Analyses of Protease Resistance and Aggregation State of Abnormal Prion Protein across the Spectrum of Human Prions

Prion diseases are characterized by tissue accumulation of a misfolded, β-sheet-enriched isoform (scrapie prion protein (PrP Sc )) of the cellular prion protein (PrP C ). At variance with PrP C , PrP Sc shows a partial resistance to protease digestion and forms highly aggregated and detergent-insoluble polymers, two properties that have been consistently used to distinguish the two proteins. In recent years, however, the idea that PrP Sc itself comprises heterogeneous species has grown. Most importantly, a putative proteinase K (PK)-sensitive form of PrP Sc (sPrP Sc ) is being increasingly investigated for its possible role in prion infectivity, neurotoxicity, and strain variability. The study of sPrP Sc , however, remains technically challenging because of the need of separating it from PrP C without using proteases. In this study, we have systematically analyzed both PK resistance and the aggregation state of purified PrP Sc across the whole spectrum of the currently characterized human prion strains. The results show that PrP Sc isolates manifest significant strain-specific differences in their PK digestion profile that are only partially explained by differences in the size of aggregates, suggesting that other factors, likely acting on PrP Sc aggregate stability, determine its resistance to proteolysis. Fully protease-sensitive low molecular weight aggregates were detected in all isolates but in a limited proportion of the overall PrP Sc ( i.e. <10%), arguing against a significant role of slowly sedimenting PK-sensitive PrP Sc in the biogenesis of prion strains. Finally, we highlight the limitations of current operational definitions of sPrP Sc and of the quantitative analytical measurements that are not based on the isolation of a fully PK-sensitive PrP Sc form. Background: Protease resistance and aggregation state, two fundamental properties of abnormal prion protein (PrP Sc ), remain significantly unexplored. Results: Prion isolates show differences in PrP Sc PK digestion profile that are only partially explained by differences in aggregate size. Conclusion: Molecular factors, likely acting on aggregate stability, significantly influence PrP Sc PK sensitivity. Significance: The study provides insight into prion strain variability with respect to PrP Sc PK sensitivity and aggregation state.