Amides are novel protein modifications formed by physiological sugars

Abstract

The elucidation of structure and formation of AGEs is of major interest to understand related pathophysiologies, such as the late complications in diabetes, atherosclerosis and aging, and to develop pharmaceutical strategies for their prevention [1,2]. The discrepancy between extent of protein modification and of fluorescence has led to the notion that the major sugar-derived protein modifications cannot be explained by colored, UV and fluorescence active structures. Furthermore, evidence has shown that potential structures must be labile to acid and alkaline hydrolysis and cannot be stabilized by sodium borohydride reduction [3].Following this concept, N6-{2-[(5-amino-5-carboxypentyl)amino]-2-oxoethyl}lysine (GOLA) and N6-glycoloyllysine (GALA) were identified as novel amide protein modifications mediated by physiological reducing sugars [4]. Their identity was unequivocally confirmed by independent synthesis and coupled liquid chromatography/mass spectrometry. Model reactions with Nα-t-BOC-lysine revealed glyoxal and glycolaldehyde as immediate precursors with reaction pathways directly linked to Nε-carboxymethyllysine (CML) via glyoxal–imine structures. Based on a common intermediate, the ratio of the amide crosslink GOLA to the imidazolium crosslink GOLD was favored towards the amide at lower glyoxal levels in incubations conducted at constant lysine concentrations. GOLA and GALA formation from the Amadori product of glucose with lysine was directly dependent on oxidation. Presence of aminoguanidine almost completely suppressed GALA formation, whereas GOLA was only inhibited by 16%. As the latter effect was exactly in line with changes in CML synthesis, it can be reasoned that during Amadori product degradation, 84% of GOLA originates via direct oxidative fragmentation and 16% via glyoxal–imine intermediates. In contrast, the formation of GALA must be explained solely via glyoxal–imines. An efficient enzymatic hydrolysis and analytical method was developed for the detection of labile amide modifications in highly modified proteins and in vivo. In a first attempt, GOLA was quantified in brunescent lens protein at 66 pmol/mg protein. This suggested that amide modifications are of major physiological relevance in comparison to the already established AGEs pentosidine, GOLD and MOLD.