Abstract
The prion protein (PrP), a glycolipid-anchored membrane glycoprotein, contains a conserved hydrophobic sequence that can span the lipid bilayer in either direction, resulting in two transmembrane forms designated (Ntm)PrP and (Ctm)PrP. Previous studies have shown that the proportion of (Ctm)PrP is increased by mutations in the membrane-spanning segment, and it has been hypothesized that (Ctm)PrP represents a key intermediate in the pathway of prion-induced neurodegeneration. To further test this idea, we have surveyed a number of mutations associated with familial prion diseases to determine whether they alter the proportions of (Ntm)PrP and (Ctm)PrP produced in vitro, in transfected cells, and in transgenic mice. For the in vitro experiments, PrP mRNA was translated in the presence of murine thymoma microsomes which, in contrast to the canine pancreatic microsomes used in previous studies, are capable of efficient glycolipidation. We confirmed that mutations within or near the transmembrane domain enhance the formation of (Ctm)PrP, and we demonstrate for the first time that this species contains a C-terminal glycolipid anchor, thus exhibiting an unusual, dual mode of membrane attachment. However, we find that pathogenic mutations in other regions of the molecule have no effect on the amounts of (Ctm)PrP and (Ntm)PrP, arguing against the proposition that transmembrane PrP plays an obligate role in the pathogenesis of prion diseases.
Highlights
Prion diseases are neurodegenerative disorders characterized by spongiform destruction of brain tissue and the presence of cerebral amyloid plaques (1, 2)
We confirmed that mutations within or near the transmembrane domain enhance the formation of CtmPrP, and we demonstrate for the first time that this species contains a C-terminal glycolipid anchor, exhibiting an unusual, dual mode of membrane attachment
Less than 10% of the prion protein (PrP) chains translated in the presence of canine pancreatic microsomes shifted into the aqueous phase after phosphatidylinositol-specific phospholipase C (PIPLC) treatment, indicating that very few molecules carried a glycosyl phosphatidylinositol (GPI) anchor
Summary
PrPSc, scrapie isoform of prion protein; ER, endoplasmic reticulum; GPI, glycosyl phosphatidylinositol; PIPLC, phosphatidylinositol-specific phospholipase C; PK, proteinase K; PrP, prion protein; PrPC, cellular isoform of prion protein; CtmPrP, C-terminal transmembrane form of prion protein; NtmPrP, N-terminal transmembrane form of prion protein; SecPrP, secretory form of prion protein; PBS, phosphate-buffered saline; BHK, baby hamster kidney; CHO, ally altered isoform of a normal cell-surface glycoprotein of unknown function called PrPC (3). CtmPrP has been identified in brain membranes from transgenic mice that express PrP molecules carrying mutations within or near the transmembrane domain; in vitro translation experiments had indicated that these mutations increased the relative proportion of CtmPrP (12, 22) Transgenic mice expressing such CtmPrP-favoring mutations at high levels develop a spontaneous neurodegenerative illness that bears some similarities to scrapie, but without the presence of PrPSc (12, 22). There is indirect evidence that CtmPrP accumulates in mice expressing wild-type PrP during the course of scrapie infection (22) Based on these results, it has been hypothesized that CtmPrP represents a common intermediate in the pathogenesis of both infectious and genetic prion diseases (22). We find that pathogenic mutations in other regions of the molecule have no effect on the amounts of CtmPrP and NtmPrP, arguing against the possibility that transmembrane PrP plays an obligate role in the pathogenesis of prion diseases
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