Abstract
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-gamma (rhIFN-gamma) is tightly associated with its in vivo glycation. Our results show that, at the time of isolation, rhIFN-gamma 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-gamma 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-gamma 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-gamma, whereas covalent dimerization completely abolished it.
Highlights
Until recently, nonenzymatic glycosylation was thought to affect the proteins of long living eukaryotes only
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-␥ during storage
The concentration of rhIFN-␥ in the three fractions was adjusted to 5 mg/ml based on at 280 nm (A280)
Summary
Purification of rhIFN-␥—A gene coding for cysteineless hIFN-␥ was expressed constitutively [35] in E. coli XL1-Blue (supE44 hsdR17 recA1 endA1 gyrA46 thi relA1 lacϪ FЈ (proABϩ lacIq lacZ⌬M15 Tn10 (tetr))). The peptide fraction was loaded onto an ODS HG-5 column (2.0 ϫ 150 mm) and eluted at a flow rate 0.3 ml/min isocratically for 5 min with 5% solvent B (2.0% trifluoroacetic acid in acetonitrile), followed by raising its concentration to 70% in 40 min. SDS-polyacrylamide gels were stained with a fresh solution of 0.2% Coomassie Brilliant Blue R-250 in 10% acetic acid and 25% methanol and destained with 30% methanol until bands became visible The latter were excised from the gel, washed twice with 0.5 ml of 50% acetonitrile in 0.2 M NH4HCO3 (pH 8.9) for 20 min at 30 °C and once with 0.5 ml of 0.2 M NH4HCO3 (pH 8.9), and evaporated to a semidry state in a rotary evaporator. Antiviral Assay—The antiviral assay was based on the protective effect of hIFN-␥ against the cytopathic action of the vesicular stomatitis virus on the human amnionic cell line WISH as described [43]
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