Conformational study of N(epsilon)-(carboxymethyl)lysine adducts of recombinant gammaC-crystallin

N -(carboxymethyl)lysine, an advanced glycation end product, is present in the human lens. The effects of CML formation on protein conformation and stability were studied using the recombinant C-crystallin as a model. Conformational change was studied by spectroscopic measurements such as fluorescence and circular dichroism. Conformational stability was determined by unfolding with heat. The results indicated that no conformational change was observed due to CML formation, but conformational stability decreased. These observations can be explained in terms of the relatively stable structure of -crystallin, especially when compared with other crystallins. The lens nucleus is rich in -crystallin and its stable conformation can assist -crystallin sustained insults and remain soluble.     

Conversion of lysine to N(epsilon)-(carboxymethyl)lysine increases susceptibility of proteins to metal-catalyzed oxidation.

Metal-catalyzed oxidation (MCO) of proteins leads to the conversion of some amino acid residues to carbonyl derivatives, and may result in loss of protein function. It is well documented that reactions with oxidation products of sugars, lipids, and amino acids can lead to the conversion of some lysine residues of proteins to N(epsilon)-(carboxymethyl)lysine (CML) derivatives, and that this increases their metal binding capacity. Because post-translational modifications that enhance their metal binding capacity should also increase their susceptibility to MCO, we have investigated the effect of lysine carboxymethylation on the oxidation of bovine serum albumin (BSA) by the Fe(3+)/ascorbate system. Introduction of approximately 10 or more mol CML/mol BSA led to increased formation of carbonyls and of the specific oxidation products glutamic and adipic semialdehydes. These results support the view that the generation of CML derivatives on proteins may contribute to the oxidative damage that is associated with aging and a number of age-related diseases.     

Active glycation in neurofibrillary pathology of Alzheimer disease: N(epsilon)-(carboxymethyl) lysine and hexitol-lysine.

Advanced glycation end products are a diverse class of posttranslational modifications, stemming from reactive aldehyde reactions, that have been implicated in the pathogenesis of a number of degenerative diseases. Because advanced glycation end products are accelerated by, and result in formation of, oxygen-derived free radicals, they represent an important component of the oxidative stress hypothesis of Alzheimer disease (AD). In this study, we used in situ techniques to assess N(epsilon)-(Carboxymethyl)lysine (CML), the predominant advanced glycation end product that accumulates in vivo, along with its glycation-specific precursor hexitol-lysine, in patients with AD as well as in young and aged-matched control cases. Both CML and hexitol-lysine were increased in neurons, especially those containing intracellular neurofibrillary pathology in cases of AD. The increase in hexitol-lysine and CML in AD suggests that glycation is an early event in disease pathogenesis. In addition, because CML can result from either lipid peroxidation or advanced glycation, while hexitol-lysine is solely a product of glycation, this study, together with studies demonstrating the presence of 4-hydroxy-2-nonenal adducts and pentosidine, provides evidence of two distinct oxidative processes acting in concert in AD neuropathology. Our findings support the notion that aldehyde-mediated modifications, together with oxyradical-mediated modifications, are critical pathogenic factors in AD.     

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.     

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.     

Receptor for advanced glycation end products (RAGE) signaling induces CREB-dependent chromogranin expression during neuronal differentiation

Receptor for advanced glycation end products (RAGE) mediates neurite outgrowth and cell migration upon stimulation with its ligand, amphoterin. We show here that RAGE-dependent changes in cell morphology are associated with proliferation arrest and changes in gene expression in neuroblastoma cells. Chromogranin B, a component of secretory vesicles in endocrine cells and neurons, was found to be up-regulated by RAGE signaling during differentiation of neuroblastoma cells along with the two other members of the chromogranin family, chromogranin A and secretogranin II. Ligation of RAGE by amphoterin lead to rapid phosphorylation and nuclear localization of cyclic AMP response element-binding protein (CREB), a major regulator of chromogranin expression. Furthermore, inhibition of ERK1/2-Rsk2-dependent CREB phosphorylation efficiently inhibited up-regulation of chromogranin gene expression upon RAGE activation. To further study the effects of RAGE and amphoterin on cellular differentiation, we stimulated embryonic stem cells expressing RAGE or a signaling-deficient mutant of RAGE with amphoterin. Amphoterin was found to promote RAGE-dependent neuronal differentiation of embryonic stem cells characterized by up-regulation of neuronal markers light neurofilament protein and beta-III-tubulin, activation of CREB, and increased expression of chromogranins A and B. These data suggest that RAGE signaling is capable of driving neuronal differentiation involving CREB activation and induction of chromogranin expression.     

