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 proinflammatory 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.

TNF Trafficking to Human Mast Cell Granules: Mature Chain-Dependent Endocytosis

“…We have shown recently that, in rodent mast cells, TNF trafficking to cytoplasmic granules is dependent on N-linked glycosylation (10). This mechanism however does not seem to be evolutionarily conserved, because it is predominantly found in Rodentia, while in Artiodactyla N86 residue is replaced by alanine, which excludes any glycosylation.…”

“…We have shown previously that in rodent mast cells, TNF delivery to mast cell granules largely depends on N-linked glycosylation (10). Phylogenetic protein sequence analysis showed, however, that the N-terminal region of sTNF is one of the least conserved and that the N-linked glycosylation motif NSS is unique to rodents and, in primates, is replaced by the RTP motif (Fig.…”

“…We have shown previously (10) that, in rodent mast cells, TNF sorting to cytoplasmic granules largely depends on N-linked glycosylation and a mannose-6-phosphate receptor-based system. Although major pieces of evidence for specific biological function of TNF stored in mast cell granules come from a rodent model, the existence of a similar pool of this cytokine in many subsets of human mast cells has been reported (11)(12)(13).…”

Cytokine release from innate immune cells: association with diverse membrane trafficking pathways

“…40,41 Cytokines are released during classical degranulation, shown by the rapid secretion of IL-4 and TNF during receptor-mediated exocytosis by crosslinking cell surface complexes of IgE and Ag. [42][43][44][45] However, cytokine release from mast cells may involve trafficking of a distinct population of secretory vesicles independently of the granules, in a process similar to piecemeal degranulation or constitutive exocytosis (Figure 1). Piecemeal degranulation is a type of regulated exocytosis that is considered to be a general secretory pathway used by immune cells and other cell types, including enteroendocrine cells of the gastrointestinal tract, adrenal chromaffin cells, and chief cells of the parathyroid gland.…”

VAMP-8 segregates mast cell–preformed mediator exocytosis from cytokine trafficking pathways

“…by guest www.bloodjournal.org From an N-linked glycosylation-dependent sorting mechanism. 22 In macrophages, TNF secretion involves a vesicular compartment containing syntaxin-6, syntaxin-7, and Vti1b (vesicle transport through interaction with t-SNARE homologue 1b) that fuses with VAMP-3-positive recycling endosomes to mediate specific delivery at the phagocytotic cup. 6 The v-SNARE VAMP-8, also known as endobrevin, is expressed in many organs and tissues including lungs, kidney, heart, and salivary glands, 23 but not in neurons.…”