Membrane topology influences N-glycosylation of the prion protein.

The glycosylation state of the glycosyl-phosphatidylinositol (GPI) anchored cellular prion protein (PrPC) can influence the formation of the disease form of the protein responsible for the neurodegenerative spongiform encephalopathies. We have investigated the role of membrane topology in the N-glycosylation of PrP by expressing a C-terminal transmembrane anchored form, PrP-CTM, an N-terminal transmembrane anchored form, PrP-NTM, a double-anchored form, PrP-DA, and a truncated form, PrPDeltaGPI, in human neuroblastoma SH-SY5Y cells. Wild-type PrP, PrP- CTM and PrP-DA were membrane anchored and present on the cell surface as glycosylated forms. In contrast, PrP-NTM, although membrane anchored and localized at the cell surface, was not N-glycosylated. PrPDeltaGPI was secreted from the cells into the medium in a hydrophilic form that was unglycosylated. The 4-fold slower rate at which PrPDeltaGPI was trafficked through the cell compared with wild-type PrP was due to the absence of the GPI anchor not the lack of N-glycans. Retention of PrPDeltaGPI in the endoplasmic reticulum did not lead to its glycosylation. These results indicate that C-terminal membrane anchorage is required for N-glycosylation of PrP.