASEB J. 2000; 14: 2559-2568Crossref PubMed Scopus (48) Google Scholar). The diameter of each cell cyst was measured, calculated, and presented as described (25Kang T. Yi J. Yang W. Wang X. Jiang A. Pei D. FASEB J. 2000; 14: 2559-2568Crossref PubMed Scopus (48) Google Scholar). Growth of MDCK Cells and Derivatives in Three-dimensional Collagen Gel—Cells (1.2 × 103) were mixed with collagen solutions (1.5 ml at 2.5 mg/ml; Collaborative Research), which were allowed to gel at 37 °C in 6-well plates to give rise to three-dimensional collagen matrices. Fresh medium containing 90% DMEM and 10% fetal bovine serum was added to the wells and changed every 2 days. BB94 (10 mm) was added as needed. After 11 days, the MDCK cells and derivatives on the three-dimensional collagen gel were photographed (magnification ×100) with a video camera and analyzed as described above. Multivariant Analysis: Hierarchical Clustering, K-means Clustering, and Principal Component Analysis—The percentages of MMP-2 activation from transient transfection or in the stable cell line and the average diameters of cell cysts in the collagen assay were analyzed by clustering principles. Because the scales and S.E. values are different among these measurements, the original data were transformed to standardized data (x′ = (x - x bar)/S.D.) before clustering analysis. All of the clustering analyses were done with J-Express Version 2.1 (Molmine Bioinformatics Software Solutions). Euclidean was chosen as the distance measure for both hierarchical and K-means clustering. For hierarchical clustering, all four cluster similarity methods available in J-Express were used, including single linkage, unweighted average linkage (UPGMA), weighted average linkage (WPGMA), and complete linkage. For K-means clustering, the number of clusters was designed as 4, and the maximum iteration number was chosen as 200. The initialization method was the most widely used one: random. For principal component analysis, the repeated information among different characters was removed from analysis. The high dimensional data were transformed into lower dimensional data when the most variance information was retained. The lower dimensional data became obvious for visualization. Construction of MT1-MMP and MT3-MMP Chimeras—The type I transmembrane MMPs, MT1-, MT2-, MT3-, and 5-MMPs, share the same domain structure with extensive homology, suggesting that they perform similar biological functions in vivo. Indeed, all have been shown to be able to activate pro-MMP-2 at the cellular level (6Sato H. Takino T. Okada Y. Cao J. Shinagawa A. Yamamoto E. Seiki M. Nature. 1994; 370: 61-65Crossref PubMed Scopus (2377) Google Scholar, 8Takino T. Sato H. Shinagawa A. Seiki M. J. Biol. Chem. 1995; 270: 23013-23020Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar, 10Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar, 28Tanaka M. Sato H. Takino T. Iwata K. Inoue M. Seiki M. FEBS Lett. 1997; 402: 219-222Crossref PubMed Scopus (55) Google Scholar). Yet, recent in vivo evidence suggests that these enzymes may perform different functions both developmentally and pathologically (14Holmbeck K. Bianco P. Caterina J. Yamada S. Kromer M. Kuznetsov S.A. Mankani M. Robey P.G. Poole A.R. Pidoux I. Ward J.M. Birkedal-Hansen H. Cell. 1999; 99: 81-92Abstract Full Text Full Text PDF PubMed Scopus (1111) Google Scholar, 15Zhou Z. Apte S.S. Soininen R. Cao R. Baaklini G.Y. Rauser R.W. Wang J. Cao Y. Tryggvason K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4052-4057Crossref PubMed Scopus (690) Google Scholar). We hypothesize that the functional differences of MT-MMPs are encoded in their structural domains, which specify unique enzymatic properties against specific substrates. To ascertain the functional difference between analogous domains among MT-MMPs, we paired MT1-MMP with MT3-MMP in a domain exchange approach. In the phylogenetic tree of MT-MMPs constructed by similarities between their catalytic domains, MT1-MMP and MT2-MMP are most closely related, MT3-MMP and MT5-MMP are in the same sub-branch, and the glycosylphosphatidylinositol-anchored MT-MMPs, MT4-MMP and MT6-MMP, compose the last group (Fig. 1A). It is predicted that there is a functional difference between the MT1-MMP/MT2-MMP subgroup and the MT3-MMP/MT5-MMP subgroup. Furthermore, we anticipate that a comparison between MT1-MMP and MT3-MMP should yield informative data on the functional differences between the MT-MMPs. Despite their structural similarities, MT1-MMP and MT3-MMP differ functionally in at least two areas: 1) activation of pro-MMP-2 into distinct patterns and 2) degradation of type I collagen efficiency. We hypothesize that the functional differences between the MT-MMPs are determined by both the catalytic and pexin-like domains. In an effort to address the structural basis of these functional differences, we constructed chimeras by shuffling domains between these two genes: (a) exchanging the ectoenzymes at the end of the pexin-like domain, (b) exchanging the hinge/pexin-like domains, and (c) exchanging the pro-catalytic domains. As illustrated in Fig. 1B, these constructs were made by a two-step PCR strategy and confirmed by sequencing as described (27Pei D. Weiss S.J. Nature. 