Identification of Aortic Endothelial Cell Binding Proteins for Amadori Adducts in Glycated Albumin

Abstract

The ability of glycated serum proteins, which exist in vivo predominantly as Amadori adducts, to influence cell biology suggests the existence of cell binding proteins that recognize glucose adducts in nonenzymatically glycated proteins. To explore this possibility, we applied detergent extracts of aortic endothelial cells to HPLC and to an affinity column of glycated human albumin immobilized onto Sepharose, which was eluted with alkaline pH. HPLC fractionation revealed a peak reactive in ELISA with glycated but not nonglycated albumin. Two polypeptides of ≍ 110 kDa and 205 kDa were identified when the affinity bound proteins were electrophoresed, transferred to immunoblotting membranes, and reacted with an enzyme conjugate of glycated albumin containing Amadori adducts. These polypeptides did not react with enzyme conjugate of carbohydrate-free albumin, confirming their binding specificity for glucose modified albumin and absence of co-identity with previously described albumin-binding proteins. The molecular weights of the glycated albumin binding polypeptides are distinct from those described for bovine albumin modified by advanced glycation end products or by formaldehyde, indicating that Amadori adducts in glycated albumin are recognized by unique endothelial cell binding sites.