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The hydrophobic tunnel present in LOX-1 is essential for oxidized LDL recognition and binding

2008; Elsevier BV; Volume: 50; Issue: 3 Linguagem: Inglês

10.1194/jlr.m800474-jlr200

1539-7262

Omar L. Francone, Meihua Tu, Lori Royer, Jian Zhu, Kimberly A. Stevens, Joseph J. Oleynek, Zhiwu Lin, Lorraine Shelley, Thomas Sand, Yi Luo, Christopher D. Kane,

Microbial Metabolism and Applications

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 (I149) 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. 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 (I149) 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. Lectin-like oxidized LDL receptor-1 (LOX-1) is a member of the class E scavenger receptor family, a structurally diverse group of cell surface receptors of the innate immune system that recognize and internalize oxidized LDL (ox-LDL) in endothelial cells of large arteries (1Sawamura T. Kume N. Aoyama T. Moriwaki H. Hosikawa H. Aiba Y. Tanaka T. Miwa S. Katsura Y. Kita T. et al.An endothelial receptor for oxidized low-density lipoprotein-receptor in human atherosclerosis lesions.Nature. 1997; 386: 73-77Crossref PubMed Scopus (1156) Google Scholar). More recent studies have indicated that LOX-1 is expressed in other cells types, including macrophages (2Yoshida H. Kondratenko N. Green S. Steinberg D. Quehenberger O. Identification of the lectin-like receptor for oxidized low-density lipoprotein in human macrophages and its potential role as a scavenger receptor.Biochem. J. 1998; 334: 9-13Crossref PubMed Scopus (208) Google Scholar), vascular smooth-muscle cells (3Draude G. Hrboticky N. Lorenz R.L. The expression of the lectin-like oxidized low-density lipoprotein receptor (LOX-1) on human vascular smooth muscle cells and monocytes and its down-regulation by lovastatin.Biochem. Pharmacol. 1999; 57: 383-386Crossref PubMed Scopus (126) Google Scholar), and platelets (4Chen M. Kakutani M. Naruko T. Ueda M. Narumiya S. Masaki T. Sawamura T. Activation-dependent surface expression of LOX-1 in human platelets.Biochem. Biophys. Res. Commun. 2001; 282: 153-158Crossref PubMed Scopus (133) Google Scholar). Its expression is not constitutive, but rather, markedly induced by proinflammatory, oxidative, and mechanical stimuli (5Kume N. Murase T. Moriwaki H. Aoyama T. Sawamara T. Masaki T. Kita T. Inducible expression of lectin-like oxidized LDL receptor-1 in vascular endothelial cells.Circ. Res. 1998; 83: 322-327Crossref PubMed Scopus (277) Google Scholar, 6Murase T. Kume N. Korenaga R. Ando J. Sawamara T. Masaki T. Kita T. Fluid shear stress transcriptionally induces lectin-like oxidized LDL receptor-1 in vascular endothelial cells.Circ. Res. 1998; 83: 328-333Crossref PubMed Scopus (187) Google Scholar), which leads to the activation of endothelial cells, transformation of smooth-muscle cells, and accumulation of lipids in macrophages, resulting in cellular injury and the development of atherosclerosis. Studies in animal models have provided further evidence in support of a role for LOX-1 in atherosclerosis. Overexpression of LOX-1 in mice leads to the formation of atheroma-like lesion areas (7Inoue K. Arai Y. Kurihara H. Kita T. Sawamara T. Overexpression of lectin-like oxidized low-density lipoprotein receptor-1 induces intramyocardial vasculopathy in apolipoprotein E-null mice.Circ. Res. 2005; 97: 176-184Crossref PubMed Scopus (155) Google Scholar). Conversely, its deletion sustains endothelial function and confers protection in the development of atherosclerosis in association with decreased inflammatory and pro-oxidant markers (8Mehta J.L. Sanada N. Hu C.P. Chen J. Dandapat A. Sugawara F. Satoh H. Inoue K. Kawase Y. Jishage K. et al.Deletion of LOX-1 reduces atherogenesis in LDLR knockout mice fed high cholesterol diet.Circ. Res. 2007; 100: 1634-1642Crossref PubMed Scopus (372) Google Scholar). Finally, human genetic studies strengthen the role of this receptor in cardiovascular disease (9Tatsuguchi M. Furutani M. Hinagata J. Tanaka T. Furutani Y. Imamura S. Kawana M. Masaki T. Kasanuki H. Sawamura T. et al.Oxidized LDL receptor gene (ORL1) is associated with the risk of myocardial infarction.Biochem. Biophys. Res. Commun. 