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.
LOX-1 (lectin-like oxidized 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 with N-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 an N-glycosylation inhibitor, tunicamycin, both tumor necrosis factor-alpha-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-alpha-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.
Kataoka
H
H
Department of Geriatric Medicine, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan.
Kume
N
N
Miyamoto
S
S
Minami
M
M
Murase
T
T
Sawamura
T
T
Masaki
T
T
Hashimoto
N
N
Kita
T
T
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002417:Cattle; D002478:Cells, Cultured; D006224:Cricetinae; D004730:Endothelium, Vascular; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D006031:Glycosylation; D007457:Iodine Radioisotopes; D008077:Lipoproteins, LDL / Q000378:metabolism; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011485:Protein Binding; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011973:Receptors, LDL / Q000096:biosynthesis / Q000378:metabolism*; D051131:Receptors, Oxidized LDL; D051127:Scavenger Receptors, Class E; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology
D007457:Iodine Radioisotopes; D008077:Lipoproteins, LDL; D011498:Protein Precursors; D011973:Receptors, LDL; D051131:Receptors, Oxidized LDL; D051127:Scavenger Receptors, Class E; D014409:Tumor Necrosis Factor-alpha; C099252:oxidized low density lipoprotein; D014415:Tunicamycin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase