Cell-specific differences in the utilization of the two N-glycosylation sequons (Asn180-Ile-Thr and Asn196-Phe-Thr) of the prion protein (PrP) have been proposed to influence the aetiology of the neurodegenerative prion diseases. As the N-glycosylation of PrP is ablated by deletion of the C-terminal glycosyl-phosphatidylinositol (GPI) anchor signal sequence, we have investigated the determinants for PrP sequon utilization in human neuronal cells using the novel approach of restoring N-glycosylation to secreted forms of PrP lacking a GPI anchor. N-glycosylation was restored to an efficiency comparable with that of GPI anchored PrP when the distance of the sequon to the C-terminus was increased so that it was sufficient to reach the active site of oligosaccharyltransferase before chain termination. Our findings indicate that sequon utilization in PrP is a co-translational process that precedes GPI anchor addition and, as such, will be greatly influenced by the dynamics of the translocon—oligosaccharyltransferase complex.
- endoplasmic reticulum
Abbreviations used: ER, endoplasmic reticulum; GPI, glycosyl-phosphatidylinositol; OST, oligosaccharyltransferase; PI-PLC, phosphatidylinositol-specific phospholipase C; PNGase F, peptide N-glycosidase F; PrP, prion protein; PrPC, normal cellular isoform of PrP; PrPSc, pathogenic isoform of PrP; PrPΔGPI, secreted, non-N-glycosylated isoform of PrP; wtPrP, wild-type transfected PrP; mutations are given using the single-letter amino acid code, where, for example, V241D means Val241→Asp.
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