Methylglyoxal Modification of Protein

Abstract

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 adducts using monoclonal antibody directed to MG-modified protein. On the other hand, as 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.Nonenzymatic glycation (Maillard reaction) is a complex series of reactions between reducing sugars and amino groups of proteins, which leads to browning, fluorescence, and crosslinking of the proteins. The reaction is initiated with the reversible formation of a Schiff's base, which undergoes a rearrangement to form a relatively stable Amadori product. The Amadori product further undergoes a series of reactions through dicarbonyl intermediates to form advanced glycation end products (AGEs) 1 (1). It has been shown that the formation of AGEs in vivo contributes to the pathophysiologies associated with aging and the long term complications of diabetes (2).A number of aldehydes and ketones, in addition to sugars, are known to form AGEs. Methylglyoxal (MG), among them, has recently received considerable attention as a mediator to form AGEs. MG is known to be formed nonenzymically by amine-catalyzed sugar fragmentation reactions (3-5) and by spontaneous decomposition of triose phosphate intermediates in glycolysis (5). It is also a product of the metabolism of acetol, an intermediate in the catabolism of both threonine (6) and the ketone body acetone (7). A recent study on the formation of AGEs in endothelial cells cultured under hyperglycemic conditions indicated that MG was the major precursor of AGEs (8). Chaplen et al. (9) have shown that high levels of MG are indeed present in cultured Chinese hamster ovary cells. In addition, increased levels of MG are also found in blood from diabetic patients and in the lens of streptozotosin-induced diabetic rats (10, 11). Che et al. (12) have reported that MG induces gene expression of heparin-binding epidermal growth factor-like growth factor by provoking oxidative stress. It is also known ...

Inflammatory mediators are induced by dietary glycotoxins, a major risk factor for diabetic angiopathy

“…Thus, it is conceivable that the frequent intake of dietary § Lin, J., Alt, A., Liersch, J., Reinhard, G. B., Brownlee AGE promotes a sustained low-grade inf lammatory state. In this sense, other markers of immune response such as cytokines and adhesion molecules are inducible by AGEs via reactive oxygen species production and nuclear factor B activation (7)(8)(9)(10)(11)(12)(13)(14)44), properties which are also exhibited by dietary AGEs (25). These reactive molecules (ranging between 12 and 22 million AGE units in a ''healthy meal''), which are ''seeding'' the systemic circulation regularly, could, together with hyperglycemia, contribute to the subtle inf lammatory state associated with diabetes (23,(41)(42)(43)45).…”

“…Immunoreagents to MG derivatives and CML-like products, however, have presented invaluable means for animal and clinical correlations (10,11,24,25). It thus has been confirmed that two-thirds of orally absorbed dietary AGEs (Ϸ10% of the amount ingested) are retained in tissues in bioreactive forms (26,27).…”

“…The nature of pathogenic AGEs is not known as yet (10)(11)(12), and multiple derivatives other than those of MG or CML moieties have yet to be identified. Immunoreagents to MG derivatives and CML-like products, however, have presented invaluable means for animal and clinical correlations (10,11,24,25).…”

Tumor necrosis factor-induced modulation of glyoxalase I activities through phosphorylation by PKA results in cell death and is accompanied by the formation of a specific methylglyoxal-derived AGE

“…After TNF incubations (1.5 or 2.5 h), the cells were rinsed three times with ice-cold PBS buffer, and cell lysates were prepared in a 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonatecontaining cytosol extraction buffer (29). MG-derived AGEs were detected by Western blotting by using the mAb6B (19). To use the antibody sparingly, SDS-polyacrylamide gels were only run across a distance of 5 cm.…”

“…Although the reaction with Cys is considered reversible, elevated concentrations of MG can lead to irreversible modifications of Lys and Arg residues through formation of advanced glycation end products (AGEs) (18). AGE formation is thought to contribute to several pathophysiological conditions, such as tissue damage after ischemia͞ reperfusion (19), to aging (20), and to the development of complications in diabetic patients such as blindness, neuropathy, and diabetic vascular diseases (21). vol͞vol), heat-inactivated newborn calf serum (5% vol͞vol), penicillin (100 units͞ml), streptomycin (0.1 mg͞ml), and Lglutamine (2 mM), at 37°C in a humidified incubator under a 5% CO 2 atmosphere.…”