NR ACKC
AU Caughey,B.W.; Neary,K.; Buller,R.; Ernst,D.; Perry,L.L.; Chesebro,B.; Race,R.E.
TI Normal and scrapie-associated forms of prion protein differ in their sensitivities to phospholipase and proteases in intact neuroblastoma cells
QU Journal of Virology 1990 Mar; 64(3): 1093-101
PT journal article
AB 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 (PrPres) in scrapie-infected neuroblastoma cells in vitro and to compare properties of PrPres with those of its normal proteinase K-sensitive homolog, PrP-sen. PrPres was detected by immunoblot in scrapie-infected but not uninfected neuroblastoma clones. Densitometry of immunoblots indicated that there was two- to threefold more PrPres 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, PrPres was not removed after reaction with these enzymes. Thus, either the scrapie-associated PrPres was not on the cell surface or it was there in a form that is resistant to these hydrolytic enzymes. Attempts to detect intracellular PrPres 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 PrPres, the scrapie agent was not removed from the infected cells by any of these enzymes.
MH Animal; Cell Line; Endopeptidase K; Flow Cytometry; Fluorescent Antibody Technique; Immunoblotting; Neuroblastoma; Phospholipase C/*metabolism; PrPsc Proteins; Prions/*metabolism; Serine Endopeptidases/*metabolism; Trypsin/*metabolism; Viral Proteins/analysis/*metabolism
AD Laboratory of Persistent Viral Diseases, National Institute of Allergy and Infectious Diseases, Hamilton, Montana 59840.
SP englisch
PO USA