"Affimer" synthetic protein scaffolds block oxidized LDL binding to the LOX-1 scavenger receptor and inhibit ERK1/2 activation.

In multicellular organisms, a variety of lipid-protein particles control the systemic flow of triacylglycerides, cholesterol, and fatty acids between cells in different tissues. The chemical modification by oxidation of these particles can trigger pathological responses, mediated by a group of membrane proteins termed scavenger receptors. The lectin-like oxidized low-density lipoprotein (LOX-1) scavenger receptor binds to oxidized low-density lipoprotein (oxLDL) and mediates both signaling and trafficking outcomes. Here, we identified five synthetic proteins termed Affimers from a phage display library, each capable of binding recombinant LOX-1 extracellular (oxLDL-binding) domain with high specificity. These Affimers, based on a phytocystatin scaffold with loop regions of variable sequence, were able to bind to the plasma membrane of HEK293T cells exclusively when human LOX-1 was expressed. Binding and uptake of fluorescently labeled oxLDL by the LOX-1-expressing cell model was inhibited with subnanomolar potency by all 5 Affimers. ERK1/2 activation, stimulated by oxLDL binding to LOX-1, was also significantly inhibited (p < 0.01) by preincubation with LOX-1-specific Affimers, but these Affimers had no direct agonistic effect. Molecular modeling indicated that the LOX-1-specific Affimers bound predominantly via their variable loop regions to the surface of the LOX-1 lectin-like domain that contains a distinctive arrangement of arginine residues previously implicated in oxLDL binding, involving interactions with both subunits of the native, stable scavenger receptor homodimer. These data provide a new class of synthetic tools to probe and potentially modulate the oxLDL/LOX-1 interaction that plays an important role in vascular disease.

Multiple molecular dynamics simulations of human LOX-1 and Trp150Ala mutant reveal the structural determinants causing the full deactivation of the receptor.

Multiple classical molecular dynamics simulations have been applied to the human LOX-1 receptor to clarify the role of the Trp150Ala mutation in the loss of binding activity. Results indicate that the substitution of this crucial residue, located at the dimer interface, markedly disrupts the wild-type receptor dynamics. The mutation causes an irreversible rearrangement of the subunits interaction pattern that in the wild-type protein allows the maintaining of a specific symmetrical motion of the monomers. The subunits dislocation determines a loss of linearity of the arginines residues composing the basic spine and a consequent alteration of the long-range electrostatic attraction of the substrate. Moreover, the anomalous subunits arrangement observed in the mutated receptor also affects the integrity of the hydrophobic tunnel, actively involved in the short-range hydrophobic recognition of the substrate. The combined effect of these structural rearrangements generates the impairing of the receptor function.

LOX-1 and Its Splice Variants: A New Challenge for Atherosclerosis and Cancer-Targeted Therapies.

Alternative splicing (AS) is a process in which precursor messenger RNA (pre-mRNA) splicing sites are differentially selected to diversify the protein isoform population. Changes in AS patterns have an essential role in normal development, differentiation and response to physiological stimuli. It is documented that AS can generate both "risk" and "protective" splice variants that can contribute to the pathogenesis of several diseases including atherosclerosis. The main endothelial receptor for oxidized low-density lipoprotein (ox-LDLs) is LOX-1 receptor protein encoded by the OLR1 gene. When OLR1 undergoes AS events, it generates three variants: OLR1, OLR1D4 and LOXIN. The latter lacks exon 5 and two-thirds of the functional domain. Literature data demonstrate a protective role of LOXIN in pathologies correlated with LOX-1 overexpression such as atherosclerosis and tumors. In this review, we summarize recent developments in understanding of OLR1 AS while also highlighting data warranting further investigation of this process as a novel therapeutic target.

Lectin-like Oxidized Low-Density Lipoprotein (LDL) Receptor (LOX-1): A Chameleon Receptor for Oxidized LDL.