1995; 375: 244-247Crossref PubMed Scopus (534) Google Scholar). Specifically, the constructs contain the following detailed sequences from MT1-MMP and MT3-MMP: MT1/MT3-1 with MT1-MMP-(1-508) and MT3-MMP-(533-607); MT1/MT3-2 with MT3-MMP-(1-532) and MT1-MMP-(509-582); MT1/MT3-3 with MT1-MMP-(1-284), MT3-MMP-(392-532), and MT1-MMP-(509-582); MT1/MT3-4 with MT3-MMP-(1-291), MT1-MMP-(284-532), and MT3-MMP-(533-607); MT1/MT3-5 with MT1-MMP-(1-284) and MT3-MMP-(392-607); MT1/MT3-6 with MT3-MMP-(1-291) and MT1-MMP-(284-582); MT1/MT3-C1 with MT3-MMP-(1-587) and MT1-MMP-(563-582); and MT1ΔC with MT1-MMP-(1-562). Segregation of Pro-MMP-2 Activation Pattern with Only the Catalytic Domain—Upon confirmation of all constructs to be error-free and configured as designed, we transiently transfected these constructs into MDCK cells and monitored their expression by Western blotting and their ability to activate pro-MMP-2 by zymography. The constructs expressed protein species as expected as judged by Western blotting using two anti-MT1-MMP antibodies (Fig. 2, C and D) and one anti-MT3-MMP antibody (Fig. 2E). In addition to the specific protein species, there were nonspecific bands cross-reacting with anti-MT1-MMP antibody 879 and anti-MT3-MMP antibody (Fig. 2, D and E, lane 1). Some of the specific species may represent different glycosylation forms as described previously for MT1-MMP and MT3-MMP (e.g. species in Fig. 2E, lanes 4, 5, 9, and 10) (25Kang T. Yi J. Yang W. Wang X. Jiang A. Pei D. FASEB J. 2000; 14: 2559-2568Crossref PubMed Scopus (48) Google Scholar). The conditioned medium was analyzed for pro-MMP-2 activation. As shown in Fig. 2A, MT1-MMP and MT3-MMP activated pro-MMP-2 with different patterns (lane 2 versus lane 10). Specifically, MT1-MMP converted more MMP-2 into the final active species compared with MT3-MMP such that the ratio between active and intermediate MMP-2 converted by MT1-MMP was significantly greater than that converted by MT3-MMP (Fig. 2A, lane 2 versus lane 10). Given the purported role of the pexin domains in mediating pro-MMP-2 activation (24Itoh Y. Takamura A. Ito N. Maru Y. Sato H. Suenaga N. Aoki T. Seiki M. EMBO J. 2001; 20: 4782-4793Crossref PubMed Scopus (342) Google Scholar), we anticipated that the observed difference in pro-MMP-2 activation mediated by MT1-MMP and MT3-MMP would segregate with their respective pexin domains. Surprisingly, when all chimeras were compared for their pro-MMP-2 activation (Fig. 2A, lanes 2-11), a trend emerged to suggest that the patterns of pro-MMP-2 activation segregate only with the catalytic domain of each MT-MMP. The rest of the domains appear to play a very small role in determining the ratio between active and intermediate MMP-2 (Fig. 2A, lanes 2-11). Because transient transfection may generate an abnormally high level of protein for each construct, the observed results in Fig. 2 may not be reflective of the activities under more stable conditions such as those in vivo. Therefore, it is imperative to validate the findings in stable cell lines as shown in Fig. 2. We generated stable cell lines from each construct as described previously (10Pei D. J. Biol. Chem. 1999; 274: 8925-8932Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). Briefly, we picked 24 clones from each construct and screened these clones for pro-MMP-2 activation by zymography and MT1-MMP/MT3-MMP protein expression by Western blotting as described in the legend to Fig. 2. On average, we obtained at least six stable clones expressing varying amounts of recombinant protein on Western blots that mediated corresponding levels of pro-MMP-2 activation (data not shown). To analyze these constructs in the same experimental settings, we selected one representative clone from each construct that expressed similar levels of recombinant proteins upon Western blotting. As shown in Fig. 3 (C-E), we determined the amounts of the recombinant proteins in those selected stable cell lines. The stable lines harboring MT1-MMP, MT1/MT3-1 to MT1/MT3-6, MT1/MT3-C1, and MT3-MMP appeared to express the recombinant proteins at comparable levels (Fig. 3, C-E, lanes 2-10). The only exception is the one expressing MT1ΔC, with about twice as much protein as the rest of the constructs (Fig. 3C, lane 11 versus lanes 2, 3, 5, 7). As shown in Fig. 2 (C-E), there were multiple species observed, presumably representing different" @default.
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