Abstract
The prion protein (PrP) can adopt multiple membrane topologies, including a fully translocated form (SecPrP), two transmembrane forms (NtmPrP and CtmPrP), and a cytosolic form. It is important to understand the factors that influence production of these species, because two of them, CtmPrP and cytosolic PrP, have been proposed to be key neurotoxic intermediates in certain prion diseases. In this paper, we perform a mutational analysis of PrP synthesized using an in vitro translation system in order to further define sequence elements that influence the formation of CtmPrP. We find that substitution of charged residues in the hydrophobic core of the signal peptide increases synthesis of CtmPrP and also reduces the efficiency of translocation into microsomes. Combining these mutations with substitutions in the transmembrane domain causes the protein to be synthesized exclusively with the CtmPrP topology. Reducing the spacing between the signal peptide and the transmembrane domain also increases CtmPrP. In contrast, topology is not altered by mutations that prevent signal peptide cleavage or by deletion of the C-terminal signal for glycosylphosphatidylinositol anchor addition. Removal of the signal peptide completely blocks translocation. Taken together, our results are consistent with a model in which the signal peptide and transmembrane domain function in distinct ways as determinants of PrP topology. We also present characterization of an antibody that selectively recognizes CtmPrP and cytosolic PrP by virtue of their uncleaved signal peptides. By using this antibody, as well as the distinctive gel mobility of CtmPrP and cytosolic PrP, we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions. We conclude that CtmPrP and cytosolic PrP are unlikely to be obligate neurotoxic intermediates in familial or infectiously acquired prion diseases.
Highlights
Prion diseases are fatal neurological disorders of humans and animals that appear in sporadic, familial, and infectiously acquired forms
As well as the distinctive gel mobility of CtmPrP and cytosolic prion protein (PrP), we show that the amounts of these two forms in cultured cells and rodent brain are not altered by infection with scrapie prions
We discovered that a non-conservative substitution (L9R) within the hydrophobic core of the signal sequence dramatically increased the proportion of CtmPrP (13)
Summary
PrPC, cellular isoform of PrP; PrPSc, scrapie isoform of PrP; CHO, Chinese hamster ovary; GPI, glycosylphosphatidylinositol; PK, proteinase K; PrP, prion protein; SP, signal peptide; WT, wild type; ER, endoplasmic reticulum; PNGase, peptide:N-glycosidase. Mice have been constructed in which a wild-type hamster PrP transgene serves as a reporter of CtmPrP formation (12) When these animals are inoculated with mouse prions, the amounts of CtmPrP as well as PrPSc in the brain are found to increase during the course of the infection. A key gap in the experimental evidence supporting roles for CtmPrP and cytosolic PrP in prion-induced neurodegeneration is the lack of data demonstrating that the amounts of these forms increase during the course of a prion infection In part, this difficulty is due to the absence of direct methods for detecting CtmPrP and cytosolic PrP in infected cells and tissues. We carry out a mutational analysis of several sequence determinants in PrP to better understand the factors that influence the topology of the protein
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