title: Sorting of soluble TNF-receptor for granule storage in hematopoietic cells as a principle for targeting of selected proteins to inflamed sites.
abstract: Hematopoietic cells have secretory lysosomes that degranulate at the inflammatory site upon stimulation. We asked whether one could target exogenous proteins with a therapeutic potential to secretory lysosomes in hematopoietic cells. For this purpose, we expressed a soluble tumor necrosis factor (TNF) receptor form (sTNFR1) in hematopoietic cell lines. In order to accomplish targeting to secretory lysosomes, both endoplasmic reticulum (ER) retention and constitutive secretion have to be prevented. ER export was facilitated by addition of a transmembrane (tm) sequence, and constitutive secretion was overcome by incorporating a cytosolic sorting signal (Y) from CD63. This signal directed the resulting sTNFR1-tm-Y to secretory lysosomes. Confirmation of these results was provided by biosynthetic radiolabeling, subcellular fractionation, immunofluorescence microscopy, and immunoelectron microscopy. The tm-Y fragment was cleaved by proteolysis, resulting in generation of the membrane-free sTNFR1 in secretory lysosomes. Our results suggest a potential for using the storage organelles of hematopoietic cells as vehicles for targeting sites of inflammation with therapeutically active agents.
authors.affiliation: Department of Hematology, Lund, Sweden.
authors.forename: Ying
authors.initials: Y
authors.lastname: Gao
authors.forename: Hanna
authors.initials: H
authors.lastname: Rosén
authors.forename: Ellinor
authors.initials: E
authors.lastname: Johnsson
authors.forename: Jero
authors.initials: J
authors.lastname: Calafat
authors.forename: Hans
authors.initials: H
authors.lastname: Tapper
authors.forename: Inge
authors.initials: I
authors.lastname: Olsson
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000235:genetics / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism*; D010450:Endopeptidases / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D007249:Inflammation / Q000378:metabolism*; D007962:Leukocytes / Q000378:metabolism*; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals; D021381:Protein Transport; D051381:Rats; D018124:Receptors, Tumor Necrosis Factor / Q000235:genetics / Q000378:metabolism*; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012995:Solubility; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism
chemical_list: D015703:Antigens, CD; D021382:Protein Sorting Signals; D018124:Receptors, Tumor Necrosis Factor; D047888:Receptors, Tumor Necrosis Factor, Type I; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases