N(epsilon)-(carboxymethyl)lysine adducts of proteins are ligands for receptor for advanced glycation end products that activate cell signaling pathways and modulate gene expression.

Recent studies suggested that interruption of the interaction of advanced glycation end products (AGEs), with the signal-transducing receptor receptor for AGE (RAGE), by administration of the soluble, extracellular ligand-binding domain of RAGE, reversed vascular hyperpermeability and suppressed accelerated atherosclerosis in diabetic rodents. Since the precise molecular target of soluble RAGE in those settings was not elucidated, we tested the hypothesis that predominant specific AGEs within the tissues in disorders such as diabetes and renal failure, N(epsilon)-(carboxymethyl)lysine (CML) adducts, are ligands of RAGE. We demonstrate here that physiologically relevant CML modifications of proteins engage cellular RAGE, thereby activating key cell signaling pathways such as NF-kappaB and modulating gene expression. Thus, CML-RAGE interaction triggers processes intimately linked to accelerated vascular and inflammatory complications that typify disorders in which inflammation is an established component.     

How to create a specific recognition for an unspecific interaction

The structure of the receptor for advanced glycation end products (RAGE) in complex with a peptide containing an N(?)-carboxy-ethyl-lysine (CEL) (Xue et al., 2011) shows how the modification of the lysine side chain is recognized without specific interaction with the peptide.     

Advanced glycation end product ligands for the receptor for advanced glycation end products: biochemical characterization and formation kinetics

Advanced glycation end products (AGEs) accumulate with age and at an accelerated rate in diabetes. AGEs bind cell-surface receptors including the receptor for advanced glycation end products (RAGE). The dependence of RAGE binding on specific biochemical characteristics of AGEs is currently unknown. Using standardized procedures and a variety of AGE measures, the present study aimed to characterize the AGEs that bind to RAGE and their formation kinetics in vitro. To produce AGEs with varying RAGE binding affinity, bovine serum albumin (BSA) AGEs were prepared with 0.5M glucose, fructose, or ribose at times of incubation from 0 to 12 weeks or for up to 3 days with glycolaldehyde or glyoxylic acid. The AGE-BSAs were characterized for RAGE binding affinity, fluorescence, absorbance, carbonyl content, reactive free amine content, molecular weight, pentosidine content, and N-epsilon-carboxymethyl lysine content. Ribose-AGEs bound RAGE with high affinity within 1 week of incubation in contrast to glucose- and fructose-AGE, which required 12 and 6 weeks, respectively, to generate equivalent RAGE ligands (IC50=0.66, 0.93, and 1.7 microM, respectively). Over time, all of the measured AGE characteristics increased. However, only free amine content robustly correlated with RAGE binding affinity. In addition, detailed protocols for the generation of AGEs that reproducibly bind RAGE with high affinity were developed, which will allow for further study of the RAGE-AGE interaction.     

Internalization of the receptor for advanced glycation end products (RAGE) is required to mediate intracellular responses

To dissect the rat receptor for advanced glycation end products (RAGE) subcellular distribution and trafficking in eukaryotic cells, an expression system coding for a fusion protein between the RAGE and an enhanced green fluorescent protein (EGFP) has been used. The RAGE-EGFP protein is expressed at the plasma membrane of CHO-k1 and Neuro-2a (N2a) cells and retains the capacity to bind Texas Red-labelled advanced glycation end products (AGEs). AGEs addition to the cell cultures induced a change in the subcellular distribution of the fluorescent RAGE-EGFP protein compatible with an internalization of the AGEs-RAGE complex. Furthermore, while N2a cells expressing the RAGE-EGFP showed an increase in ERK1/2 phosphorylation and NF-kappaB DNA binding in response to AGEs, pre-incubation with dansyl-cadaverine or phenylarsine oxide, inhibitors of receptors internalization, blocked the activation of ERKs and other intracellular responses mediated by AGEs. These results suggest that internalization plays a key role in the signal transduction mediated by RAGE.     

