Purification and characterization of a bovine acetyl low density lipoprotein receptor.

Abstract

The acetyl low density lipoprotein (LDL) receptor is expressed on macrophages and some endothelial cells and mediates macrophage-foam cell formation in culture. A 2Z0-kDa acetyl LDL binding protein was partially purified from bovine liver membranes and was used to make a specific monoclonal antibody. The 220-kDa protein immunoprecipitated by this antibody retained binding activity, and the antibody was used to detect this protein in cells lining bovine liver sinusoids and on the surface of cultured bovine alveolar macrophages. In the human monocytic cell line THP-1, the expression of both acetyl LDL receptor activity and a 220-kDa acetyl LDL binding protein were dramatically induced in parallel after differentiation to a macrophage-like state induced by phorbol ester. The ligand specificity, tissue and cell-type specificity, and coinduction data indicated that this 220-kDa cell-surface binding protein is probably a receptor that mediates acetyl LDL endocytosis. The 220-kDa protein, which was purified 238,000-fold from bovine lung membranes to near homogeneity using monoclonal antibody affinity chromatography, is a trimer of 77-kDa subunits that contain asparaginelinked carbohydrate chains.A hallmark of atherosclerosis is the deposition of plasma cholesterol in atherosclerotic plaques in arterial walls (1). Key cellular components of plaques are foam cells, which are derived from monocytes-macrophages (2-4). Chemically modified low density lipoprotein (LDL), such as acetyl LDL, can be rapidly taken up by cultured macrophages via receptor-mediated endocytosis resulting in foam cell formation. This suggested that the acetyl LDL receptor system may play an important role in the formation offoam cells in vivo (5-10). Acetyl LDL receptor activity has also been detected in endothelial cells but is usually not expressed at substantial levels in other cell types. This receptor activity is induced during differentiation of cultured human monocytes into macrophages (11) and by the phorbol ester-induced differentiation of the human monocytic cell line THP-1 (12). In vivo, high levels of acetyl LDL receptor activity have been detected in the liver, especially in cells lining liver sinusoids, adrenal gland, spleen, and several other organs (13-15). A ligand blotting assay of unreduced specimens has been used by Via and colleagues (16,17) to detect acetyl LDL binding proteins of 200-260 kDa in a murine macrophage cell line, P388dl, and in rat liver. To obtain further information about the acetyl LDL receptor, we purified and made a monoclonal antibody against a 220-kDa bovine liver protein that exhibited acetyl LDL binding activity. Several lines of evidence, including ligand specificity and cell-type and tissue specificity, indicate that this 220-kDa protein is probably an acetyl LDL receptor. The 220-kDa protein was purified to near homogeneity and was shown to be a trimer of 77-kDa glycoprotein subunits that contain asparagine-linked carbohydrate chains. METHODSMaterials. All reagents were obtained as described ...

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