Immunochemical detection of advanced glycosylation end products in vivo.

Reducing sugars react with protein amino groups to form a diverse group of protein-bound moieties with fluorescent and cross-linking properties. These compounds, called advanced glycosylation end products (AGEs), have been implicated in the structural and functional alterations of proteins that occur during aging and long-term diabetes. Although several AGEs have been identified on the basis of de novo synthesis and tissue isolation procedures, the measurement of AGE compounds in vivo has remained difficult. As an approach to the study of AGE formation in vivo, we prepared polyclonal antiserum to an AGE epitope(s) which forms in vitro after incubation of glucose with ribonuclease (RNase). This antiserum proved suitable for the detection of AGEs which form in vivo. Both diabetic tissue and serum known to contain elevated levels of AGEs readily competed for antibody binding. Cross-reactivity studies revealed the presence of a common AGE epitope(s) which forms after the incubation of diverse proteins with glucose. Cross-reactive epitopes also formed with glucose 6-phosphate or fructose. These data suggest that tissue AGEs which form in vivo appear to contain a common immunological epitope which cross-reacts with AGEs prepared in vitro, supporting the concept that immunologically similar AGE structures form from the incubation of sugars with different proteins (Horiuchi, S., Araki, N., and Morino, Y. (1991) J. Biol. Chem. 266, 7329-7332). None of the known AGEs, such as 4-furanyl-2-furoyl-1H-imidazole, 1-alkyl-2-formyl-3,4-diglycosylpyrrole, pyrraline, carboxymethyllysine, or pentosidine, were found to compete for binding to anti-AGE antibody. These data further suggest that the dominant AGE epitope which forms from the reaction of glucose with proteins under native conditions is immunologically distinct from the structurally defined AGEs described to date.     

Characterization of a solubilized cell surface binding protein on macrophages specific for proteins modified nonenzymatically by advanced glycosylated end products

Glucose can react nonenzymatically with free protein amino groups to form Amadori products, 1-amino-1-deoxyketose residues. These adducts can undergo subsequent rearrangements and dehydrations to form a complex group of brown, fluorescent pigments collectively referred to as advanced glycosylation end products (AGE). One AGE has been identified as 2-(2-furoyl)-4(5)-(2-furanyl)-1H-imidazole (FFI). The AGE-protein adducts accumulate with time and are implicated in irreversible tissue damage. We have previously demonstrated that macrophages bind and degrade AGE-proteins via a specific cell surface binding protein, thus selectively removing senescent macromolecules. In the present communication, we have solubilized this binding protein from the membranes of the murine macrophage cell line RAW 264.7. We have characterized the nature of binding protein-ligand interaction by competition studies using modified ligands. The data indicate that the carbonyl group, the furan ring(s), and the central imidazole structure are all important in the binding protein-ligand interaction. We have established that the binding constant (Ka) of binding protein for the ligand FFI-BA is 3.1 X 10(7) M-1. Chemical crosslinking studies have demonstrated that the molecular weight of the binding protein is 90,000.     

Cell surface expression of membrane-anchored v-sis gene products: glycosylation is not required for cell surface transport

The v-sis gene is able to transform cells by production of a growth factor that is structurally related to platelet-derived growth factor. This growth factor has been detected in the conditioned media of v-sis transformed cells, and is able to stimulate the autophosphorylation of the platelet-derived growth factor receptor. We have used the v-sis gene product to analyze the role of protein-encoded signals in cell surface transport. We constructed several gene fusions that encode transmembrane forms of the v-sis gene product. These membrane-anchored forms of the v-sis gene product are properly folded into a native structure, as indicated by their dimerization, glycosylation, and NH2- terminal proteolytic processing. Indirect immunofluorescence demonstrated that several of these membrane-anchored gene products are transported to the cell surface. Removal of the N-linked glycosylation site from the v-sis gene product did not prevent cell surface transport. Several of these mutant genes are able to induce focus formation in NIH3T3 cells, providing further evidence that the membrane- anchored proteins are properly folded. These results demonstrate that N- linked glycosylation is not required for the cell surface transport of a protein that is in a native, biologically active conformation. These results provide a correlation between cell surface expression of the membrane-anchored v-sis gene products and transformation.     

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.     

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.     

Overexpression of receptor of advanced glycation end products hypersensitizes cells for amyloid beta peptide-induced cell death

Receptor of advanced glycation end products (RAGE) was identified as one of the receptors for amyloid beta peptide (Abeta). There is evidence for controversial functions of RAGE such as a mediator of cell death or differentiation. In this report, we demonstrate that RAGE mediates Abeta toxicity. Transient transfection of RAGE already induced cell death. For further analysis, stable clones of hemagglutinin (HA)-tagged RAGE were selected. Analysis of cellular localization of HA-tagged RAGE protein revealed, in addition to the expected cell surface expression, a novel intracellular localization. Stable RAGE-expressing cells were hypersensitive to nanomolar amounts of Abeta. Only cells expressing RAGE at the cell surface showed hypersensitivity to Abeta.     

