Immunological Evidence for Methylglyoxal-derived Modifications in vivo

Abstract

The Maillard reaction, a non-enzymatic reaction of ketones and aldehydes with amino groups of proteins, contributes to the aging of proteins and to complications associated with diabetes. Methylglyoxal (MG) is a 2-oxoaldehyde derived from glycolytic intermediates and produced during the Maillard reaction. We reported previously the formation of a lysine-lysine protein crosslinking structure (imidazolysine) and a fluorescent arginine modification (argpyrimidine) from the Maillard reaction of MG. Here we show that rabbit antibodies to MG-modified ribonuclease A identify proteins modified by the Maillard reaction of glucose, fructose, ribose, glyceraldehyde, glyoxal, ascorbate, and ascorbate oxidation products (dehydroascorbate, 2,3-diketogulonate, L-xylosone, and L-threose) in addition to those modified by MG. The antibody recognized imidazolysine and argpyrimidine and a glyoxal-derived lysine-lysine cross-link. It did not react with N⑀-carboxymethyllysine. Incubations with amino acids revealed strongest reactivity with N␣-tbutoxycarbonylarginine and MG, and we identified argpyrimidine as one of the epitopes from this incubation mixture. Serum proteins from human diabetics reacted more strongly with the antibody than those from normal individuals, and the levels correlated with glycemic control. Collagen from human corneas contained MG-derived modifications, with those from older subjects containing higher levels of modified proteins than those from younger ones. An immunoaffinity-purified antibody showed higher reactivity with old corneas than with younger ones and localized the antigens primarily within the stromal region of the cornea. These results confirm reported MG-derived modifications in tissue proteins and show that dicarbonyl-mediated protein modification occurs during Maillard reactions in vivo.

PP2A contributes to endothelial death in high glucose: inhibition by benfotiamine

“…Then dot blots were treated similarly to Western blots. The antibody against argpyrimidine (41), an indicator of advanced glycation end-product levels, was generously donated by Dr. R. Nagaraj (Case Western Reserve University, Cleveland, OH).…”

Analysis of Methylglyoxal Metabolism in CHO Cells Grown in Culture

“…Arginine and lysine modification results in AGE formation via Schiff's base and Amadori rearrangement product intermediates and is believed to be an important signal for senescent protein degradation (Westwood and Thornalley 1996). The initial reaction steps are reversible and involve non-enzymatic Schiff's base formation between the aldehyde or ketone moiety of MG and protein amine groups (Shamsi et al 1998), followed by a rearrangement to more stable, covalently bonded Amadori products. These then undergo further irreversible rearrangements to form AGEs with a broad range of structures.…”

3-Hydroxykynurenine-mediated Modification of Human Lens Proteins

“…The most striking changes in these proteins include yellowing and browning of proteins, intra-and intermolecular cross-linking, and cross-linking with fiber cell membrane proteins (1)(2)(3)(4). Several mechanisms have been proposed for such changes, including oxidation (5,6) and glycation (7)(8)(9). Recent studies suggest that these two processes are interrelated (10,11) and may thus synergistically contribute to the observed lens protein modifications in aging and cataract formation.…”