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.

Tetracysteine‐tagged prion protein allows discrimination between the native and converted forms

“…This is in agreement with the results of antibody-binding studies [43,44]. Additionally, a GPI anchor attached to the C-terminus of PrP Sc cannot be cleaved by phospholipase C, indicating that this PrP segment is protected after formation of the protease-resistant form of PrP [45], which is in accordance with our results. An MS study showed that the flexible N-terminus is highly protected in fibrils in comparison to native PrP [46].…”

The scrapie prion protein is present in flotillin‐1‐positive vesicles in central‐ but not peripheral‐derived neuronal cell lines

“…PrP c can be degraded by proteases (Luhr et al, 2004) and, actually, this could explain why it is undetectable into the LYAAT-1-lysosomal compartment. Despite the fact that PrP Sc is resistant to proteolysis (Caughey et al, 1990;Taraboulos et al, 1990a), it is not found in LYAAT-1 lysosomes, indicating that it does not enter this compartment. Actually, PrP Sc mostly accumulates in LAMP-1-vesicles in both GT1-7 and N2a cell lines.…”

Cellular Biology of Prion Diseases

“…Chemical analysis of the purified protein demonstrates that PrP Sc , like PrP C , possesses a C-terminal GPI anchor (161). Unlike PrP C , however, PrP Sc is not releasable from brain membranes with PI-PLC (24,141,164). The lack of release is not due to sequestration in the lumen of a vesicular compartment, since membrane-bound PrP Sc is accessible to biotinylation and protease digestion.…”

“…Because it is attached exclusively by its GPI anchor, it can be released from the cell surface by treatment with the bacterial enzyme PI-specific phospholipase C (PI-PLC), which cleaves off the diacylglycerol portion of the anchor (see Fig. 7B) (13,24,94).…”