Assay for advanced glycation end products generating intracellular oxidative stress through binding to its receptor.

Advanced glycation end products (AGEs) are a heterogenous group of glycation adducts on amino acids produced with sugars or dicarbonyls. Intracellular inflammation triggered by binding of AGEs to receptor for AGEs (RAGE) is linked to some chronic diseases. Here, we established a competitive assay format to comprehensively quantify AGEs which bound to RAGE. RAGE-binding activities of sugar- and dicarbonyl-derived AGEs were correlated with oxidative stress in cultured cells generated by the respective AGEs, suggesting that this would be a promising method for evaluating AGEs which could affect cellular functions despite limited information on individual glycation adducts.

Microplate-based Assay for Screening of Advanced Glycation End Products Binding to Its Receptor.

Advanced Glycation End products (AGEs) are a group of amino-acid modifications produced with sugars or di-carbonyls. Some AGEs are known to affect health through binding to the receptor of AGEs (RAGE). Here, we propose a method for screening RAGE-binding AGEs by a competitive assay using purified RAGE and AGEs-specific antibody. This method has clarified that at least carboxyethyl lysine and pentosidine among methylglyoxal-derived AGEs are involved in RAGE binding, suggesting that this would be a promising method for classifying RAGE-binding AGEs.

Co-expression of BirA with biotin bait achieves in vivo biotinylation of overexpressed stable N-glycosylated sRAGE in transgenic silkworms.

Here, we demonstrated the expression of the N-glycosylated extracellular ligand binding domain of receptor for advanced glycation end products (sRAGE) in middle silk glands (MSGs) of transgenic silkworms using the GAL4/UAS system. Over 1 mg of sRAGE was obtained from one transgenic silkworm. sRAGE purified from the silkworm exhibited good stability and maintained specific ligand-binding ability. In addition, N-glycan analysis of sRAGE revealed that N-glucan completely lacked potentially allergenic fucose. Moreover, co-expression of biotin ligase (BirA) with C-terminal BioEase-tagged sRAGE in MSGs resulted in efficient biotinylation of sRAGE after addition of biotin bait. C-terminal biotinylated sRAGE could be immobilized onto a solid surface in one direction through binding to streptavidin without any loss of ability. The dissociation constant of sRAGE with fructose-BSA, a typical RAGE ligand, was 7.25 × 10-7 M, consistent with that on the mammalian cell surface. Thus, we developed a novel, innovative silkworm expression system for efficient expression of recombinant sRAGE, which could serve as a basis for the elucidation of RAGE-ligand interactions and facilitate the search for new ligands and inhibitors.

Screening, expression, and characterization of an anti-human oxidized low-density lipoprotein single-chain variable fragment.

Increased levels of oxidized low-density lipoprotein (OxLDL) in the blood circulation are correlated with atherosclerosis. Monoclonal antibody-based detection systems have been reported for OxLDL. We identified novel single-chain variable fragments (scFvs) having affinity for human OxLDL and related ligands. We constructed an scFv library from nonimmunized human spleen mRNA. Two types (γ+κ and µ+λ) of scFv phage libraries were enriched by biopanning, and five scFv clones with affinity for OxLDL were identified. The γκ5 scFv, which showed the highest affinity for OxLDL, was cloned into pET-22b(+) and expressed in Escherichia coli BL21(DE3). γκ5, expressed as an inclusion body in BL21(DE3), was refolded and purified. The specificity and sensitivity of γκ5 were analyzed using enzyme-linked immunosorbent assays (ELISAs). The γκ5 scFv showed affinity for OxLDL and acetylated LDL. The sensitivity of γκ5 to low concentrations (1-2 µg/mL) of OxLDL was higher than that to AcLDL and LDL. Finally, we developed a sandwich ELISA using γκ5 and CTLD14 (a lectin-like OxLDL receptor-1 ligand recognition region), which allowed specific detection of OxLDL at a level below 0.1 µg/mL. Our results indicated that the γκ5 scFv was a promising molecule for the detection of modified LDL at very low concentrations.

