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.     

Participation of the receptor for advanced glycation end products in efferocytosis

Clearance of apoptotic cells by macrophages and other phagocytic cells, called efferocytosis, is a central process in the resolution of inflammation. Although the receptor for advanced glycation end products (RAGE) has been shown to participate in a variety of acute and chronic inflammatory processes in the lungs and other organs, a role for RAGE in efferocytosis has not been reported. In the present studies, we examined the potential involvement of RAGE in efferocytosis. Macrophages from transgenic RAGE(-/-) mice showed a decreased ability to engulf apoptotic neutrophils and thymocytes. Pretreatment of RAGE(+/+) macrophages with advanced glycation end products, which competitively bind to RAGE, or Abs against RAGE diminished phagocytosis of apoptotic cells. Overexpression of RAGE in human embryonic kidney 293 cells resulted in an increased ability to engulf apoptotic cells. Furthermore, we found that incubation with soluble RAGE enhances phagocytosis of apoptotic cells by both RAGE(+/+) and RAGE(-/-) macrophages. Direct binding of RAGE to phosphatidylserine (PS), an "eat me" signal highly expressed on apoptotic cells, was shown by using solid-phase ELISA. The ability of RAGE to bind to PS on apoptotic cells was confirmed in an adhesion assay. Decreased uptake of apoptotic neutrophils by macrophages was found under in vivo conditions in the lungs and peritoneal cavity of RAGE(-/-) mice. These results demonstrate a novel role for RAGE in which it is able to enhance efferocytosis through binding to PS on apoptotic cells.     

Inflammation-induced chondrocyte hypertrophy is driven by receptor for advanced glycation end products

The multiligand receptor for advanced glycation end products (RAGE) mediates certain chronic vascular and neurologic degenerative diseases accompanied by low-grade inflammation. RAGE ligands include S100/calgranulins, a class of low-molecular-mass, calcium-binding polypeptides, several of which are chondrocyte expressed. Here, we tested the hypothesis that S100A11 and RAGE signaling modulate osteoarthritis (OA) pathogenesis by regulating a shift in chondrocyte differentiation to hypertrophy. We analyzed human cartilages and cultured human articular chondrocytes, and used recombinant human S100A11, soluble RAGE, and previously characterized RAGE-specific blocking Abs. Normal human knee cartilages demonstrated constitutive RAGE and S100A11 expression, and RAGE and S100A11 expression were up-regulated in OA cartilages studied by immunohistochemistry. CXCL8 and TNF-alpha induced S100A11 expression and release in cultured chondrocytes. Moreover, S100A11 induced cell size increase and expression of type X collagen consistent with chondrocyte hypertrophy in vitro. CXCL8-induced, IL-8-induced, and TNF-alpha-induced but not retinoic acid-induced chondrocyte hypertrophy were suppressed by treatment with soluble RAGE or RAGE-specific blocking Abs. Last, via transfection of dominant-negative RAGE and dominant-negative MAPK kinase 3, we demonstrated that S100A11-induced chondrocyte type X collagen expression was dependent on RAGE-mediated p38 MAPK pathway activation. We conclude that up-regulated chondrocyte expression of the RAGE ligand S100A11 in OA cartilage, and RAGE signaling through the p38 MAPK pathway, promote inflammation-associated chondrocyte hypertrophy. RAGE signaling thereby has the potential to contribute to the progression of OA.     

Septic shock is associated with receptor for advanced glycation end products ligation of LPS

Septic shock is a severe systemic response to bacterial infection. Receptor for advanced glycation end products (RAGE) plays a role in immune reactions to recognize specific molecular patterns as pathogen recognition receptors. However, the interaction between LPS, the bioactive component of bacterial cell walls, and RAGE is unclear. In this study, we found direct LPS binding to RAGE by a surface plasmon resonance assay, a plate competition assay, and flow cytometry. LPS increased TNF-α secretion from peritoneal macrophages and an NF-κB promoter-driven luciferase activity through RAGE. Blood neutrophils and monocytes expressed RAGE, and TLR2 was counterregulated in RAGE(-/-) mice. After LPS injection, RAGE(+/+) mice showed a higher mortality, higher serum levels of IL-6, TNF-α, high mobility group box 1, and endothelin-1, and severe lung and liver pathologies compared with RAGE(-/-) mice without significant differences in plasma LPS level. Administration of soluble RAGE significantly reduced the LPS-induced cytokine release and tissue damage and improved the LPS-induced lethality even in RAGE(-/-) as well as RAGE(+/+) mice. The results thus suggest that RAGE can associate with LPS and that RAGE system can regulate inflammatory responses. Soluble RAGE would be a therapeutic tool for LPS-induced septic shock.     

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.     

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.     

