Identification of soluble forms of lectin-like oxidized LDL receptor-1

Arterioscler Thromb Vasc Biol. 2000 Mar;20(3):715-20. doi: 10.1161/01.atv.20.3.715.

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

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Aorta / cytology
  • Aprotinin / pharmacology
  • Arteriosclerosis / metabolism*
  • Biotinylation
  • CHO Cells
  • Cattle
  • Cell Membrane / drug effects
  • Cell Membrane / metabolism
  • Cricetinae
  • Culture Media, Conditioned / pharmacology
  • Cysteine Proteinase Inhibitors / pharmacology
  • Endopeptidases / metabolism
  • Endothelium, Vascular / chemistry
  • Endothelium, Vascular / cytology
  • Endothelium, Vascular / enzymology*
  • Glycoproteins / pharmacology
  • Lectins
  • Leupeptins / pharmacology
  • Lipoproteins, LDL / metabolism
  • Membrane Proteins / analysis
  • Membrane Proteins / isolation & purification
  • Membrane Proteins / metabolism
  • Molecular Sequence Data
  • Pepstatins / pharmacology
  • Protease Inhibitors / pharmacology
  • Protein Structure, Tertiary
  • Receptors, LDL / analysis
  • Receptors, LDL / blood*
  • Receptors, LDL / chemistry
  • Receptors, Oxidized LDL
  • Serine Proteinase Inhibitors / pharmacology
  • Solubility
  • Tosyl Compounds / pharmacology
  • Tosyllysine Chloromethyl Ketone / pharmacology
  • Tumor Necrosis Factor-alpha / pharmacology

Substances

  • Culture Media, Conditioned
  • Cysteine Proteinase Inhibitors
  • Glycoproteins
  • Lectins
  • Leupeptins
  • Lipoproteins, LDL
  • Membrane Proteins
  • Pepstatins
  • Protease Inhibitors
  • Receptors, LDL
  • Receptors, Oxidized LDL
  • Serine Proteinase Inhibitors
  • Tosyl Compounds
  • Tumor Necrosis Factor-alpha
  • calpain inhibitors
  • oxidized low density lipoprotein
  • Streptomyces pepsin inhibitor
  • Tosyllysine Chloromethyl Ketone
  • 4-toluenesulfonyl fluoride
  • Aprotinin
  • Endopeptidases
  • leupeptin
  • pepstatin