Abstract
One of the main characteristics of the transmissible isoform of the prion protein (PrPSc) is its partial resistance to proteinase K (PK) digestion. Diagnosis of prion disease typically relies upon immunodetection of PK-digested PrPSc following Western blot or ELISA. More recently, researchers determined that there is a sizeable fraction of PrPSc that is sensitive to PK hydrolysis (sPrPSc). Our group has previously reported a method to isolate this fraction by centrifugation and showed that it has protein misfolding cyclic amplification (PMCA) converting activity. We compared the infectivity of the sPrPSc versus the PK-resistant (rPrPSc) fractions of PrPSc and analyzed the biochemical characteristics of these fractions under conditions of limited proteolysis. Our results show that sPrPSc and rPrPSc fractions have comparable degrees of infectivity and that although they contain different sized multimers, these multimers share similar structural properties. Furthermore, the PK-sensitive fractions of two hamster strains, 263K and Drowsy (Dy), showed strain-dependent differences in the ratios of the sPrPSc to the rPrPSc forms of PrPSc. Although the sPrPSc and rPrPSc fractions have different resistance to PK-digestion, and have previously been shown to sediment differently, and have a different distribution of multimers, they share a common structure and phenotype.
Highlights
The prion (PrPSc) is the infectious agent responsible for a suite of different rare animal and human diseases known as transmissible spongiform encephalopathies (TSEs) [1,2,3,4,5]
We developed methods to isolate the sPrPSc from resistant PrPSc (rPrPSc) fraction of the 263K strain of hamster-adapted scrapie
We investigated first the infectivity of the sPrPSc vs. the proteinase K (PK)-resistant fraction of hamster PrPSc (263K strain). sPrPSc was further characterized by limited proteolysis and mass spectrometry
Summary
The prion (PrPSc) is the infectious agent responsible for a suite of different rare animal and human diseases known as transmissible spongiform encephalopathies (TSEs) [1,2,3,4,5]. The conversion of PrPC into PrPSc involves a conformational change of the protein in which the total amount of b-sheet increases and that of a-helical secondary structure decreases or perhaps disappears [6,7]. PrPSc is a multimer, while PrPC is monomeric [8,9]. These conformational differences are the only demonstrated structural differences between PrPSc and PrPC [10]. No post-translational differences have been found between PrPSc and PrPC: both share one disulfide bond, two or less sugar antennae and a single glycophosphatidylinositol (GPI) anchor [12]. The composition of the sugar antennae and the GPI anchor vary in both PrPSc and PrPC [13]
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