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Molecular Cloning of cDNA for Matriptase, a Matrix-degrading Serine Protease with Trypsin-like Activity

1999; Elsevier BV; Volume: 274; Issue: 26 Linguagem: Inglês

10.1074/jbc.274.26.18231

1083-351X

Chen‐Yong Lin, Joanna Anders, Michael D. Johnson, Qing‐Xiang Amy Sang, Robert B. Dickson,

Blood Coagulation and Thrombosis Mechanisms

A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease. A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease. Elevated proteolytic activity has been implicated in neoplastic progression. Although the exact role(s) of proteolytic enzymes in the progression of tumor remains unclear, it seems that proteases may be involved in almost every step of the development and spread of cancer. A widely proposed view is that proteases contribute to the degradation of extracellular matrix and to tissue remodeling and are necessary for cancer invasion and metastasis. A wide array of extracellular matrix-degrading proteases have been discovered, the expression of some of which correlates with tumor progression, as reviewed by Magnatti and Rifkin (1Mignatti P. Rifkin D.B. Physiol. Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar). The plasmin/urokinase-type plasminogen activator system and the 72-kDa gelatinase (MMP-2)/membrane-type MMP system have received the most attention for their potential roles in the process of invasion of breast cancer and other carcinomas. However, both systems appear to be largely synthesized by stromal cells in vivo (2Nielsen B.S. Sehested M. Timshel S. Pyke C. Dano K. Lab. Invest. 1996; 74: 168-177PubMed Google Scholar, 3Pyke C. Graem N. Ralfkiaer E. Ronne E. Hoyer-Hansen G. Brunner N. Dano K. Cancer Res. 1993; 53: 1911-1915PubMed Google Scholar, 4Polette M. Gilbert N. Stas I. Nawrocki B. Noel A. Remacle A. Stetler-Stevenson W.G. Birembaut P. Foidart M. Virchows Arch. 1994; 424: 641-645Crossref PubMed Google Scholar, 5Okada 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) and require indirect mechanisms for their recruitment and activation on the surfaces of cancer cells. The stromal origins of these well characterized extracellular matrix-degrading proteases may suggest that cancer invasion is an event that either depends entirely upon stromal-epithelial cooperation or is controlled by some other unknown epithelium-derived protease(s). A search for these epithelium-derived proteolytic systems that may interact with the plasmin/urokinase-type plasminogen activator system and/or with the MMP family could provide a missing link in our understanding of malignant invasion.We have pursued studies of a novel protease with the hypothesis that a tumor itself may be a major source of proteases important for multiple aspects of malignant behavior, including invasion and metastasis. To this end, we systematically altered several conditions such as the pH using gelatin zymography to search for potentially important breast cancer cell-derived gelatinases. This search led us to the discovery of a major protease, which on a gelatin zymogram had a slightly alkaline pH optimum and a size between those of MMP-2 and MMP-9 in T-47D human breast cancer cells (6Shi Y.E. Torri J. Yieh L. Wellstein A. Lippman M.E. Dickson R.B. Cancer Res. 1993; 53: 1409-1415PubMed Google Scholar). We now propose to call this protease matriptase. Matriptase has been purified from T-47D cell-conditioned medium and has been used as an immunogen to produce monoclonal antibodies (7Lin C.-Y. Wang J.K. Torri J. Dou L. Sang Q.A. Dickson R.B. J. Biol. Chem. 1997; 272: 9147-9152Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Although matriptase was initially isolated from cell-conditioned medium, three lines of evidence, including immunofluorescence staining, surface biotinylation, and subcellular fractionation, suggested that a portion of the enzyme molecules were localized on the surfaces of cells. Given its extracellular matrix-degrading activity and presentation on the surfaces of breast cancer cells, we hypothesize that matriptase may be involved in breast cancer invasion. To further characterize the newly discovered matrix-degrading protease in this study, we have purified the enzyme and its binding protein from human