Biochemical and Biophysical Research Communications
Prion proteins from susceptible and resistant sheep exhibit some distinct cell biological features
Section snippets
Experimental procedures
Reagents. Protein-A–Sepharose was from Pharmacia Biotech and PNGase F was purchased from New England BioLabs.
Antibodies. The monoclonal antibodies against PrP were 4F2 [13], ICSM 18 [14]. MH48 is a rabbit polyclonal serum raised against recombinant ovine PrP [15]. Monoclonal antibody CTR433 [16] was used to stain the Golgi apparatus.
Scrapie strain. The isolate originally used to infect Rov cells derives from a naturally scrapie-affected sheep. This isolate was serially propagated and
An improved cell model to study the cell biological properties of the VRQPrP and ARRPrP variants
To minimize as much as possible cell clone-specific versus allele-specific differences, we exploited a recently designed regulatory expression system to express both PrP variants in the same Rov clone. This “dual” regulatory system is based on tetracycline-controlled transactivators that reversibly regulate the activity of two transfected genes in a mutually exclusive manner [19]. In such Rov clones, VRQPrP is expressed in the absence of dox. Addition of 1 μg/ml dox in the cell culture medium
Discussion
Much experimental data indicate that PrP conversion and prion multiplication are complex biological processes that require the coordinated involvement of several cellular and molecular factors [9]. Possible distinct cell biological properties of two PrP polymorphic variants may therefore contribute to the differential capacity of the protein to support prion multiplication. In this study, the cell biology of two ovine PrP variants (ARRPrP and VRQPrP) associated with sheep scrapie susceptibility
Acknowledgments
We thank S. Hawke and J. Collinge (Imperial College, London, United Kingdom) and M. Bornens (Institut Curie, Paris, France) for ICSM18 and CTR433 mAbs, respectively. We acknowledge O. Andreoletti and SAGA for providing us with PrP-genotyped sheep, V. Setola and V. Beringue for critical reading of the manuscript, and R. Morris for helpful discussions. E.S. was partly supported by a fellowship from INRA.
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The PrP<sup>C</sup> C1 fragment derived from the ovine A<inf>136</inf>R<inf>154</inf>R<inf>171</inf> PRNP allele is highly abundant in sheep brain and inhibits fibrillisation of full-length PrP<sup>C</sup> protein in vitro
2013, Biochimica et Biophysica Acta - Molecular Basis of DiseaseCitation Excerpt :The C1 fragment is produced by the action of an unknown ‘α-secretase’ and high C1 levels in ARR/ARR sheep may result from an increased proteolytic processing of PrPARR compared to PrPARQ or PrPVRQ or due to differences in the half-lives of the allotypic C1 fragments. The former may be explained by conformational differences leading to increased presentation of the cleavage site for the α-secretase or by the extended presence of PrPARR within the Golgi apparatus increasing the chance of interacting with the α-secretase [49]. Setting the reason for the differences aside, the correlation between a high C1 level and scrapie resistance is supported by similar evidence from cell culture assays and transgenic mouse models.
Glycan chains modulate prion protein binding to immobilized metal ions
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These authors contributed equally to the work.