UC San Diego

The receptor for advanced glycation end products, RAGE, is a protein receptor localized on the cellular plasma membrane which binds to various ligands such as AGEs or HMGB1. RAGE activates downstream inflammatory signaling associated with diabetes and atherosclerosis. Our recent studies suggest heparan sulfate, a negatively charged linear polysaccharide, directly interacts with RAGE to promote receptor signaling and oligomerization. The objective of this study is to characterize the structural details of RAGE-heparan sulfate interactions and RAGE oligomerization. Site directed mutagenesis, heparin sepharose chromatography, and filter binding assays shows that basic residues on the V domain (K39, K43, K44, R104, and K107) and C1 domain (R216 and R218) of RAGE are essential for binding to heparin and heparan sulfate. Gel filtration chromatography demonstrated heparin-derived oligosaccharides with a minimal length of a dodecasaccharide (12-mer) are capable of inducing a stable tetramer of RAGE. Further testing suggests the tetramer complex consists of four RAGE monomers with two dodecasaccharides. A second site directed mutagenesis study identifies a patch of hydrophobic residues on the V domain (V35, V78-L79 and F85-V86) affecting RAGE oligomerization. Our findings show critical residues important for heparan sulfate binding and a subset of hydrophobic binding residues necessary for RAGE oligomerization. The construction of a preliminary crystal structure of RAGE with heparin-derived oligosaccharide is in progress. Lastly, the heparan sulfate and hydrophobic binding surface of RAGE appears to be separate from its ligand binding surface based on a ligand binding assay. Thus, understanding these RAGE-heparan sulfate interactions could lead to the development of pharmacological agents reducing the progression of certain pathological conditions in cardiovascular diseases and diabetes