Localization of the ezrin binding epitope for advanced glycation endproducts

Glycated proteins/advanced glycation endproducts contribute to the development of diabetic complications but the precise pathway from glycated proteins to complications is still being delineated. The ezrin, radixin and moesin protein family is a new class of advanced glycation endproduct-binding protein and we hypothesize that advanced glycation endproducts mediate some of their detrimental effects leading to diabetic complications by inhibiting ezrin's actions. Our previous study revealed that glycated proteins bind to the N-terminal domain of ezrin (aa 1-324) and this study further defines the ezrin binding epitope. Binding of glycated albumin to recombinant N-ezrin deletion constructs (aa 1-280, 1-170 and 1-144) and glutathione-S-transferase-N-ezrin fusion proteins, (aa 200-324 and 270-324) was analysed using ligand and far Western blotting, and surface plasmon resonance. Glycated albumin binding was markedly reduced on removal of amino acids 280-324, while binding was preserved in the fusion proteins. A series of peptides based on residues 280-324 was synthesized and those containing residues 277-299 of ezrin bound maximally. Peptide binding to glycated albumin was glycation-specific. An ezrin peptide (aa 277-299) dose-dependently reversed the inhibitory effect of glycated albumin on ezrin (1-324) phosphorylation in vitro, suggesting that binding of advanced glycation endproducts to ezrin changes the conformation of the latter sufficiently to alter binding interactions distant from the advanced glycation endproduct-binding site. This may have consequences for subcellular ezrin localization and signalling pathways. Altogether, these studies provide important structural knowledge for developing peptide antagonists that may be therapeutically useful in preventing advanced glycation endproduct:ezrin interactions in diabetes.