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* Department of Molecular Biology, International Institute of Molecular and Cell Biology, Warsaw, Poland;
Department of Dermatology/Allergology and
Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands; and
Centre for Medical Biology, Polish Academy of Sciences, Lodz, Poland; and
¶ Laboratory of Molecular Immunology, International Institute of Molecular and Cell Biology, Warsaw, Poland
Mast cells play a crucial role at the early stages of immune response against bacteria and parasites where their functionality is based on their capability of releasing highly bioactive compounds, among them TNF. Mast cells are considered the only cells storing preformed TNF, which allows for the immediate release of this cytokine upon contact with pathogens. We approached the question of mechanisms and amino acid motifs directing newly synthesized TNF for storage in cytoplasmic granules by analyzing the trafficking of a series of TNF-enhanced GFP fusion proteins in human mast cell lines HMC-1 and LAD2. Protein covering the full TNF sequence was successfully sorted into secretory granules in a process involving transient exposure on the outer membrane and re-endocytosis. In human cells, contrary to results previously obtained in a rodent model, TNF seems not to be glycosylated and, thus, trafficking is carbohydrate independent. In an effort to localize the amino acid motif responsible for granule targeting, we constructed additional fusion proteins and analyzed their trafficking, concluding that granule-targeting sequences are localized in the mature chain of TNF and that the cytoplasmic tail is expendable for endocytotic sorting of this cytokine, thus excluding direct interactions with intracellular adaptor proteins.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 M.B.O. was supported by the European Commission 5th Framework Programme Project "Center of Excellence in Molecular Bio-Medicine Contract" QLK6-CT-2002-90363, EMBO short-term fellowship, and FEBS Collaborative Experimental Scholarship for Central and Eastern Europe.
2 Address correspondence and reprint requests to Dr. Maciej Olszewski, Department of Molecular Biology, International Institute of Molecular and Cellular Biology, 4 Ksiecia Trojdena Street, Warsaw, Poland. E-mail address: maciej{at}olszewski.name
3 Abbreviations used in this paper: sTNF, soluble form TNF; ER, endoplasmic reticulum; EGFP, enhanced GFP; ORF, open reading frame; CHX, cycloheximide; TACE, TNF-converting enzyme.
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