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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:715.)
© 2000 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Identification of Soluble Forms of Lectin-Like Oxidized LDL Receptor-1

Takatoshi Murase; Noriaki Kume; Hiroharu Kataoka; Manabu Minami; Tatsuya Sawamura; Tomoh Masaki; Toru Kita

From the Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University (N.K., H.K., M.M., T.K.), and the Department of Bioscience, National Cardiovascular Center Research Institute, Suita, Osaka (T.S., T.M.), Japan. T. Murase is now at Biological Science Laboratories, Kao Corp, Ichikaimachi, Tochigi, Japan.

Correspondence to Noriaki Kume, MD, PhD, Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail nkume{at}kuhp.kyoto-u.ac.jp

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-–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-–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 Arg⁸⁶-Ser⁸⁷ and Lys⁸⁹-Ser⁹⁰, 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.

Key Words: LOX-1 • oxidized LDL • proteolysis • atherosclerosis • endothelial cells

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