Identification of C1q as a Binding Protein for Advanced Glycation End Products.

Advanced glycation end products (AGEs) make up a heterogeneous group of molecules formed from the nonenzymatic reaction of reducing sugars with the free amino groups of proteins. The abundance of AGEs in a variety of age-related diseases, including diabetic complications and atherosclerosis, and their pathophysiological effects suggest the existence of innate defense mechanisms. Here we examined the presence of serum proteins that are capable of binding glycated bovine serum albumin (AGEs-BSA), prepared upon incubation of BSA with dehydroascorbate, and identified complement component C1q subcomponent subunit A as a novel AGE-binding protein in human serum. A molecular interaction analysis showed the specific binding of C1q to the AGEs-BSA. In addition, we identified DNA-binding regions of C1q, including a collagen-like domain, as the AGE-binding site and established that the amount of positive charge on the binding site was the determining factor. C1q indeed recognized several other modified proteins, including acylated proteins, suggesting that the binding specificity of C1q might be ascribed, at least in part, to the electronegative potential of the ligand proteins. We also observed that C1q was involved in the AGEs-BSA-activated deposition of complement proteins, C3b and C4b. In addition, the AGEs-BSA mediated the proteolytic cleavage of complement protein 5 to release C5a. These findings provide the first evidence of AGEs as a new ligand recognized by C1q, stimulating the C1q-dependent classical complement pathway.

Fucoxanthin Derivatives: Synthesis and their Chemical Properties.

Novel fucoxanthin derivatives that could change the size of mixed micelles were synthesized. The mixed micelles under consideration consist of a bile acid and some additives. To change the affinity against a bile acid, we designed the synthesis of a fucoxanthin-lithocholic acid complex. Lithocholic acid is one of the bile acids. The 3-OH on lithocholic acid was protected by a levulinyl group, and the protected lithocholic acid was selectively coupled via an ester linkage to the 3-OH on fucoxanthin to obtain levulinyl-protected lithocholyl fucoxanthin (LevLF). The levulinyl group was then selectively deprotected using hydrazine to obtain a lithocholyl fucoxanthin (LF). The average sizes of the micelles that contained these compounds (fucoxanthin, LevLF, and LF) with a bile acid (sodium taurocholate) were measured. The LevLF induced larger micelles than fucoxanthin or LF. Interestingly, the addition of 1-oleoyl-rac-glycerol induced a more efficient change in the micelle size. The large micelles grew larger, and the small micelles became smaller. Triple-mixed micelles with LevLF, sodium taurocholate, and 1-oleoyl-rac-glycerol formed the largest micelle with a diameter of 68 nm. On the other hand, triple-mixed micelles using LF, sodium taurocholate, and 1-oleoyl-rac-glycerol made the smallest micelles with diameters as low as 12 nm. We also investigated the hydrolysis of these compounds with enzymes (esterase from porcine liver, lipase from porcine pancreas, and cholesterol esterase from Pseudomonas sp.). The ester linkage between the lithocholic acid and fucoxanthin of LevLF was hydrolyzed with cholesterol esterase. In addition, the intestinal absorption was examined with Caco-2 cells, and no advantageous change in absorption efficiency was observed by chemically modifying the fucoxanthin unless different micelles sizes and increasing hydrophobicity are induced.

Lectin-like oxidized LDL receptor-1 is palmitoylated and internalizes ligands via caveolae/raft-dependent endocytosis.

Lectin-like oxidized low-density lipoprotein (LDL) receptor-1 (LOX-1) is an endothelial scavenger receptor that is important for oxidized low-density lipoprotein uptake. LOX-1 functions as an oligomer; however, little is known about the oligomeric complex and ligand processing after recognition by LOX-1. Here, we found that LOX-1 recognized and internalized ligands through the caveolae/raft-dependent endocytosis pathway in human coronary artery endothelial cells. Furthermore, we demonstrated that LOX-1 was palmitoylated and that both cysteine 36 and cysteine 46 were necessary for the recruitment of LOX-1 into raft microdomains and for its ligand uptake ability.

