Abstract
The membrane-anchored protease regulator RECK plays important roles in mammalian development and tumor suppression. The biochemical bases of these bioactivities, however, remain poorly understood. Here we report on the properties of a recombinant RECK protein expressed in mouse fibroblasts and purified to near homogeneity. Multiple lines of evidence indicate that RECK forms dimers. Single particle reconstruction using transmission electron microscopy revealed a unique cowbell-like shaped RECK dimer. RECK is cleaved by MMP-2 and MMP-7 and competitively inhibits MMP-7-catalyzed cleavage of fibronectin. Forced RECK expression in HT1080 cells, whose endogenous RECK expression is minimal, leads to an increase in the amount of fibronectin associated with the cell. Our data demonstrate the ability of RECK to protect fibronectin from MMP-mediated degradation.
Highlights
RECK was initially identified as a transformation suppressor gene [1]
Some Matrix metalloproteinases (MMPs) family members are up-regulated in cancer cells, and their expression is often associated with poor prognosis [12]
Expression and Purification of Recombinant Human RECK Protein—Our preliminary experiments indicated that a recombinant human RECK protein (RECK-His), in which the C-terminal GPI-anchoring signal (28 residues) was replaced by six
Summary
Plasmid Construction—The retroviral vector, pL(RECKHis)SB, was constructed by inserting, by three parts ligation, the MluI-EagI fragment of human RECK cDNA, and the double-stranded oligonucleotides encoding the C-terminal 7 amino acids (954AHHHHHH) between the MluI and ApaI site of pLXSB, a derivative of pLXSN [19] carrying the bsd marker. A transfectant clone (TOM2– 6) secreting RECK-His at a high level was expanded to 20 – 40 150-mm dishes using selection medium. Cell lysate (16.7 g of protein) or purified RECK-His (0.14 pmol) was heat-denatured (100 °C, 10 min) in 0.5% SDS, 40 mM dithiothreitol and incubated with N-glycosidase F (1,000 units; New England Biolabs) (37 °C, 1 h) in 30 l of reaction mixture in 50 mM NaPO4 (pH 7.5), 1% Nonidet P-40. The cells (5 ϫ 105) were plated onto 6-well dishes, incubated for 17–24 h, and homogenized in lysis buffer (50 mM Tris-HCl (pH 6.8), 1% SDS, protease inhibitor mixture (Nakalai Tesque)). For blue native-PAGE, the cells were homogenized in 1ϫ Native-PAGE Sample Buffer (Invitrogen) containing 1% digitonin. The samples were mounted with VECTASHIELD mounting medium with 4Ј,6-diamidino-2phenylindole (Vector Laboratories) and recorded with a fluorescence microscope (Axioplan, Zeiss) or a confocal microscope (FV300, Olympus)
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