Intracellular glycation of nuclear DNA, mitochondrial DNA, and cytosolic proteins during senescence-like growth arrest

To investigate the accumulation of intracellular advanced glycation end products (AGEs), a method was established for the simultaneous analysis of glycation products of cytosolic proteins, nuclear DNA, and mitochondrial DNA (mtDNA). Nuclear DNA, mtDNA, and cytosolic proteins were simultaneously isolated from one cell lysate by differential centrifugation and combined mechanical and chemical cell disruption methods. The major DNA-AGE N(2)-carboxyethyl-2'-deoxyguanosine (CEdG) was quantified in nuclear DNA and mtDNA by ELISA, whereas the protein-AGEs N(?)-(carboxymethyl)lysine (CML) and N(?)-(carboxyethyl)lysine (CEL) were determined by western blot. The method was used to analyze NIH3T3 fibroblasts. In untreated cells, CEdG levels of mtDNA (14.84?±?3.07?pg CEdG/μg mtDNA) were significantly higher compared with nuclear DNA (4.40?±?0.64?pg CEdG/μg DNA; p?相似文献   

Structural basis for pattern recognition by the receptor for advanced glycation end products (RAGE)

The receptor for advanced glycated end products (RAGE) is a multiligand receptor that is implicated in the pathogenesis of various diseases, including diabetic complications, neurodegenerative disorders, and inflammatory responses. The ability of RAGE to recognize advanced glycated end products (AGEs) formed by nonenzymatic glycoxidation of cellular proteins places RAGE in the category of pattern recognition receptors. The structural mechanism of AGE recognition was an enigma due to the diversity of chemical structures found in AGE-modified proteins. Here, using NMR spectroscopy we showed that the immunoglobulin V-type domain of RAGE is responsible for recognizing various classes of AGEs. Three distinct surfaces of the V domain were identified to mediate AGE-V domain interactions. They are located in the positively charged areas of the V domain. The first interaction surface consists of strand C and loop CC ', the second interaction surface consists of strand C ', strand F, and loop FG, and the third interaction surface consists of strand A ' and loop EF. The secondary structure elements of the interaction surfaces exhibit significant flexibility on the ms-micros time scale. Despite highly specific AGE-V domain interactions, the binding affinity of AGEs for an isolated V domain is low, approximately 10 microm. Using in-cell fluorescence resonance energy transfer we show that RAGE is a constitutive oligomer on the plasma membrane. We propose that constitutive oligomerization of RAGE is responsible for recognizing patterns of AGE-modified proteins with affinities less than 100 nm.     

An autoantibody against N(ε)-(carboxyethyl)lysine (CEL): possible involvement in the removal of CEL-modified proteins by macrophages

Advanced glycation end products (AGEs) are believed to play a significant role in the development of diabetic complications. In this study, we measured the levels of autoantibodies against several AGE structures in healthy human plasma and investigated the physiological role of the autoantibodies. A high titer of the autoantibody against N(ε)-(carboxyethyl)lysine (CEL) was detected in human plasma compared with other AGE structures such as CML and pentosidine. The purified human anti-CEL autoantibody reacted with CEL-modified human serum albumin (CEL-HSA), but not CML-HSA. A rabbit polyclonal anti-CEL antibody, used as a model autoantibody against CEL, accelerated the uptake of CEL-HSA by macrophages, but did not enhance the uptake of native HSA. Furthermore, when (125)I-labeled CEL-HSA was injected into the tail vein of mice, accumulation of (125)I-CEL-HSA in the liver was accelerated by co-injection of the rabbit anti-CEL antibody. These results demonstrate that the autoantibody against CEL in plasma may play a role in the macrophage uptake of CEL-modified proteins.     

Blockade of receptor for advanced glycation endproducts: a new target for therapeutic intervention in diabetic complications and inflammatory disorders

The glycation and oxidation of proteins/lipids leads to the generation of a new class of biologically active moieties, the advanced glycation endproducts (AGEs). Recent studies have elucidated that carboxymethyllysine (CML) adducts of proteins/lipids are a highly prevalent AGE in vivo. CML-modified adducts are signal transduction ligands of the receptor for AGE (RAGE), a member of the immunoglobulin superfamily. Importantly, CML-modified adducts accumulate in diverse settings. In addition to enhanced formation in settings of high glucose, these adducts form in inflammatory milieu. Studies performed both in vitro and in vivo have suggested that the proinflammatory/tissue destructive consequences of RAGE activation in the diabetic/inflamed environment may be markedly attenuated by blockade of the ligand-RAGE axis. Here, we will summarize the known consequences of RAGE activation in the tissues and highlight novel areas for therapeutic intervention in these disease states.     

Minimum stable structure of the receptor for advanced glycation end product possesses multi ligand binding ability

The receptor for advanced glycation end products (RAGE) is a multi-ligand receptor involved in the development of diabetic complications. Although the soluble form of the extracellular domain maintains the ability to bind multi-ligands, it is unstable and degrades into several peptide species during storage. Proteolysis with thrombin or factor Xa revealed several protease sensitive sites. Most sensitive site is located between Arg228 and Val229, and peptide bond next to Arg216, Arg116, Arg114 and Trp271 are also cleaved. Seven truncated extracellular domains of RAGE were engineered in order to obtain a stable soluble fragment. RAGE 143 (Ala23-Thr143) is not only protease resistant but also shows the same ligand-binding ability as that of the full-length extracellular domain. The resultant minimum RAGE 143 works as a stable recognition devise to detect advanced glycation end products (AGEs).     

