Evidence for non-enzymatic glycosylation in Escherichia coli.
Non-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-gamma) 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 non-enzymatic 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-gamma, such as covalent dimerization and truncation.
Mironova
R
R
Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. rumym@obzor.bio21.bas.bg
Niwa
T
T
Hayashi
H
H
Dimitrova
R
R
Ivanov
I
I
D001426:Bacterial Proteins / Q000378:metabolism; D003847:Deoxyglucose / Q000031:analogs & derivatives / Q000032:analysis; D019281:Dimerization; D004926:Escherichia coli / Q000378:metabolism*; D005453:Fluorescence; D006031:Glycosylation; D006801:Humans; D007093:Imidazoles / Q000276:immunology; D007371:Interferon-gamma / Q000276:immunology / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives* / Q000276:immunology; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins; D013050:Spectrometry, Fluorescence / Q000379:methods
D001426:Bacterial Proteins; D007093:Imidazoles; D011994:Recombinant Proteins; C117197:imidazolone; C048496:N(6)-carboxymethyllysine; D007371:Interferon-gamma; D003847:Deoxyglucose; C016350:3-deoxyglucosone; D008239:Lysine