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.

Lipid peroxidation modification of protein generates Nepsilon-(4-oxononanoyl)lysine as a pro-inflammatory ligand.

4-Oxo-2(E)-nonenal (ONE), a peroxidation product of ω-6 polyunsaturated fatty acids, covalently reacts with lysine residues to generate a 4-ketoamide-type ONE-lysine adduct, N(ε)-(4-oxononanoyl)lysine (ONL). Using an ONL-coupled protein as the immunogen, we raised the monoclonal antibody (mAb) 9K3 directed to the ONL and conclusively demonstrated that the ONL was produced during the oxidative modification of a low density lipoprotein (LDL) in vitro. In addition, we observed that the ONL was present in atherosclerotic lesions, in which an intense immunoreactivity was mainly localized in the vascular endothelial cells and macrophage- and vascular smooth muscle cell-derived foam cells. Using liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for quantification of the ONL and confirmed that the ONL was indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. To evaluate the biological implications for ONL formation, we examined the recognition of ONL by the scavenger receptor lectin-like oxidized LDL receptor-1 (LOX-1). Using CHO cells stably expressing LOX-1, we evaluated the ability of ONL to compete with the acetylated LDL and found that both the ONE-modified and ONL-coupled proteins inhibited the binding and uptake of the modified LDL. In addition, we demonstrated that the ONL-coupled protein was incorporated into differentiated THP-1 cells via LOX-1. Finally, we examined the effect of ONL on the expression of the inflammation-associated gene in THP-1 and observed that the ONL-coupled proteins significantly induced the expression of atherogenesis-related genes, such as the monocyte chemoattractant protein-1 and tumor necrosis factor-α, in a LOX-1-dependent manner. Thus, ONL was identified to be a potential endogenous ligand for LOX-1.

Lipid peroxidation generates body odor component trans-2-nonenal covalently bound to protein in vivo.

trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis- and trans-N(epsilon)-3-[(hept-1-enyl)-4-hexylpyridinium]lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis- and trans-HHP-lysine and confirmed that the 2-nonenal-lysine adducts were indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand.

Silicone-urethane adhesive for improved coverslip mounting and leak-free preparation of living cell observation chambers.

Using a combination of silicone and urethane resin, we established a rapid technique for preparing living specimens for microscopy. One major advantage of this technique is that the coverslip is rigidly attached and does not detach during handling. As a result, it is possible to continuously observe living cells at high magnification and resolution using an oil immersion objective. Another advantage is that living cells are quickly confined to the space between the glass slide and coverslip, protecting them from environmental changes, which can cause serious effects on cell function and morphology. Moreover, high-resolution observations of real-time responses of cells are possible, using the combination of the mounting technique and a simple flow chamber.

Oxidized LDL receptor LOX-1 binds to C-reactive protein and mediates its vascular effects.

BACKGROUND: C-reactive protein (CRP) exerts biological activity on vascular endothelial cells. This activity may promote atherothrombosis, but the effects of this activity are still controversial. Lectin-like oxidized LDL receptor-1 (LOX-1), the oxidized LDL receptor on endothelial cells, is involved in endothelial dysfunction induced by oxidized LDL. METHODS: We used laser confocal microscopy to examine and fluorescence cell image analysis to quantify the binding of fluorescently labeled CRP to cells expressing LOX-1. We then examined the binding of unlabeled CRP to recombinant human LOX-1 in a cell-free system. Small interfering RNAs (siRNAs) against LOX-1 were applied to cultured bovine endothelial cells to analyze the role of LOX-1 in native cells. To observe its in vivo effects, we injected CRP intradermally in stroke-prone spontaneously hypertensive (SHR-SP) rats and analyzed vascular permeability. RESULTS: CRP bound to LOX-1-expressing cells in parallel with the induction of LOX-1 expression. CRP dose-dependently bound to the cell line and recombinant LOX-1, with significant binding detected at 0.3 mg/L CRP concentration. The K(d) value of the binding was calculated to be 1.6 x 10(-7) mol/L. siRNA against LOX-1 significantly inhibited the binding of fluorescently labeled CRP to the endothelial cells, whereas control RNA did not. In vivo, intradermal injection of CRP-induced vascular exudation of Evans blue dye in SHR-SP rats, in which expression of LOX-1 is greatly enhanced. Anti-LOX-1 antibody significantly suppressed vascular permeability. CONCLUSIONS: CRP and oxidized LDL-receptor LOX-1 directly interact with each other. Two risk factors for ischemic heart diseases, CRP and oxidized LDL, share a common molecule, LOX-1, as their receptor.

