Identification of soluble forms of lectin-like oxidized LDL receptor-1.
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.
Murase
T
T
Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, Japan.
Kume
N
N
Kataoka
H
H
Minami
M
M
Sawamura
T
T
Masaki
T
T
Kita
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000166:cytology; D007611:Aprotinin / Q000494:pharmacology; D001161:Arteriosclerosis / Q000378:metabolism*; D019921:Biotinylation; D016466:CHO Cells; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D017077:Culture Media, Conditioned / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D010450:Endopeptidases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000737:chemistry / Q000166:cytology / Q000201:enzymology*; D006023:Glycoproteins / Q000494:pharmacology; D037102:Lectins; D007976:Leupeptins / Q000494:pharmacology; D008077:Lipoproteins, LDL / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010436:Pepstatins / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011973:Receptors, LDL / Q000032:analysis / Q000097:blood* / Q000737:chemistry; D051131:Receptors, Oxidized LDL; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012995:Solubility; D014105:Tosyl Compounds / Q000494:pharmacology; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D017077:Culture Media, Conditioned; D015853:Cysteine Proteinase Inhibitors; D006023:Glycoproteins; D037102:Lectins; D007976:Leupeptins; D008077:Lipoproteins, LDL; D008565:Membrane Proteins; D010436:Pepstatins; D011480:Protease Inhibitors; D011973:Receptors, LDL; D051131:Receptors, Oxidized LDL; D015842:Serine Proteinase Inhibitors; D014105:Tosyl Compounds; D014409:Tumor Necrosis Factor-alpha; C039304:calpain inhibitors; C099252:oxidized low density lipoprotein; C014195:Streptomyces pepsin inhibitor; D014107:Tosyllysine Chloromethyl Ketone; C003614:4-toluenesulfonyl fluoride; D007611:Aprotinin; D010450:Endopeptidases; C032854:leupeptin; C031375:pepstatin