Biosynthesis and N‐glycosylation of Human Interferon‐γ

Abstract

Interferon-y (IFN-y) 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-y in human CD8' T lymphocytes stimulated via T-cell receptor. Highly elevated IFN-y mRNA levels were found as early as 1 h after stimulation. Maximal IFN-y protein synthesis was observed 2-4 h after induction and appeared to correlate to steady-state IFN-y mRNA levels. As analyzed by pulse/chase experiments, the secretion of IFN-y 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-y, but did not block its secretion. Natural IFN-y is heterogeneously glycosylated and doubly, singly, and unglycosylated forms exist. Experiments performed in a cell-free translation/glycosylation system with mutated IFN-y constructs lacking either one of the potential glycosylation sites suggested that Am25 is more efficiently glycosylated than Asn97. Site-specific oligosaccharide analysis of natural IFN-y 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 Am25 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-y and show that there is a considerable heterogeneity in the individual sugar chains of this important human cytokine.

Kaposi's Sarcoma-associated Herpesvirus-encoded Viral Interleukin-6 Is Secreted and Modified Differently Than Human Interleukin-6

“…Interferon-␥ from primary human T-cell culture supernatants also has a very high proportion of complex N-glycans at two sites. Only a very small percentage of molecules contain Endo H-sensitive hybrid (13%) or high-mannose (10%) structures at a single site (53). Some other types of secreted proteins, such as immunoglobulins IgM and IgA, do contain a very small proportion of high-mannose glycans, but they have been shown to be inaccessible to exoglycosidases that would normally modify them in the Golgi apparatus due to blocking by polymerization or association with J chain that occurs in early secretory compartments, respectively (54,55).…”

“…Although it is secreted much slower than its cellular counterpart, its signal sequence efficiently targets hIL-6 for secretion at nearly wild-type levels. The protein is decorated with a high proportion of high-mannose N-glycans compared with other cytokines (39,41,52,53) and must be present at extraordinarily high extracellular levels to promote growth of IL-6-dependent myeloma cells (22,23,70). The immature glycosylation of vIL-6 could be explained by intracellular interactions with its receptor, gp130, that render the protein inaccessible to Golgi exoglycosidases.…”

Nuclear Cotransport Mechanism of Cytoplasmic Human MxB Protein

“…All the DNA manipulations were performed according to standard protocols (27), and the newly created gene constructs were partially sequenced. Various MxB gene constructs were also cloned into the BamHI site of pGEM-3Zf(ϩ) (Promega) vector, and in vitro translation was carried out as previously decribed (28), using T7 Cap-Scribe and reticulocyte translation kits (Boehringer Mannheim GmbH, Mannheim, Germany).…”

Sialic Acids on Varicella-Zoster Virus Glycoprotein B Are Required for Cell-Cell Fusion

“…Indeed, glycoproteins often have a range of different N-glycans on a particular N-glycosylation site (58,59). Different sites in a molecule have different subsets of N-glycans with different numbers of SAs (58,60,61). Therefore, the amount of SAs on each gB glycosylation site might be different, and Asn-557 of gB seems to be the major glycosylation site for sialylated N-glycans.…”