Partial Deletion of the Carboxyl-Terminal Signal Sequence of the Cellular Prion Protein Alters Protein Expression via Endoplasmic Reticulum-Associated Degradation.

Cellular prion protein (PrP) is a glycoprotein tethered to the plasma membrane via a GPI-anchor, and it plays a crucial role in prion diseases by undergoing conformational change to PrP. To generate a knock-in (KI) mouse model expressing bank vole PrP (BVPrP), a KI targeting construct was designed. However, a Prnp gene sequence that encodes PrP lacking seven C-terminal amino acid residues of the GPI-anchoring signal sequence (GPI-SS) was unintentionally introduced into the construct. The resulting KIBVPrP248 mice exhibited very low PrP expression and resistance to prion infection. To investigate the underlying mechanism of reduced PrP expression, RK13 cells expressing either full-length GPI-SS (BVPrP255) or truncated GPI-SS (BVPrP248) and KIBVPrP248 mice were analyzed. In RK13-BVPrP248 cells, PrP protein levels were nearly ten-fold lower than in RK13-BVPrP255 cells, mimicking the extremely low PrP expression of the KIBVPrP248 mice. The abundance, stability, and translational efficiency of the Prnp mRNA were not the primary causes for the low PrP expression in RK13-BVPrP248 cells. A pharmacological analysis revealed that BVPrP248 underwent enhanced degradation via the ER-associated degradation pathway, with increased PrP ubiquitination detected in both the cell and animal models. An immunofluorescence analysis showed that BVPrP248 was mislocalized to the ER, co-localizing with Grp78, an ER chaperone. Although mislocalization of BVPrP248 under the transient overexpression condition led to mild activation of the unfolded protein response in RK13-BVPrP248 cells, low-level chronic expression of BVPrP248 in stable transfectants and KIBVPrP248 mice did not facilitate such events. These findings suggested that the C-terminal GPI-SS of PrP plays a critical role in PrP biogenesis.