SummaryNon-enzymatic glycosylation (glycation) is a chain of chemical reactions affecting free amino groups in proteins of long-living eukaryotes. It proceeds in several steps leading to the consecutive formation of Schiff bases, Amadori products and advanced glycation end-products (AGEs). To our knowledge, this process has not been observed in prokaryotes so far. However, the present study provides clear-cut evidence that glycation takes place in bacteria despite their short life span. We have detected AGEs in recombinant human interferon gamma (rhIFN-g) produced in Escherichia coli as well as in total protein of the same bacterium using three different approaches: (i) Western blotting using two monoclonal antibodies raised against AGEs; (ii) fluorescent spectroscopy; and (iii) investigation of the effect of known AGE inhibitors (such as acetyl salicylic acid and thiamine) on the glycation reaction. Our study shows that nonenzymatic glycosylation is initiated during the normal growth of E. coli and results in AGE formation even after isolation of proteins. This process seems to be tightly associated with some post-translational modifications observed in the cysteineless rhIFN-g, such as covalent dimerization and truncation.

SummaryWe have recently shown that the process of nonenzymatic glycosylation (glycation) takes place in Escherichia coli under physiological conditions and affects both recombinant and endogenous bacterial proteins. In this study, we further demonstrate that E. coli chromosomal DNA is also subjected to glycation under physiological growth conditions. The E. coli DNA accumulates early glycation (Amadori) products as proven by the nitroblue tetrazolium (NBT) reduction assay. It showed also immunoreactivity to a monoclonal antibody raised against N OE OE OE OE -(carboxymethyl)lysine and fluorescent properties indicative of modifications with advanced glycation end-products. Two types of fluorophores were detected in the E. coli DNA with excitation maxima at 360 nm and 380 nm and emission maxima at 440 nm and 410 nm. Using the NBT reduction assay, fluorescence spectroscopy and enzyme-linked immunosorbent assay we revealed that glycation adducts accumulate in DNA predominantly in the stationary phase of growth, although they could be detected also in exponentialphase cells. Besides on the growth phase, the extent of DNA glycation depends also on the nutrient broth composition being more extensive in rich media. Thiamine was found to inhibit both DNA glycation and spontaneous point mutations as judged by the decreased rate of the argE 3 to Arg + reversions in the E. coli strain AB1157.

Until recently, nonenzymatic glycosylation (glycation) was thought to affect the proteins of long living eukaryotes only. However, in a recent study (Mironova, R., Niwa, T., Hayashi, H., Dimitrova, R., and Ivanov, I.