Biosynthesis and Post-translational Processing of Lectin-like Oxidized Low Density Lipoprotein Receptor-1 (LOX-1)
N-LINKED GLYCOSYLATION AFFECTS CELL-SURFACE EXPRESSION AND LIGAND BINDING*
- Hiroharu Kataoka‡,
- Noriaki Kume‡§,
- Susumu Miyamoto¶,
- Manabu Minami‡,
- Takatoshi Murase‡,
- Tatsuya Sawamura‖,
- Tomoh Masaki‖,
- Nobuo Hashimoto¶ and
- Toru Kita‡
- From the Departments of ‡Geriatric Medicine and¶Neurosurgery, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan and the ‖Department of Bioscience, National Cardiovascular Center Research Institute, Osaka 565-8565, Japan
Abstract
LOX-1 (lectin-likeoxidized low density lipoprotein receptor-1) is a type II membrane protein belonging to the C-type lectin family that can act as a cell-surface receptor for atherogenic oxidized low density lipoprotein (Ox-LDL) and may play crucial roles in atherogenesis. In this study, we show, by pulse-chase labeling and glycosidase digestion, that LOX-1 is synthesized as a 40-kDa precursor protein withN-linked high mannose carbohydrate chains (pre-LOX-1), which is subsequently further glycosylated and processed into the 48-kDa mature form within 40 min. Furthermore, when treated with anN-glycosylation inhibitor, tunicamycin, both tumor necrosis factor-α-activated bovine aortic endothelial cells and CHO-K1 cells stably expressing bovine LOX-1 (BLOX-1-CHO) exclusively produced a 32-kDa deglycosylated form of LOX-1. Cell enzyme-linked immunosorbent assay, flow cytometry, and immunofluorescence confocal microscopy demonstrated that the deglycosylated form of LOX-1 is not efficiently transported to the cell surface, but is retained in the endoplasmic reticulum or Golgi apparatus in tumor necrosis factor-α-activated bovine aortic endothelial cells, but not in BLOX-1-CHO cells. Radiolabeled Ox-LDL binding studies revealed that the deglycosylated form of LOX-1 expressed on the cell surface of BLOX-1-CHO cells has a reduced affinity for Ox-LDL binding. Taken together,N-linked glycosylation appears to play key roles in the cell-surface expression and ligand binding of LOX-1.
Footnotes
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↵* This work was supported by Research Grants 11307018, 11838008, 09281104, and 09281103 from the Ministry of Education, Science, and Culture of Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵§ To whom correspondence should be addressed: Dept. of Geriatric Medicine, Graduate School of Medicine, Kyoto University, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Tel.: 81-75-751-3465; Fax: 81-75-751-3574; E-mail: nkume@kuhp.kyoto-u.ac.jp.
- Abbreviations:
- Ox-LDL
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oxidized low density lipoprotein
- TNF-α
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tumor necrosis factor-α
- CHO
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Chinese hamster ovary
- BAEC
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bovine aortic endothelial cell
- DMEM
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Dulbecco's modified Eagle's medium
- FCS
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fetal calf serum
- ELISA
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enzyme-linked immunosorbent assay
- PBS
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phosphate-buffered saline
- BSA
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bovine serum albumin
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- Received August 26, 1999.
- Revision received December 8, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
