Biosynthesis and N‐glycosylation of Human Interferon‐γ

Publisher Website
Google Scholar
Abstract
Interferon‐γ (IFN‐γ) 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‐γ in human CD8+ T lymphocytes stimulated via T‐cell receptor. Highly elevated IFN‐γ mRNA levels were found as early as 1 h after stimulation. Maximal IFN‐γ protein synthesis was observed 2–4 h after induction and appeared to correlate to steady‐state IFN‐γ mRNA levels. As analyzed by pulse/chase experiments, the secretion of IFN‐γ 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‐γ, but did not block its secretion. Natural IFN‐γ is heterogeneously glycosylated and doubly, singly, and unglycosylated forms exist. Experiments performed in a cell‐free translation/glycosylation system with mutated IFN‐γ 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‐γ 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‐γ and show that there is a considerable heterogeneity in the individual sugar chains of this important human cytokine.