N-glycosylation negatively regulates the expression of tumor necrosis factor (TNF) in mouse macrophage

Tumor necrosis factor alpha (TNF) is a potent inflammatory cytokine and is also involved in the pathogenesis of various diseases such as inflammatory bowel disease and rheumatoid arthritis. While the intracellular signal cascades of TNF stimulation have been extensively studied, the regulatory mechanism of TNF production is still largely unknown. In this study, we investigated the role of N-glycosylation of TNF in its production. First, an inducible-TNF expression model was established based on the newly created TNF-knockout cells where TNF expression is induced only by doxycycline. We further analyzed the effect of N-glycosylation by testing mutant TNF proteins in which a single amino acid of the putative glycosylation site was substituted with alanine. The resulting mutant TNF (N86A) exhibited enhanced protein expressions both in the cells and in cell culture supernatants while the level of TNF mRNA remained constant. Our results indicate that N-glycosylation suppresses the production of TNF.