LOX-1, one of the main receptors for oxLDL, is found mainly on the surface of endothelial cells. It is a multifacet 52 kDa type II transmembrane protein that structurally belongs to the C-type lectin family. It exists with short intracellular N-terminal and long extracellular C-terminal hydrophilic domains separated by a hydrophobic domain of 26 amino acids. LOX-1 acts like a bifunctional receptor either showing pro-atherogenicity by activating the NFκB-mediated down signaling cascade for gene activation of pro-inflammatory molecules or playing an atheroprotective agent by receptor-mediated uptake of oxLDL in the presence of an anti-inflammatory molecule like IL-10. Mildly, moderately, and highly oxidized LDL show their characteristic features upon LOX-1 activation and its ligand binding indenture. The polymorphic LOX-1 genes are intensively associated with increased susceptibility to myocardial diseases. The splicing variant LOX IN dimerizes with the native form of LOX-1 and protects cells from damage by oxidized LDL. In the developing field of regenerating medicine, LOX-1 is a potential target for therapeutic intervention.

Structure-based Design Targeted at LOX-1, a Receptor for Oxidized Low-Density Lipoprotein.

Atherosclerosis related cardiovascular diseases continue to be the primary cause of mortality in developed countries. The elevated level of low density lipoprotein (LDL) is generally considered to be the driver of atherosclerosis, but recent years have seen a shift in this perception in that the vascular plaque buildup is mainly caused by oxidized LDL (ox-LDL) rather than native-LDL. The scavenger receptor LOX-1 found in endothelial cells binds and internalizes ox-LDL which leads to the initiation of plaque formation in arteries. Using virtual screening techniques, we identified a few potential small molecule inhibitors of LOX-1 and tested their inhibitory potential using differential scanning fluorimetry and various cellular assays. Two of these molecules significantly reduced the uptake of ox-LDL by human endothelial cells, LOX-1 transcription and the activation of ERK1/2 and p38 MAPKs in human endothelial cells. In addition, these molecules suppressed ox-LDL-induced VCAM-1 expression and monocyte adhesion onto human endothelial cells demonstrating their therapeutic potential.

Exploration of binding site pattern in arachidonic acid metabolizing enzymes, Cyclooxygenases and Lipoxygenases.

BACKGROUND: Cyclooxygenase (COXs) and Lipoxygenase (LOXs) pathways are the two major enzymatic pathways in arachidonic acid (AA) metabolism. The term eicosanoid is used to describe biologically active lipid mediators including prostaglandins, thromboxanes, leukotrienes and other oxygenated derivatives, which are produced primarily from AA. Eicosanoids generated in a tissue specific manner play a key role in inflammation and cancer. As AA is the substrate common to variety of COXs and LOXs, inhibition of one pathway results in diversion of the substrate to other pathways, which often is responsible for undesirable side effects. Hence there is need for development of not only isozyme specific inhibitors but also dual/multi enzyme inhibitors. Understanding the interactions of AA and characterizing its binding sites in these enzymes therefore is crucial for developing enzyme specific and multi enzyme inhibitors for enhancing therapeutic efficacy and/or overcoming side effects. RESULTS: AA binding sites in COXs and LOXs are identified and compared by the development of receptor based pharmacophore using MultiBind. Physico chemical properties were compared to understand the details of the binding sites in all the enzymes and to elucidate important amino acids that can be targeted for drug design. The alignment of AA binding sites in the seven enzymes COX-1, COX-2, 5-LOX, 12-LOX, 15-LOX and plant soybean LOX-1 and LOX-3 indicated a common pattern of five common interacting groups. In the same way, comparison of AA binding sites was done pair wise and by multiple alignment in various combinations. It has been identified that aliphatic and aromatic interactions are the most common in all the enzymes. In addition interactions unique to each one of these enzymes were identified. CONCLUSION: The complete analysis of AA binding sites in the seven enzymes was performed; 120 combinations for the seven enzymes were studied in detail. All the seven enzymes are structurally quite different, yet they share AA as the common binding partner. Comparisons in various combinations showed how they are similar and dissimilar with each other. This information will be helpful in designing specific as well as common inhibitors.

Functional expression of the Fc-fused extracellular domains of group II membrane proteins.

The complicated delivery mechanism of group II membrane proteins makes it difficult to decide the fusion pattern of their extracellular domains (ECDs) with Fc moiety. In this study, we compared the expression of ECDs of three group II membrane proteins including CLEC-2, Dectin-1, and LOX-1 by fusion of Fc moiety. We found that the pattern of ECD-Fc fusion order produced the functionally active recombinant proteins while the pattern of Fc-ECD fusion order led to the altered glycosylation which abolished the binding of these proteins with their ligands. Meanwhile, our results indicated that the secretion of mouse Fc (mFc)-fused ECD of CLEC-2 was more efficient than that of rabbit Fc (rFc)-fused protein, while rFc moiety was more sensitive for detection compared with mFc moiety. Altogether, we provide a favorable fusion pattern of Fc moiety with the ECDs of group II transmembrane proteins.

