Pathogenic mutations in the glycosylphosphatidylinositol signal peptide of PrP modulate its topology in neuroblastoma cells

Abstract

Point mutations M232R (PrP(232R)), M232T (PrP(232T)), and P238S (PrP(238S)) in the glycosylphosphatidylinositol signal peptide (GPI-SP) of the prion protein (PrP(C)) segregate with familial Creutzfeldt-Jakob disease (CJD). However, the mechanism by which these mutations induce cytotoxicity is unclear since the GPI-SP is replaced by a GPI anchor within 5 min of PrP synthesis and translocation into the endoplasmic reticulum (ER). To examine if mutations in this region interfere with translocation of nascent PrP into the ER or anchor addition, the metabolism of PrP(232R) and PrP(232T) was investigated in transfected human neuroblastoma cells. In this report, we demonstrate that PrP mutations M232R and M232T do not interfere with GPI anchor addition. Instead, these mutations increase the stability and transport of GPI-SP mediated post-translationally translocated PrP to the plasma membrane, where it is linked to the lipid bilayer in a potentially neurotoxic C-transmembrane ((Ctm)PrP) orientation. Furthermore, we demonstrate that the GPI-SP of PrP functions as an efficient co-translational and inefficient post-translational ER translocation signal when tagged to an unrelated protein, underscoring the functional versatility of this peptide. These data uncover an alternate pathway of ER translocation for nascent PrP, and provide information on the possible mechanism(s) of neurotoxicity by mutations in the GPI-SP.

Residues Surrounding the Glycosylphosphatidylinositol Anchor Attachment Site of PrP Modulate Prion Infection: Insight from the Resistance of Rabbits to Prion Disease

“…Since RbPrP exhibits a large amino acid sequence difference around the site of PrP compared with those from other mammalian species, this may have a significant impact on the GPI anchoring of RbPrP. Moreover, many pathogenic mutations within PrP have been shown to alter the localization and membrane topology of PrP expressed in cultured cells (14,20,40). In this study, we have shown that despite exhibiting a significant amino acid difference around the site compared to PrPs from other mammals, RbPrP is anchored to the plasma membrane through a GPI anchor and is localized to lipid rafts.…”

The GPI-anchoring of PrP

“…42 It has been proposed that the two first mutations are related to an increased transmembrane orientation which could explain neurotoxicity. 43 More recently, Guizzunti et al used a chimeric protein expressing EGFP with the SS-GPI of PrP linked to a Myc tag to demonstrate that the P238S mutation in the SS-GPI of PrP prevents degradation of the SS-GPI by the proteasome. As a consequence the cleaved SS-GPI peptide tends to accumulate in the ER.…”

Cytosolically expressed PrP GPI-signal peptide interacts with mitochondria

“…Specifically, PrP GPI-SP has been shown to work as an ER-signal sequence when linked to the N-term of GFP. 33 However, when expressed in HeLa cells, cPrP-GPI-SP localized to discrete tubular elements (Fig. 1d), reminiscent of the mitochondrial network.…”