Expression and purification of recombinant human receptor for advanced glycation endproducts in Escherichia coli

The receptor for advanced glycation endproducts (RAGE) is a multiligand receptor that binds a variety of structurally and functionally unrelated ligands, including advanced glycation endproducts (AGEs), amyloid fibrils, amphoterin, and members of the S100 family of proteins. The receptor has been implicated in the pathology of diabetes as well as in inflammatory processes and tumor cell metastasis. For the present study, the extracellular region of RAGE (exRAGE) was expressed as a soluble, C-terminal hexahistidine-tagged fusion protein in the periplasmic space of Escherichia coli. Proper processing and folding of the purified protein, predicted to contain three immunoglobulin-type domains, was supported by the results of electrospray mass spectroscopy and circular dichroism experiments. Sedimentation velocity experiments showed that exRAGE was primarily monomeric in solution. Binding to several RAGE ligands, including AGE-BSA, immunoglobulin light chain amyloid fibrils, and glycosaminoglycans, was demonstrated using pull-down, dot-blot, or enzyme-linked microplate assays. Using surface plasmon resonance, the interaction of exRAGE with AGE-BSA was shown to fit a two-site model, with KD values of 88 nM and 1.4 microM. The E. coli-derived exRAGE did not bind the advanced glycation endproduct Nepsilon-(carboxymethyl)lysine, as reported for the cellular receptor, and the possible role of RAGE glycosylation in recognition of this ligand is discussed. This new RAGE construct will facilitate detailed studies of RAGE-ligand interactions and provides a platform for preparation of site-directed mutants for future structure/function studies.     

Advanced Glycation End Product (AGE)-Receptor for AGE (RAGE) Signaling and Up-regulation of Egr-1 in Hypoxic Macrophages

Receptor for advanced glycation end product (RAGE)-dependent signaling has been implicated in ischemia/reperfusion injury in the heart, lung, liver, and brain. Because macrophages contribute to vascular perturbation and tissue injury in hypoxic settings, we tested the hypothesis that RAGE regulates early growth response-1 (Egr-1) expression in hypoxia-exposed macrophages. Molecular analysis, including silencing of RAGE, or blockade of RAGE with sRAGE (the extracellular ligand-binding domain of RAGE), anti-RAGE IgG, or anti-AGE IgG in THP-1 cells, and genetic deletion of RAGE in peritoneal macrophages, revealed that hypoxia-induced up-regulation of Egr-1 is mediated by RAGE signaling. In addition, the observation of increased cellular release of RAGE ligand AGEs in hypoxic THP-1 cells suggests that recruitment of RAGE in hypoxia is stimulated by rapid production of RAGE ligands in this setting. Finally, we show that mDia-1, previously shown to interact with the RAGE cytoplasmic domain, is essential for hypoxia-stimulated regulation of Egr-1, at least in part through protein kinase C βII, ERK1/2, and c-Jun NH₂-terminal kinase signaling triggered by RAGE ligands. Our findings highlight a novel mechanism by which an extracellular signal initiated by RAGE ligand AGEs regulates Egr-1 in a manner requiring mDia-1.     

RAGE and soluble RAGE: potential therapeutic targets for cardiovascular diseases

Receptor for advanced glycation end-products (RAGE) is known to be involved in microvascular complications in diabetes. RAGE is also profoundly associated with macrovascular complications in diabetes through regulation of atherogenesis, angiogenic response, vascular injury, and inflammatory response. The potential significance of RAGE in the pathogenesis of cardiovascular disease appears not to be confined solely to nondiabetic rather than diabetic conditions. Numerous truncated forms of RAGE have recently been described, and the C-terminally truncated soluble form of RAGE has received much attention. Soluble RAGE consists of several forms, including endogenous secretory RAGE (esRAGE), which is a spliced variant of RAGE, and a shedded form derived from cell-surface RAGE. These heterogeneous forms of soluble RAGE, which carry all of the extracellular domains but are devoid of the transmembrane and intracytoplasmic domains, bind ligands including AGEs and can antagonize RAGE signaling in vitro and in vivo. ELISA systems have been developed to measure plasma esRAGE and total soluble RAGE, and the pathophysiological roles of soluble RAGE have begun to be unveiled clinically. In this review, we summarize recent findings regarding pathophysiological roles in cardiovascular disease of RAGE and soluble RAGE and discuss their potential usefulness as therapeutic targets and biomarkers for the disease.     

