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The receptor for advanced glycation end products (RAGE) plays a key role in mammal physiology and in the etiology and progression of inflammatory and oxidative stress-based diseases. In adults, RAGE expression is normally high only in the lung where the protein concentrates in the basal membrane of alveolar Type I epithelial cells. In diseases, RAGE levels increase in the affected tissues and sustain chronic inflammation. RAGE exists as a membrane glycoprotein with an ectodomain, a transmembrane helix, and a short carboxyl-terminal tail, or as a soluble ectodomain that acts as a decoy receptor (sRAGE). VC1 domain is responsible for binding to the majority of RAGE ligands including advanced glycation end products (AGEs), S100 proteins, and HMGB1. To ascertain whether other ligands exist, we analyzed by MS the material pulled down by VC1 from human plasma. Twenty of 295 identified proteins were selected and associated to coagulation and complement processes and to extracellular matrix. Four of them contained a g-carboxyl glutamic acid (Gla) domain, a calcium-binding module, and prothrombin (PT) was the most abundant. Using MicroScale thermophoresis, we quantified the interaction of PT with VC1 and sRAGE in the absence or presence of calcium that acted as a competitor. PT devoid of the Gla domain (PT des-Gla) did not bind to sRAGE, providing further evidence that the Gla domain is critical for the interaction. Finally, the presence of VC1 delayed plasma clotting in a dose-dependent manner. We propose that RAGE is involved in modulating blood coagulation presumably in conditions of lung injury. [ABSTRACT FROM AUTHOR]

Serum levels of early-glycated albumin are significantly increased in patients with diabetes mellitus and may play a role in worsening inflammatory status and sustaining diabetes-related complications. To investigate possible pathological recognition involving early-glycated albumin and the receptor for advanced glycation end products (RAGE), an early-glycated human serum albumin (HSAgly), with a glycation pattern representative of the glycated HSA form abundant in diabetic patients, and the recombinant human RAGE ectodomain (VC1) were used. Biorecognition between the two interactants was investigated by combining surface plasmon resonance (SPR) analysis and affinity chromatography coupled with mass spectrometry (affinity-MS) for peptide extraction and identification. SPR analysis proved early-glycated albumin could interact with the RAGE ectodomain with a steady-state affinity constant of 6.05 ± 0.96 × 10−7 M. Such interaction was shown to be specific, as confirmed by a displacement assay with chondroitin sulfate, a known RAGE binder. Affinity-MS studies were performed to map the surface area involved in the recognition. These studies highlighted that a region surrounding Lys525 and part of subdomain IA were involved in VC1 recognition. Finally, an in silico analysis highlighted (i) a key role for glycation at Lys525 (the most commonly glycated residue in HSA in diabetic patients) through a triggering mechanism similar to that previously observed for AGEs or advanced lipoxidation end products and (ii) a stabilizing role for subdomain IA. Albeit a moderate affinity for complex formation, the high plasma levels of early-glycated albumin and high percentage of glycation at Lys525 in diabetic patients make this interaction of possible pathological relevance. [ABSTRACT FROM AUTHOR]

Background: The AGE-RAGE-oxidative stress (AROS) axis is involved in the onset and progression of metabolic syndrome induced by a high-fructose diet (HFD). PPARγ activation is known to modulate metabolic syndrome; however a systems-level investigation looking at the protective effects of PPARγ activation as related to the AROS axis has not been performed. The aim of this work is to simultaneously characterize multiple molecular parameters within the AROS axis, using samples taken from different body fluids and tissues of a rat model of HFD-induced metabolic syndrome, in the presence or absence of a PPARγ agonist, Rosiglitazone (RGZ). Methods: Rats were fed with 60% HFD for the first half of the treatment duration (21 days) then continued with either HFD alone or HFD plus RGZ for the second half. Results: Rats receiving HFD alone showed metabolic syndrome manifestations including hypertension, dyslipidemia, increased glucose levels and insulin resistance, as well as abnormal kidney and inflammatory parameters. Systolic blood pressure, plasma triglyceride and glucose levels, plasma creatinine, and albuminuria were significantly improved in the presence of RGZ. The following molecular parameters of the AROS axis were significantly upregulated in our rat model: carboxymethyl lysine (CML) in urine and liver; carboxyethyl lysine (CEL) in urine; advanced glycation end products (AGEs) in plasma; receptor for advanced glycation end products (RAGE) in liver and kidney; advanced oxidation protein products (AOPP) in plasma; and 4-hydroxynonenal (HNE) in plasma, liver, and kidney. Conversely, with RGZ administration, the upregulation of AOPP and AGEs in plasma, CML and CEL in urine, RAGE in liver as well as HNE in plasma and liver was significantly counteracted/prevented. Conclusions: Our data demonstrate (i) the systems-level regulatory landscape of HFD-induced metabolic syndrome involving multiple molecular parameters, including HNE, AGEs and their receptor RAGE, and (ii) attenuation of metabolic syndrome by PPARγ modulation. [ABSTRACT FROM AUTHOR]

