RELEASE OF THE CELLULAR PRION PROTEIN FROM CULTURED-CELLS AFTER LOSS OF ITS GLYCOINOSITOL PHOSPHOLIPID ANCHOR

Secreted forms of the sialoglycoprotein designated cellular prion protein (PrP(C)) have been identified that cannot be explained by alternative splicing. We report that secreted forms of PrP(C) derive from precursors that are bound to the plasma membrane by glycoinositol phospholipid (GPI) anchors. Secreted PrP(C) slowly appeared in the culture medium of metabolically radiolabelled cells after incubations of 8-24 h. Digestion of nascent PrP(C) with phosphatidylinositol-specific phospholipase C (PIPLC) prevented the appearance of secreted PrP(C). Secreted PrP(C) partitioned into the aqueous phase of Triton X-114 like PIPLC-released PrP(C). While the M(r) of PIPLC-released PrP(C) was reduced 2-4 kDa after treatment with aqueous hydroflouric acid, which removes the entire GPI anchor modification, the M(r) of secreted PrP(C) was unchanged. Both PIPLC-released and secreted PrP(C) were recognized by antiserum raised against a synthetic C-terminal peptide corresponding to residues 220-233 (amino acid 231 is the site of GPI attachment). We conclude that GPI-anchored PrP(C) is post-translationally processed to remove most, if not all, of the GPI modification and then shed into culture medium. Whether PrP(C) is shed after proteolysis near the C-terminus remains to be established. A minority of PrP(C) in normal Syrian hamster brain partitioned into the aqueous phase of Triton X-114 like shed PrP(C), suggesting physiological significance.