Abstract
Ubiquitously expressed membrane type-1 matrix metalloproteinase (MT1-MMP), an archetype member of the MMP family, binds tissue inhibitor of metalloproteinases-2 (TIMP-2), activates matrix metalloproteinase-2 (MMP-2), and stimulates cell migration in various cell types. In contrast with MT1-MMP, the structurally similar MT6-MMP associates with the lipid raft compartment of the plasma membrane using a GPI anchor. As a result, MT6-MMP is functionally distinct from MT1-MMP. MT6-MMP is insufficiently characterized as yet. In addition, a number of its biochemical features are both conflicting and controversial. To reassess the biochemical features of MT6-MMP, we have expressed the MT6-MMP construct tagged with a FLAG tag in breast carcinoma MCF-7 and fibrosarcoma HT1080 cells. We then used phosphatidylinositol-specific phospholipase C to release MT6-MMP from the cell surface and characterized the solubilized MT6-MMP fractions. We now are confident that cellular MT6-MMP partially exists in its complex with TIMP-2. Both TIMP-1 and TIMP-2 are capable of inhibiting the proteolytic activity of MT6-MMP. MT6-MMP does not stimulate cell migration. MT6-MMP, however, generates a significant level of gelatinolysis of the fluorescein isothiocyanate-labeled gelatin and exhibits an intrinsic, albeit low, ability to activate MMP-2. As a result, it is exceedingly difficult to record the activation of MMP-2 by cellular MT6-MMP. Because of its lipid raft localization, cellular MT6-MMP is inefficiently internalized. MT6-MMP is predominantly localized in the cell-to-cell junctions. Because MT6-MMP has been suggested to play a role in disease, including cancer and autoimmune multiple sclerosis, the identity of its physiologically relevant cleavage targets remains to be determined.
Highlights
Org. 2 The abbreviations used are: MMP, matrix metalloproteinase; at Molecular Probes. Human ␣1-antitrypsin (AAT), ␣1-antitrypsin; GPI, glycosylphosphatidylinositol; DAPI, 4Ј,6-diamidino-2phenylindole; EZ-link sulfo-NHS-SS-biotin, sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; HT cells, human fibrosarcoma HT1080 cells; Mca-PLGL-Dpa-AR-NH2, methoxycoumarin-4-yl)-acetylPro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-l-2,3-diaminopropionyl)-Ala-ArgNH2; MCF7 cells, human breast carcinoma MCF-7 cells; Mercaptoethane Sulfonic Acid (MESNA), 2-mercaptoethane sulfonic acid; MT-MMP, membrane-type MMP; membrane type-1 matrix metalloproteinase (MT1-MMP) and MT6-MMP, membrane type-1 and membrane type-6 MMP, respectively; growth factors, and cell receptors and play important roles in multiple diseases [1,2,3]
Recombinant tissue inhibitors of MMPs (TIMPs)-2 and the TIMP-2-free matrix metalloproteinase-2 (MMP-2) proenzyme were isolated from the conditioned medium of CHO cells and p2AHT2A72 cells, respectively. p2AHT2A72 cells were derived from the fibrosarcoma HT1080 cell line sequentially transfected with E1A and MMP-2 cDNAs, respectively [22, 23]
There was conflicting evidence about the ability of MT6MMP to be regulated by TIMPs, to play a role in the activation of MMP-2, and to support cell migration
Summary
Org. 2 The abbreviations used are: MMP, matrix metalloproteinase; AAT, ␣1-antitrypsin; GPI, glycosylphosphatidylinositol; DAPI, 4Ј,6-diamidino-2phenylindole; EZ-link sulfo-NHS-SS-biotin, sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate; HT cells, human fibrosarcoma HT1080 cells; Mca-PLGL-Dpa-AR-NH2, methoxycoumarin-4-yl)-acetylPro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-l-2,3-diaminopropionyl)-Ala-ArgNH2; MCF7 cells, human breast carcinoma MCF-7 cells; MESNA, 2-mercaptoethane sulfonic acid; MT-MMP, membrane-type MMP; MT1-MMP and MT6-MMP, membrane type-1 and membrane type-6 MMP, respectively; growth factors, and cell receptors and play important roles in multiple diseases [1,2,3]. MMPs, especially membrane-type MMPs (MT-MMPs), have been proposed to play key roles in tumor invasion and metastasis [4]. MT1-MMP, MT2-MMP, MT3MMP, and MT5-MMP are anchored to the plasma membrane via a transmembrane domain [1, 4]. There is a significant level of knowledge of the structural-functional relationships and regulation of MT1-MMP, an archetype member of the subfamily, exceedingly little is known about the biochemical and cellular properties of GPI-linked MT4-MMP and especially MT6-MMP. MT6-MMP is a membrane proteinase with an extracellular prodomain followed by the catalytic domain, the hinge region, the hemopexin domain, the stalk region, and a GPI anchor attached to the carboxyl end of the stalk. The relevance of MT6-MMP proteolysis of these target proteins to the enzyme functionality in both normal development and disease progression, is not entirely clear as of
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