Abstract
Lectin-like oxidized LDL (ox-LDL) receptor-1 (LOX-1) is a type-II transmembrane protein that belongs to the C-type lectin family of molecules. LOX-1 acts as a cell surface endocytosis receptor and mediates the recognition and internalization of ox-LDL by vascular endothelial cells. Internalization of ox-LDL by LOX-1 results in a number of pro-atherogenic cellular responses implicated in the development and progression of atherosclerosis. In an effort to elucidate the functional domains responsible for the binding of ox-LDL to the receptor, a series of site-directed mutants were designed using computer modeling and X-ray crystallography to study the functional role of the hydrophobic tunnel present in the LOX-1 receptor. The isoleucine residue (I(149)) sitting at the gate of the channel was replaced by phenylalanine, tyrosine, or glutamic acid to occlude the channel opening and restrict the docking of ligands to test its functional role in the binding of ox-LDL. The synthesis, intracellular processing, and cellular distribution of all mutants were identical to those of wild type, whereas there was a marked decrease in the ability of the mutants to bind ox-LDL. These studies suggest that the central hydrophobic tunnel that extends through the entire LOX-1 molecule is a key functional domain of the receptor and is critical for the recognition of modified LDL.
Highlights
Lectin-like oxidized LDL receptor-1 (LOX-1) is a type-II transmembrane protein that belongs to the C-type lectin family of molecules
The increased binding of oxidized LDL (ox-LDL) to cells expressing LOX-1 led to a 1.6-fold accumulation of cholesteryl esters when compared with control cells (75.9 6 6.4 ng/105 cells and 48.4 6 12.7 ng/105 cells, n 5 4, P, 0.005, for LOX-1-transfected and wild-type cells, respectively; Fig. 1C)
To assess the role that the hydrophobic tunnel plays in the recognition and binding of ox-LDL, we focused on isoleucine 149 (I149), which forms the portal to the channel, with its side chain pointing to the empty space in the center of the tunnel (Fig. 3)
Summary
Lectin-like oxidized LDL (ox-LDL) receptor-1 (LOX-1) is a type-II transmembrane protein that belongs to the C-type lectin family of molecules. LOX-1 acts as a cell surface endocytosis receptor and mediates the recognition and internalization of ox-LDL by vascular endothelial cells. In an effort to elucidate the functional domains responsible for the binding of ox-LDL to the receptor, a series of site-directed mutants were designed using computer modeling and X-ray crystallography to study the functional role of the hydrophobic tunnel present in the LOX-1 receptor. The synthesis, intracellular processing, and cellular distribution of all mutants were identical to those of wild type, whereas there was a marked decrease in the ability of the mutants to bind ox-LDL These studies suggest that the central hydrophobic tunnel that extends through the entire LOX-1 molecule is a key functional domain of the receptor and is critical for the recognition of modified LDL.—Francone, O. Site-directed mutagenesis demonstrated that the conserved positively charged residues R208, R209, H226, R229, and R231 and the uncharged hydrophilic residues Q193, S198, S199, and N210 are involved in ligand binding [17, 18], suggesting that ligand recogni-
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