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

Daniela Saverioni,Silvio Notari,Sabina Capellari,Ilaria Poggiolini,Armin Giese,Hans A Kretzschmar,Piero Parchi

doi:10.1074/jbc.m113.477547

Abstract

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.

Highlights

Protease resistance and aggregation state, two fundamental properties of abnormal prion protein (PrPSc), remain significantly unexplored
The known truncated species associated with the non-CJD brain include a major 18-kDa C-terminal fragment, known as C1, which is generated by the cleavage of full-length PrPC between residues 111 and 112, and a minor ϳ20-kDa C-terminal fragment (CTF) [38]
We have carried out the first systematic analysis of PrPSc protease resistance and aggregation state across the spectrum of human sporadic and variant CJD (vCJD) prions

Summary

Background

Protease resistance and aggregation state, two fundamental properties of abnormal prion protein (PrPSc), remain significantly unexplored. PrPSc associated with atypical animal and human prion diseases such as Nor or subtypes of Gerstmann-SträusslerScheinker disease did not exhibit a higher proportion of slowly sedimenting species when compared with classic strains [29, 35], arguing that, at least in these atypical prionopathies, the pronounced PK sensitivity of PrPSc is not due to low size aggregates but rather to its tertiary structure or other factors To further address these issues and gain insights into the molecular basis of phenotypic variability and disease pathogenesis in human prion disease, in the present study we have characterized the detergent-insoluble PrPSc fraction in relation to both PK resistance and the aggregation state across the whole spectrum of the currently characterized human prion strains. For each strain or disease subtype we have searched for and quantified the low sedimenting sPrPSc fraction, defined the sedimenting velocity profile of PrPSc, studied the overall resistance of PrPSc to PK digestion, and compared the resistance of PrPSc to proteolysis between strains in fractions with sized aggregates

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION