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 beta-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.
Highlights
Misfolding of the mammalian prion protein (PrP) is implicated in the pathogenesis of prion diseases
Our study provides experimental evidence that deletion of the N-terminal domain of PrPC including the putative transmembrane region and the first  strand have no impact on the formation of complex glycosylated and GPI-anchored PrPC
Whereas the GPI anchor signal sequence allows core glycosylation to occur, whether or not attachment of the GPI anchor takes place, membrane anchorage of PrPC is necessary for the terminal processing of glycans (Fig. 8)
Summary
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, helix 1 induced the formation of unglycosylated and partially protease-resistant PrP aggregates. 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. Neuropathologic, genetic, and transgenic studies argue strongly in favor of a causal role of protein misfolding in the pathogenic process. Beyond this conceptual framework, prion diseases are exceptional in that they exist as sporadic, genetic, and infectious forms. Prion diseases are characterized by the formation of PrPSc, an abnormally folded isoform of the cellular prion protein PrPC, a highly conserved protein mainly pres-
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