Polymorphism of the OLR1 3'UTR potential microRNA binding site and risk of Alzheimer's disease: a meta-analysis.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that contributes to dementia in the elderly population. Genome-wide linkage analysis has identified chromosome 12p as the AD-susceptible region, which includes lectin-like oxidized low-density lipoprotein receptor 1 (OLR1). The OLR1 +1073 C/T single-nucleotide polymorphism is located in the 3'-untranslated region of the gene and may influence the binding of regulatory microRNAs (miRNAs) and OLR1 protein homeostasis. A number of studies have reported an association between this variant and AD. However, the results are controversial. A meta-analysis of case-control studies examining the relationship between the OLR1 +1073 C/T single-nucleotide polymorphism and AD risk was performed. Five studies were selected that included 2419 cases and 2381 controls. The results revealed a significantly decreased AD risk in the recessive model (TT vs TC + CC: odds ratio (OR) = 0.79, 95% confidence interval (CI) = 0.65-0.96). The control group in one of the studies was in Hardy-Weinberg disequilibrium, so we performed additional meta-analysis excluding this study. The significance was much more pronounced in the recessive model (TT vs TC + CC: OR = 0.72, 95%CI = 0.62-0.85). Using miRanda and RNA hybrid methods, the polymorphic allele was shown to influence the binding of various miRNAs. Our results suggested that the +1073 C/T polymorphism decreased the risk of AD. The polymorphic allele was also predicted to affect the binding site of many miRNAs, which may explain the relationship between the +1073 C/T variant and AD.

Manganese-mefenamic acid complexes exhibit high lipoxygenase inhibitory activity.

The coordination of non-steroidal anti-inflammatory drugs (NSAIDs) to metal ions could improve the pharmaceutical efficacy of NSAIDs due to the unique characteristics of metal complexes. However, the structures of many metal-NSAID complexes are not well characterized; the functional mechanism and pharmaceutical effect of these complexes thus are not fully understood. In this work, three manganese-mefenamic acid (Mn-mef) complexes were synthesized and structurally characterized, and their pharmaceutical effect was investigated. We found that the three Mn-mef complexes exhibit higher lipoxygenase (LOX-1) inhibitory activity (IC50 values are 16.79, 38.63 and 28.06 µM, respectively) than the parent ligand mefenamic acid (78.67 µM). More importantly, the high inhibitory activity of the Mn-mef complexes is closely related to their spatial arrangements, which determine their interaction with LOX-1. Computer docking of the Mn-mef complexes with the LOX-1 confirms the experimental results: smaller Mn-mef complexes tend to bind competitively to LOX-1 at the substrate binding site, which is also analogous to the binding of the ligand mefenamic acid, while the bulky metal complexes inhibit the enzyme activity un-competitively. In addition, the Mn-mef complexes exhibit higher anti-oxidant activity than the ligand mefenamic acid. The higher anti-oxidant activity of the Mn-mef complexes apparently originated from the manganese centre of the complexes. We thus conclude that Mn-mef complexes enhance the anti-inflammatory activity of mefenamic acid by increasing their activity via changing their interaction mode with the enzymes, and/or by improving their anti-oxidant ability using metal ions. This work provides experimental evidence that with the unique spatial arrangements, metal-NSAID complexes could interact with the target enzymes more specifically and efficiently, which is superior to their parent NSAID ligand.

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.