Cellular signalling of the receptor for advanced glycation end products (RAGE)

The receptor for advanced glycation end-product (RAGE) is the signal transduction receptor which senses a variety of signalling molecules including advanced glycation end products (AGEs), HMGB1, S100/calgranulins, β-amyloid, phosphatidylserine, C3a and advanced oxidation protein products (AOPPs). It is usually abnormally up-regulated and plays crucial roles during the development of many human diseases such as diabetes, cardiovascular diseases, osteoarthritis and cancer. RAGE regulates a number of cell processes of pivotal importance like inflammation, apoptosis, proliferation and autophagy. Therapeutic strategies to block RAGE may represent great therapeutic potentials and therefore it has been under extensive investigation during the last decade. Accordingly, there is a growing interest of unraveling the intracellular signalling pathways by which RAGE controls these disease-related processes. Early studies are mainly focused on inflammatory pathways involving the NFκB and the MAPK pathways. Nevertheless, many novel signalling pathways implicated in other cell processes, such as autophagy, have also recently been found to be activated upon RAGE stimulation and contribute to the detrimental effects of RAGE. In this review, we aim to provide a comprehensive summary of previous and recent studies relating to the complex molecular network of RAGE signalling, with a particular emphasis on RAGE transgenic mouse models.     

Hypochlorous acid generates Nε-(carboxymethyl)lysine from Amadori products

Since the accumulation of N^ε-(carboxymethyl)lysine (CML), a major antigenic advanced glycation end product, is implicated in tissue disorders in hyperglycemia and inflammation, the identification of the pathway of CML formation will provide important information regarding the development of potential therapeutic strategies for these complications. The present study was designed to measure the effect of hypochlorous acid (HOCl) on CML formation from Amadori products. The incubation of glycated human serum albumin (glycated-HSA), a model of Amadori products, with HOCl led to CML formation, and an increasing HOCl concentration and decreasing pH, which mimics the formation of these products in inflammatory lesions. CML formation was also observed when glycated-HSA was incubated with activated neutrophils, and was completely inhibited in the presence of an HOCl scavenger. These data demonstrated that HOCl-mediated CML formation from Amadori products plays a role in CML formation and tissue damage at sites of inflammation.     

Solution structure of the soluble receptor for advanced glycation end products (sRAGE)

The receptor for advanced glycation end products (RAGE) is a multiligand cell surface receptor involved in various human diseases, as it binds to numerous molecules and proteins that modulate the activity of other proteins. Elucidating the three-dimensional structure of this receptor is therefore most important for understanding its function during activation and cellular signaling. The major alternative splice product of RAGE comprises its extracellular region that occurs as a soluble protein (sRAGE). Although the structures of sRAGE domains were available, their assembly into the functional full-length protein remained unknown. We observed that the protein has concentration-dependent oligomerization behavior, and this is also mediated by the presence of Ca(2+) ions. Moreover, using synchrotron small angle x-ray scattering, the solution structure of human sRAGE was determined in the monomeric and dimeric forms. The model for the monomer displays a J-like shape, whereas the dimer is formed through the association of the two N-terminal domains and has an elongated structure. These results provide insights into the assembly of the RAGE homodimer, which is essential for signal transduction, and the sRAGE:RAGE heterodimer that leads to blockage of the receptor signaling, paving the way for the design of therapeutic strategies for a large number of different pathologies.     

The receptor for advanced glycation end products influences the expression of its S100 protein ligands in melanoma tumors

Recent studies have suggested that the receptor for advanced glycation end products (RAGE) participates in melanoma progression by promoting tumor growth. However, the mechanisms of RAGE activation in melanoma tumors are not clearly understood. To get deeper insights into these mechanisms, we transfected a melanoma cell line, which was established from a human melanoma primary tumor, with RAGE, and studied the effect of RAGE overexpression on cell proliferation and migration in vitro. We observed that overexpression of RAGE in these cells not only resulted in significantly increased migration rates compared to control cells, but also in decreased proliferation rates (Meghnani et al., 2014).In the present study, we compared the growth of xenograft tumors established from RAGE overexpressing WM115 cells, to that of control cells. We observed that when implanted in mice, RAGE overexpressing cells generated tumors faster than control cells. Analysis of protein tumor extracts showed increased levels of the RAGE ligands S100B, S100A2, S100A4, S100A6 and S100A10 in RAGE overexpressing tumors compared to control tumors. We show that the tumor growth was significantly reduced when the mice were treated with anti-RAGE antibodies, suggesting that RAGE, and probably several S100 proteins, were involved in tumor growth. We further demonstrate that the anti-RAGE antibody treatment significantly enhanced the efficacy of the alkylating drug dacarbazine in reducing the growth rate of RAGE overexpressing tumors.     