2003; 303: 247-250Crossref PubMed Scopus (89) Google Scholar, 10Mango R. Biocca S. del Vecchio F. Clementi F. Sangiuolo F. Amati F. Filareto A. Grelli S. Spitalieri P. Filesi I. et al.In vivo and in vitro studies support that a new splicing isoform of OLR1 gene is protective against acute myocardial infarction.Circ. Res. 2005; 97: 152-158Crossref PubMed Scopus (103) Google Scholar–11Puccetti L. Pasqui A.L. Bruni F. Pastorelli M. Ciani F. Palazzuoli A. Pontani A. Ghezzi A. Auteri A. Lectin-like oxidized LDL receptor-1 (LOX-1) polymorphisms influence cardiovascular events rate during statin treatment.Int. J. Cardiol. 2006; 119: 41-47Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar).LOX-1 is a disulfide-linked homodimeric type II transmembrane protein with a short 34-residue cytoplasmic tail, a single transmembrane domain, and an extracellular region consisting of an 80-residue domain predicted to be a coil followed by a 130-residue C-terminal C-type lectin-like domain (CTLD) responsible for ox-LDL recognition (12Chen M. Inoue K. Narumiya S. Masaki T. Sawamura T. Requirements of basic amino acids residues within the lectin-like domain of LOX-1 for the binding of oxidized low-density lipoprotein.FEBS Lett. 2001; 499: 215-219Crossref PubMed Scopus (45) Google Scholar, 13Chen M. Narumiya S. Masaki T. Sawamura T. Conserved C-terminal residues within the lectin-like domain of LOX-1 are essential for oxidized low-density-lipoprotein binding.Biochem. J. 2001; 355: 289-296Crossref PubMed Scopus (0) Google Scholar–14Shi X. Ogawa S. Otani T. Machida S. Involvement of conserved hydrophobic residues in the CTLD of human lectin-like oxidized LDL receptor in ligand binding.Mol. Cell Biol. Res. Commun. 2001; 4: 292-298Crossref PubMed Scopus (4) Google Scholar). Homodimers are formed via an interchain disulfide bond between Cys140 residues (15Xie Q. Matsunaga S. Niimi S. Ogawa S. Tokuyasu K. Sakakibara Y. Machida S. Human lectin-like oxidized low-density lipoprotein receptor-1 functions as a dimer in living cells.DNA Cell Biol. 2004; 23: 111-117Crossref PubMed Scopus (47) Google Scholar) that promotes noncovalent interactions leading to multimerization and ox-LDL binding facilitated by the neck region (16Ishigaki T. Ohki I. Utsunomiya-Tate N. Tate S.I. Chimeric structural stabilities in the coiled-coil structure of the NECK domain in human lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1).J. Biochem. 2007; 141: 855-866Crossref PubMed Scopus (23) Google Scholar) and receptor density at the plasma membrane (17Matsunaga S. Xie Q. Kumano M. Niimi S. Sekizawa K. Sakakibara Y. Komba S. Machida S. Lectin-like oxidized low-density lipoprotein receptor (LOX-1) functions as an oligomer and oligomerization is dependent on receptor density.Exp. Cell Res. 2007; 313: 1203-1214Crossref PubMed Scopus (27) Google Scholar).The structural domains within LOX-1 involved in the substrate recognition and binding remain unknown. Site-directed mutagenesis and X-ray crystallography (18Park H. Adsit F.G. Boyington J.C. The 1.4 angstrom crystal structure of the human oxidized low density lipoprotein receptor lox-1.J. Biol. Chem. 2005; 280: 13593-13599Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 19Ohki I. Ishigaki T. Oyama T. Matsunaga S. Xie Q. Ohsishi-Kameyama M. Murata T. Tsuchiya D. Machida S. Morikawa K. et al.Crystal structure of human lectin-like, oxidized low-density lipoprotein receptor 1 ligand binding domain and its ligand recognition mode to oxLDL.Structure. 2005; 13: 905-917Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar) have provided important clues as to how this receptor might interact with ox-LDL. 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 (17Matsunaga S. Xie Q. Kumano M. Niimi S. Sekizawa K. Sakakibara Y. Komba S. Machida S. Lectin-like oxidized low-density lipoprotein receptor (LOX-1) functions as an oligomer and oligomerization is dependent on receptor density.Exp. Cell Res. 2007; 313: 1203-1214Crossref PubMed Scopus (27) Google Scholar, 18Park H. Adsit F.G. Boyington J.C. The 1.4 angstrom crystal structure of the human oxidized low density lipoprotein receptor lox-1.J. Biol. Chem. 2005; 280: 13593-13599Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), suggesting that ligand recognition is dependent on electrostatic interaction and tridimensional conformation, which involves hydrophobic residues (14Shi X. Ogawa S. Otani T. Machida S. Involvement of conserved hydrophobic residues in the CTLD of human lectin-like oxidized LDL receptor in ligand binding.Mol. Cell Biol. Res. Commun. 