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 k inetics of the synthesis, N-glycosylation, and secretion of IFN-gamma in human CD8(+) T lymphocytes stimulated via T-cell receptor. Highly e levated IFN-gamma mRNA levels were found as early as 1 h after stimula tion. Maximal IFN-gamma protein synthesis was observed 2-4 h after ind uction and appeared to correlate to steady-state IFN-gamma mRNA levels . As analyzed by pulse/chase experiments, the secretion of IFN-gamma f rom T cells was Very rapid, the secretion half-time being approximatel y 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 t ranslation/glycosylation system with mutated IFN-gamma constructs lack ing either one of the potential glycosylation sites suggested that Asn 25 is more efficiently glycosylated than Asn97. Site-specific oligosac charide analysis of natural IFN-gamma by glycosidase treatment followe d by matrix-assisted-laser-desorption-ionization mass spectrometry rev ealed considerable variation in the carbohydrate structures, with more than 30 different forms. The glycans at Asn25 consisted of fucosylate d, 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 biolog y of IFN-gamma and show that there is a-considerable heterogeneity in the individual sugar chains of this important human cytokine.