Requirements of basic amino acid residues within the lectin-like domain of LOX-1 for the binding of oxidized low-density lipoprotein.

Lectin-like OxLDL receptor-1 (LOX-1) was identified as the major receptor for oxidized low-density lipoprotein (OxLDL) in aortic endothelial cells. LOX-1 is a type II membrane protein that structurally belongs to the C-type lectin family. Here, we found that the lectin-like domain of LOX-1 is essential for ligand binding, but the neck domain is not. In particular, the large loop between the third and fourth cysteine of the lectin-like domain plays a critical role for OxLDL binding as well as C-terminal end residues. Alanine-directed mutagenesis of the basic amino acid residues around this region revealed that all of the basic residues are involved in OxLDL binding. Simultaneous mutations of these basic residues almost abolished the OxLDL-binding activity of LOX-1. Electrostatic interaction between basic residues in the lectin-like domain of LOX-1 and negatively charged OxLDL is critical for the binding activity of LOX-1.     

Structural implication for the impaired binding of W150A mutant LOX-1 to oxidized low density lipoprotein, OxLDL

Lectin-like oxidized lipoprotein (OxLDL) receptor 1, LOX-1, is the major OxLDL receptor expressed on vascular endothelial cells. We have previously reported the ligand-recognition mode of LOX-1 based on the crystal structure of the ligand binding domain (C-type lectin-like domain, CTLD) and surface plasmon resonance analysis, which suggested that the functional significance of the CTLD dimer (the 'canonical' dimer) is to harbor the characteristic "basic spine" on its surface. In this study, we have identified the key inter-domain interactions in retaining the canonical CTLD dimer by X-ray structural analysis of the inactive mutant W150A CTLD. The canonical CTLD dimer forms through tight hydrophobic interactions, in which W150 engages in a lock-and-key manner and represents the main interaction. The loss of the Trp ring by mutation to Ala prevents the formation of the canonical dimer, as elucidated from docking calculations using the crystal structure of W150A CTLD. The results emphasize that the canonically formed CTLD dimer is essential for LOX-1 to bind to OxLDL, which supports our proposed view that the basic spine surface present in the correctly formed dimer plays a primal role in OxLDL recognition. This concept provides insight into the pathogenic pattern recognized by LOX-1 as a member of the pattern recognition receptors.     

Crystal structure of human lectin-like, oxidized low-density lipoprotein receptor 1 ligand binding domain and its ligand recognition mode to OxLDL

Lectin-like, oxidized low-density lipoprotein (LDL) receptor 1, LOX-1, is the major receptor for oxidized LDL (OxLDL) in endothelial cells. We have determined the crystal structure of the ligand binding domain of LOX-1, with a short stalk region connecting the domain to the membrane-spanning region, as a homodimer linked by an interchain disulfide bond. In vivo assays with LOX-1 mutants revealed that the "basic spine," consisting of linearly aligned arginine residues spanning over the dimer surface, is responsible for ligand binding. Single amino acid substitution in the dimer interface caused a severe reduction in LOX-1 binding activity, suggesting that the correct dimer arrangement is crucial for binding to OxLDL. Based on the LDL model structure, possible binding modes of LOX-1 to OxLDL are proposed.     

Chaperonin-containing TCP-1 complex directly binds to the cytoplasmic domain of the LOX-1 receptor

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) is a scavenger receptor that binds oxidized low-density lipoprotein (OxLDL) and has a role in atherosclerosis development. The N-terminus intracellular region (cytoplasmic domain) of LOX-1 mediates receptor internalization and trafficking, potentially through intracellular protein interactions. Using affinity isolation, we identified 6 of the 8 components of the chaperonin-containing TCP-1 (CCT) complex bound to LOX-1 cytoplasmic domain, which we verified by coimmunoprecipitation and immunostaining in human umbilical vein endothelial cells. We found that the interaction between CCT and LOX-1 is direct and ATP-dependent and that OxLDL suppressed this interaction. Understanding the association between LOX-1 and the CCT complex may facilitate the design of novel therapies for cardiovascular disease.     