Atorvastatin exerts its anti-atherosclerotic effects by targeting the receptor for advanced glycation end products

Recent studies demonstrated the beneficial role of atorvastatin in reducing the risk of cardiovascular morbidity and mortality in patients with diabetes mellitus and/or metabolic syndrome. To investigate the mechanisms underlying the anti-atheroscleroic action of atorvastatin, we examined the expression of the receptor for advanced glycation end products (RAGE) and its downstream target gene, monocyte chemoattractant protein-1 (MCP-1) using real-time PCR. In in vitro studies, exposure to high glucose or AGE induced oxidative stress and activation of the AGE/RAGE system in human umbilical vein endothelial cells. Treatment of the cells with atorvastatin significantly released the oxidative stress by restoring the levels of glutathione and inhibited the RAGE upregulation. In diabetic Goto Kakisaki (GK) rats fed with a high-fat diet for 12 weeks, RAGE and MCP-1 were upregulated in the aortas, and there was a significant correlation between RAGE and MCP-1 mRNA abundance (r = 0.482, P = 0.031). Treatment with atorvastatin (20 mg/kg qd) significantly downregulated the expression of RAGE and MCP-1. These data thus demonstrate a novel “pleiotropic” activity of atorvastatin in reducing the risk of cardiovascular diseases by targeting RAGE expression.     

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.     

Modulation of high sensitivity C-reactive protein by soluble receptor for advanced glycation end products

High sensitivity C-reactive protein (hs-CRP) is synthesized mainly by hepatocytes in response to tumor necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6). The interaction of advanced glycation end products (AGEs) with the receptor for advanced glycation end products (RAGE) increases the expression of the cytokines TNF-α, IL-1, and IL-6. Soluble receptor for advanced glycation end products (sRAGE) competes with RAGE for binding with AGEs. Hence, low sRAGE levels may increase interaction of AGEs with RAGE resulting in the increased production of cytokines. It is hypothesized that serum levels of sRAGE modulate serum levels of hs-CRP. The objectives are to determine if (i) serum levels of sRAGE are lower and those of TNF-α and hs-CRP are higher in non-ST-segment elevation myocardial infarction (NSTEMI) patients compared to control subjects; (ii) serum levels of TNF-α and hs-CRP are positively correlated; and (iii) sRAGE is negatively correlated with hs-CRP and TNF-α. The study consisted of 36 patients with NSTEMI and 30 age-matched healthy male subjects. Serum levels of sRAGE and TNF-α were determined by enzyme-linked immunoassay and hs-CRP was measured using near infrared immunoassay. Serum levels of sRAGE were lower, while those of TNF-α and hs-CRP were higher in patients with NSTEMI compared to controls. The levels of sRAGE were negatively correlated with those of TNF-α and hs-CRP, while TNF-α was positively correlated with hs-CRP in both the control subjects and NSTEMI patients. The data suggest that sRAGE modulates the synthesis of hs-CRP through TNF-α.     

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.     

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.     

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.     

Cd36, a member of the class b scavenger receptor family, as a receptor for advanced glycation end products

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. Five AGE receptors identified so far are RAGE (receptor for AGE), galectin-3, 80K-H, OST-48, and SRA (macrophage scavenger receptor class A types I and II). Since SRA is known to belong to the class A scavenger receptor family, and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36, although belonging to the class B scavenger receptor family, can recognize AGE proteins as ligands. This was tested at the cellular level in this study using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CD36-CHO cells). Cellular expression of CD36 was confirmed by immunoblotting and immunofluorescent microscopy using anti-CD36 antibody. Upon incubation at 37 degrees C, (125)I-AGE-bovine serum albumin (AGE-BSA) and (125)I-oxidized low density lipoprotein (LDL), an authentic ligand for CD36, were endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CD36-CHO cells, but not wild-type CHO cells. In binding experiments at 4 degrees C, (125)I-AGE-BSA exhibited specific and saturable binding to CD36-CHO cells (K(d) = 5.6 microg/ml). The endocytic uptake of (125)I-AGE-BSA by these cells was inhibited by 50% by oxidized LDL and by 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of oxidized LDL. Our results indicate that CD36 expressed by these cells mediates the endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major oxidized LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, these results suggest that, like oxidized LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.     

Decreased levels of soluble receptor for advanced glycation end products in patients with rheumatoid arthritis indicating deficient inflammatory control

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily being expressed as a cell surface molecule and binding a variety of ligands. One of these ligands is high-mobility group box chromosomal protein 1, a potent proinflammatory cytokine, expression of which is increased in synovial tissue and in synovial fluid of rheumatoid arthritis (RA) patients. The interaction of high-mobility group box chromosomal protein 1 with cell-surface RAGE leads to an inflammatory response. In contrast, the presence of soluble RAGE (sRAGE) may abrogate cellular activation since the ligand is bound prior to interaction with the surface receptor.