title: Removal of the glycosylphosphatidylinositol anchor from PrP(Sc) by cathepsin D does not reduce prion infectivity.
abstract: According to the protein-only hypothesis of prion propagation, prions are composed principally of PrP(Sc), an abnormal conformational isoform of the prion protein, which, like its normal cellular precursor (PrP(C)), has a GPI (glycosylphosphatidylinositol) anchor at the C-terminus. To date, elucidating the role of this anchor on the infectivity of prion preparations has not been possible because of the resistance of PrP(Sc) to the activity of PI-PLC (phosphoinositide-specific phospholipase C), an enzyme which removes the GPI moiety from PrP(C). Removal of the GPI anchor from PrP(Sc) requires denaturation before treatment with PI-PLC, a process that also abolishes infectivity. To circumvent this problem, we have removed the GPI anchor from PrP(Sc) in RML (Rocky Mountain Laboratory)-prion-infected murine brain homogenate using the aspartic endoprotease cathepsin D. This enzyme eliminates a short sequence at the C-terminal end of PrP to which the GPI anchor is attached. We found that this modification has no effect (i) on an in vitro amplification model of PrP(Sc), (ii) on the prion titre as determined by a highly sensitive N2a-cell based bioassay, or (iii) in a mouse bioassay. These results show that the GPI anchor has little or no role in either the propagation of PrP(Sc) or on prion infectivity.
authors.affiliation: MRC Prion Unit, Department of Neurodegenerative Disease, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK.
authors.forename: Patrick A
authors.initials: PA
authors.lastname: Lewis
authors.forename: Francesca
authors.initials: F
authors.lastname: Properzi
authors.forename: Kanella
authors.initials: K
authors.lastname: Prodromidou
authors.forename: Anthony R
authors.initials: AR
authors.lastname: Clarke
authors.forename: John
authors.initials: J
authors.lastname: Collinge
authors.forename: Graham S
authors.initials: GS
authors.lastname: Jackson
mesh_terms: D000818:Animals; D001681:Biological Assay; D002402:Cathepsin D / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004492:Edetic Acid / Q000494:pharmacology; D005784:Gene Amplification / Q000235:genetics; D017261:Glycosylphosphatidylinositols / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D008670:Metals / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D018620:PrPSc Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D011485:Protein Binding / Q000187:drug effects; D012608:Scrapie / Q000378:metabolism / Q000473:pathology
chemical_list: D017261:Glycosylphosphatidylinositols; D008670:Metals; D018620:PrPSc Proteins; D004492:Edetic Acid; D002402:Cathepsin D