Identification of 4-hydroxy-2-nonenal-histidine adducts that serve as ligands for human lectin-like oxidized LDL receptor-1

LOX-1 (lectin-like oxidized low-density lipoprotein receptor-1) is an endothelial scavenger receptor that is important for the uptake of OxLDL (oxidized low-density lipoprotein) and contributes to the pathogenesis of atherosclerosis. However, the precise structural motifs of OxLDL that are recognized by LOX-1 are unknown. In the present study, we have identified products of lipid peroxidation of OxLDL that serve as ligands for LOX-1. We used CHO (Chinese-hamster ovary) cells that stably express LOX-1 to evaluate the ability of BSA modified by lipid peroxidation to compete with AcLDL (acetylated low-density lipoprotein). We found that HNE (4-hydroxy-2-nonenal)-modified proteins most potently inhibited the uptake of AcLDL. On the basis of the findings that HNE-modified BSA and oxidation of LDL resulted in the formation of HNE-histidine Michael adducts, we examined whether the HNE-histidine adducts could serve as ligands for LOX-1. The authentic HNE-histidine adduct inhibited the uptake of AcLDL in a dose-dependent manner. Furthermore, we found the interaction of LOX-1 with the HNE-histidine adduct to have a dissociation constant of 1.22×10(-8) M using a surface plasmon resonance assay. Finally, we showed that the HNE-histidine adduct stimulated the formation of reactive oxygen species and activated extracellular-signal-regulated kinase 1/2 and NF-κB (nuclear factor κB) in HAECs (human aortic endothelial cells); these signals initiate endothelial dysfunction and lead to atherosclerosis. The present study provides intriguing insights into the molecular details of LOX-1 recognition of OxLDL.     

Surface plasmon resonance study on functional significance of clustered organization of lectin-like oxidized LDL receptor (LOX-1)

Lectin-like oxidized low-density lipoprotein (OxLDL) receptor 1 (LOX-1) is the major OxLDL receptor of vascular endothelial cells and is involved in an early step of atherogenesis. LOX-1 exists as a disulfide-linked homodimer on the cell surface, which contains a pair of the ligand-binding domains (CTLD; C-type lectin-like domain). Recent research using living cells has suggested that the clustered state of LOX-1 dimer on the cell is functionally required. These results questioned how LOX-1 exists on the cell to achieve OxLDL binding. In this study, we revealed the functional significance of the clustered organization of the ligand-binding domain of LOX-1 with surface plasmon resonance. Biotinylated CTLD was immobilized on a streptavidin sensor chip to make CTLD clusters on the surface. In this state, the CTLD had high affinity for OxLDL with a dissociation constant (K(D)) in the nanomolar range. This value is comparable to the K(D) measured for LOX-1 on the cell. In contrast, a single homodimeric LOX-1 extracellular domain had lower affinity for OxLDL in the supra-micromolar range of K(D). Monomeric CTLD showed marginal binding to OxLDL. In combination with the analyses on the loss-of-binding mutant W150A, we concluded that the clustered organization of the properly formed homodimeric CTLD is essential for the strong binding of LOX-1 to OxLDL.     

The LOX-1 scavenger receptor cytoplasmic domain contains a transplantable endocytic motif

Oxidized low-density lipoprotein particles is a pro-atherogenic factor implicated in atherosclerotic plaque formation. The LOX-1 scavenger receptor binds OxLDL and is linked to atherosclerotic plaque initiation and progression. We tested the hypothesis that the LOX-1 cytoplasmic domain contains a transplantable signal for membrane protein endocytosis. Structural modeling of the LOX-1 cytoplasmic domain reveals that a tripeptide motif (DDL) implicated in LOX-1 endocytosis is part of a curved β-pleated sheet structure. The two aspartic acid residues within this structural model are highly solvent-accessible enabling recognition by cytosolic factor(s). A triple alanine substitution of the DDL motif within the LOX-1 scavenger receptor substantially reduced endocytosis of OxLDL. Transplantation of the LOX-1 cytoplasmic domain into a transferrin receptor reporter molecule conferred efficient endocytosis on this hybrid protein. Mutation of the DDL motif within the hybrid LOX-1-TfR protein also substantially reduced receptor-mediated endocytosis. Thus a transplantable endocytic motif within the LOX-1 cytoplasmic domain is needed to ensure efficient internalization of pro-atherogenic OxLDL particles.     