Detection of diabetic nephropathy from advanced glycation endproducts (AGEs) differs in plasma and urine,and is dependent on the method of preparation

Increased advanced glycation endproducts (AGEs) and oxidation products (OPs) have been proposed as pathogenic for diabetic nephropathy (DN). We investigated the relationship between AGEs and OPs measured in different plasma and urine preparations, and progression of DN in 103 young, normoalbuminuric, normotensive participants with type 1 diabetes in the Natural History of Diabetic Nephropathy Study. The primary endpoint was electron microscopy-measured change in glomerular basement membrane (GBM) width from baseline to 5 years; change in mesangial fractional volume was a secondary endpoint. Fast progressors (FP) were defined as the upper quartile (n = 24) of rate of GBM thickening; slow progressors (SP) were the remainder (n = 79). Four AGEs [3-deoxyglucosone and methylglyoxal hydroimidazolones (DG3H1, MGH1) and carboxymethyl and ethyl lysine (CML, CEL)], and two oxidation products methionine sulfoxide and aminoadipic acid were measured by liquid chromatography, triple quadrupole mass spectrometry. Measurements were done on 10 K plasma filtrates and plasma proteolytic digests (PPD) at year 5, and at four time points over 5 years for urinary 10 K filtrates. Urinary filtrate CEL levels were significantly higher in FP, but not after adjustment for HbA1c, sex, and duration of diabetes. MGHI, CEL, and CML plasma filtrate levels were significantly higher in FP relative to SP (p < 0.05). In PPD, only MGHI showed borderline significantly higher levels in FP relative to SP (p = 0.067), while no other product showed correlation. AGE and OP measurements were not correlated with mesangial expansion. In plasma filtrates, HbA1c at year 5 accounted for 4.7 % of the variation in GBM width. The proportion of variation in GBM width was increased to 11.6 % when MGHI, CEL, and CML were added to the model (6.9 % increase).     

Chemoselective synthesis of peptides containing major advanced glycation end-products of lysine and arginine.

Useful methodologies have been developed, enabling the straightforward synthesis of peptides containing N(epsilon)-(carboxymethyl)-L-lysine (CML) and N(epsilon)-(carboxyethyl)-L-lysine (CEL), the major glycation end-products of lysine. These lysine derivatives were successfully incorporated into growing peptide chains via standard Fmoc/Ot-Bu peptide synthesis procedures. For the synthesis of peptides containing major glycation end-products of arginine, synthetic routes have been developed enabling the transformation of ornithine residues in peptides into the well-known arginine-derived advanced glycation end-products (AGEs) Glarg, carboxymethyl-L-arginine (CMA), MG-H1, MG-H2, MG-H3, and carboxyethyl-L-arginine (CEA), respectively, by means of special modifying agents. Furthermore, it was shown that Glarg-containing peptides become quantitatively hydrolyzed into CMA-peptides under physiologic conditions. A similar reaction was observed in case of a MG-H3-peptide, which turned into a CEA-peptide under these conditions.     

Advanced glycation endproducts and their pathogenic roles in neurological disorders

Glycation is implicated in neurological disorders. In some cases it plays a key role in the pathogenesis, in others it plays a co-adjuvant role or it appears as a consequence of degenerative changes and protein accumulation stemming from other pathways. In this work, we attempt to provide a concise, updated review of the major recent findings concerning glycation in neurological diseases. After a short introduction covering advanced glycation endproducts (AGEs) and the receptor for AGEs (RAGE), we will discuss the impact of glycation in central nervous system disorders including Alzheimer’s, Parkinson’s and Creutzfeldt–Jakob disease, as well as peripheral diabetic polyneuropathies. Therapies directed at lowering the concentrations of RAGE ligands including AGEs, blocking RAGE signaling, preventing oxidative stress or lowering methylglyoxal (MGO) levels may significantly decrease the development of AGE-related pathologies in patients with neurological disorders. Many drugs are on the pipeline and the future clinical trials will reveal if the promising results translate into clinical application.     

