Glycosylphosphatidylinositol Anchor-Dependent Stimulation Pathway Required for Generation of Baculovirus-Derived Recombinant Scrapie Prion Protein

Abstract

ABSTRACT The pathogenic isoform (PrP Sc ) of the host-encoded cellular prion protein (PrP C ) is considered to be an infectious agent of transmissible spongiform encephalopathy (TSE). The detailed mechanism by which the PrP Sc seed catalyzes the structural conversion of endogenous PrP C into nascent PrP Sc in vivo still remains unclear. Recent studies reveal that bacterially derived recombinant PrP (recPrP) can be used as a substrate for the in vitro generation of protease-resistant recPrP (recPrP res ) by protein-misfolding cyclic amplification (PMCA). These findings imply that PrP modifications with a glycosylphosphatidylinositol (GPI) anchor and asparagine (N)-linked glycosylation are not necessary for the amplification and generation of recPrP Sc by PMCA. However, the biological properties of PrP Sc obtained by in vivo transmission of recPrP res are unique or different from those of PrP Sc used as the seed, indicating that the mechanisms mediated by these posttranslational modifications possibly participate in reproductive propagation of PrP Sc . In the present study, using baculovirus-derived recombinant PrP (Bac-PrP), we demonstrated that Bac-PrP is useful as a PrP C substrate for amplification of the mouse scrapie prion strain Chandler, and PrP Sc that accumulated in mice inoculated with Bac-PrP res had biochemical and pathological properties very similar to those of the PrP Sc seed. Since Bac-PrP modified with a GPI anchor and brain homogenate of Prnp knockout mice were both required to generate Bac-PrP res , the interaction of GPI-anchored PrP with factors in brain homogenates is essential for reproductive propagation of PrP Sc . Therefore, the Bac-PMCA technique appears to be extremely beneficial for the comprehensive understanding of the GPI anchor-mediated stimulation pathway.