Methylglyoxal-Modification of Human Serum Albumin Alters Bilirubin Binding

Abstract

The glycation of protein is a non-enzymatic post-translational modification targeting terminal amines of positively-charged amino acids, and has been linked to the progression of diabetic complications. Methylglyoxal (MG) is a potent glycating agent elevated in individuals with prolonged hyperglycemia and in the elderly, with the ability to change both the structure and function of protein. Due to its abundance and high molar-ratio of lysine and arginine, human serum albumin (HSA) is highly susceptible to the effects of pathological glycaemia. Glycation of HSA by MG has resulted in decreased affinity for several drugs and cellular metabolites, possibly due to the glycation of amino acids responsible for electrostatic interaction and hydrogen bonding within the ligand binding site. Among the molecules affected is bilirubin, a tetrapyrrolic metabolic of heme, which binds to circulating HSA in a binding pocket housing the potential glycation site R218. Recombinant R218 variants, wtHSA, R218M, R218H, R218A, and R218E, were synthesized to mimic the effects of glycation at this site, and used to study the how bilirubin binding is altered. Our results suggest positively-charged and sterically hindering residues at position 218 discourage association. R218 unfavorably influence the binding of bilirubin, but its presence is required in the event of glycation for proper function. Unglycated R218M and R218E had a Kd two-folds greater than wtHSA while R218A had a 10-fold increase. Glycation severely depressed affinity of bilirubin in all non-wtHSA. This suggests multiple site modification per mole of HSA, and conformational changes resulting from allosterism is responsible the decreased functionality. Although the absence of R218 is clearly implicated in the function of glycated albumin, further studies will be needed to determine the exact mechanism of functional impairment.