Roumyana Mironova

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‐γ) 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‐γ, such as covalent dimerization and truncation.

Evidence for non-enzymatic glycosylation of Escherichia coli chromosomal DNA

We have recently shown that the process of non‐enzymatic 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∈‐(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 exponential‐phase 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 argE3 to Arg+ reversions in the E. coli strain AB1157.

Glycation and Post-translational Processing of Human Interferon-γ Expressed in Escherichia coli

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. (2001) Mol. Microbiol. 39, 1061-1068), we have shown that glycation takes place in Escherichia coli as well. In the present study, we demonstrate that the post-translational processing (proteolysis and covalent dimerization) observed with cysteineless recombinant human interferon-γ (rhIFN-γ) is tightly associated with its in vivo glycation. Our results show that, at the time of isolation, rhIFN-γ contained early (but not advanced) glycation products. Using reverse phase high performance liquid chromatography in conjunction with fluorescence measurements, enzyme-linked immunosorbent assay, and mass spectrometry, we found that advanced glycation end products arose in rhIFN-γ during storage. The latter were identified mainly in the Arg/Lys-rich C terminus of the protein, which was also the main target of proteolysis. Mass spectral analysis and N-terminal sequencing revealed four major (Arg140↓Arg141, Phe137↓Arg138, Met135↓Leu136, and Lys131↓Arg132) and two minor (Lys109↓Ala110 and Arg90↓Asp91) cleavage sites in this region. Tryptic peptide mapping indicated that the covalent dimers of rhIFN-γ originating during storage were formed mainly by lateral cross-linking of the monomer subunits. Antiviral assay showed that proteolysis lowered the antiviral activity of rhIFN-γ, whereas covalent dimerization completely abolished it.

N-terminal methionine in recombinant proteins expressed in two different Escherichia coli strains.

Two genes coding for chloramphenicol acetyltransferase and human interferon gamma, respectively, were overexpressed constitutively in two different strains of Escherichia coli (E. coli LE392 and E. coli XL1). The N-terminal amino acid analysis of the purified proteins showed that: (a) the N-terminal methionine is processed more efficiently in E. coli LE392 rather than in E. coli XL1 cells; (b) the N-terminal methionine is removed better from the heterologous human interferon gamma in comparison with the homologous chloramphenicol acetyltransferase protein: and (c) there is no strong correlation between the efficiency of N-terminal procession and the yield of recombinant protein.

Non-Enzymatic Glycosylation and Deglycating Enzymes

ABSTRACT Biological amines react with reducing sugars to form a heterogeneous group of compounds, called advanced glycation end products, a process known as the Maillard reaction. Glycation of proteins starts with formation of Shiffs base, which rearrange into Amadori product. The Amadori products then undergo a series of chemical modifications to form advanced glycation end products (AGEs). Many advanced glycation end products are capable of forming cross-links between proteins and many of them are fluorophores. The formation of AGEs is an irreversible process and glycation is a major cause of spontaneous damage to proteins in physiological systems. AGEs accumulate in tissues with age and their rate of accumulation is accelerated in diabetes. Hyperglycemia in diabetes causes accelerated AGEs formation and has been linked to various diabetic complications like nephropathy, retinopathy, angiopathy, and neuropathy. Nature has several defense mechanisms to protect tissues from AGEs accumulation. Among them are amadoriases, which can remove the Amadori product and interrupt the glycation cascade in the early steps of the Maillard reaction. To date three classes of deglycating enzymes were found-fructosamine oxidases, fructosamine-3-kinases, and fructoselysine 6-phosphate deglycase. These enzymes are known to occur in mammalian, fungal and other eukaryotic and prokaryotic cells.

Comparative molecular analysis of bacterial communities inhabiting pristine and polluted with polycyclic aromatic hydrocarbons Black Sea coastal sediments

Molecular analysis was applied to characterize bacterial community structure in sediment samples collected from pristine site and oil-polluted Black Sea harbor. Amplified Ribosomal DNA Restriction Analysis (ARDRA) revealed a high similarity in the restriction patterns of both samples thus not demonstrating the effect of the pollutant on the structure of the bacterial communities. Constructed 16S rRNA gene libraries gave more detailed assessment of members. Results showed that α- and γ-Proteobacteria were dominant in the oil polluted site, whereas the pristine site was characterized by prevalence of Actinobacteria. The biodegradative potential of the adapted bacterial community in the oil-polluted sediments was demonstrated by the presence of the aromatic ring hydroxylating dioxygenase genes.

GLYCATION OF PROTEINS IN ESCHERICHIA COLI: EFFECT OF NUTRIENT BROTH INGREDIENTS ON GLYCATION

ABSTRACT Glycation is a condensation reaction between reducing sugars and primary amino groups in proteins. It starts with formation of Schiff bases and Amadori products (called early glycation products), which are later converted into complex heterocyclic compounds designated as advanced glycation end (AGE) products. Until recently glycation was studied mainly in higher eukaryotes and only our recent studies [6] showed that glycation took place also bacteria. The aim of this study is to investigate the effect of growth media and nutrient broth ingredients on glycation of proteins in Escherichia coli. The obtained results indicate that glycation is more extensive in rich media and depends on the type and source of nutrient broth ingredients (yeast extract and protein hydrolysate). Sometimes different batches of one ingredient produced by the same manufacturer might have different effect on glycation. Since the increased glycation decreases stability and biological activity of recombinant proteins, we highly recommend all nutrient broth ingredients to be tested for their effect on glycation before use for manufacturing of such proteins.

Molecular heterogeneity of amyloid β2-microglobulin and modification with advanced glycation end products

By using liquid chromatography-electrospray ionization mass spectrometry, Western blotting and N-terminal amino acid sequence analysis, we characterized the molecular heterogeneity and advanced glycation end product (AGE) modification of beta2-microglobulin (beta2m) extracted from the amyloid tissue of a hemodialysis patient. Amyloid beta2m was composed of full-length beta2m, truncated beta2m and dimer beta2m. Truncated beta2m and dimer beta2m were modified with AGEs such as imidazolone and N(e)-(carboxymethyl)lysine, and showed fluorescence characteristic of AGE. Truncated beta2m species were formed by cleavage between amino acid residues of Pro6/Ile7, Gln/Val9 and Val9/Tyr10. Heterogeneous dimer beta2m species showed the molecular masses of 22,591 and 22 675, which resulted from cross-linking between truncated beta2m.

Maillard Reaction Products in the Escherichia coli–derived Therapeutic Protein Interferon Alfacon-1

We have recently shown that recombinant human interferon‐γ is affected by early stages of the Maillard reaction during its production in Escherichia coli. Over time, advanced glycation end products accumulated in the purified protein, accompanied with degradation, cross‐linking, and a drop in the proteins biologic activity. Here, we provide further evidence for the presence of Maillard reaction products in another E. coli–derived therapeutic protein, interferon alfacon‐1. These products might interfere with both treatment efficacy and patient safety.

Evidence for the Presence of Glycation Adducts in Protein Therapeutics

ABSTRACT Glycation is a non-emzymatic reaction between free amino groups and reducing sugars (16), which was shown to take place also in human (13). It causes severe complications in diabetic and uremic patients, whereas in normal subjects contributes to senescence and aging. This study points to another negative aspect of glycation concerning the quality of protein therapeutics. Although therapeutic proteins are designed to be equivalent to their natural human counterparts, the development of antidrug antibodies in patients treated with proteins appears to be a rule rather than the exception (5, 21, 23). The anti-drug antibodies may sometimes cause serious complications such as allergic reactions and anaphylaxis (15). In addition, severe clinical consequences might be expected with those therapeutic proteins, whose endogenous counterparts are endowed with essential biologic functions. For example, neutralizing antibodies to megakaryocyte-derived growth factor and recombinant human erythropoietin have been found to cause severe thrombocytopenia (30) and pure red cell aplasia (14), respectively. The reasons for the immunogenicity of protein therapeutics still remain unresolved. This study provides evidence for the presence of potentially immunogenic glycation adducts in widely used protein drugs that could compromise therapeutic efficacy and patient safety.