Peptide Mapping Identifies Hotspot Site of Modification in Human Serum Albumin by Methylglyoxal Involved in Ligand Binding and Esterase Activity

Abstract

Methylglyoxal is a potent glycating agent under physiological conditions. Human serum albumin is modified by methylglyoxal in vivo. The glycation adducts formed and structural and functional changes induced by methylglyoxal modification have not been fully disclosed. Methylglyoxal reacted with human serum albumin under physiological conditions to form mainly the hydroimidazolone N ␦ -(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (92% of total modification) with a minor formation of argpyrimidine, N ⑀ -(1-carboxyethyl)lysine, and methylglyoxal lysine dimer. When human serum albumin was modified minimally with methylglyoxal, tryptic peptide mapping indicated a hotspot of modification at Arg-410 located in drug-binding site II and the active site of albumin-associated esterase activity. Modification of Arg-410 by methylglyoxal was found in albumin glycated in vivo. Other sites of minor modification were: Arg-114, Arg-186, Arg-218, and Arg-428. Hydroimidazolone formation at Arg-410 inhibited ketoprofen binding and esterase activity; correspondingly, glycation in the presence of ketoprofen inhibited Arg-410 modification and loss of esterase activity. The pH dependence of esterase activity indicated a catalytic group with pK a ‫؍‬ 7.9 ؎ 0.1, assigned to the catalytic base Tyr-411 with the conjugate base stabilized by interaction with the guanidinium group of Arg-410. Modification by methylglyoxal destabilized Tyr-411 and increased the pK a to 8.8 ؎ 0.1. Molecular dynamics and modeling studies indicated that hydroimidazolone formation caused structural distortion leading to disruption of argininedirected hydrogen bonding and loss of electrostatic interactions. Methylglyoxal modification of critical arginine residues, therefore, whether experimental or physiological, is expected to disrupt protein-ligand interactions and inactivate enzyme activity by hydroimidazolone formation.

Degradation products of proteins damaged by glycation, oxidation and nitration in clinical type 1 diabetes

“…In diabetic patients, the largest quantitative increases in protein glycation adduct residues of plasma protein occurred for MG-H1 and 3DG-H. The major site of MG-H1 residue formation in human serum albumin was identified recently as arg-410 [27]. The lack of increase in CML residues in the plasma protein of diabetic patients was unexpected and discordant with the prior report by Schleicher et al [28], who found increased CML residue concentration.…”

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay for organophosphorus toxicants bound to human albumin at Tyr411

“…The specific labeling of one tyrosine in a molecule that contains 18 tyrosines suggests that Tyr 411 is in a special environment. This tyrosine has an unusually low pKa of 7.9-8.3 (Means and Wu, 1979;Ahmed et al, 2005), in contrast to the pKa of 10 for the average tyrosine.…”

Increased Dicarbonyl Metabolism in Endothelial Cells in Hyperglycemia Induces Anoikis and Impairs Angiogenesis by RGD and GFOGER Motif Modification

“…residues, an advanced glycation end product (AGE) (17,18), with loss of associated side chain positive charge (19) (Fig. 1B).…”

“…Increased concentration of MG-H1 residues in plasma protein of diabetic patients was not linked directly to A1C (22), probably because methylglyoxal formation is increased in both fasting and postprandial hyperglycemia (16,23) and influenced by factors other than hyperglycemia (low glyceraldehyde-3-phosphate dehydrogenase activity [24]). MG-H1 residue formation occurred at susceptible hotspot sites in proteins with loss of functional activity (19). The surface sheath network of type IV collagen in blood vessels (25) binds integrins of vascular endothelial cells, anchoring and sustaining the vascular endothelium by interaction with integrins at GFOGER and RGD sites of the triple helical domain (9,26).…”