Isolation of Proteinase K-Sensitive Prions Using Pronase E and Phosphotungstic Acid

Laura D'Castro,Nathalie Gros,Adam Wenborn,Sabrina Cronier,Jonathan D F Wadsworth,John Collinge,Susan Joiner,Sergio T Ferreira

doi:10.1371/journal.pone.0015679

Abstract

Disease-related prion protein, PrPSc, is classically distinguished from its normal cellular precursor, PrPC, by its detergent insolubility and partial resistance to proteolysis. Molecular diagnosis of prion disease typically relies upon detection of protease-resistant fragments of PrPSc using proteinase K, however it is now apparent that the majority of disease-related PrP and indeed prion infectivity may be destroyed by this treatment. Here we report that digestion of RML prion-infected mouse brain with pronase E, followed by precipitation with sodium phosphotungstic acid, eliminates the large majority of brain proteins, including PrPC, while preserving >70% of infectious prion titre. This procedure now allows characterization of proteinase K-sensitive prions and investigation of their clinical relevance in human and animal prion disease without being confounded by contaminating PrPC.

Highlights

Prion diseases are fatal neurodegenerative conditions including kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-StrausslerScheinker syndrome and fatal familial insomnia in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and chronic wasting disease in cervids [1,2,3,4]
The activity of pronase E is similar to thermolysin and preserves disease-related PrP in a full length form rather than producing the N-terminally truncated fragments that are generated by proteinase K (PK)
Prion diseased brain contains a highly heterogeneous population of abnormal PrP isoforms that may differ with respect to their conformation, aggregate size, potential neurotoxicity and specific prion infectivity [5] and prion strains, rather than constituting a molecular clone, appear to comprise an ensemble or quasispecies maintained under host selection pressure [5,37]

Summary

Introduction

Prion diseases are fatal neurodegenerative conditions including kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-StrausslerScheinker syndrome and fatal familial insomnia in humans, scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle and chronic wasting disease in cervids [1,2,3,4]. They are characterized by the post-translational conversion of host cellular prion protein (PrPC) into an abnormal disease-related isoform designated PrPSc [1,2,3,5]. Under conditions in which PrPC exists as a detergent-soluble monomer and is completely degraded by the non-specific protease, proteinase K (PK), PrPSc exists in an aggregated form with the C-terminal two thirds of the protein showing marked resistance to proteolytic degradation leading to the generation of aminoterminally truncated fragments of di-, mono- and non-glycosylated PrP [1,7]

Methods

Results

Conclusion