Biosynthesis and N-glycosylation of human interferon-gamma. Asn25 and Asn97 differ markedly in how efficiently they are glycosylated and in their oligosaccharide composition.
Interferon-gamma (IFN-gamma) is a secretory glycoprotein produced by T cells in response to antigenic or mitogenic stimuli. We studied the kinetics of the synthesis, N-glycosylation, and secretion of IFN-gamma in human CD8+ T lymphocytes stimulated via T-cell receptor. Highly elevated IFN-gamma mRNA levels were found as early as 1 h after stimulation. Maximal IFN-gamma protein synthesis was observed 2-4 h after induction and appeared to correlate to steady-state IFN-gamma mRNA levels. As analyzed by pulse/chase experiments, the secretion of IFN-gamma from T cells was very rapid, the secretion half-time being approximately 20-25 min. Inhibition of N-glycosylation by tunicamycin dramatically reduced the expression of IFN-gamma, but did not block its secretion. Natural IFN-gamma is heterogeneously glycosylated and doubly, singly, and unglycosylated forms exist. Experiments performed in a cell-free translation/glycosylation system with mutated IFN-gamma constructs lacking either one of the potential glycosylation sites suggested that Asn25 is more efficiently glycosylated than Asn97. Site-specific oligosaccharide analysis of natural IFN-gamma by glycosidase treatment followed by matrix-assisted-laser-desorption-ionization mass spectrometry revealed considerable variation in the carbohydrate structures, with more than 30 different forms. The glycans at Asn25 consisted of fucosylated, mainly complex-type oligosaccharides, whereas the glycans at Asn97 were more heterogeneous, with hybrid and high-mannose structures. Our results emphasize the essential role of N-linked glycans in the biology of IFN-gamma and show that there is a considerable heterogeneity in the individual sugar chains of this important human cytokine.
Sareneva
T
T
Department of Virology, National Public Health Institute, Helsinki, Finland.
Mørtz
E
E
Tölö
H
H
Roepstorff
P
P
Julkunen
I
I
D000595:Amino Acid Sequence; D001216:Asparagine; D015153:Blotting, Western; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002240:Carbohydrate Sequence; D002474:Cell-Free System; D002478:Cells, Cultured; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis*; D007700:Kinetics; D008213:Lymphocyte Activation; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D010449:Peptide Mapping; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011948:Receptors, Antigen, T-Cell / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D014158:Transcription, Genetic
D006020:Glycopeptides; D009844:Oligosaccharides; D011948:Receptors, Antigen, T-Cell; D001216:Asparagine; D007371:Interferon-gamma