Oxidized low density lipoprotein impairs endothelial progenitor cells by regulation of endothelial nitric oxide synthase

Oxidized low density lipoprotein (OxLDL) is one of the most important risk factors of cardiovascular disease. Here, we study the impact of OxLDL on endothelial progenitor cells (EPCs) and determine whether OxLDL affects EPCs by an inhibitory effect on endothelial nitric oxide synthase (eNOS). It was found that OxLDL decreased EPC survival and impaired its adhesive, migratory, and tube-formation capacities in a dose-dependent manner. However, all of the detrimental effects of OxLDL were attenuated by pretreatment of EPCs with lectin-like oxidized low density lipoprotein receptor (LOX-1) monoclonal antibody or l-arginine. Western blot analysis revealed that OxLDL dose-dependently decreased Akt phosphorylation and eNOS protein expression and increased LOX-1 protein expression. Furthermore, OxLDL caused a decrease in eNOS mRNA expression and an increase in LOX-1 mRNA expression. These data indicate that OxLDL inhibits EPC survival and impairs its function, and this action is attributable to an inhibitory effect on eNOS.     

Asymmetric dimethylarginine upregulates LOX-1 in activated macrophages: role in foam cell formation

Intimal infiltration by monocytes and accumulation of lipids represent a critical step in the formation of fatty streaks during atherogenesis. Because elevated plasma levels of asymmetric dimethylarginine (ADMA), a potent nitric oxide (NO) synthase (NOS) inhibitor, are prevalent in diverse cardiovascular diseases, the goal of this study was to examine the contribution of NO deficiency to macrophage lipid accumulation. Inhibition of NO synthesis in PMA-primed human monocytic leukemia HL-60 cells resulted in a twofold increase in expression of the receptor for oxidized LDL (OxLDL), termed the lectin-like OxLDL receptor (LOX-1). Blockade of inducible NOS in activated macrophages resulted in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-OxLDL accumulation and imparted macrophages with a foamy appearance as detected with oil-red O lipid staining. ADMA (15 microM) or N(G)-nitro-l-arginine methyl ester (l-NAME, 300 microM), both of which suppress inducible NOS activity, increased oil-red staining 1.9- and 2.8-fold, respectively. Macrophages treated with ADMA or l-NAME showed a 2.4-fold increase in accumulation of DiI-OxLDL. To examine the role of LOX-1 in this process, we used small interfering RNA (siRNA) duplex-mediated LOX-1 gene silencing. LOX-1 expression was suppressed twofold by siRNA as shown by Western blot analysis. This suppression was associated with a two- to fourfold decrease in DiI-OxLDL uptake as identified by fluorescence microscopy and decreased oil-red O staining by activated macrophages. In conclusion, accumulation of ADMA (a competitive inhibitor of NOS) in patients with chronic renal failure may be responsible for upregulation of LOX-1 receptor and increased OxLDL uptake, thus contributing to lipidosis and foam cell formation. The data illustrate an additional nonendothelial mode of antiatherogenic action of NO: prevention of LOX-1 induction and lipid accumulation by macrophages.     

Low-density lipoproteins induce the renin-angiotensin system and their receptors in human endothelial cells.

