Analytical Strategies for the Identification and Characterization of RAGE Binders of Proinflammatory mediators. AGEs and ALES

INTRODUCTION AGEs and ALEs (Advanced Glycoxidation/Lipoxidation End products) are covalently modified proteins that can act as pathogenic factors in several chronic diseases, like diabetes and cardiovascular diseases. These covalent adducts are formed by different mechanisms. AGEs are proteins covalently modified by reducing sugars or their oxidative degradation products, involving the Maillard reaction. ALEs are proteins modified by reactive carbonyl species (RCS) generated by lipid peroxidation. AGEs/ALEs can be the basis of many different pathologies, underlining the importance for good analytical methods for identification and characterization for the use of biomarkers, but also as a drug target. However, the identification, characterization and quantification of AGEs/ALEs remains to be very challenging due to heterogeneous precursors (sugars, lipids) leading to heterogeneous AGEs/ALEs, present in low concentrations and being very complex analytes. Various techniques to identify and characterize AGEs/ALEs have been described, making use of an isolation/enrichment step based on reactive groups, like carbonyls. However, not all AGEs/ALEs retain reactive groups and therefore can not be isolated and identified using these techniques, indicating the need for a new strategy. The strategy that has been employed in our laboratory is to use the soluble domain of the RAGE receptor, VC1, to affinity enrich AGEs. Using this approach, AGEs/ALEs will be enriched independently of the protein and type of modification. Moreover, a ligand of RAGE can be identified, which could be a potential biomarker of a disease caused by oxidative stress. RAGE is a type I cell surface receptor that is expressed in several cells, such as endothelial cells, smooth muscle cells, but also dendritic cells and T-lymphocytes and is predominantly located in the lungs. The receptor has been implicated in many different pathologies with a marked oxidative base, such as diabetes, atherosclerosis and neurodegenerative diseases. One of the pathways that can be activated is the Nf-κB pathway. The Nf-κB pathway is the ideal signaling pathway to investigate the binding and activation of RAGE by AGEs or ALEs. For this purpose, a cell line was obtained with and without overexpression of RAGE. Furthermore, the cell lines were transfected with a Nf-κB reporter gene, providing us with a fast and high-throughput assay for the evaluation of a pro-inflammatory response upon stimulation with AGEs/ALEs. AIM OF THE PROJECT The identification and characterization of AGEs/ALEs has proven to be crucial in the onset and development of many pathologies. Therefore, good analytical strategies need to be developed/optimized for better understanding of the exact nature of modification, to understand the role they play in disease progression. Identified AGEs/ALEs can serve as biomarker, as well as drug targets. The VC1 technique was proven to be a promising technique to accommodate the need for enrichment of AGEs for better characterization. The first aim of the project was therefore to investigate whether also ALEs are binder of RAGE, since they share the same structural properties than AGEs, and also have been shown to activate the Nf-κB pathway, implicating a role for receptors, like RAGE. Furthermore, to gain a deeper insight into the molecular mechanisms involved in the protein-protein engagement. Since a successful enrichment strategy was developed, the second aim of this project was focused on identifying AGEs/ALEs in biological samples. The first part was focused on oxidizing healthy human plasma in-vitro using AAPH as a radical initiator, and the incubation of plasma directly with RCS, anticipating the production of AGEs/ALEs. The VC1 technique was then used to identify which AGEs/ALEs are produced. Simultaneously, other variables during the sample preparation and analysis were optimized. As explained before, AGEs/ALEs are present in very low concentrations in biological samples, hence the need for very sensitive methods and instrumentation allowing identification. Since human serum albumin (HSA) is the main protein present in plasma, around 50-60%, and has multiple nucleophilic targets, it represents the best model for characterizing AGEs/ALEs. For this reason, the focus was on extracting HSA from