Abstract
Lectin-like oxidized LDL receptor-1 (LOX-1) is a type II membrane protein belonging to the C-type lectin family molecules, which can act as a cell-surface endocytosis receptor for atherogenic oxidized LDL. In this study, we show that soluble forms of LOX-1 are present in conditioned media of cultured bovine aortic endothelial cells (BAECs) and CHO-K1 cells stably transfected with LOX-1 cDNA. Immunoblot analysis of conditioned media from TNF-alpha-activated BAECs and CHO-K1 cells stably expressing LOX-1 revealed that soluble LOX-1 has an approximate molecular mass of 35 kDa. In TNF-alpha-activated BAECs, cell-surface expression of LOX-1 precedes soluble LOX-1 production. Cell-surface biotinylation followed by immunoprecipitation and immunoblotting showed that soluble LOX-1 in cell-conditioned media is derived from LOX-1 expressed on the cell surface. Production of soluble LOX-1 was inhibited by PMSF, suggesting that PMSF-sensitive proteases may be involved in this process. Purification of soluble LOX-1 by high-performance liquid chromatography and N-terminal amino acid sequencing of soluble LOX-1 identified the 2 cleavage sites between Arg(86)-Ser(87) and Lys(89)-Ser(90), which were located in the membrane proximal extracellular domain of LOX-1. The data demonstrate that cell-surface LOX-1 can be cleaved at 2 different sites and transformed into soluble forms. Further studies may explore therapeutic and diagnostic applications of soluble LOX-1 in atherosclerotic diseases.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Animals
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Aorta / cytology
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Aprotinin / pharmacology
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Arteriosclerosis / metabolism*
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Biotinylation
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CHO Cells
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Cattle
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Cell Membrane / drug effects
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Cell Membrane / metabolism
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Cricetinae
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Culture Media, Conditioned / pharmacology
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Cysteine Proteinase Inhibitors / pharmacology
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Endopeptidases / metabolism
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Endothelium, Vascular / chemistry
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Endothelium, Vascular / cytology
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Endothelium, Vascular / enzymology*
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Glycoproteins / pharmacology
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Lectins
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Leupeptins / pharmacology
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Lipoproteins, LDL / metabolism
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Membrane Proteins / analysis
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Membrane Proteins / isolation & purification
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Membrane Proteins / metabolism
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Molecular Sequence Data
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Pepstatins / pharmacology
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Protease Inhibitors / pharmacology
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Protein Structure, Tertiary
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Receptors, LDL / analysis
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Receptors, LDL / blood*
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Receptors, LDL / chemistry
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Receptors, Oxidized LDL
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Serine Proteinase Inhibitors / pharmacology
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Solubility
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Tosyl Compounds / pharmacology
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Tosyllysine Chloromethyl Ketone / pharmacology
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Tumor Necrosis Factor-alpha / pharmacology
Substances
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Culture Media, Conditioned
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Cysteine Proteinase Inhibitors
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Glycoproteins
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Lectins
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Leupeptins
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Lipoproteins, LDL
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Membrane Proteins
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Pepstatins
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Protease Inhibitors
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Receptors, LDL
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Receptors, Oxidized LDL
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Serine Proteinase Inhibitors
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Tosyl Compounds
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Tumor Necrosis Factor-alpha
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calpain inhibitors
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oxidized low density lipoprotein
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Streptomyces pepsin inhibitor
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Tosyllysine Chloromethyl Ketone
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4-toluenesulfonyl fluoride
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Aprotinin
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Endopeptidases
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leupeptin
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pepstatin