Efficient sorting of TNF-alpha to rodent mast cell granules is dependent on N-linked glycosylation

Abstract

Mast cells play an important role at the early stages of immunological response to bacterial infections and parasite infestations. One of the major mast cell pro-inflammatory mediators is TNF-α. Mast cells are considered the only cells capable of storing TNF-α in cytoplasmic granules and rapidly releasing it upon activation. To determine what pathway is utilized to direct TNF-α to cytoplasmic granules and what motifs are responsible for the sorting process, we constructed a fusion protein covering the full sequence of TNF-α, N-terminally fused to enhanced green fluorescent protein (EGFP). In rodent mast cells, such protein was sorted to secretory granules, and this process was inhibited by both brefeldin A and monensin. Considering the relationship between lysosomes and secretory granules and following TNF-α sequence analysis, it was determined whether TNF-α is sorted through the mannose-6-phosphate receptor (MPR)-dependent pathway. We observed that ammonium chloride and tunicamycin blocked TNF-α-EGFP fusion protein delivery to secretory granules. In situ mutagenesis experiments confirmed the necessity of N-linked glycosylation for efficient sorting of TNF-α into rodent mast cell granules. In this work we established that TNF-α travels from the ER to mast cell granules via a brefeldin A- and monensin-sensitive route, utilizing the MPR-dependent pathway, although this dependency does not seem to be absolute. © 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.