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

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.