Conformational variation between allelic variants of cell-surface ovine prion protein

Abstract

The distribution of prion infectivity and PrPSc between peripheral lymphoid tissues suggests their possible haematogenic spread during the progression of natural scrapie in susceptible sheep. Since ovine PBMCs (peripheral blood mononuclear cells) express PrPC, they have the potential to carry or harbour disease-associated forms of PrP. To detect the possible presence of disease-associated PrP on the surface of blood cells, an understanding is required of the conformations that normal ovine cell-surface PrPC may adopt. In the present study, we have used monoclonal antibodies that recognize epitopes in either the N- or C-terminal portions of PrP to probe the conformations of PrPC on ovine PBMCs by flow cytometry. Although PBMCs from scrapie-susceptible and -resistant genotypes of sheep expressed similar levels of cell-surface PrPC, as judged by their reactivity with N-terminal-specific anti-PrP monoclonal antibodies, there was considerable genotypic heterogeneity in the region between helix-1 and residue 171. Cells from PrP-VRQ (V136R154Q171) sheep showed uniform reactivity with monoclonal antibodies that bound to epitopes around helix-1, whereas cells from PrP-ARQ (A136R154Q171) and PrP-ARR (A136R154R171) sheep showed variable binding. The region between b-strand-2 and residue 171, which includes a YYR motif, was buried or obscured in cell-surface PrPC on PBMCs from scrapie-susceptible and -resistant sheep. However, an epitope of PrPC that is influenced by residue 171 was more exposed on PBMCs from PrP-VRQ sheep than on PBMCs from the PrP-ARQ genotype. Our results highlight conformational variation between scrapie-susceptible and -resistant forms of cell-surface PrPC and also between allelic variants of susceptible genotypes.

Molecular and Transmission Characteristics of Primary-Passaged Ovine Scrapie Isolates in Conventional and Ovine PrP Transgenic Mice

“…The anti-PrP monoclonal antibodies used here have been described in detail elsewhere. Monoclonal antibody V24 recognizes an undefined epitope in the C-terminal region of PrP (66 (67) and was purchased from RBiopharm, Darmstadt, Germany. Monoclonal antibodies were biotinylated as described in detail previously (63).…”

Mouse-Adapted Ovine Scrapie Prion Strains Are Characterized by Different Conformers of PrPSc

“…Full-length murine recombinant PrP protein (amino acid residues 23 to 231) was generated as described previously (51). PrP was verified by mass spectroscopy to confirm the correct protein sequence and the presence of a disulfide bond.…”

“…The C-terminus-specific anti-PrP monoclonal antibodies A516 and V24 were generated from Prnp o/o mice immunized with ovine recombinant ARR and VRQ PrP (amino acid residues 23 to 231), respectively (51). Monoclonal antibody 245 was generated from Prnp o/o mice immunized with copper-refolded murine recombinant PrP (amino acid residues 23 to 231) (50).…”

“…Monoclonal antibody 245 was generated from Prnp o/o mice immunized with copper-refolded murine recombinant PrP (amino acid residues 23 to 231) (50). Monoclonal antibody 683 was generated from Prnp o/o mice immunized with a peptide of murine recombinant PrP (amino acid residues 161 to 231) (51). The specificities of the anti-PrP monoclonal antibodies were determined by epitope mapping using a panel of overlapping 15-mer peptides that spanned the entire length of mature ovine PrP.…”

Mechanisms of Cell Death in the Transmissible Spongiform Encephalopathies

“…Amino acid substitutions in the prion protein affect susceptibility of animals to prion disease and mutations in the human PRNP gene (encoding the prion protein) appear to be a direct cause of familial prion diseases. In general, those amino acid substitutions associated with resistance to prion disease in animals appear to decrease the stability of recombinant prion proteins in vitro (Bujdoso et al, 2005, Paludi et al, 2007, Thackray et al, 2004 potentially leading to differing levels of cellular toxicity. By contrast, there is conflicting data on the ability of mutations associated with human familial disease to affect the structure and stability of PrP C (e.g.…”