Catalytic Activities and Substrate Specificity of the Human Membrane Type 4 Matrix Metalloproteinase Catalytic Domain

Abstract

Matrix metalloproteinases are members of a family of homologous Zn 2ϩ -dependent endoproteinases that participate in physiological and pathological processes involving tissue remodeling or cell migration (1, 2). MMP 1 gene expression is highly regulated in different cell types in response to various physiological stimuli (3), and dysregulated MMP activity is implicated in the development of many diseases involving matrix remodeling, including cancer (4, 5), arthritis (6), and cardiovascular disease (7). With nearly 20 MMPs identified and more likely to be discovered, delineation of the functional capabilities of these proteases becomes crucial as MMP inhibitors are developed as therapeutic agents. Human MT4-MMP (i.e. MMP-17) was recently cloned from a breast carcinoma cDNA library (11) using primers that were directed to the conserved propeptide region and catalytic Zn 2ϩ -binding site of MMPs (1, 2). In addition to being present in breast cancers, MT4-MMP mRNA was found highly expressed in brain, colon, ovary, and testis tissues as well as in leukocytes (11). The deduced amino acid sequence of MT4-MMP contained regions homologous to all of the domains conserved in MMPs, but two invariant aspartic acid residues that ligate structural Ca 2ϩ ions in related MMPs were replaced by Tyr-137 and Asn-141 in MT4-MMP (11). Although antibodies to MT4-MMP detected a ϳ70-kDa protein in human brain extracts, the catalytic competence of this putative protease was not evaluated (11).The expression and characterization of MMP catalytic domains has facilitated structure-function studies of these proteases (15, 16, 18 -22). For example, three-dimensional structures of the human MMP-3CD (stromelysin-1) in complex with small molecule inhibitors and TIMP-1 have been solved by x-ray crystallography and NMR methods (23-26). Furthermore, MMP CDs designed without a propeptide domain obviate activation steps and simplify their use.In our continuing studies of MMPs, we set out to characterize the catalytic capabilities of MT4-MMP. We cloned the MT4-MMPCD DNA from a breast carcinoma cDNA library, purified the bacterially expressed protein, and defined its catalytic competence with synthetic peptide substrates and extracellular matrix components. Consistent with the expectation that MT4-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ These authors contributed equally to this work. § Present address: Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 294 Tai-Yuan Rd., Shanghai, China 200031.¶ To whom correspondence should be addressed: Biochemistry Dept., Parke-Davis Pharmaceutical Research Division, Warner-Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 734-622-5163; Fax: 734-622-1355; E-mail: Richard.Dyer@wl.com. 1 The abbreviations used are: MMP, matrix metalloproteinase; MT, membrane type; MT1-, MT2-, MT3, MT4-, and MT5-MMPCD, hu...

Mutation of the Matrix Metalloproteinase At2-MMP Inhibits Growth and Causes Late Flowering and Early Senescence in Arabidopsis

“…Degradation of the extracellular matrix has an important role for physiological processes as embryogenesis, organ morphogenesis, and bone remodeling, for example (5). Unregulated enzyme activity of matrix metalloproteinases is involved in the development of diseases including cancer, arthritis, and atherosclerosis (4).…”

“…The enzymes play a role in development, embryogenesis, and organ morphogenesis but also in wound healing in vertebrates (4). They also participate in pathological processes such as cancer and arthritis (5). In vertebrates, the enzyme activity of matrix metalloproteinases is transcriptionally regulated as well by proteolytic activation of the mature enzyme from the inactive pro-enzyme and by co-secretion with endogenous inhibitors (4).…”

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

“…Specifically, soluble MT4-MMP was found to be active only against gelatin, fibrin, and fibrinogen (English et al, 2000;Kolkenbrock et al, 1999;Rozanov et al, 2004;Wang et al, 1999b). As a matter of fact, MT4-MMP has been observed to display ADAM-17-like activity (see Section 1.1.2), since it can behave as a sheddase of tumor necrosis factor (TNF)-a when co-transfected with pro-TNF-a in Cos-7 cells (English et al, 2000).…”

Membrane type-matrix metalloproteinases and tumor progression

“…MT2-MMP cleaves fibronectin, laminin-1, gelatin, tenascin, perlecan and nidogen in identical fragment as MT1-MMP [48,49]. MT4-MMP has a lower activity against extracellular matrix components, but its catalytic domain significantly degrades gelatin, fibrinogen and fibrin [53,54]. The role of MT4-MMP in tissue extracellular matrix degradation seems to be indirectly mediated by its ability to activate aggrecanase-1 (ADAM-TS4) enzyme [55].…”