Isolation and characterization of a proteinase K-sensitive PrPSc fraction

Requena, Jesus R.; Castilla, Joaquin; Pastrana, Miguel A.; Morales, Rodrigo; Onisko, Bruce; Soto, Claudio; Sajnani, Gustavo

Abstract

Recent studies have shown that a sizable fraction of PrPSc present in prion-infected tissues is, contrary to previous conceptions, sensitive to digestion by proteinase K (PK). This finding has important implications in the context of diagnosis of prion disease, as PK has been extensively used in attempts to distinguish between PrPSc and PrPC. Even more importantly, PK-sensitive PrPSc (sPrP(Sc)) might be essential to understand the process of conversion and aggregation of PrPC leading to infectivity. We have isolated a fraction of sPrP(Sc). This material was obtained by differential centrifugation at an intermediate speed of Syrian hamster PrPSc obtained through a conventional procedure based on ultracentrifugation in the presence of detergents. PK-sensitive PrPSc is completely degraded under standard conditions (50 mu g/mL of proteinase K at 37 degrees C for 1 h) and can also be digested with trypsin. Centrifugation in a sucrose gradient showed sPrP(Sc) to correspond to the lower molecular weight fractions of the continuous range of oligomers that constitute PrPSc. PK-sensitive PrPSc has the ability to convert PrPC into protease-resistant PrPSc, as assessed by the protein misfolding cyclic amplification assay (PMCA). Limited proteolysis of sPrP(Sc) using trypsin allows for identification of regions that are particularly susceptible to digestion, i.e., are partially exposed and flexible; we have identified as such the regions around residues K110, R136, R151, K220, and R229. PK-sensitive PrPSc isolates should prove useful for structural studies to help understand fundamental issues of the molecular biology of PrPSc and in the quest to design tests to detect preclinical prion disease.

Más información

Título según WOS: ID WOS:000242935600059 Not found in local WOS DB
Título de la Revista: BIOCHEMISTRY
Volumen: 45
Número: 51
Editorial: AMER CHEMICAL SOC
Fecha de publicación: 2006
Página de inicio: 15710
Página final: 15717
DOI:

10.1021/bi0615442

Notas: ISI