The protease-sensitive N-terminal polybasic region of prion protein modulates its conversion to the pathogenic prion conformer

Abstract

Conversion of normal prion protein (PrP^C) to the pathogenic PrP^Sc conformer is central to prion diseases such as Creutzfeldt-Jakob disease and scrapie; however, the detailed mechanism of this conversion remains obscure. To investigate how the N-terminal polybasic region of PrP (NPR) influences the PrP^C-to-PrP^Sc conversion, we analyzed two PrP mutants: ΔN6 (deletion of all six amino acids in NPR) and Met4-1 (replacement of four positively charged amino acids in NPR with methionine). We found that ΔN6 and Met4-1 differentially impacted the binding of recombinant PrP (recPrP) to the negatively charged phospholipid 1-palmitoyl-2-oleoylphosphatidylglycerol, a nonprotein cofactor that facilitates PrP conversion. Both mutant recPrPs were able to form recombinant prion (recPrP^Sc) in vitro, but the convertibility was greatly reduced, with ΔN6 displaying the lowest convertibility. Prion infection assays in mammalian RK13 cells expressing WT or NPR-mutant PrPs confirmed these differences in convertibility, indicating that the NPR affects the conversion of both bacterially expressed recPrP and post-translationally modified PrP in eukaryotic cells. We also found that both WT and mutant recPrP^Sc conformers caused prion disease in WT mice with a 100% attack rate, but the incubation times and neuropathological changes caused by two recPrP^Sc mutants were significantly different from each other and from that of WT recPrP^Sc. Together, our results support that the NPR greatly influences PrP^C-to-PrP^Sc conversion, but it is not essential for the generation of PrP^Sc. Moreover, the significant differences between ΔN6 and Met4-1 suggest that not only charge but also the identity of amino acids in NPR is important to PrP conversion.

Keywords: N-terminal polybasic region; POPG; conformational change; convertibility; prion; prion disease.