milk, a biological source of relatively high abundance. A cDNA clone for matriptase has now been generated and characterized. Elevated proteolytic activity has been implicated in neoplastic progression. Although the exact role(s) of proteolytic enzymes in the progression of tumor remains unclear, it seems that proteases may be involved in almost every step of the development and spread of cancer. A widely proposed view is that proteases contribute to the degradation of extracellular matrix and to tissue remodeling and are necessary for cancer invasion and metastasis. A wide array of extracellular matrix-degrading proteases have been discovered, the expression of some of which correlates with tumor progression, as reviewed by Magnatti and Rifkin (1Mignatti P. Rifkin D.B. Physiol. Rev. 1993; 73: 161-195Crossref PubMed Scopus (1179) Google Scholar). The plasmin/urokinase-type plasminogen activator system and the 72-kDa gelatinase (MMP-2)/membrane-type MMP system have received the most attention for their potential roles in the process of invasion of breast cancer and other carcinomas. However, both systems appear to be largely synthesized by stromal cells in vivo (2Nielsen B.S. Sehested M. Timshel S. Pyke C. Dano K. Lab. Invest. 1996; 74: 168-177PubMed Google Scholar, 3Pyke C. Graem N. Ralfkiaer E. Ronne E. Hoyer-Hansen G. Brunner N. Dano K. Cancer Res. 1993; 53: 1911-1915PubMed Google Scholar, 4Polette M. Gilbert N. Stas I. Nawrocki B. Noel A. Remacle A. Stetler-Stevenson W.G. Birembaut P. Foidart M. Virchows Arch. 1994; 424: 641-645Crossref PubMed Google Scholar, 5Okada 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) and require indirect mechanisms for their recruitment and activation on the surfaces of cancer cells. The stromal origins of these well characterized extracellular matrix-degrading proteases may suggest that cancer invasion is an event that either depends entirely upon stromal-epithelial cooperation or is controlled by some other unknown epithelium-derived protease(s). A search for these epithelium-derived proteolytic systems that may interact with the plasmin/urokinase-type plasminogen activator system and/or with the MMP family could provide a missing link in our understanding of malignant invasion. We have pursued studies of a novel protease with the hypothesis that a tumor itself may be a major source of proteases important for multiple aspects of malignant behavior, including invasion and metastasis. To this end, we systematically altered several conditions such as the pH using gelatin zymography to search for potentially important breast cancer cell-derived gelatinases. This search led us to the discovery of a major protease, which on a gelatin zymogram had a slightly alkaline pH optimum and a size between those of MMP-2 and MMP-9 in T-47D human breast cancer cells (6Shi Y.E. Torri J. Yieh L. Wellstein A. Lippman M.E. Dickson R.B. Cancer Res. 1993; 53: 1409-1415PubMed Google Scholar). We now propose to call this protease matriptase. Matriptase has been purified from T-47D cell-conditioned medium and has been used as an immunogen to produce monoclonal antibodies (7Lin C.-Y. Wang J.K. Torri J. Dou L. Sang Q.A. Dickson R.B. J. Biol. Chem. 1997; 272: 9147-9152Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Although matriptase was initially isolated from cell-conditioned medium, three lines of evidence, including immunofluorescence staining, surface biotinylation, and subcellular fractionation, suggested that a portion of the enzyme molecules were localized on the surfaces of cells. Given its extracellular matrix-degrading activity and presentation on the surfaces of breast cancer cells, we hypothesize that matriptase may be involved in breast cancer invasion. To further characterize the newly discovered matrix-degrading protease in this study, we have purified the enzyme and its binding protein from human milk, a biological source of relatively high abundance. A cDNA clone for matriptase has now been generated and characterized. We thank Dr. Henry Yang for the automated DNA sequencing that was performed at the Lombardi Cancer Center Macromolecular Synthesis and Sequencing Shared Resource. We thank the Lombardi Cancer Center Tissue Culture Resource for cells and reagents.