A new high resolution mass spectrometry application for the comprehensive analysis of the ligand-binding properties of rage

The receptor for advanced glycation end products (RAGE) is a type I transmembrane glycoprotein of the immunoglobulin superfamily of cell surface receptors [1]. RAGE is able to bind a class of heterogeneous compounds such as advanced glycation end products (AGEs) formed as a result of nonenzymatic biochemical reactions involving glucose and cheto-aldehydes derived from glucose and lipid oxidation. The accumulation of these compounds is accelerated in the presence of hyperglycemia and oxidative stress. AGEs are involved in the onset and progression of different oxidativebased diseases, including diabetic vascular complications. The engagement of RAGE by ligands triggers the oligomerization of RAGE and activation of key signaling pathways resulting in reprogramming of gene expression and release of proinflammatory molecules. Long term-activation of RAGE also elicits oxidative stress thus contributing to the pathological changes observed in diabetic vascular complications [2]. Hence, the inhibition of AGEs formation as well as the blockade of AGEs-RAGE interaction represents a promising drug target for therapeutic interventions in complications of diabetes. In order to exploit RAGE as a therapeutic target, a comprehensive analysis of the structure and ligand-binding properties of RAGE is required. For this purpose, the research was firstly focused on two different steps: (i) RAGE (sRAGE) expression as recombinant proteins and (ii) set-up of a MS method to study the non covalent interactions between low molecular weight AGEs and recombinant sRAGE. The RAGE extracellular portion is involved in ligand binding and contains one “V”-type followed by two “C”-type immunoglobulin-like domains (V-C1-C2 structure). The V-C1 domains form an autonomous structural and functional unit. The V-C1 protein was expressed in E.coli with a C-terminal tag of 6 Histidine residues and a thrombin cleavage site to facilitate the purification procedures and the removal of the tag. In the second step, a high resolution mass spectrometric (MS) approach was applied in native conditions to study the non covalent interactions of sRAGE. The method is based on an orbitrap as mass analyzer working in positive ion conditions. The method was validated by using well known low- and high-molecular weight sRAGE ligands such as carboxymethyl lysine (CML), peptides containing a CML modification and glycoxidated proteins. The method we set-up is suitable to test a library of peptides bearing a variety of AGEs modification including carboxyethyl lysine, imidazolone, methylimidazolone, and the Michael adducts with alfa,beta-unsaturated aldehydes, in order to understand the structure requirements of AGEs for RAGE recognition. References [1] Dattilo BM, Fritz G, Leclerc E, et al. Biochemistry 2007,46, 6957-70. [2] Barlovic DP, Soro-Paavonen A, Jandeleit-Dahm KA. Clin Sci (Lond). 2011, 121(2), 43-55.