Targeting receptor for advanced glycation end products (RAGE) expression induces apoptosis and inhibits prostate tumor growth

Expression of receptor for advanced glycation end products (RAGE) plays a key role in the progression of prostate cancer. However, the therapeutic potential of targeting RAGE expression in prostate cancer is not yet evaluated. Therefore in this study, we have investigated the effects of silencing the expression of RAGE by RNAi approach both in vitro and in vivo. The results of this study showed that down regulation of RAGE expression by RNAi inhibited the cell proliferation of androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells. Furthermore, targeting RAGE expression resulted in apoptotic elimination of these prostate cancer cells by activation of caspase-8 and caspase-3 death signaling. Of note, the levels of prostate specific antigen (PSA) were also reduced in LNCaP cells transfected with RAGE RNAi constructs. Importantly, the RAGE RNAi constructs when administered in nude mice bearing prostate tumors, inhibited the tumor growth by targeting the expression of RAGE, and its physiological ligand, HMGB1 and by up regulating death receptors DR4 and DR5 expression. Collectively, the results of this study for the first time show that targeting RAGE by RNAi may be a promising alternative therapeutic strategy for treating prostate cancer.     

Impact of advanced glycation end products (AGEs) signaling in coronary artery disease

Coronary artery disease remains the leading cause of mortality in adult diabetic population with however, a high predominance also in non-diabetic subjects. In search of common molecular mechanisms and metabolic by-products with potential pathogenic role, increased advanced glycation end products (AGEs) present a critical biomarker for CAD development in both cases. Interaction of AGEs with their transmembrane cell receptor, RAGE in endothelial and smooth muscle cells as well as in platelets, activates intracellular signaling that leads to endothelial injury, modulation of vascular smooth muscle cell function and altered platelet activity. Furthermore, tissue accumulation of AGEs affects current treatment approaches being involved in stent restenosis. The present review provides an update of AGE-induced molecular mechanisms involved in CAD pathophysiology while it discusses emerging therapeutic interventions targeting AGE reduction and AGE-RAGE signaling with beneficial clinical outcome.     

Purification and characterization of mouse soluble receptor for advanced glycation end products (sRAGE)

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface proteins that has been implicated as a progression factor in a number of pathologic conditions from chronic inflammation to cancer to Alzheimer's disease. In such conditions, RAGE acts to facilitate pathogenic processes. Its secreted isoform, soluble RAGE or sRAGE, has the ability to prevent RAGE signaling by acting as a decoy. sRAGE has been used successfully in animal models of a range of diseases to antagonize RAGE-mediated pathologic processes. In humans, sRAGE results from alternative splicing of RAGE mRNA. This study was aimed to determine whether the same holds true for mouse sRAGE and, in addition, to biochemically characterize mouse sRAGE. The biochemical characteristics examined include glycosylation and disulfide patterns. In addition, sRAGE was found to bind heparin, which may mediate its distribution in the extracellular matrix and cell surfaces of tissues. Finally, our data indicated that sRAGE in the mouse is likely produced by carboxyl-terminal truncation, in contrast to the alternative splicing mechanism reported in humans.     

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.     

Association between the receptor for advanced glycation end products gene polymorphisms and coronary artery disease

Receptor for advanced glycation end products (RAGE) is a cell-surface molecule member of the immunoglobulin superfamily and thought to play a critical role in diabetic atherosclerosis. A growing body of studies has been conducted to determine the extent to which the variants of RAGE gene influence the risk of coronary artery disease (CAD). However, these have reported conflicting results. To investigate this inconsistency, we performed a comprehensive meta-analysis on the associations between the RAGE ?374T/A, ?429T/C, and Gly82Ser polymorphisms and the risk of CAD. A total of 4,402 cases and 6,081 controls from 17 published case–control studies were included. The overall odds ratio (OR) of CAD was 0.99 (95 % CI 0.87–1.13), 1.06 (95 % CI 0.95–1.18) and 1.12 (95 % CI 0.90–1.39) for ?374A, ?429C, and the minor S allele of the Gly82Ser polymorphism, respectively. Similarly, no significant results were observed for these polymorphisms using dominant model. However, when stratified by diabetic/non-diabetic status of the CAD patients, we found significant association among Caucasian type two diabetic CAD patients with the ?374A allele [OR 1.39, 95 % CI 1.10–1.76, P(Z) = 0.006], while no association was detected between the ?374T/A polymorphism and non-diabetic CAD in Caucasians [OR 0.79, 95 % CI 0.58–1.07, P(Z) = 0.13]. In conclusion, this meta-analysis suggested that possession of the ?374A allele may be a risk factor in CAD among Caucasian patients with type two diabetes.     

