Molecular Profiling of Ovine Prion Diseases by Using Thermolysin-Resistant PrP ^Sc and Endogenous C2 PrP Fragments

Abstract

Disease-associated PrP fragments produced upon in vitro or in vivo proteolysis can provide significant insight into the causal strain of prion disease. Here we describe a novel molecular strain typing assay that used thermolysin digestion of caudal medulla samples to produce PrP res signatures on Western blots that readily distinguished experimental sheep bovine spongiform encephalopathy (BSE) from classical scrapie. Furthermore, the accumulation of such PrP res species within the cerebellum also appeared to be dependent upon the transmissible spongiform encephalopathy (TSE) strain, allowing discrimination between two experimental strains of scrapie and grouping of natural scrapie isolates into two profiles. The occurrence of endogenously produced PrP fragments, namely, glycosylated and unglycosylated C2, within different central nervous system (CNS) regions is also described; this is the first detailed description of such scrapie-associated fragments within a natural host. The advent of C2 fragments within defined CNS regions, compared between BSE and scrapie cases and also between two experimental scrapie strains, appeared to be largely dependent upon the TSE strain. The combined analyses of C2 fragments and thermolysin-resistant PrP species within caudal medulla, cerebellum, and spinal cord samples allowed natural scrapie isolates to be separated into four distinct molecular profiles: most isolates produced C2 and PrP res in all CNS regions, a second group lacked detectable cerebellar C2 fragments, one isolate lacked both cerebellar PrP res and C2, and a further isolate lacked detectable C2 within all three CNS regions and also lacked cerebellar PrP res . This CNS region-specific deposition of disease-associated PrP species may reflect the natural heterogeneity of scrapie strains in the sheep population in the United Kingdom.

Strain-specific proteolytic processing of the prion protein in prion diseases of ruminants transmitted in ovine transgenic mice

“…1a) that included classical and 'atypical' BSEs (H-type and L-type), as well as transmissible mink encephalopathy (TME) experimentally transmitted to cattle, showed (i) that the PrP pattern in BSE-H inoculated mice and uninfected mice were similar, in relation to the Chandler Baron & Biacabe, 2007C506M3 Baron & Biacabe, 2007Baron et al, , 2008 absence of transmission of H-type BSE in TgOvPrP4 mice , (ii) that the C2 fragment, of~19 kDa, was predominant in BSE-C, BSE-L and TME, whereas C1 was only present in small amounts, or undetectable. These observations matched with previously reported studies of TSE-infected human, murine and ovine tissues or cellculture models (Chen et al, 1995;Jiménez-Huete et al, 1998;Owen et al, 2007b;Yadavalli et al, 2004). The levels of C2 and of PrP res are known to increase during prion infection (Yadavalli et al, 2004).…”

“…In sheep, immunohistochemistry studies indicated that the N-terminal end of PrP d from BSE and from CH1641, compared with other scrapie sources such as SSBP/1, was more strongly digested (Jeffrey et al, 2006). Consistently, Western blot analyses in sheep recently showed that C2 fragments could be detected in SSBP/1, but not in CH1641 and BSE, when an N-terminal (P4) antibody was used (Owen et al, 2007b). However, the novelty in our study is the description of another endogenous, more C-terminally cleaved, PrP fragment of 14 kDa in its unglycosylated form, which we have called CTF14.…”

“…It has been suggested that such an approach might facilitate the molecular discrimination of TSE sources, especially BSE and an unusual form of scrapie known as CH1641 that shares some molecular similarities with BSE (Hope et al, 1999). The accumulation of endogenous prion fragments in the brain of TSE-affected animals or humans, in the absence of in vitro proteolysis, has also been investigated in several studies, by Western blot analysis and also by immunohistochemistry (Chen et al, 1995;Jeffrey et al, 2003Jeffrey et al, , 2006Jiménez-Huete et al, 1998;Owen et al, 2007b;Yadavalli et al, 2004).…”

“…Proteinase K (PK) digestion is generally used for the diagnosis of prion diseases and for molecular discrimination of TSE strains, such as BSE and scrapie, by Western blot analysis (Baron et al, 2008). Another method, based on protease thermolysin (TL), has recently been reported to permit the isolation of a PK-sensitive fraction of PrP d in its full-length form (Cronier et al, 2008;Owen et al, 2007b). It has been suggested that such an approach might facilitate the molecular discrimination of TSE sources, especially BSE and an unusual form of scrapie known as CH1641 that shares some molecular similarities with BSE (Hope et al, 1999).…”

Misfolding of the prion protein: linking biophysical and biological approaches

“…This lack in information is mostly due to the absence of the PrP Sc high resolution 3D structure. Proteolysis fingerprinting performed on extractive PrP Sc reveals that the N-terminal region is completely accessible to proteases [89]. Furthermore, transgenic mice whose Nterminal PrP domain was deleted, were able to replicate the infectious agent [36,102].…”