2001; 4: 292-298Crossref PubMed Scopus (4) Google Scholar, 19Ohki I. Ishigaki T. Oyama T. Matsunaga S. Xie Q. Ohsishi-Kameyama M. Murata T. Tsuchiya D. Machida S. Morikawa K. et al.Crystal structure of human lectin-like, oxidized low-density lipoprotein receptor 1 ligand binding domain and its ligand recognition mode to oxLDL.Structure. 2005; 13: 905-917Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). Truncation at the utmost C-terminal domain (13Chen M. Narumiya S. Masaki T. Sawamura T. Conserved C-terminal residues within the lectin-like domain of LOX-1 are essential for oxidized low-density-lipoprotein binding.Biochem. J. 2001; 355: 289-296Crossref PubMed Scopus (0) Google Scholar) and mutations of basic amino acid residues located between the third and fourth cysteine of the CTLD confirmed that the binding of ox-LDL requires the interaction between basic arginine residues and negatively charged domains within ox-LDL.The crystal structure of LOX-1 has confirmed the findings from mutagenesis studies and provided new and important insights into the domains involved in the recognition of ox-LDL (18Park H. Adsit F.G. Boyington J.C. The 1.4 angstrom crystal structure of the human oxidized low density lipoprotein receptor lox-1.J. Biol. Chem. 2005; 280: 13593-13599Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 19Ohki I. Ishigaki T. Oyama T. Matsunaga S. Xie Q. Ohsishi-Kameyama M. Murata T. Tsuchiya D. Machida S. Morikawa K. et al.Crystal structure of human lectin-like, oxidized low-density lipoprotein receptor 1 ligand binding domain and its ligand recognition mode to oxLDL.Structure. 2005; 13: 905-917Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). As expected from the sequence, the CTLD monomer is composed of two antiparallel B-sheets flanked by two α helices stabilized by three conserved intrachain disulfide bonds. The disulfide bridge between Cys140 present in monomers, together with the conserved W150 side chain, are key to the LOX-1 structure, and function by contributing to the formation of a dimer and favoring proper folding. The extracellular CTLD domain forms a heart-shaped homodimer with a ridge of six basic arginine residues extending diagonally across the apolar top of molecule. These residues, often referred to as the basic spine, play a key biological role in the binding of ox-LDL, as demonstrated by site-directed mutagenesis.Interestingly, the X-ray structure of the LOX-1 dimer also reveals the presence of a central hydrophobic tunnel that extends through the entire molecule. This tunnel, first discovered by Park, Adsit, and Boyington (18Park H. Adsit F.G. Boyington J.C. The 1.4 angstrom crystal structure of the human oxidized low density lipoprotein receptor lox-1.J. Biol. Chem. 2005; 280: 13593-13599Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), is ∼7–8 Å in diameter and ∼380 A3 in volume. Interestingly, the volume and architecture of this channel are sufficient to accommodate the components of LDL, such as cholesterol, long-chain FAs, or possibly a nonpolar peptide. Hydrogen bonds to the main-chain residues F158, D147, A194, and Y197, and to side-chain residues I149 and Y197 determine the conformation of the central channel and restrict its opening to 4 Å. Although the topology of the channel suggests a prominent role in substrate binding and recognition, no study to date has addressed the significance and biological relevance of the portal to the binding of ox-LDL. Therefore, we attempted to gain insight into this key structural domain of LOX-1 protein, replacing I149 with structurally distinct amino acids to assess the functional significance of the hydrophobic tunnel in the binding of ox-LDL.MATERIALS AND METHODSLipoprotein and ligand preparationHuman LDL (1.019 to 1.063 g/ml) and HDL (1.063 to 1.21 g/ml) were isolated from the plasma of healthy subjects by sequential ultracentrifugation at 15°C. Ox-LDL and ox-HDL were purchased from Intracell, Frederik, MD. The degree of oxidation was determined by measuring the amount of thiobarbituric acid-reactive substances (TBARS). Values for TBARS in ox-LDL, ox-HDL, and native LDL were 17.0 nM, 5.2 nM, and 0.01 nM malonyldialdehyde/mg protein, respectively. Ox-LDL was biotinylated following the ECL protein biotinylation procedure (Amersham Biosciences, Piscataway, NJ) recommended by the manufacturer.Cloning of human LOX-1 and generation of I149 mutantsA full-length human LOX-1 cDNA was cloned by RT-PCR using specific 5′ and 3′ terminal primers to the published LOX-1 cDNA sequence (accession number AB010710) and subsequently cloned into the BamHI/NotI site of pcDNA5/FRT (Invitrogen, Carlsbad, CA). An additional expression vector was built to attach a V5-His epitope to the LOX-1 cDNA for detection by inserting the entire LOX-1 cDNA from the start codon to the 3′ end without the endogenous stop codon into pcDNA5/FRT/V5-His-Topo from Invitrogen.Site-directed mutagenesis was conducted using the Quick-Change Site-Directed Mutagenesis Kit (Strategene, La Jolla, CA) according to the manufacturer's protocol. Synthetic oligonucleotides 30–33 bases long carrying the mismatched bases were used to mutagenize nucleotides and replace isoleucine 149 (I149) with phenylalanine (F), tyrosine (Y), or glutamic acid (E) using pcDNA5/FRT.hsLox-1 as a template. Full-length sequence confirmation of wild-type and mutants was done by dideoxynucleotide sequencing.Transfection of wild-type and LOX-1 mutant cDNA into Chinese hamster ovary cellsChinese hamster ovary (CHO) cells were maintained in DMEM/F12 medium (Gibco, Carlsbad, CA) with 10% FBS. pcDNA5 plasmids containing full-length wild-type or mutant human LOX-1 cDNAs were transfected into ChoFlp-in by using Lipofectamine according to the manufacturer's instructions. ChoFlp-in cells stably expressing human LOX-1 were maintained in DMEM/F12 with 10% FBS supplemented with 200 μg/ml of Hygomycin B (Invitrogen).Immunofluorescent staining and confocal microscopyFluorescence microscopy of wild-type and I149 LOX-1 mutants was carried out in cells grown to 50% confluency in chamber culture slides (BD Biosciences, San Jose, CA) maintained in DMEM/F12 medium. The media was removed and cells were washed twice in PBS and then fixed for 5 min in 100% methanol, washed five times with PBS, and blocked for 20 min at room temperature in PBS containing 10% FBS. Blocking solution was removed, and anti-V5-FITC-conjugated antibody (Invitrogen) in PBS/10% FBS was added overnight at a 1:500 dilution in the dark at 4°C. Finally, cells were washed twice with PBS and observed using confocal microscopy.Preparation and isolation of plasma membranesThe isolation of plasma membranes from wild-type and ChoFlp-in cells expressing LOX-1 was conducted according to the protocol described in the literature (20Nagamatsu S. Kornhauser J.M. Burant C.F. Seino S. Mayo K.E. Bell G.I. Glucose transporter expression in brain. cDNA sequence of mouse GLUT3, the brain facilitative glucose transporter isoform, and identification of sites of expression by in situ hybridization.J. Biol. Chem. 1992; 267: 467-472Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were grown until 70% confluency in DMEM/F12 medium containing 10% FBS, harvested, and homogenized in ice-cold HEPES medium containing 1 mM EDTA and protease inhibitors. Sucrose was added to a final concentration of 200 mM. The homogenate was centrifuged at 900 g for 5 min at 4°C, and the resulting supernatant was centrifuged at 110,000 g for 1 h at 4°C. The membrane pellets were washed with PBS containing 5 mM CaCl2, and aliquots were kept frozen at −80°C. The concentration of the solution of solubilized membrane proteins was determined using a Bio-Rad protein assay kit.Binding of wild-type and mutant LOX-1 to ox-LDLBinding of ox-LDL to wild-type and mutant LOX-1 receptors was carried out using the Meso Scale Discovery (MSD) technology platform utilizing a Sector Imager 6000 (Meso Scale Discovery, Gaithersburg, MD). The MSD platform is essentially an immunoassay that utilizes electrochemiluminescence to measure binding. Three micrograms of cell membrane (in 30 μl PBS) was added to each well of a 96-well, high-bind MSD plate (MSD L11XB-1) and incubated at room temperature with shaking for 1 h. Plates were then blocked for 1 h at room temperature with 50 μl PBS containing 3% BSA (essential FA-free; Sigma, St. Louis, MO) and 200 μg/ml of human LDL. After the blocking step, plates were washed twice with PBS. Binding buffer (Kreb's Ringer phosphate buffer plus 0.5% FA-free BSA) containing 30 μl/well of biotin-labeled human ox-LDL (10 μg/ml) and varied amounts of unlabeled ox-LDL (Intracel) were then added, and plates were incubated at 4°C, overnight. The next morning, the plates were washed five times with PBS, after which 30 μl/well of ruthenylated streptavidin (SA, MSD SULFO-TAG Streptavidin R32AD-1, and diluted 1:1,000 in MSD antibody diluent) was added to the wells for 1 h at room temperature. Plates