Normal and scrapie-assciated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells

B. Caughey, K. Neary, R. Buller, D. Ernst, L. L. Perry, B. Chesebro, R. E. Race

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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 P-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.

Original languageEnglish
Pages (from-to)1093-1101
Number of pages9
JournalJournal of virology
Volume64
Issue number3
StatePublished - Mar 14 1990

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    Caughey, B., Neary, K., Buller, R., Ernst, D., Perry, L. L., Chesebro, B., & Race, R. E. (1990). Normal and scrapie-assciated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells. Journal of virology, 64(3), 1093-1101.