plasma, using the newest generation of tribrid MS for the analysis of AGEs/ALEs in plasma samples. AGEs are ligands for RAGE, meaning, they can bind and activate the receptor, inducing a signaling pathway and pro-inflammatory response. ALEs have also been shown to induce a pro-inflammatory response; however, no specific receptor has been linked to this cellular event. Using a cell line with and without RAGE, we aimed to determine whether ALEs can bind and activate the Nf-κB pathway through RAGE. RESULTS AND DISCUSSION ALEs as binder of RAGE In order to investigate the interaction between RAGE and ALEs, different ALEs were produced in-vitro by incubating HSA with different concentrations of well-known lipid derived RCS and in particular: ACR, MDA and HNE. After 24, 48 and 72 h, aliquots of the incubation mixtures were withdrawn, and the reaction was stopped by removing the excess of RCS by ultrafiltration. Intact protein analysis by direct infusion MS was used to evaluate the extent of HSA modifications and demonstrated that by using a wide range of molar ratios and different time-points a quite wide array of ALEs for each tested RCS was generated. In order to characterize ALEs selectively enriched by RAGE, a VC1 pull-down assay was performed as previously described. HSA and HSA treated with MDA, ACR or HNE were assayed for binding to VC1-resins and to control resin. As expected, unmodified HSA was not retained by the VC1-resin. At increasing molar ratios and incubation time, higher amounts of albumin modified with MDA or ACR were eluted from the VC1 resin, with a predominance of the high molecular weight (HMW) species. The modified albumins were retained by the VC1-resin, but not by the control resin. ALEs in the reaction mixtures and those enriched by VC1 were analyzed by bottom-up MS in order to identify the PTMs and to localize the amino acid residues involved in the protein adduct formation. With regard to MDA, only di-hydropyridine adducts on lysines (DHPK), and N-2-pyrimidyl-ornithine adducts on arginines (NPO) were retained by VC1-domain. The n-propenal modifications of lysine (NPK), largely identified before enrichment, were not identified after the enrichment. ACR induced a set of modifications which were identified only after VC1 enrichment and in particular the N-(3-formyl-3,4-dehydro-piperidinyl) lysine (FDPK) modifications, the Michael adduct on cysteines, the double Michael adduct of lysines, the Michael adduct of histidine, the N-2-(4 hydroxy-tetrahydro-pyrimidyl) ornitine (propane-arginine, HTPO) and the Nε-(3-methylpyridinium)-lysine (MP-lysine). Most of the ALEs generated by HNE were found both before or after enrichment, with the exception of a few Michael adducts which were selectively retained by VC1 (not detected before enrichment). With a view to rationalizing the key factors influencing the RAGE binding of the monitored adducts, in silico studies were performed. They were focused on the adducts on arginine and lysine residues as formed by ACR and MDA since they are numerous, with a very broad range of affinity, thus allowing the development of clear structure-affinity relationships. RAGE-ligand interacting regions are characterized by a rich set of positively charged residues which can bind acidic regions of a protein. The mechanism identified using in silico studies, involves a basic amino acid at the center of carboxylic acids like glutamate and aspartate, which forms a set of ionic bridges. Once the basic amino acid is modified by ACR or MDA to an adduct with a neutral charge, the carboxylic acids become available to freely contact the RAGE positive residues. Identification of AGEs/ALEs in biological samples The VC1 technique has proven to be successful in enriching AGEs and ALEs, so the next step was to exploit this technique in biological samples. In order to identify proteins prone to be modified due to oxidative pathways, and possibly serve as biomarker, healthy human plasma was oxidized using the radical initiator AAPH. Different concentrations of AAPH and different timepoints were tested for the presence of protein carbonyl groups, an indicator for protein oxidation and possibly the formation of AGEs/ALEs. A time and concentration dependent formation of carbonyl groups is observed in plasma. Next, samples were analyzed using a bottom-up approach. Results obtained were showing many oxidation products, such as amino side chain oxidation, however no