cells as a principle for targeting of selected proteins to inflamed sites Sorting of soluble TNF-receptor for granule storage in hematopoietic

AbstractHematopoietic 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 TNF receptor form (sTNFR1) in hematopoietic cell lines. In order to accomplish targeting to secretory lysosomes both ER-retention and constitutive secretion has to be prevented. ER-retention 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 from 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.Inge.Olsson@hematologi.lu.seFrom bloodjournal.hematologylibrary.org by guest on June 1, 2013. For personal use only.