were then washed five times with PBS. Read buffer (150 μl, MSD R92TC-3) was added to each well, and plates were read on a Sector Imager 6000. The Sector Imager 6000 applies an electric potential to each well, inducing emitted light (electrochemiluminescence) proportionate to the amount of ruthenylated streptavidin/biotin-labeled human ox-LDL/receptor complex.1-Palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine, 1-O-hexadecyl-2-azelaoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine, and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine were purchased from Avanti Polar Lipids (Alabaster, AL), dried under nitrogen, resuspended in chloroform-methanol-water (65:25:4), and further diluted in binding buffer containing biotin-labeled ox-LDL to achieve the desired final concentration. Their ability to block the binding of ox-LDL to LOX-1 was examined as described above.Structure prediction and molecular modelingThe wild-type LOX-1 protein structure was downloaded from Protein Data Bank (PDB ID, 1YPQ). The structures of the mutant LOX-1 proteins (I149F, I149Y, and I149E) were built using the Maestro molecular modeling package (Schrodinger, Inc., New York, NY). Nonpolar surface area and residue volume were obtained from the literature (21Karplus P.A. Hydrophobicity regained.Protein Sci. 1997; 6: 1302-1307Crossref PubMed Scopus (189) Google Scholar). The interactions between oxidized phospholipids and LOX-1 were modeled using Gold3.0 software (CCDC, Cambridge, United Kingdom). To prevent bias toward any particular binding site and binding mode, we defined the binding site as 20 Å around the dioxane molecule in the 1YPQ structure. The defined binding site was large enough to cover almost all of the protein surface. We also defined the number of genetic algorithm runs in GOLD as 1,000, because of the flexibility of the oxidized phospholipids.RESULTSCloning and expression of wild-type human LOX-1Expression of wild-type human LOX-1 was achieved by transfecting a full-length human LOX cDNA into ChoFlp-in cells as described in Materials and Methods. Cell surface expression of functional LOX-1 protein was demonstrated by the increased binding to labeled ox-LDL (Fig. 1A). Binding of labeled ox-LDL was displaced in the presence of a 50-fold excess of cold, unlabeled oxLDL (Fig. 1B). The increased binding of ox-LDL to cells expressing LOX-1 led to a 1.6-fold accumulation of cholesteryl esters when compared with control cells (75.9 ± 6.4 ng/105 cells and 48.4 ± 12.7 ng/105 cells, n = 4, P < 0.005, for LOX-1-transfected and wild-type cells, respectively; Fig. 1C). Cellular levels of unesterified cholesterol were not significantly changed, consistent with the tight regulation of cholesterol levels in mammalian cells.A membrane binding assay was developed and optimized to assess the binding properties and substrate specificity of the human LOX-1 receptor. In agreement with the literature, incubation of ox-LDL with plasma membranes isolated from cells expressing LOX-1 demonstrated a dose-response relationship between human LOX-1 and ox-LDL. Binding of labeled ox-LDL was displaced by using increasing amounts of cold, unlabeled ox-LDL, acetylated-LDL, and ox-HDL (Fig. 2). In contrast, no inhibitory effect was observed using native HDL (Fig. 2) or native LDL (data not shown).Fig. 2Effects of an excess amount of HDL, ox-LDL, ox-HDL, and acetylated LDL on ox-LDL binding to LOX-1. Plasma membranes isolated from LOX-1-expressing cells were incubated with biotin-labeled ox-LDL in the presence or absence of excess amounts of HDL, ox-LDL, ox-HDL, and acetylated LDL, as described in Materials and Methods. Each data point represents the average of three independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Computational properties of I149-LOX-1 mutantsTo assess the role that the hydrophobic tunnel plays in the recognition and binding of ox-LDL, we focused on 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). A series of single point mutations were designed using computational modeling to replace the isoleucine residue in position 149 with phenylalanine, tyrosine, or glutamic acid. These new residues were predicted to block the channel entrance because of their increased side-chain volume (F149 and Y149) or polarity (E149) without destabilizing the protein (Table 1and Fig. 3). The structures of the mutants (I149F, I149Y, and I149E) were built using Maestro software, and the mutated