IDENTIFICATION OF N-2-(1-CARBOXYETHYL)GUANINE (CEG) AS A GUANINE ADVANCED GLYCOSYLATION END-PRODUCT

Reducing sugars such as glucose react nonenzymatically with protein amino groups to initiate a posttranslational modification process known as advanced,glycosylation. Nucleotide bases also participate in advanced, glycosylation reactions, producing DNA-linked advanced glycosylation endproducts (AGEs) that cause mutations and DNA transposition. Although several protein-derived AGEs have been isolated and structurally characterized, AGE-modified nucleotides have not yet been reported. We systematically examined the reactivities of the model nucleotide bases 9-methylguanine (9-mG), 9-methyladenine (9-mA), and 1-methylcytosine (1-mC) toward glucose and several glucose-derived reactants. In ''fast'' reactions performed at refluxing temperature and physiological pH, 1 equiv of nucleotide base was reacted with 10 equiv of D-glucose, D-glucose 6-phosphate (G-6-P), D-glucose 6-phosphate/lysine (G-6-P/Lys), the Schiff base 1-n-propylamino-N-D-,glucoside (SE), or the Amadori product 1-n-propylamino-N-D-fructose (AP). In every reaction involving 9-mG, N-2-(1-carboxyethyl)-9-methylguanine (CEmG) was a major product which was produced. N-2-(1-carboxyethyl)-9-methylguanine also formed from 9-mG and AP in longterm incubations performed at 37 degrees C. Direct treatment of 9-mG with methylglyoxal (MG), a Maillard reaction propagator that forms from the decomposition of AP, also produced CEmG in high yield. N-2-(1-carboxyethyl)-9-methylguanine appears to result from the nucleophilic addition of the primary amino group of guanine to the ketone group of MG followed by an intramolecular rearrangement. Methylglyoxal is a known prokaryotic mutagen and was shown additionally to be mutagenic in a eukaryotic shuttle vector assay system. These data implicate MG as a glucose-derived mutagen and suggest that CEmG is a major product that forms as a result of DNA-advanced glycosylation.