Characterization of a Novel, Membrane-bound, 80-kDa Matrix-degrading Protease from Human Breast Cancer Cells

Abstract

A major, apparently novel extracellular matrix-degrading protease was previously identified and partially isolated from hormone-dependent but not from hormone-independent human breast cancer cells (Shi, Y. E., Torri, J., Yieh, L., Wellstein, A., Lippman, M. E., and Dickson, R. B. (1993) Cancer Res. 53, 1409 -1415). Although initially the 80-kDa protease was identified from breast cancer cell-conditioned medium, immunofluorescence staining of breast cancer cells with anti-80-kDa protease monoclonal antibody 21-9 showed that in addition to its detection in intracellular compartments, the protease was uniformly localized around periphery of the cells with more intensive staining on the pseudopodia and membrane ruffles. A surface biotinylation technique confirmed the plasma membrane localization of the protease. In addition, the 80-kDa protease could not be washed from the membrane fraction of homogenized breast cancer cells with high concentrations of salts or with EDTA.The 80-kDa protease may noncovalently associate with other protein(s) to form complexes, the 95-and 110-kDa proteases. Both complexes showed gelatinolytic activity and bore the epitopes recognized by monoclonal antibody 21-9. Furthermore, both complexes could be converted to 80-kDa forms by boiling in SDS in the absence of reducing agents. Expression of this novel, integral membrane gelatinase could allow breast cancer cells an alternative to other previously described matrix-degrading enzymes for degradation of the extracellular matrix in close proximity to their surfaces.Fatality in breast cancer is the result of metastatic lesions. Degradation of the extracellular matrix (ECM), 1 including the basement membrane and interstitial stroma, an important aspect of metastasis, is required for metastatic cancer cells to migrate through anatomical barriers and to invade tissues. Matrix-degrading proteases, including the plasminogen activator-plasmin system of serine proteases, 72-kDa gelatinase A (MMP-2) and 92-kDa gelatinase B (MMP-9) of the matrix metalloprotease (MMP) family, and cathepsins B (a cysteine protease) and D (an aspartic protease), have been implicated in tumor cell invasion (2). Morphological studies show that degradation of the ECM occurs in close proximity to the cell surface (3, 4), and that matrix-degrading proteases must be localized on the cell surface to perform their functions. This theory was further supported by the observation that the cancer cellconditioned medium alone failed to degrade the ECM (5). Studies of two matrix-degrading proteases, the plasminogen activator-plasmin system and the activation of 72-kDa gelatinase A (MMP-2), clearly demonstrate that localized ECM degradation is achieved by protease binding to a membrane-bound protease receptor and/or activator followed by a proteolytic activation cascade. The urokinase-type plasminogen activator (uPA) is secreted as a soluble, inactive precursor by tumor cells and/or tumor stromal cells and recruited to tumor cell surfaces by binding to the membrane-bound uPA receptor. ...

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

“…Purification of Matriptase from Human Milk-In our previous study (7), a small proportion of the matriptase molecules were identified as complexes in human breast cancer cells. We have subsequently found human milk to be a good source for isolation of larger quantities of the matriptase complexes (39).…”

“…Immunoblot Analysis-Immunoblotting was conducted as described previously (7). Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membrane, and subsequently probed with anti-matriptase mAb 1 M32.…”

“…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 (7). 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.…”

Matriptase Activation, an Early Cellular Response to Acidosis

“…Matriptase is known as an integral membrane protein that is localized at the plasma membrane, however, as we have shown before, the majority of matriptase resides inside the cell (18,35). This intracellular pool of matriptase is anchored to the intracellular membranes surrounding the nucleus, and is the pool of enzyme that is assayed in the in vitro, cell-free activation system (16).…”

Functional Characterization of Kunitz Domains in Hepatocyte Growth Factor Activator Inhibitor Type 1

“…HAI-1 was also identified from human breast milk in a complex with another serine protease, named matriptase (10). Matriptase is an epithelial cell-derived serine protease that has extracellular matrix-degrading activity and has been proposed to play a role in breast cancer invasion (11,12). Thus, HAI-1 may also function as a regulator of tumor progression by controlling the activity of an extracellular matrix-degrading enzyme.…”