Glycation and post-translational processing of human interferon-gamma 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-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.
Mironova
Roumyana
R
Department of Gene Regulations, Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria. rumym@obzor.bio21.bas.bg
Niwa
Toshimitsu
T
Dimitrova
Rositsa
R
Boyanova
Maya
M
Ivanov
Ivan
I
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019281:Dimerization; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis* / Q000494:pharmacology; D010449:Peptide Mapping; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D014780:Viruses / Q000187:drug effects
D011994:Recombinant Proteins; D007371:Interferon-gamma; D010450:Endopeptidases