The biology of the receptor for advanced glycation end products and its ligands

Receptor for advanced glycation end products (RAGE) is a multiligand member of the immunoglobulin superfamily of cell surface molecules whose repertoire of ligands includes advanced glycation end products (AGEs), amyloid fibrils, amphoterins and S100/calgranulins. The overlapping distribution of these ligands and cells overexpressing RAGE results in sustained receptor expression which is magnified via the apparent capacity of ligands to upregulate the receptor. We hypothesize that RAGE-ligand interaction is a propagation factor in a range of chronic disorders, based on the enhanced accumulation of the ligands in diseased tissues. For example, increased levels of AGEs in diabetes and renal insufficiency, amyloid fibrils in Alzheimer's disease brain, amphoterin in tumors and S100/calgranulins at sites of inflammation have been identified. The engagement of RAGE by its ligands can be considered the 'first hit' in a two-stage model, in which the second phase of cellular perturbation is mediated by superimposed accumulation of modified lipoproteins (in atherosclerosis), invading bacterial pathogens, ischemic stress and other factors. Taken together, these 'two hits' eventuate in a cellular response with a propensity towards tissue destruction rather than resolution of the offending pathogenic stimulus. Experimental data are cited regarding this hypothesis, though further studies will be required, especially with selective low molecular weight inhibitors of RAGE and RAGE knockout mice, to obtain additional proof in support of our concept.     

Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE

Engagement of the receptor for advanced glycation end products (RAGE) by products of nonenzymatic glycation/oxidation triggers the generation of reactive oxygen species (ROS), thereby altering gene expression. Because dissection of the precise events by which ROS are generated via RAGE is relevant to the pathogenesis of complications in AGE-related disorders, such as diabetes and renal failure, we tested the hypothesis that activation of NADPH oxidase contributed, at least in part, to enhancing oxidant stress via RAGE. Here we show that incubation of human endothelial cells with AGEs on the surface of diabetic red blood cells, or specific AGEs, (carboxymethyl)lysine (CML)-modified adducts, prompted intracellular generation of hydrogen peroxide, cell surface expression of vascular cell adhesion molecule-1, and generation of tissue factor in a manner suppressed by treatment with diphenyliodonium, but not by inhibitors of nitric oxide. Consistent with an important role for NADPH oxidase, although macrophages derived from wild-type mice expressed enhanced levels of tissue factor upon stimulation with AGE, macrophages derived from mice deficient in a central subunit of NADPH oxidase, gp91phox, failed to display enhanced tissue factor in the presence of AGE. These findings underscore a central role of NADPH oxidase in AGE-RAGE-mediated generation of ROS and provide a mechanism for altered gene expression in AGE-related disorders.     

Age dependent accumulation patterns of advanced glycation end product receptor (RAGE) ligands and binding intensities between RAGE and its ligands differ in the liver,kidney, and skeletal muscle

Background

Much evidence indicates receptor for advanced glycation end products (RAGE) related inflammation play essential roles during aging. However, the majority of studies have focused on advanced glycation end products (AGEs) and not on other RAGE ligands. In the present study, the authors evaluated whether the accumulation of RAGE ligands and binding intensities between RAGE and its ligands differ in kidney, liver, and skeletal muscle during aging.

Results

In C57BL/6 N mice aged 12 weeks, 12 months, and 22 months, ligands accumulation, binding intensities between RAGE and its ligands, activated macrophage infiltration, M1/M2 macrophage expression, glyoxalase-1expression, and signal pathways related to inflammation were evaluated. The RAGE ligands age-associated accumulation patterns were found to be organ dependent. Binding intensities between RAGE and its ligands in kidney and liver increased with age, but those in skeletal muscle were unchanged. Infiltration of activated macrophages in kidney and liver increased with age, but infiltration in the skeletal muscle was unchanged. M1 expression increased and M2 and glyoxalase-1 expression decreased with age in kidney and liver, but their expressions in skeletal muscle were not changed.

Conclusion

These findings indicate patterns of RAGE ligands accumulation, RAGE/ligands binding intensities, or inflammation markers changes during aging are organs dependent.

     

Advanced glycation end products (AGEs), free radicals and diabetes

Nonenzymatic glycation, i.e. binding of monosaccharides to amino groups of proteins, gives rise to complex components called "advanced glycation end-products" (AGEs), which alter protein structure and functions, and participate in diabetic long-term complications. Glycation and oxidative stress are closely linked, and are often referred to as "glycoxidation" processes. Experimental data support these interactions. a) All glycation steps generate oxygen free radicals, some of these steps being common with these of lipid peroxidation. b) AGEs bind to membrane receptors such as RAGE, and induce an oxidative stress and a pro-inflammatory status. c) Glycated proteins modulate cellular oxidative functions: glycated collagens induce an inappropriate oxidative response in PMNs. d) Products of lipid peroxidation (MDA) bind to proteins and amplify glycoxidation-induced damages. Glycoxydation intensity increases in diabetes mellitus, ageing, renal failure and other pathological states with oxidative stress. Therapies aiming at limiting glycoxidation take into account its oxidative part.