Abstract
Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrPSc, a disease-associated isoform of the host-encoded cellular protein (PrPC). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrPSc. However, PrPSc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrPSc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrPC and PrPSc by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrPSc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl]1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl]1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrPSc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrPSc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrPSc conformational stabilities of protease-resistant and protease-sensitive PrPSc and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.
Highlights
Transmissible spongiform encephalopathies (TSEs), or prion diseases, are neurodegenerative disorders that afflict humans and others mammals
It has been previously reported that normal PrP is composed of full-length PrP (FL-PrP) as well as of 2 C-terminal fragments derived from physiological cleavage at the a and b sites: C1, which is the most represented, derives from a PrP cleavage at position 111/112, while C2 is usually barely detectable and is cleaved around the octarepeat region [27]
We have shown that voles infected with MM1/MV1 sCJD and E200K gCJD isolates were characterised by a PrPres fragment of,19 kDa, MM2 sCJD showed a PrPres fragment of,17 kDa, while natural scrapie isolates and murine scrapie ME7 were characterised by a PrPres fragment of,18 kDa, intermediate between types 1 and 2 Creutzfeldt-Jakob disease (CJD)
Summary
Transmissible spongiform encephalopathies (TSEs), or prion diseases, are neurodegenerative disorders that afflict humans and others mammals. Creutzfeldt-Jakob disease (CJD) is the most common TSE in humans and may be sporadic (sCJD), genetic (gCJD), or acquired (iatrogenic CJD). A novel human acquired prion disease, variant CJD (vCJD), appeared from 1995 onwards and was postulated to be caused by consumption of beef from cows infected with bovine spongiform encephalopathy (BSE). The most common forms of TSE in animals, scrapie in small ruminants, BSE in cattle and chronic wasting disease (CWD) in deer, are all acquired. New atypical forms of BSE in cattle, namely BSE-H [1] and BSE-L or BASE [2] and atypical scrapie in small ruminants, namely Nor98 [3], are supposed to be sporadic
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