title: Isolation and characterization of a proteinase K-sensitive PrPSc fraction.
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 (sPrPSc) might be essential to understand the process of conversion and aggregation of PrPC leading to infectivity. We have isolated a fraction of sPrPSc. 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 mug/mL of proteinase K at 37 degrees C for 1 h) and can also be digested with trypsin. Centrifugation in a sucrose gradient showed sPrPSc 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 sPrPSc 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.
authors.affiliation: Prion Research Unit, Department of Medicine, School of Medicine, University of Santiago de Compostela, Rue de S. Francisco s/n, Santiago de Compostela, Galiza, Spain 15782.
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Pastrana
authors.forename: Gustavo
authors.initials: G
authors.lastname: Sajnani
authors.forename: Bruce
authors.initials: B
authors.lastname: Onisko
authors.forename: Joaquín
authors.initials: J
authors.lastname: Castilla
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Morales
authors.forename: Claudio
authors.initials: C
authors.lastname: Soto
authors.forename: Jesús R
authors.initials: JR
authors.lastname: Requena
mesh_terms: D000818:Animals; D001923:Brain Chemistry; D002499:Centrifugation, Density Gradient; D005591:Chemical Fractionation; D006224:Cricetinae; D019286:Endopeptidase K / Q000378:metabolism / Q000494:pharmacology*; D006868:Hydrolysis; D008647:Mesocricetus; D018620:PrPSc Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D011328:Prions / Q000378:metabolism; D011489:Protein Denaturation; D012608:Scrapie / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
chemical_list: D018620:PrPSc Proteins; D011328:Prions; D014357:Trypsin; D019286:Endopeptidase K