title: Normal and scrapie-associated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells.
abstract: Previous studies have indicated that scrapie infection results in the accumulation of a proteinase K-resistant form of an endogenous brain protein generally referred to as prion protein (PrP). The molecular nature of the scrapie-associated modification of PrP accounting for proteinase K resistance is not known. As an approach to understanding the cellular events associated with the PrP modification in brain tissue, we sought to identify proteinase K-resistant PrP (PrP-res) in scrapie-infected neuroblastoma cells in vitro and to compare properties of PrP-res with those of its normal proteinase K-sensitive homolog, PrP-sen. PrP-res was detected by immunoblot in scrapie-infected but not uninfected neuroblastoma clones. Densitometry of immunoblots indicated that there was two- to threefold more PrP-res than PrP-sen in one infected clone. Metabolic labeling and membrane immunofluorescence experiments indicated that PrP-sen was located on the cell surface and could be removed from intact cells by phosphatidylinositol-specific phospholipase C and proteases. In contrast, PrP-res was not removed after reaction with these enzymes. Thus, either the scrapie-associated PrP-res was not on the cell surface or it was there in a form that is resistant to these hydrolytic enzymes. Attempts to detect intracellular PrP-res by immunofluorescent staining of fixed and permeabilized cells revealed that PrP was present in discrete perinuclear Golgi-like structures. However, the staining pattern was similar in both scrapie-infected and uninfected clones, and thus the intracellular staining may have represented only PrP-sen. Analysis of scrapie infectivity in cells treated with extracellular phospholipase, proteinase K, and trypsin indicated that, like PrP-res, the scrapie agent was not removed from the infected cells by any of these enzymes.
authors.affiliation: Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840.
authors.forename: B
authors.initials: B
authors.lastname: Caughey
authors.forename: K
authors.initials: K
authors.lastname: Neary
authors.forename: R
authors.initials: R
authors.lastname: Buller
authors.forename: D
authors.initials: D
authors.lastname: Ernst
authors.forename: L L
authors.initials: LL
authors.lastname: Perry
authors.forename: B
authors.initials: B
authors.lastname: Chesebro
authors.forename: R E
authors.initials: RE
authors.lastname: Race
mesh_terms: D000818:Animals; D002460:Cell Line; D019286:Endopeptidase K; D005434:Flow Cytometry; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D009447:Neuroblastoma; D018620:PrPSc Proteins; D011328:Prions / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism*; D014764:Viral Proteins / Q000032:analysis / Q000378:metabolism*
chemical_list: D018620:PrPSc Proteins; D011328:Prions; D014764:Viral Proteins; D010738:Type C Phospholipases; D012697:Serine Endopeptidases; D014357:Trypsin; D019286:Endopeptidase K