AGEs/ALEs were identified. Thus, a new approach was adopted, including the incubation of plasma directly with RCS, such as HNE, MDA and ACR. This resulted in the formation of AGEs/ALEs in plasma samples, however, they could not be retained by the VC1 domain. Instead of using the VC1 technique to enrich AGEs/ALEs from biological samples, other variables throughout the experimental set-up were optimized. Previously, peptides were analyzed using the Orbitrap LTQ XL, a very powerful instrument. Nonetheless, the newest generation of tribrid MS offers even higher resolution, and it increases protein coverage due to parallel isolation and detection, and faster analyzers. Moreover, we focused on AGEs/ALEs from HSA and using NaBH4 to reduce and stabilize adducts throughout the analysis. This new approach permitted us to identify many AGEs/ALEs in both healthy human plasma samples, but also AGEs/ALEs only present in heart failure samples. Glycation on lysine residues was the main modification identified, present in both healthy and heart failure plasma samples. Important is the HNE Michael adduct, specifically identified in only heart failure samples. Moreover, the importance of stabilizing adducts is underlined by the fact that the acrolein Michael adduct could only be identified after reduction with NaBH4. Development of a cellular assay to determine pro-inflammatory activity of RAGE binders Another part of this project was focused on elucidating whether AGEs/ALEs induce an inflammatory response in cells. For this purpose, a collaboration was started with the Laboratory of Vascular Biology and Regenerative Medicine, Centro Cardiologico Monzino. Using a rat epithelial lung cell line overexpressing RAGE, and a control cell line not expressing RAGE, it could be detected if AGEs/ALEs exhibit an effect by binding to RAGE. Moreover, both cell lines were transfected with a Nf-κB reporter gene allowing us a fast and sensitive method for determining if binding of RAGE induces a down-stream signaling pathway. This system implies a firefly luciferase gene downstream from the Nf-κB gene. When the Nf-κB pathway is activated, independently from RAGE, it produces the firefly luciferase enzyme. After adding a luciferin substrate, firefly luciferase is able to convert this substrate into another substrate with light as by-product, which can be measured by a luminometer. IL-1α was used as a positive control, since it induces a strong inflammatory response through Nf-κB. Moreover, known ligands of RAGE able to activate the Nf-κB pathway, were used to validate the cellular experiment, including HSA modified with fructose (AGE), and HMGB1. Results show that Nf-κB is already increased in untreated cells with RAGE and that AGEs induce the Nf-κB pathway independently from RAGE. Moreover, the difference between control and RAGE cells is not significantly increased in the presence of HMGB1 compared to untreated. However, the positive control seemed to induce a much stronger activity in cells with RAGE. Overall, this cellular assay is good for assessing pro-inflammatory activity, however, it is not optimized yet for distinguishing a RAGE-dependent mechanism. CONCLUSION In summary, by using an integrated MS (intact protein and bottom-up approach) and computational approach we have found that some ALEs generated from lipid peroxidation RCS are RAGE binders. We have also found the basic features that ALEs from HNE, MDA and ACR must have to be a RAGE binder: 1) the covalent adducts should greatly reduce or abolish the basicity of the target amino acid, 2) the basic amino acid should be at the center of a set of carboxylic acids which, once the residue is modified, become available to freely contact the RAGE positive residues. Next step was to use the VC1 technique to enrich AGEs/ALEs in biological samples. First, oxidized human plasma was used, however, using the Orbitrap LTQ XL, it was not sufficient to identify AGEs/ALEs. Therefore, analysis was moved to a higher resolution mass spectrometer, which allowed us to identify AGEs/ALEs in plasma samples of heart failure patients, showing the powerfulness of this new generation MS. Important was to understand whether ALEs could induce pro-inflammatory activity through RAGE, since we showed that ALEs are RAGE binders. Unfortunately, the cellular assay that was set up is efficiently in determining Nf-κB dependent pro-inflammatory activity, but not if it is RAGE dependent.