Abstract
The cellular prion protein (PrP(C)) is essential for the pathogenesis and transmission of prion diseases. Whereas the majority of PrP(C) is bound to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor, a secreted form of the protein has been identified. Here we show that PrP(C) can be shed into the medium of human neuroblastoma SH-SY5Y cells by both protease- and phospholipase-mediated mechanisms. The constitutive shedding of PrP(C) was inhibited by a range of hydroxamate-based zinc metalloprotease inhibitors in a manner identical to the alpha-secretase-mediated shedding of the amyloid precursor protein, indicating a proteolytic shedding mechanism. Like amyloid precursor protein, this zinc metalloprotease-mediated shedding of PrP(C) could be stimulated by phorbol myristate acetate and by copper ions. The lipid raft-disrupting agents filipin and methyl-beta-cyclodextrin promoted the shedding of PrP(C) via a distinct mechanism that was not inhibited by hydroxamate-based inhibitors. Filipin-mediated shedding of PrP(C) is likely to occur via phospholipase cleavage of the GPI anchor, since a transmembrane polypeptide-anchored PrP construct was not shed in response to filipin treatment. Collectively, our data indicate that shedding of PrP(C) can occur via both secretase-like proteolytic cleavage of the protein and phospholipase cleavage of the GPI anchor moiety.
Highlights
Transmissible spongiform encephalopathies encompass a group of neurodegenerative diseases including scrapie in sheep, bovine spongiform encephalopathy in cattle, and human pathologies such as Creutzfeldt-Jakob disease and GerstmannStraussler-Scheinker syndrome (1–3)
We show for the first time that a significant proportion of PrPC is shed from the cell surface by the proteolytic action of a zinc metalloprotease with similarities to the ␣-secretase involved in the shedding of the amyloid precursor protein (APP)
Metalloprotease-mediated Constitutive Shedding of prion protein (PrP)—In order to elucidate the mechanism involved in constitutive PrP secretion, we compared the shedding of overexpressed wildtype PrP from human neuroblastoma SH-SY5Y cells with that of two mutant PrP constructs (Fig. 1A)
Summary
PrP, prion protein; PrPC, cellular prion protein; PrPSc, pathologic conformation of PrP; wt-PrP, wild-type PrP; APP, amyloid precursor protein; GPI, glycosylphosphatidylinositol; MCD, methyl--cyclodextrin; PI-PLC, phosphatidylinositol-specific phospholipase C; PMA, phorbol myristate acetate
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