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.