High resolution mass spectrometry based approach to investigate RAGE-ligand interactions

The receptor for advanced glycation end products (RAGE) is a type I transmembrane glycoprotein of the immunoglobulin superfamily of cell surface receptors (1). The engagement of RAGE by ligands triggers key signalling pathways resulting in oxidative damage and release of pro-inflammatory and pro-fibrotic molecules (2). 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 oxidative derived cheto-aldehydes. AGEs are involved in the onset and progression of different oxidative based diseases, including diabetic vascular complications and atherosclerosis. Hence, the inhibition of AGEs formation as well as the blockade of AGEs-RAGE interaction represents a promising drug target. 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, a MS method was firstly set up to study the non covalent interactions between ligands and recombinant sRAGEs (V1-C1), which represents the ligand binding domain of RAGE. The V1-C1 protein target was expressed in E. coli and the ligand-protein binding properties (stoichiometry and Kd values) were determined by a high resolution mass spectrometric (orbitrap) approach carried out in non denaturating conditions. Denaturing-nondenaturing experiments indicate that the V1-C1 RAGE portion is maintained in a native-folded conformation during the ion gas phase ESI-MS experiments. The method was then validated by using well known low molecular weight sRAGE ligands such as carboxymethyl lysine (CML) and carboxyethyl lysine (CEL) derived FKDGLEE peptide. In particular the Kd values of these peptides as well as that of the native peptide were in line with those previously reported by fluorescence titration experiments (3). In conclusion the high resolution ESI-MS method here reported is suitable to detect V1- C1 RAGE portion-ligand complexes and to determine accurate Kd. The method we set up is suitable to test a library of peptides bearing a variety of AGEs modifications including imidazolinone, methyl-imidazolinone, and the Michael adducts with alfa,betaunsaturated aldehydes, in order to understand the structure requirements for RAGE recognition. (1) Dattilo BM, Fritz G, Leclerc E, et al. Biochemistry 2007,46, 6957-6970. (2) Barlovic DP, Soro-Paavonen A, Jandeleit-Dahm KA. Clin Sci (Lond). 2011, 121,43-55. (3) Xue J, Shekhtman A, et al. Cell Press 2011, 19, 722-73