Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro

Abstract

Alteration in cellular prion protein (PrP^C) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrP^Sc). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrP^Sc and cellular trafficking of PrP^Q227X remain to be determined. Here, we show that PrP^Sc in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrP^Wt), suggesting pathological PrP^Q227X is incapable of recruiting PrP^Wt in vivo. This mutant anchorless protein, however, is able to recruit PrP^Wt from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrP^Sc-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrP^Q227X or PrP^Wt were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrP^Wt is recovered in the cell lysate fraction, while most of the mutant PrP^Q227X is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrP^Q227X spots clustered at molecular weights of 22-25 kDa with an isoelectric point (pI) of 3.5-5.5, whereas protein spots from the medium are at 18-26 kDa with a pI of 7-10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.