Methylglyoxal modification of protein - Chemical and immunochemical characterization of methylglyoxal-arginine adducts

Methylglyoxal (MG), an endogenous metabolite that increases in diabetes and is a common intermediate in the Maillard reaction(glycation), reacts with proteins and forms advanced-glycation end products. In the present study, we identify a novel MG-arginine adduct and also characterize the structure of a major fluorescent adduct, In addition we describe the immunochemical study on the MG-arginine addicts using monoclonal antibody directed to MG-modified protein. Upon incubation of N(alpha)-acetyl-L-arginine with MG at 37 degrees C, two non-fluorescent products and one-fluorescent product were detected as the-major products, The nonfluorescent products were identified as the N(delta)-(5-hydro-5-methyl-4-imidazolon-2-yl)-L-ornithine derivatives (5-hydro-5-methylimidazolone) and a novel MG-arginine adduct having a tetra-hydropyrimidine -moiety; N(delta)-(4-carboxy-4,6-dimethyl-5,6-dihydroxy-1,4,5,6-tetrahydropyrimidine-2 -L-ornithine). On the basis of the following chemical and spectroscopic evidence; the-major fluorescent product, putatively identified as N(delta)-(5-methylimidazolon-2-yl)-L-ornithithine (5-methylimidazolone), was found to-be identical to N(delta)-(5-hydroxy4,6-dimethylpyrimidine-2-yl)-L-ornithine (argpyrimidine): (i) the low and-high resolution fast atom bombardment mass spectrometry gave a molecular ion peak at m/z of 297 (M+H) and:a molecular formula of C(10)H(25)O(6)N(4), respectively, which coincided with pyrimidine; (ii) the (1)H NMR spectrum of this product in. d6-Me(2)SO showed a singlet at 2.10 ppm corresponding to six protons; (iii) the peak corresponding to the 5-methylimidazolone derivative was not detected by the liquid chromatography-mass spectrometry with the mode of selected ion monitoring; (iv) incubation of 5-hydro-5-methylimidazolone, a putative precursor of 5 methylimidazolone, at 37 degrees C for 14 days scarcely generated 5-methylimidazolone. On the other hand, ras an immunochemical approach to the-detection-of these MG adducts, we raised the monoclonal antibodies (mAb3C and mAb6B) directed to the MG-modified protein and found that they specifically recognized the major, fluorescent product, argpyrimidine; as the dominant epitope, The immunohistochemical analysis of-the kidneys from diabetic patients revealed the localization of argpyrimidine in intima and media of small artery walls. Furthermore, the accumulation of argpyrimidine was also observed in some arterial walls of the rat brain after middle cerebral artery occlusion followed by reperfusion. These results suggest that argpyrimidine may contribute to the progression of not only long term diabetic complications, such as nephropathy and atherosclerosis, but also the tissue injury caused by ischemia/reperfusion.