title: A capture method based on the VC1 domain reveals new binding properties of the human receptor for advanced glycation end products (RAGE).
abstract: The Advanced Glycation and Lipoxidation End products (AGEs and ALEs) are a heterogeneous class of compounds derived from the non-enzymatic glycation or protein adduction by lipoxidation break-down products. The receptor for AGEs (RAGE) is involved in the progression of chronic diseases based on persistent inflammatory state and oxidative stress. RAGE is a pattern recognition receptor (PRR) and the inhibition of the interaction with its ligands or of the ligand accumulation have a potential therapeutic effect. The N-terminal domain of RAGE, the V domain, is the major site of AGEs binding and is stabilized by the adjacent C1 domain. In this study, we set up an affinity assay relying on the extremely specific biological interaction AGEs ligands have for the VC1 domain. A glycosylated form of VC1, produced in the yeast Pichia pastoris, was attached to magnetic beads and used as insoluble affinity matrix (VC1-resin). The VC1 interaction assay was employed to isolate specific VC1 binding partners from in vitro generated AGE-albumins and modifications were identified/localized by mass spectrometry analysis. Interestingly, this method also led to the isolation of ALEs produced by malondialdehyde treatment of albumins. Computational studies provided a rational-based interpretation of the contacts established by specific modified residues and amino acids of the V domain. The validation of VC1-resin in capturing AGE-albumins from complex biological mixtures such as plasma and milk, may lead to the identification of new RAGE ligands potentially involved in pro-inflammatory and pro-fibrotic responses, independently of their structures or physical properties, and without the use of any covalent derivatization process. In addition, the method can be applied to the identification of antagonists of RAGE-ligand interaction.
authors.affiliation: University of Milan, Department of Biosciences, Via Celoria 26, 20133 Milano, Italy; Department of Pharmaceutical Sciences, Via Mangiagalli 25, 20133 Milano, Italy. Electronic address: genny.degani@unimi.it.
authors.forename: Genny
authors.initials: G
authors.lastname: Degani
authors.affiliation: Department of Pharmaceutical Sciences, Via Mangiagalli 25, 20133 Milano, Italy. Electronic address: alessandra.altomare@unimi.it.
authors.forename: Alessandra A
authors.initials: AA
authors.lastname: Altomare
authors.affiliation: Department of Pharmaceutical Sciences, Via Mangiagalli 25, 20133 Milano, Italy. Electronic address: mara.colzani@unimi.it.
authors.forename: Mara
authors.initials: M
authors.lastname: Colzani
authors.affiliation: University of Milan, Department of Biosciences, Via Celoria 26, 20133 Milano, Italy. Electronic address: caterina.martino1@studenti.unimi.it.
authors.forename: Caterina
authors.initials: C
authors.lastname: Martino
authors.affiliation: Department of Pharmaceutical Sciences, Via Mangiagalli 25, 20133 Milano, Italy. Electronic address: angelica.mazzolari@unimi.it.
authors.forename: Angelica
authors.initials: A
authors.lastname: Mazzolari
authors.affiliation: University of Freiburg, Institute of Neuropathology Neurozentrum, Breisacher Straße 64, 79106 Freiburg, Germany. Electronic address: guenter.fritz@uniklinik-freiburg.de.
authors.forename: Guenter
authors.initials: G
authors.lastname: Fritz
authors.affiliation: Department of Pharmaceutical Sciences, Via Mangiagalli 25, 20133 Milano, Italy. Electronic address: giulio.vistoli@unimi.it.
authors.forename: Giulio
authors.initials: G
authors.lastname: Vistoli
authors.affiliation: University of Milan, Department of Biosciences, Via Celoria 26, 20133 Milano, Italy. Electronic address: laura.popolo@unimi.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Popolo
authors.affiliation: Department of Pharmaceutical Sciences, Via Mangiagalli 25, 20133 Milano, Italy. Electronic address: giancarlo.aldini@unimi.it.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: Aldini
mesh_terms: D000418:Albumins / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D001681:Biological Assay; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D015227:Lipid Peroxidation; D008315:Malondialdehyde / Q000737:chemistry*; D008958:Models, Molecular; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D017433:Protein Structure, Secondary; D000067759:Receptor for Advanced Glycation End Products / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C000593871:AGER protein, human; D000418:Albumins; D008024:Ligands; D000067759:Receptor for Advanced Glycation End Products; D011994:Recombinant Proteins; D008315:Malondialdehyde
keywords: AGEs and ALEs; MDA; Pull-down assay; RAGE; Reactive carbonyl species; VC1 domain