Sorting of soluble TNF-receptor for granule storage in hematopoietic cells as a principle for targeting of selected proteins to inflamed sites.
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.
Gao
Ying
Y
Department of Hematology, Lund, Sweden.
Rosén
Hanna
H
Johnsson
Ellinor
E
Calafat
Jero
J
Tapper
Hans
H
Olsson
Inge
I
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
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