Maya Boyanova

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.

SIGNIFICANCE OF THE PUTATIVE UPSTREAM POLYBASIC NUCLEAR LOCALISATION SEQUENCE FOR THE BIOLOGICAL ACTIVITY OF HUMAN INTERFERON-GAMMA

ABSTRACT Interferon-gamma (IFNγ) accomplishes its multiple biological effects by activating STAT transcription factors, which are translocated to the nucleus through a specific nuclear localization sequence(s) (NLS) in the IFNγ molecule. Two putative NLS have been pointed out in the human interferon gamma (hIFNγ): an upstream NLS located in helix E (residues 83–89) and a downstream NLS located at the C-terminal unstructured region (residues 124–132). To investigate the significance of the putative upstream NLS for the biological activity of hIFNγ we have introduced a point mutation in the hIFNγ gene to disturb the polybasic sequence typical for the conventional NLS. In the new gene a Gln codon was substituted for the Lys88 codon and the mutated gene was cloned and expressed in E. coli LE392. This mutation led to a 1000-fold decrease in both hIFNγ antiviral and antiproliferative activities. When co-incubated with the wild-type hIFNγ (standard), the mutant hIFNγ competed for the cellular receptors that led to a 30% inhibition of the standard activity. This indicates that the mutation does not interfere with the interaction of the protein to the receptor but probably affects the intracellular signal transduction pathway. To avoid any putative compensatory effect in the function of the upstream NLS caused by the downstream C-terminal NLS, 21 C-terminal codons were deleted from the mutant hIFNγ gene. The latter resulted in only 10-fold additional decrease in biological activity and 50% inhibition of the standard activity in the competition assay. Our data indicate that the upstream NLS is endowed with a greater functional significance for the hIFNγ mediated signal transduction rather than the downstream NLS.

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.

FLUORESCENCE ANALYSIS OF INTERFERON-GAMMA

ABSTRACT Equations were derived describing the fluorescence intensity of IFNy solutions on the basis of a dimer-monomers equilibrium of the native protein. The experimental data were found to fit very well the theoretical values predicted by these equations. Thus they open the possibility of studying the effect of different factors on parameters reflecting the conformational state and the biological activity of IFNy. The dimer-monomers dissociation upon dilution of the interferon solution opens the possibility to assess its native state. The presence and the size of a fraction unable to dissociate and to form active dimers (aggregates due to denaturation or covalent dimers due to glycation) can be revealed by the relative fluorescence decrease upon dilution of the hIFNy solution (“dilution test”).

Impact of Glycation Inhibitors on the Biologic Activity of Recombinant Human Interferon-Gamma

ABSTRACT Glycation of recombinant human interferon-gamma (rhIFN-γ) causes conformational alterations of the molecule and results in reduction of its biologic activity. The aim of this study was to find adequate approaches for prevention of glycation in order to obtain a stable recombinant protein with sustained biologic activity. To this end we investigated the effect of seven chemical compounds (acetylsalicylic acid, vitamin B1, aminoguanidine, arginine, pyridoxine, pyridoxal 5′-phosphate and pyridoxamine) on the biologic activity of rhIFN-γ produced in Escherichia coli. The obtained results showed that rhIFN-γ isolated from bacterial cells grown in the presence of 0.1 mM acetylsalicylic acid was the least affected by glycation. It demonstrated high stability in solution and biologic activity of about 2×106 IU/mg over three months of storage at -20°C.

Effect of Arginine on Glycation and Stability of Recombinant Human Interferon-Gamma

ABSTRACT Recombinant human interferon-gamma (rhIFN-γ) produced in Escherichia coli (E. coli) undergoes structural and functional alterations as a result of two different but parallel processes—aggregation and non-enzymatic glycosylation (glycation). Finding approaches for their inhibition is of great importance for the quality of the rhIFN-γ. In this study we used arginine for this purpose. We found that arginine added to the E. coli culture medium, inhibits formation of fluorescent glycation adducts and imidazolone in the total bacterial protein but does not interfere with the early glycation stages. In addition, refolding and storage of rhIFN-γ in the presence of arginine led to delayed accumulation of Nε-(carboxymethyl)lysine and structural stabilization of the recombinant protein.

Effect of Some Physical Factors on Fluorescence and Biological Activity of Human Interferon Gamma

ABSTRACT The effect of temperature, mechanical stress and surface tension on fluorescence and biological activity of recombinant human interferon gamma (rhIFN-γ) was studied. All three physical factors caused a similar decrease of fluorescence intensity and biological activity, the latter being more strongly affected—a 10–15% fall of fluorescence intensity was paralleled by 50–60 % decrease of activity. All data show that the link between fluorescence intensity and biological activity is complicated and depends on many factors such as the nature of the denaturing factor, the time period after its action, the conditions of storage etc. Thus fluorescence intensity cannot be directly used as a measure of activity. The most rapid denaturing effect (in 2–3 min.) has the surface tension. An expression is derived to determine the percentage of native interferon in the droplets of interferon when given in the form of aerosols.