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.     

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.     

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.     

Regulation of susceptibility and cell surface receptor for the B-lymphotropic papovavirus by N glycosylation.

The host range of the B-lymphotropic papovavirus (LPV) in cultured human cells is limited to a few B-lymphoma-derived cell lines. The constitutively expressed cell surface receptor for the virus is a major determinant restricting the LPV host range (G. Haun, O. T. Keppler, C. T. Bock, M. Herrmann, H. Zentgraf, and M. Pawlita, J. Virol. 67:7482-7492, 1993). Here we show that human B-lymphoma cells with low-level susceptibility are rendered highly susceptible to LPV infection by pretreatment with the N glycosylation inhibitor tunicamycin but remain nonsusceptible to infection by the related polyomavirus simian virus 40. Among the selective N glycosylation processing inhibitors, deoxymannojirimycin, but not deoxynojirimycin, swainsonine, or castanospermine, could mimic the effect of tunicamycin. Tunicamycin treatment also induced a drastic enhancement of the cells' LPV-binding capacity, indicating that the induction of LPV susceptibility might be mediated by an increase in the number of functional cell surface receptors and/or by increased receptor affinity. Sialidase sensitivity of the tunicamycin-induced LPV receptor showed that oligosaccharides carrying terminal sialic acids are necessary for binding and are likely to be O linked. The constitutive LPV receptor is also sialic acid dependent, which points to a possible identity with the sialic acid-dependent tunicamycin-induced LPV receptor. We conclude that removal or modification of certain N-linked oligosaccharides in human B-lymphoma cells can enhance expression or functional activity of the sialylated LPV receptor.     

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.     

Aldose reductase (AKR1B3) regulates the accumulation of advanced glycosylation end products (AGEs) and the expression of AGE receptor (RAGE)

Diabetes results in enhanced chemical modification of proteins by advanced lipoxidation end products (ALEs) and advanced glycation end products (AGEs) precursors. These modifications have been linked to the development of several secondary diabetic complications. Our previous studies showed that aldose reductase (AR; AKR1B3) catalyzes the reduction of ALEs and AGEs precursors; however, the in vivo significance of this metabolic pathway during diabetes and obesity has not been fully assessed. Therefore we examined the role of AR in regulating ALEs and AGEs formation in murine models of diet-induced obesity and streptozotocin-induced diabetes. In comparison with wild-type (WT) and AR-null mice fed normal chow, mice fed a high-fat (HF) diet (42% kcal fat) showed increased accumulation of AGEs and protein-acrolein adducts in the plasma. AGEs and acrolein adducts were also increased in the epididymal fat of WT and AR-null mice fed a HF diet. Deletion of AR increased the accumulation of 4-hydroxy-trans-2-nonenal (HNE) protein adduct in the plasma and increased the expression of the AGE receptor (RAGE) in HF fed mice. No change in AGEs formation was observed in the kidneys of HF-fed mice. In comparison, renal tissue from AR-null mice treated with streptozotocin showed greater AGE accumulation than streptozotocin-treated WT mice. These data indicated that AR regulated the accumulation of lipid peroxidation derived aldehydes and AGEs under conditions of severe, but not mild, hyperglycemia and that deletion of AR increased RAGE-induction via mechanisms that were independent of AGEs accumulation.     

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.     

糖基化终产物对人肾小球系膜细胞氧化应激及MCP-1表达的影响及机制

目的：探讨体外培养条件下糖基化终产物（AGEs）对人肾小球系膜细胞（HRMCs）中糖基化终产物受体（RAGE）、氧化应激及单核细胞趋化因子-1（MCP-1）表达的影响。方法：将HRMCs与不同浓度的糖化牛血清白蛋白（AGE-BSA）和牛血清白蛋白（BSA）共同培养,或与同一质量浓度的AGE-BSA和BSA共同培养不同时间,以中和抗RAGE抗体封闭细胞膜上RAGE;采用细胞免疫化学法检测AGEs对HRMCs中RAGE表达的影响,流式细胞术检测细胞内活性氧（ROS）,半定量逆转录-聚合酶链反应（RT-PCR）法检测MCP-1 mRNA的表达。结果：在HRMCs中AGE-BSA能够促进RAGE的表达,并以时间和剂量依赖方式促进HRMCs中ROS及MCP-1的表达;ROS及MCP-1的表达水平在加入不同浓度（50、100、200、400 mg/L）的AGE-BSA作用48 h后以及加入质量浓度为200 mg/L的AGE-BSA作用不同时间（12、24、48、72 h）后,较相应质量浓度或时间的BSA组和对照组均明显升高（P〈0.05）;抗RAGE抗体干预后能够部分抑制AGE-BSA诱导ROS及MCP-1的表达,而人IgG没有这种作用。结论：AGEs通过RAGE激活氧化应激效应诱导MCP-1的表达上调,是糖尿病肾病发生发展的可能机制。     

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.