Simulative and experimental investigation on the cleavage site that generates the soluble human LOX-1.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor that mediates the recognition, the binding and internalization of ox-LDL. A truncated soluble form of LOX-1 (sLOX-1) has been identified that, at elevated levels, has been associated to acute coronary syndrome. Human sLOX-1 is the extracellular part of membrane LOX-1 which is cleaved in the NECK domain with a mechanism that has not yet been identified. Purification of human sLOX-1 has been carried out to experimentally identify the cleavage site region within the NECK domain. Molecular modelling and classical molecular dynamics simulation techniques have been used to characterize the structural and dynamical properties of the LOX-1 NECK domain in the presence and absence of the CTLD recognition region, taking into account the obtained proteolysis results. The simulative data indicate that the NECK domain is stabilized by the coiled-coil heptad repeat motif along the simulations, shows a definite flexibility pattern and is characterized by specific electrostatic potentials. The detection of a mobile inter-helix space suggests an explanation for the in vivo susceptibility of the NECK domain to the proteolytic cleavage, validating the assumption that the NECK domain sequence is composed of a coiled-coil motif destabilized in specific regions of functional significance.

LOX-1 is a novel marker for peripheral artery disease in patients with type 2 diabetes.

OBJECTIVE: The aim of this study was to investigate whether serum soluble lectin-like oxidized low-density lipoprotein receptor-1 (sLOX-1), which mediates initiation and progression of atherosclerosis in endothelial cells, could be a novel marker for peripheral artery disease (PAD) in patients with type 2 diabetes. METHODS: We evaluated relationships of serum sLOX-1 to ankle-brachial index (ABI) and examined the association of serum sLOX-1 with PAD in 410 patients with type 2 diabetes. RESULTS: Serum sLOX-1 was inversely correlated with ABI (r=-0.197, P<0.0001). Stepwise regression analysis demonstrated that serum sLOX-1 (ß=-0.168, F=5.571, P<0.05) was independently associated with ABI, and multiple logistic regression analysis demonstrated that serum sLOX-1 (16.254 (1.237-213.651), P=0.0339) was independently associated with PAD. CONCLUSIONS: Serum sLOX-1 is associated with ABI and it could be a novel marker for PAD in patients with type 2 diabetes.

Site-specific N-glycosylation identification of recombinant human lectin-like oxidized low density lipoprotein receptor-1 (LOX-1).

Human LOX-1/OLR 1 plays a key role in atherogenesis and endothelial dysfunction. The N-glycosylation of LOX-1 has been shown to affect its biological functions in vivo and modulate the pathogenesis of atherosclerosis. However, the N-glycosylation pattern of LOX-1 has not been described yet. The present study was aimed at elucidating the N-glycosylation of recombinant human LOX-1 with regard to N-glycan profile and N-glycosylation sites. Here, an approach using nonspecific protease (Pronase E) digestion followed by MALDI-QIT-TOF MS and multistage MS (MS(3)) analysis is explored to obtain site-specific N-glycosylation information of recombinant human LOX-1, in combination with glycan structure confirmation through characterizing released glycans using tandem MS. The results reveal that N-glycans structures as well as their corresponding attached site of LOX-1 can be identified simultaneously by direct MS analysis of glycopeptides from non-specific protease digestion. With this approach, one potential glycosylation site of recombinant human LOX-1 on Asn(139) is readily identified and found to carry heterogeneous complex type N-glycans. In addition, manual annotation of multistage MS data utilizing diagnostic ions, which were found to be particularly useful in defining the structure of glycopeptides and glycans was addressed for proper spectra interpretation. The findings described herein will shed new light on further research of the structure-function relationships of LOX-1 N-glycan.

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.

An alternative protein standard to measure activity of LOX-1 ligand containing apoB (LAB) - utilization of anti-LOX-1 single- chain antibody fused to apoB fragment.

AIM: We have recently demonstrated that the circulating level of LOX-1 ligand containing apoB (LAB) predicts the risk of cardiovascular events; however, as is the case in other assays measuring oxidized LDL (oxLDL), chemical unstability and inter-lot variance of standard oxLDL may limit the utility of measuring LAB. This study aimed to develop an alternative protein standard that is simultaneously recognized by LOX-1 and anti-apoB antibody instead of copper-oxidized LDL. METHODS AND RESULTS: cDNAs encoding the variable regions of anti-LOX-1 monoclonal antibody were cloned from hybridomas and reorganized to express anti-LOX-1 single-chain variable fragment (Fv). cDNAs of four regions of human apoB (B1 to B4), which were reported to be epitopes of many anti-apoB antibodies, were also cloned. After confirming the respective reactivity of Fv and apoB fragments to LOX-1 and anti-apoB antibodies, cDNAs of Fv and apoB fragments were connected to express Fv-ApoB chimeric proteins. These fusion proteins were found to be recognized by both LOX-1 and anti-apoB antibodies. Among them, the fusion proteins of Fv-B1 and Fv-B3 gave saturable binding curves against immobilized LOX-1 when detected by anti-apoB antibodies. The binding curves of different Fv-B1 preparations to LOX-1 were almost identical while those of oxLDL varied among the preparations, suggesting better quality control of Fv-B1 preparations. CONCLUSIONS: The fusion proteins composed of Fv-form anti-LOX-1 antibody and apoB fragment are useful alternatives to copper-oxidized LDL in determining LAB, which would facilitate the application of modified LDL analyses to the clinical diagnosis and risk evaluation of cardiovascular disease.