Advanced glycation end-products (AGEs) and advanced lipoxidation end-products (ALEs), particularly carboxymethyl-lysine (CML), have been largely proposed as factors involved in the establishment and progression of heart failure (HF). Despite this evidence, the current literature lacks the comprehensive identification and characterization of the plasma AGEs/ALEs involved in HF (untargeted approach). This work provides the first ex vivo high-resolution mass spectrometry (HR-MS) profiling of AGEs/ALEs occurring in human serum albumin (HSA), the most abundant protein in plasma, characterized by several nucleophilic sites and thus representing the main protein substrate for AGE/ALE formation. A set of AGE/ALE adducts in pooled HF-HSA samples was defined, and a semi-quantitative analysis was carried out in order to finally select those presenting in increased amounts in the HF samples with respect to the control condition. These adducts were statistically confirmed by monitoring their content in individual HF samples by applying a targeted approach. Selected AGEs/ALEs proved to be mostly CML derivatives on Lys residues (i.e., CML-Lys12, CML-Lys378, CML-Lys402), and one deoxy-fructosyl derivative on the Lys 389 (DFK-Lys 389). The nature of CML adducts was finally confirmed using immunological methods and in vitro production of such adducts further confirmed by mass spectrometry. [ABSTRACT FROM AUTHOR]

Highlights: [•] AGEs of β2-m were identified and characterized in in vitro conditions. [•] Carboxymethyl, carboxyethyl and methyl-imidazolinone Arg3 were identified in β2-m isolated from the urine of uremic subjects. [•] AGEs content of urinary β2-m were found to range from 0.2 to 1% in uremic subjects. [Copyright &y& Elsevier]

Advanced Lipoxidation Endproduct (ALEs) are modified proteins that can act as pathogenic factors in several chronic diseases, like diabetes and cardiovascular diseases [1]. These covalent adducts belong to a heterogeneous class of compounds derived from the protein adduction by reactive carbonyl species (RCS), which are generated upon lipid peroxidation. A similar class of compounds, Advanced Glycation Endproducts (AGEs), exhibit the same damaging effects as ALEs, partly due to binding to the Receptor for Advanced Glycation End products (RAGE). Using this receptor as a stationary phase for affinity chromatography, modified proteins could be potentially entrapped and enriched from any sample, to identify and characterize the origin of the modification. Applying this innovative approach, it has been shown already that AGEs can be captured, enabling the full characterization of the adducted moieties and site of modification using a bottom-up approach [2]. In order to validate this strategy for ALEs, and with the aim to understand whether ALEs are also binder of RAGE, fully characterized ALEs were produced in-vitro, by incubating human serum albumin (HSA) with glyoxal (GO), methylglyoxal (MG), 4-hydroxynonenal (HNE), acrolein (ACR) and malondialdehyde (MDA). The formation of ALEs was confirmed using a top-down MS approach by direct infusion on a triple-quadrupole mass spectrometer and the modifications and sites of adduction fully characterized by a bottom-up approach. The in-vitro produced ALEs were then subjected to VC1 Pull-Down relying on magnetic beads bound to VC1, the domain of RAGE necessary for binding, which can easily be separated. ALEs will be retained by VC1 and unbound protein can be easily removed, enriching ALEs in the sample. After binding, ALEs are eluted from the magnetic beads and subjected to GeLC-MS/MS to identify and localize the modification. Data obtained using this method were analysed using a targeted approach based on setting known modifications. Results obtained using VC1 Pull-Down were compared to the results of the in-solution digested ALEs and showed that ALEs containing a cyclic moiety induced by the modifications, are better retained by VC1, including pyrimidine, pyridine, pyraline and imidazolone adducts. Another observation is the binding of ALEs containing a carboxy-derivative, since these adducts exhibit a negative charge and increases the binding specificity to VC1, which has a positive charge. Semi-quantitative analysis also showed an enrichment of ALEs from VC1 Pull-Down, compared to unenriched sample. Results showed that VC1 can be used as a stationary phase to selectively enrich ALEs, depending on the structure and nature of the modification. Different applications of this technique are underway to identify and characterize ALEs and AGEs from samples of patients affected by diseases involving oxidative stress. In conclusion, we have found that besides AGEs, also ALEs are RAGE binders. ALEs involvement in the RAGE dependent proinflammatory cascade is currently under investigation. [ABSTRACT FROM AUTHOR]