Increased levels of low-density lipoproteins are well-established risk factors of endothelial dysfunction and the metabolic syndrome. In this study, we evaluated the effect of native low-density lipoprotein (nLDL) and oxidized LDL (oxLDL) on the expression of genes of the renin-angiotensin system (angiotensin-converting enzyme, ACE; angiotensin II type 1 receptor, AT(1)) and their receptors (low-density lipoprotein receptor: LDLR; lectin-like oxLDL receptor: LOX-1; toll-like receptor 4: TLR4) in primary cultures of human umbilical vein endothelial cells. ACE and AT(1) expressions were significantly increased after stimulation with nLDL and oxLDL. OxLDL receptor LOX-1 showed a maximum induction after 7 hours. Increased LOX-1 protein expression in response to oxLDL could be blocked by a LOX-1-specific antibody. TLR4 expression was increased by nLDL and oxLDL as well. We conclude that LDL and oxLDL can activate the renin-angiotensin system and their receptors LDLR, LOX-1, and TLR4 in human endothelial cells. These data suggest a novel link between hypercholesterolemia and hypertension in patients with the metabolic syndrome.     

Activation-dependent surface expression of LOX-1 in human platelets

Lectin-like oxidized LDL receptor-1 (LOX-1) was initially identified as an oxidized LDL receptor in aortic endothelial cells. Here we identified LOX-1 mRNA and protein in human platelets in addition to recent findings on the expression in macrophages and smooth muscle cells. The presence of LOX-1 was further confirmed in the megakaryocytic cell lines. Flow cytometric analyses revealed that LOX-1 was exposed on the surface of platelets in an activation-dependent manner. Consistently, the activation-dependent binding of OxLDL to platelets was mostly inhibited by anti-LOX-1 antibody. Immunohistochemistry of the atherosclerotic plaque from a patient with unstable angina pectoris (UAP) revealed accumulation of LOX-1 protein at the site of thrombus. As LOX-1 recognizes and binds activated platelets, exposure of LOX-1 on activated platelets surface might assist thrombosis formation.     

Molecular dynamics simulation of human LOX-1 provides an explanation for the lack of OxLDL binding to the Trp150Ala mutant

Background  

Dimeric lectin-like oxidized low-density lipoprotein receptor-1 LOX-1 is the target receptor for oxidized low density lipoprotein in endothelial cells. In vivo assays revealed that in LOX-1 the basic spine arginine residues are important for binding, which is lost upon mutation of Trp150 with alanine. Molecular dynamics simulations of the wild-type LOX-1 and of the Trp150Ala mutant C-type lectin-like domains, have been carried out to gain insight into the severe inactivating effect.     

Design of a novel LOX-1 receptor antagonist mimicking the natural substrate

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), the major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, is overexpressed in atherosclerotic lesions. LOX-1 specific inhibitors, urgently necessary to reduce the rate of atherosclerotic and inflammation processes, are not yet available. We have designed and synthesized a new modified oxidized phospholipid, named PLAzPC, which plays to small scale the ligand-receptor recognition scheme. Molecular docking simulations confirm that PLAzPC disables the hydrophobic component of the ox-LDL recognition domain and allows the interaction of the l-lysine backbone charged groups with the solvent and with the charged/polar residues located around the edges of the LOX-1 hydrophobic tunnel. Binding assays, in a cell model system expressing human LOX-1 receptors, confirm that PLAzPC markedly inhibits ox-LDL binding to LOX-1 with higher efficacy compared to previously identified inhibitors.     

Lectin-like oxidized LDL receptor-1 as extracellular chaperone receptor: its versatile functions and human diseases

