Nanopore analysis reveals differences in structural stability of ovine PrP^C proteins corresponding to scrapie susceptible (VRQ) and resistance (ARR) genotypes

Abstract

Species, as well as individuals within species, have unique susceptibilities to prion infection that are likely based on sequence differences in cellular prion protein (PrP^C). Species barriers to transmission also reflect PrP^C sequence differences. Defining the structure-activity relationship of PrP^C/PrP^Sc with respect to infectivity/susceptibility will benefit disease understanding and assessment of transmission risks. Here, nanopore analysis is employed to investigate genotypes of sheep PrP^C corresponding to differential susceptibilities to scrapie infection. Under non-denaturing conditions scrapie resistant (ARR) and susceptible (VRQ) genotypes display similar, type I (bumping) predominant event profiles, suggesting a conserved folding pattern. Under increasingly denaturing conditions both proteins shift to type II (intercalation/translocation) events but with different sensitivities to unfolding. Specifically, when pre-incubated in 2M Gdn-HCl, the VRQ variant had more of type II events as compared with the ARR protein, suggesting a more flexible unfolding pattern. Addition of PrP^Sc-specific polyclonal antibody (YML) to the ARR variant, pre-incubated in 2M Gdn-HCl, reduced the number of type II events with no clear intercalation/translocation peak, whereas for VRQ, type II events above blockades of 90 pA bound YML. A second PrP^Sc-specific antibody (SN6b) to a different cryptic epitope reduced type II events for VRQ but not the ARR variant. Collectively, the event patterns associated with sequential denaturation, as well as interactions with PrP^Sc-specific antibodies, support unique patterns and/or propensities of misfolding between the genotypes. Overall, nanopore analysis identifies intermediate conformations that occur during the unfolding pathways of ARR and VRQ genotypes and may help to understand the correlation of structural properties that induce protein misfolding.

Keywords: :

• nanopore
• genotypes
• polymorphisms
• prion disease
• antibody

10.4161/pri.27502

Discosure of Potential Conflicts of Interest

No potential conflict of interest was disclosed.

Acknowledgments

Thank you to Dr David S Wishart (Department of Computing Science and Biological Sciences, University of Alberta, Edmonton, Alberta, T6G 2E8, Canada) for the VRQ plasmid. We also sincerely thank Dr Hugh GG Townsend (DVM, MSc), research scientist and program manager (Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, Saskatchewan, S7N 5E3, Canada).

Footnotes

This work was supported by PrioNet Canada, Saskatchewan Agriculture and Development, Alberta Prion Research Institute and NSERC.