N -Glycans on the receptor for advanced glycation end products influence amphoterin binding and neurite outgrowth

In this study we show that embryonic neurite growth-promoting protein amphoterin binds to carboxylated N -glycans previously identified on mammalian endothelial cells. Since amphoterin is a ligand for the receptor for advanced glycation end products (RAGE), and the ligand-binding V-domain of the receptor contains two potential N -glycosylation sites, we hypothesized that N -glycans on RAGE may mediate its interactions with amphoterin. In support of this, anti-carboxylate antibody mAbGB3.1 immunoprecipitates bovine RAGE, and PNGase F treatment reduces its molecular mass by 4.5 kDa, suggesting that the native receptor is a glycoprotein. The binding potential of amphoterin to RAGE decreases significantly in presence of soluble carboxylated glycans or when the receptor is deglycosylated. Oligosaccharide analysis shows that RAGE contains complex type anionic N -glycans with non-sialic acid carboxylate groups, but not the HNK-1 (3-sulfoglucuronyl beta1-3 galactoside) epitope. Consistent with the functional localization of RAGE and amphoterin at the leading edges of developing neurons, mAbGB3.1 stains axons and growth cones of mouse embryonic cortical neurons, and inhibits neurite outgrowth on amphoterin matrix. The carboxylated glycans themselves promote neurite outgrowth in embryonic neurons and RAGE-transfected neuroblastoma cells. This outgrowth requires full-length, signalling-competent RAGE, as cells expressing cytoplasmic domain-deleted RAGE are unresponsive. These results indicate that carboxylated N -glycans on RAGE play an important functional role in amphoterin-RAGE-mediated signalling.     

Cloning and expression of a cell surface receptor for advanced glycosylation end products of proteins.

Advanced glycosylation end products of proteins (AGEs) are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. A approximately 35-kDa polypeptide with a unique NH2-terminal sequence has been isolated from bovine lung and found to be present on the surface of endothelial cells where it mediates the binding of AGEs (receptor for advanced glycosylation end product or RAGE). Using an oligonucleotide probe based on the amino-terminal sequence of RAGE, an apparently full-length cDNA of 1.5 kilobases was isolated from a bovine lung cDNA library. This cDNA encoded a 394 amino acid mature protein comprised of the following putative domains: an extracellular domain of 332 amino acids, a single hydrophobic membrane spanning domain of 19 amino acids, and a carboxyl-terminal domain of 43 amino acids. A partial clone encoding the human counterpart of RAGE, isolated from a human lung library, was found to be approximately 90% homologous to the bovine molecule. Based on computer analysis of the amino acid sequence of RAGE and comparison with databases, RAGE is a new member of the immunoglobulin superfamily of cell surface molecules and shares significant homology with MUC 18, NCAM, and the cytoplasmic domain of CD20. Expression of the RAGE cDNA in 293 cells allowed them to bind 125I-AGE-albumin in a saturable and dose-dependent manner (Kd approximately 100 nM), blocked by antibody to RAGE. Western blots of 293 cells transfected with RAGE cDNA probed with anti-RAGE IgG demonstrated expression of immunoreactive protein compared to its absence in mock-transfected cells. These results suggest that RAGE functions as a cell surface receptor for AGEs, which could potentially mediate cellular effects of this class of glycosylated proteins.     

S100B and S100A6 differentially modulate cell survival by interacting with distinct RAGE (receptor for advanced glycation end products) immunoglobulin domains

S100 proteins are EF-hand calcium-binding proteins with various intracellular functions including cell proliferation, differentiation, migration, and apoptosis. Some S100 proteins are also secreted and exert extracellular paracrine and autocrine functions. Experimental results suggest that the receptor for advanced glycation end products (RAGE) plays important roles in mediating S100 protein-induced cellular signaling. Here we compared the interaction of two S100 proteins, S100B and S100A6, with RAGE by in vitro assay and in culture of human SH-SY5Y neuroblastoma cells. Our in vitro binding data showed that S100B and S100A6, although structurally very similar, interact with different RAGE extracellular domains. Our cell assay data demonstrated that S100B and S100A6 differentially modulate cell survival. At micromolar concentration, S100B increased cellular proliferation, whereas at the same concentration, S100A6 triggered apoptosis. Although both S100 proteins induced the formation of reactive oxygen species, S100B recruited phosphatidylinositol 3-kinase/AKT and NF-kappaB, whereas S100A6 activated JNK. More importantly, we showed that S100B and S100A6 modulate cell survival in a RAGE-dependent manner; S100B specifically interacted with the RAGE V and C(1) domains and S100A6 specifically interacted with the C(1) and C(2) RAGE domains. Altogether these results highlight the complexity of S100/RAGE cellular signaling.