Well-known coronary risk factors such as hyperlipidemia, hypertension, smoking, and diabetes are reported to induce the oxidative stress. Under the oxidative stress, low-density lipoprotein (LDL) is oxidatively modified in the vasculature, and formed oxidized LDL induces endothelial dysfunction, expression of adhesion molecules and apoptosis of vascular smooth muscle cells. It has become evident that these cellular responses induced by oxidized LDL are mediated by lectin-like oxidized LDL receptor-1 (LOX-1). LOX-1 was originally identified from cultured aortic endothelial cells as a receptor for oxidized LDL; however, recent investigations revealed that LOX-1 has diverse roles in the host-defense system and inflammatory responses, and it is involved in the pathogenesis of various diseases such as atherosclerosis-based cardiovascular diseases and septic shock. Beside oxidized LDL, LOX-1 recognizes multiple ligands including apoptotic cells, platelets, advanced glycation end products, bacteria, and heat shock proteins (HSPs). The HSPs function as a chaperone to affect protein folding of newly synthesized or denatured proteins. There are accumulating evidences that the HSPs released into the extracellular space have potent biological activities and it may work as a kind of cytokines. It is demonstrated that LOX-1 works as a receptor for HSP70, since it has high affinity for HSP70. The interaction of LOX-1 with HSP70 is involved in the cross-presentation of antigen. Given the potent and wide variety of biological activities, more understanding their interaction provides potential therapeutic strategy for various human diseases.     

Induction of the oxLDL receptor LOX-1 by endothelin-1 in human endothelial cells.

In this study, we analyzed the effect of endothelin-1 (ET-1) on expression of the lectin-like oxidized low-density lipoprotein (oxLDL) receptor-1 LOX-1 and on oxLDL uptake in primary cultures of human umbilical vein endothelial cells (HUVEC). LOX-1 mRNA was quantified by standard-calibrated competitive RT-PCR, LOX-1 protein expression by Western analysis and endothelial oxLDL uptake using DiI-labeled oxLDL. ET-1 induces LOX-1 mRNA expression, reaching its maximum after 1 h (160 +/- 14% of control, 100 nM ET-1, P < 0.05). This increased ET-1-mediated LOX-1 mRNA expression could be inhibited by endothelin receptor B antagonist BQ-788. In addition, ET-1 stimulates LOX-1 protein expression and oxLDL uptake in HUVEC. The augmented oxLDL uptake by ET-1 is mediated by endothelin receptor B, but not by protein kinases. These data support a new pathophysiological mechanism how locally and systemically increased ET-1 levels could promote LOX-1-mediated oxLDL uptake in human endothelial cells and the development and progression of endothelial dysfunction and atherosclerosis.     

LOX-1 supports adhesion of Gram-positive and Gram-negative bacteria

Adhesion of bacteria to vascular endothelial cells as well as mucosal cells and epithelial cells appears to be one of the initial steps in the process of bacterial infection, including infective endocarditis. We examined whether lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1), a member of scavenger receptor family molecules with C-type lectin-like structure, can support adhesion of Gram-positive and Gram-negative bacteria. Chinese hamster ovary-K1 (CHO-K1) cells stably expressing LOX-1 can support binding of FITC-labeled Staphylococcus aureus and Escherichia coli, which was suppressed by poly(I) and an anti-LOX-1 mAb. Adhesion of these bacteria to LOX-1 does not require divalent cations or serum factors and can be supported under both static and nonstatic conditions. Cultured bovine aortic endothelial cells (BAEC) can also support adhesion of FITC-labeled S. aureus, which was similarly suppressed by poly(I) and an anti-LOX-1 mAb. In contrast, binding of FITC-labeled E. coli to BAEC was partially inhibited by the anti-LOX-1 mAb, and poly(I) did not block FITC-labeled E. coli adhesion to BAEC, but, rather, enhanced it under a static condition. TNF-alpha increased LOX-1-dependent adhesion of E. coli, but not that of S. aureus, suggesting that S. aureus adhesion to BAEC may require additional molecules, which cooperate with LOX-1 and suppressed by TNF-alpha. Taken together, LOX-1 can work as a cell surface receptor for Gram-positive and Gram-negative bacteria, such as S. aureus and E. coli, in a mechanism similar to that of class A scavenger receptors; however, other unknown molecules may also be involved in the adhesion of E. coli to BAEC, which is enhanced by poly(I).     

