Determinants of the in Vivo Folding of the Prion Protein

Abstract

Misfolding of the mammalian prion protein (PrP) is implicated in the pathogenesis of prion diseases. We analyzed wild type PrP in comparison with different PrP mutants and identified determinants of the in vivo folding pathway of PrP. The complete N terminus of PrP including the putative transmembrane domain and the first ␤-strand could be deleted without interfering with PrP maturation. Helix 1, however, turned out to be a major determinant of PrP folding. Disruption of helix 1 prevented attachment of the glycosylphosphatidylinositol (GPI) anchor and the formation of complex N-linked glycans; instead, a high mannose PrP glycoform was secreted into the cell culture supernatant. In the absence of a C-terminal membrane anchor, however, helix 1 induced the formation of unglycosylated and partially protease-resistant PrP aggregates. Moreover, we could show that the C-terminal GPI anchor signal sequence, independent of its role in GPI anchor attachment, mediates core glycosylation of nascent PrP. Interestingly, conversion of high mannose glycans to complex type glycans only occurred when PrP was membrane-anchored. Our study indicates a bipartite function of helix 1 in the maturation and aggregation of PrP and emphasizes a critical role of a membrane anchor in the formation of complex glycosylated PrP.

Post-translational Import of the Prion Protein into the Endoplasmic Reticulum Interferes with Cell Viability

“…In this context it is important to note that in mammalian cells PrP⌬GPI is mainly unglycosylated, but in contrast to gPrP⌬GPI and pPrP⌬GPI expressed in yeast, it is N-terminally processed. In the same study we showed that PrP containing a nonfunctional C-terminal GPI anchor signal sequence is both N-terminally processed and core-glycosylated (10). Further biochemical analysis revealed that gPrP⌬GPI (Fig.…”

“…Endo H Digestion and Secretion Analysis in N2a Cells-For Endo H digestion, cell lysates were prepared in 0.5% SDS, boiled for 10 min, and digested with Endo H (NEB) for 1 h at 37°C (10). After boiling in Laemmli sample buffer, the lysates were examined by immunoblotting.…”

“…Mouse neuroblastoma (N2a) cells (31) were cultured as described previously (32). PrP was detected by the monoclonal anti-PrP antibody 3F4 (33) or the polyclonal anti-PrP antiserum A7 (10). The anti-Gas1p antibody was kindly provided by R. Barz, and the anti-Gim2p antibody was described earlier (34).…”

“…3B and Ref. 10). To test whether the putative transmembrane domain also had an effect on the glycosylation of PrP⌬GPI in mammalian cells we analyzed PrP⌬TM⌬GPI in N2a cells, and core glycosylation was shown to be restored (Fig.…”

“…In the case of PrP, however, we and others have shown that PrP devoid of a GPI anchor remains mainly unglycosylated but is imported efficiently into the ER and transported through the secretory pathway (10 -14). It has been found that the only specific marker for ER import of PrP is a cleaved N-terminal signal sequence (10). 2 Misfolding of PrP C in the cytosol or in the ER can induce neurodegeneration in the absence of PrP Sc .…”