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.

IVS4-14 A/G and IVS4-73 C/T polymorphisms in OLR1 gene in patients with ischemic cerebrovascular diseases.

INTRODUCTION: Oxidized low-density lipoprotein (ox-LDL) plays a key role in the processes of atherogenesis, the major cause of myocardial infarction. Increased levels of ox-LDL relate to plaque instability in human coronary atherosclerotic lesions. Moreover, ox-LDL levels show a direct correlation to the severity of coronary syndromes. Most of these effects are mediated by the interaction of ox-LDL with its major receptor, named LOX-1, that is encoded by OLR1 gene. AIMS AND METHODS: In the present study, we examined the prevalence of OLR1 gene polymorphisms, IVS4-14 A/G and IVS4-73 C/T, which regulate the expression of LOXIN, in patients with ischemic cerebrovascular diseases (ICVD). We studied 43 consecutive patients (males = 19; females = 24) aged 26 to 65 years. All the patients were from the same geographical area. They were affected by ICVD. The control group comprised 69 healthy blood donors, with age and sex comparable to those of the patients. RESULTS: The distribution of G/G genotype and A/G genotype was statistically significant between patients and controls (chi(2) = 5.87, p = 0.01 and chi(2) = 4.33, p = 0.04, respectively). CONCLUSION: These preliminary data would suggest that in ICVD patients the LOX-1 isoform that induces internalization of ox-LDL is more frequent and a cascade of events responsible for endothelial dysfunction and injury. LOX-1 might play a fundamental role in the initiation and progression of atherosclerosis and have a significant role in the pathogenesis of ICVD. Therefore, the patients with G homozygosity for IVS4-14 polymorphism and T homozygosity for IVS4-73 polymorphism have higher risk to develop ICVD. Future studies are warranted to assess whether the analysis of polymorphisms may be useful for the clinical approach to evaluate risk factors for atherosclerosis and related disorders.

Lox-1: the multifunctional receptor underlying cardiovascular dysfunction.

Oxidatively modified low-density lipoprotein (oxLDL) is implicated in the pathogenesis of atherosclerosis. Endothelial dysfunction is the initial change in the vascular wall that induces morphological changes for atheroma-formation. Lectin-like oxidized LDL receptor-1 (LOX-1) was identified as the receptor for oxLDL that was thought to be a major cause of endothelial dysfunction. LOX-1 has been demonstrated to contribute not only to endothelial dysfunction, but also to atherosclerotic-plaque formation, myocardial infarction and intimal thickening after balloon injury. Recent findings on the genetics of LOX-1 and the methodology to detect it and its ligands would further facilitate the examination of the receptor's pathophysiological contribution in atherosclerosis. Furthermore, LOX-1-related tools might open new gateways from diagnosis to therapeutics for cardiovascular diseases.

Lectin-like oxidized low density lipoprotein receptor 1 (LOX-1) in atherogenesis: a brief review.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a scavenger receptor that primarily binds and regulates oxidized low-density lipoprotein (LDL). Expression of LOX-1 is regulated by a feed-forward system stimulated by oxidized LDL (oxLDL), a major component of atherosclerosis. LOX-1 is a homodimer with a reactive backbone that can bind to a host of different ligands, including small molecules, and whole cells. LOX-1 is involved in many intercellular, intracellular, and molecular processes that are atherogenic. LOX-1 levels are elevated within atherosclerotic plaques and its expression is induced by proinflammatory cytokines. The ability of LOX-1 to bind many different ligands and control several atherogenic processes makes this receptor a likely vascular disease biomarker as well as an ideal choice for drug therapy aimed at preventing cardiovascular disease.