LOX-1 expressed in cultured rat chondrocytes mediates oxidized LDL-induced cell death-possible role of dephosphorylation of Akt

The oxidative changes of lipids in cartilage proceed with ageing and with the grade of osteoarthritis. To clarify the role of oxidatively modified lipids in articular cartilage in osteoarthritis, here, we investigated lectin-like oxidized LDL receptor (LOX-1) in rat cultured articular chondrocytes. LOX-1 expression was detectable in basal culture condition and enhanced by the treatment of oxidized LDL and interleukin-1beta. DiI-labeled oxidized LDL was bound and ingested by chondrocytes via LOX-1. Oxidized LDL dose-dependently reduced chondrocyte viability, inducing non-apoptotic cell death, which was again suppressed by anti-LOX-1 antibody treatment. Oxidized LDL reduced the amount of phosphorylated Akt, a substrate of PI3 kinase via LOX-1. Consistently, the PI3 kinase inhibitor, LY294002, decreased cell viability dose-dependently, and the PI3 kinase activator, IGF-I, reversed the effect of oxidized LDL on the cell death. LOX-1 might be involved in the pathogenesis of osteoarthritis, inducing chondrocyte death through PI3 kinase/Akt pathway.     

LOX-1 augments oxLDL uptake by lysoPC-stimulated murine macrophages but is not required for oxLDL clearance from plasma

Oxidized LDL (oxLDL) promotes lipid accumulation as well as growth and survival signaling in macrophages. OxLDL uptake is mainly due to scavenger receptors SR-AI/II and CD36. However, other scavenger receptors such as lectin-like oxLDL receptor-1 (LOX-1) may also play a role. We used mice with targeted inactivation of the LOX-1 gene to define the role of this receptor in the uptake of oxLDL and in activation of survival pathways. There was no difference in uptake or degradation of ¹²⁵I-oxLDL in unstimulated macrophages from wild-type and LOX-1 knockout mice and no difference in the rate of clearance of oxLDL from plasma in vivo. However, when expression of LOX-1 was induced with lysophosphatidylcholine, oxLDL uptake and degradation increased 2-fold in wild-type macrophages but did not change in LOX-1 knockout macrophages. Macrophages lacking LOX-1 showed the same stimulation of PKB phosphorylation and enhancement of survival by oxLDL as wild-type cells. These data show that LOX-1 does not alter the uptake of oxLDL in unstimulated macrophages and is not essential for the pro-survival effect of oxLDL in these cells. However, LOX-1 expression is highly inducible by lysophosphatidylcholine and pro-inflammatory cytokines, and if that occurred in macrophages within atheromas, LOX-1 could substantially increase oxLDL uptake by lesion macrophages.     

Molecular mechanism of statin-mediated LOX-1 inhibition

Statins are largely used in clinics in the treatment of patients with cardiovascular diseases for their effect on lowering circulating cholesterol. Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for ox-LDL, plays a central role in the pathogenesis of atherosclerosis and cardiovascular disorders. We have recently shown that chronic exposure of cells to lovastatin disrupts LOX-1 receptor cluster distribution in plasma membranes, leading to a marked loss of LOX-1 function. Here we investigated the molecular mechanism of statin-mediated LOX-1 inhibition and we demonstrate that all tested statins are able to displace the binding of fluorescent ox-LDL to LOX-1 by a direct interaction with LOX-1 receptors in a cell-based binding assay. Molecular docking simulations confirm the interaction and indicate that statins completely fill the hydrophobic tunnel that crosses the C-type lectin-like (CTLD) recognition domain of LOX-1. Classical molecular dynamics simulation technique applied to the LOX-1 CTLD, considered in the entire receptor structure with or without a statin ligand inside the tunnel, indicates that the presence of a ligand largely increases the dimer stability. Electrophoretic separation and western blot confirm that different statins binding stabilize the dimer assembly of LOX-1 receptors in vivo. The simulative and experimental results allow us to propose a CTLD clamp motion, which enables the receptor-substrate coupling. These findings reveal a novel and significant functional effect of statins.