Efficient sorting of TNF-alpha to rodent mast cell granules is dependent on N-linked glycosylation.
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-alpha. Mast cells are considered the only cells capable of storing TNF-alpha in cytoplasmic granules and rapidly releasing it upon activation. To determine what pathway is utilized to direct TNF-alpha to cytoplasmic granules and what motifs are responsible for the sorting process, we constructed a fusion protein covering the full sequence of TNF-alpha, 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-alpha sequence analysis, it was determined whether TNF-alpha is sorted through the mannose-6-phosphate receptor (MPR)-dependent pathway. We observed that ammonium chloride and tunicamycin blocked TNF-alpha-EGFP fusion protein delivery to secretory granules. In situ mutagenesis experiments confirmed the necessity of N-linked glycosylation for efficient sorting of TNF-alpha into rodent mast cell granules. In this work we established that TNF-alpha 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.
Laboratory of Molecular Immunology, International Institute of Molecular and Cell Biology, Warsaw, Poland.
Maciej B
MB
Olszewski
Dominika
D
Trzaska
Edward F
EF
Knol
Violetta
V
Adamczewska
Jaroslaw
J
Dastych
D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology; D015153:Blotting, Western; D020126:Brefeldin A / Q000494:pharmacology; D002478:Cells, Cultured; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism*; D018613:Microscopy, Confocal; D008985:Monensin / Q000494:pharmacology; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017527:Receptor, IGF Type 2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D022142:Secretory Vesicles / Q000378:metabolism*; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D000981:Antiprotozoal Agents; D011500:Protein Synthesis Inhibitors; D017527:Receptor, IGF Type 2; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D008985:Monensin