Expression and characterization of recombinant C-terminal biotinylated extracellular domain of human receptor for advanced glycation end products (hsRAGE) in Escherichia coli.

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the development of diabetic complications. Using an Escherichia coli expression system, we have successfully expressed and purified the C-terminal biotinylated extracellular domain of human RAGE (hsRAGE), which consists of three immunoglobulin-like domains carrying three putative disulfide bonds. Over 90% of hsRAGE was expressed in soluble form in trxB and gor mutant E. coli strain Origami (DE3). Most hsRAGE was biotinylated with a C-terminal AviTag, and stably immobilized onto matrix via streptavidin without any treatment. Immobilized hsRAGE without glycosylation recognized its ligands, such as AGEs. Biotinylated hsRAGE was also able to apply in the detection of AGEs on microtitre wells like antibodies used in enzyme-linked immunoassay. SPR analysis demonstrated that the dissociation constant (K(d)) of RAGE for AGE-BSA was 23.1 nM with the two-state reaction model, and 13.5 nM with the 1:1 binding model, comparable to those of RAGEs on cell surface. These results indicate that biotinylated hsRAGE must be useful not only in analysing RAGE-ligand interactions but also detect AGEs.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1) functions as an oligomer and oligomerization is dependent on receptor density.

Lectin-like oxidized low-density lipoprotein (LDL) receptor (LOX-1) exists as a homodimer formed by an intermolecular disulfide bond. Although the dimer is the minimum structural unit of LOX-1 on cell membranes, LOX-1 can form larger noncovalent oligomeric complexes. But, the functional unit of LOX-1 is not known. We quantitatively analyzed the correlation between cyan fluorescent protein-tagged LOX-1 expression and the fluorescence-labeled ligand (DiD-AcLDL) binding ability on each cell. The results clearly indicate that there is a threshold level of expression that enables LOX-1 to bind ligand. Above this threshold level, the ability of LOX-1 to bind ligand was proportional to its level of expression. Using the membrane impermeable crosslinker BS(3), we detected oligomers (primarily hexamers) only on the cell lines that stably expressed LOX-1 above the threshold level. In contrast, little oligomer or ligand binding was detected in cell lines expressing LOX-1 below the threshold level. Moreover, oligomerization was independent of ligand binding. These results indicate that the functional unit of LOX-1 is an oligomer and that oligomerization of LOX-1 is dependent on the receptor density on the plasma membrane.

In vitro system for high-throughput screening of random peptide libraries for antimicrobial peptides that recognize bacterial membranes.

Antibacterial peptides have been isolated from a wide range of species. Some of these peptides act on microbial membranes, disrupting their barrier function. With the increasing development of antibiotic resistance by bacteria, these antibacterial peptides, which have a new mode of action, have attracted interest as antibacterial agents. To date, however, few effective high-throughput approaches have been developed for designing and screening peptides that act selectively on microbial membranes. In vitro display techniques are powerful tools to select biologically functional peptides from peptide libraries. Here, we used the ribosome display system to form peptide-ribosome-mRNA complexes in vitro from nucleotides encoding a peptide library, as well as immobilized model membranes, to select specific sequences that recognize bacterial membranes. This combination of ribosome display and immobilized model membranes was effective as an in vitro high-throughput screening system and enabled us to identify motif sequences (ALR, KVL) that selectively recognized the bacterial membrane. Owing to host toxicity, it was not possible to enrich any sequence expected to show antimicrobial activity using another in vitro system, e.g. phage display. The synthetic peptides designed from these enriched motifs acted selectively on the bacterial model membrane and showed antibacterial activity. Moreover, the motif sequence conferred selectivity onto native peptides lacking selectivity, and decreased mammalian cell toxicity of native peptides without decreasing their antibacterial activity.

