IMMUNOLOGICAL EVIDENCE FOR METHYLGLYOXAL-DERIVED MODIFICATIONS IN-VIVO - DETERMINATION OF ANTIGENIC EPITOPES

The Maillard reaction, a non-enzymatic reaction of ketones and aldehyd es 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 dur ing the Maillard reaction, We reported previously the formation of a l ysine-lysine protein crosslinking structure (imidazolysine) and a fluo rescent arginine modification (argpyrimidine) from the Maillard reacti on of MG, Here we show that rabbit antibodies to MG modified ribonucle ase A identify proteins modified by the Maillard reaction of glucose, fructose, ribose, glyceraldehyde, glyoxal, ascorbate, and ascorbate ox idation products (dehydroascorbate, 2,3-diketogulonate, L-xylosone, an d L-threose) in addition to those modified by MG;, The antibody recogn ized imidazolysine and argpyrimidine and a glyoxal-derived lysine-lysi ne cross-link, It did not react with N epsilon-carboxymethyllysine. In cubations with amino acids revealed strongest reactivity with N alpha- t-butoxycarbonylarginine and MG, and we identified argpyrimidine as on e of the epitopes from this incubation mixture. Serum proteins from hu man diabetics reacted more strongly with the antibody than those from normal individuals, and the levels correlated with glycemic control, C ollagen from human corneas contained MG-derived modifications, with th ose from older subjects containing higher levels of modified proteins than those from younger ones, An immunoaffinity-purified antibody show ed higher reactivity with old corneas than with younger ones and local ized the antigens primarily within the stromal region of the cornea, T hese results confirm reported MG-derived modifications in tissue prote ins and show that dicarbonyl mediated protein modification occurs duri ng Maillard reactions in vivo.