Evaluation of Infective Property of Recombinant Prion Protein Amyloids in Cultured Cells Overexpressing Cellular Prion Protein

Misfolded isoform of prion protein (PrP), termed scrapie PrP (PrP(Sc)), tends to aggregate into various fibril forms. Previously, we reported various conditions that affect aggregation of recombinant PrP into amyloids. Because amyloidogenesis of PrP is closely associated with transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, we investigated infectivity of recombinant PrP amyloids generated in vitro. Using cultured cell lines which overexpress cellular PrP of different species, we measured the level of de novo synthesized PrP(Sc) in cells inoculated with recombinant mouse PrP amyloids. While PrP-overexpressing cells were susceptible to mouse-adapted scrapie prions used as the positive control, demonstrating the species barrier effect, infection with amyloids made of truncated recombinant PrP (PrP[89-230]) failed to form and propagate PrP(Sc) even in the cells that express mouse cellular PrP. This suggests that infectivity of PrP amyloids generated in vitro is different from that of natural prions. Recombinant PrP (89-230) amyloids tested in the current study retain no or a minute level, if any, of prion infectivity.

Characterizing affinity epitopes between prion protein and beta-amyloid using an epitope mapping immunoassay

Cellular prion protein, a membrane protein, is expressed in all mammals. Prion protein is also found in human blood as an anchorless protein, and this protein form is one of the many potential sources of misfolded prion protein replication during transmission. Many studies have suggested that beta-amyloid1-42 oligomer causes neurotoxicity associated with Alzheimer's disease, which is mediated by the prion protein that acts as a receptor and regulates the hippocampal potentiation. The prevention of the binding of these proteins has been proposed as a possible preventative treatment for Alzheimer's disease; therefore, a greater understanding of the binding hot-spots between the two molecules is necessary. In this study, the epitope mapping immunoassay was employed to characterize binding epitopes within the prion protein and complementary epitopes in beta-amyloid. Residues 23-39 and 93-119 in the prion protein were involved in binding to beta-amyloid1-40 and 1-42, and monomers of this protein interacted with prion protein residues 93-113 and 123-166. Furthermore, beta-amyloid antibodies against the C-terminus detected bound beta-amyloid1-42 at residues 23-40, 104-122 and 159-175. beta-Amyloid epitopes necessary for the interaction with prion protein were not determined. In conclusion, charged clusters and hydrophobic regions of the prion protein were involved in binding to beta-amyloid1-40 and 1-42. The 3D structure appears to be necessary for beta-amyloid to interact with prion protein. In the future, these binding sites may be utilized for 3D structure modeling, as well as for the pharmaceutical intervention of Alzheimer's disease.