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.

Purification, crystallization and preliminary X-ray analysis of the ligand-binding domain of human lectin-like oxidized low-density lipoprotein receptor 1 (LOX-1).

Two different fragments of the ligand-binding domain of LOX-1, the major receptor for oxidized low-density lipoprotein (LDL) on endothelial cells, have been crystallized in different forms. One crystal form contains the disulfide-linked dimer, which is the form of the molecule present on the cell surface; the other contains a monomeric form of the receptor that lacks the cysteine residue necessary to form disulfide-linked homodimers. The crystal of the monomeric ligand-binding domain belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 56.79, b = 67.57, c = 79.02 A. The crystal of the dimeric form belongs to space group C2, with unit-cell parameters a = 70.86, b = 49.56, c = 76.73 A, beta = 98.59 degrees. Data for the dimeric form of the LOX-1 ligand-binding domain have been collected to 2.4 A. For the monomeric form of the ligand-binding domain, native, heavy-atom derivative and SeMet-derivative crystals have been obtained; their diffraction data have been measured to 3.0, 2.4 and 1.8 A resolution, respectively.

Human lectin-like oxidized low-density lipoprotein receptor-1 functions as a dimer in living cells.

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) is a unique scavenger receptor that plays important roles in atherogenesis and has been thought to function as a monomer. Using coimmunoprecipitation studies, we demonstrate that human LOX-1 (hLOX-1) forms constitutive homo-interactions in vivo. Western blot analysis of cell lysates under nonreducing or reducing conditions revealed one clear immunoreactive species corresponding to the size of a putative receptor dimer or a monomer, respectively, consistent with the presence of disulfide-linked hLOX-1 complexes. Site-directed mutagenesis studies indicated that cysteine 140 has a key role in the formation of these disulfide-linked hLOX-1 dimers. Eliminating this intermolecular disulfide bond markedly impairs the recognition of Escherichia coli by hLOX-1. Furthermore, these dimers can act as a "structural unit" to form noncovalently associated oligomers, as demonstrated by a membrane-impermeant crosslinker, which resulted in immunoreactive species corresponding to the sizes of putative tetramers and hexamers. These results provide the first evidence for the existence of hLOX-1 dimers/oligomers.

Refolding and characterization of the functional ligand-binding domain of human lectin-like oxidized LDL receptor.

Lectin-like oxidized low-density lipoprotein receptor (LOX-1), a type II membrane protein that can recognize a variety of structurally unrelated macromolecules, plays an important role in host defense and is implicated in atherogenesis. To understand the interaction between human LOX-1 and its ligands, in this study the functional C-type lectin-like domain (CTLD) of LOX-1 was reconstituted at high efficiency from inactive aggregates in Escherichia coli using a refolding technique based on an artificial chaperone. The CD spectra of the purified domain suggested that the domain has alpha-helical structure and the blue shift of Trp residues was observed on refolding of the domain. Like wild-type hLOX-1, the refolded CTLD domain was able to bind modified LDL. Thus, even though CTLD contains six Cys residues that form disulfide bonds, it recovered its specific binding ability on refolding. This suggests that the correct disulfide bonds in CTLD were formed by the artificial chaperone technique. Although the domain lacked N-glycosylation, it showed high affinity for its ligand in surface plasmon resonance experiments. Thus, unglycosylated CTLD is sufficient for binding modified LDL.