residue side chains were optimized by Prime module. The final structures were checked by the Protein Report module in Maestro, and no steric clashes or improper torsions were found, suggesting that minimal, if any, disturbance of the overall tridimensional structure of the protein occurred, as supported by energy calculations in wild-type and mutants (Table 1).Fig. 3Hydrophobic tunnel structure in wild-type and I149-LOX-1 mutants. The protein structure of the native LOX-1 receptor was downloaded from Protein Data Bank (PDB ID, 1YPQ). The three mutant protein structures (I149F, I149Y, and I149E) were built using the Maestro molecular modeling package. Chain A of the dimer is shown in magenta, and chain B is shown in yellow.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TABLE 1Changes in residue nonpolar surface area, volume, and energy introduced by site-directed mutagenesis of I149Residue PropertiesLOX-1 EnergyNonpolar Surface AreaVolumeKcal/molA2A3I149Hydrophobic; nonpolar–10,703155167I149 → FHydrophobic; nonpolar–10,710194190I149 → YHydrophobic; polar–10,729154194I149 → ENonhydrophobic; polar–10,81769138LOX-1, lectin-like oxidized LDL receptor-1. LOX-1 energy calculations were made using Maestro software with optimized potential for liquid simultations force field. Nonpolar surface area and residue volume were obtained as described in Materials and Methods. Open table in a new tab Synthesis and intracellular processing of I149-LOX-1 mutantsStably transfected cells expressing either the wild-type or I149-LOX-1 mutants were selected and grown to assess whether the expression and cellular trafficking of LOX-1 was impaired by the mutations. Cell lysates from cells expressing wild-type and I149-LOX-1 mutants were subjected to PAGE, followed by immunoblotting to visualize either LOX-1 or actin. Actin was used as an internal standard to ensure equal protein loading. As shown in Fig. 4A, wild-type and mutant forms of LOX-1 were expressed at the same levels and have molecular weights similar to that of wild-type LOX-1. In addition, I149-LOX-1 mutants maintain the ability to form dimers and retain the same molecular weight as wild-type LOX-1, as demonstrated using nondenaturing gradient gel electrophoresis (Fig. 4B).Fig. 4Expression of wild-type and I149-LOX-1 mutants. Cells expressing wild-type and mutant forms of LOX-1 were grown in DMEM/F12 tissue culture medium as described in Materials and Methods. A: Detection of wild-type and mutants forms of LOX-1. Cell lysates were fractionated by SDS-PAGE under reducing conditions and visualized by Western blotting using a rabbit anti-human LOX-1 or actin antibody. B: Detection of disulfite-linked dimeric forms of LOX-1. Cell lysates prepared from cells expressing wild-type and LOX-1 mutants were mixed with SDS sample buffer in the absence of β-mercaptoethanol and subjected to SDS-PAGE under nonreducing conditions. LOX-1 dimers were visualized by Western blot analysis using a rabbit anti-LOX-1 antibody. The molecular masses (in kDa) of protein standards are indicated.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To examine the cellular topology of wild-type and mutant LOX-1 proteins, a V5 tag was fused to the 3′ terminus of LOX-1 that greatly facilitated the visualization and detection of the LOX-1 protein without affecting the normal cellular distribution of the protein. The cellular distribution of I149-LOX-1 mutants did not differ from that of wild-type LOX-1. Although no signal was observed in untransfected cells (insert, Fig. 5), cells expressing LOX-1 displayed a prominent fluorescence at the plasma membrane. These findings are consistent with the literature and the established role that this protein plays as a cell surface scavenger receptor. Taken together, the findings from our studies indicate that the I149-LOX-1 mutants appear to be synthesized and topologically distributed in the same way as wild-type LOX-1. This suggests that these mutants share the same properties as wild-type LOX-1 and constitute an adequate model for conducting structure-function relationship studies.Fig. 5Fluorescence microscopy of ChoFlp-in cells transfected with wild-type and I149-LOX-1 mutants. Fluorescence microscopy of wild-type and I149-LOX-1 mutants was carried out in cells grown to 50% confluency in chamber culture slides maintained in DMEM/F12 medium as described in Materials and Methods.View Large Image Figure ViewerDownload Hi-res