Overexpression of Nonconvertible PrP(c)Δ114–121 in Scrapie-Infected Mouse Neuroblastoma Cells Leads to trans-Dominant Inhibition of Wild-Type PrP(Sc) Accumulation

One hallmark of prion diseases is the accumulation of the abnormal isoform PrP(Sc) of a normal cellular glycoprotein, PrP(c), which is characterized by a high content of β-sheet structures and by its partial resistance to proteinase K. It was hypothesized that the PrP region comprising amino acid residues 109 to 122 [PrP(109–122)], which spontaneously forms amyloid when it is synthesized as a peptide but which does not display significant secondary structure in the context of the full-length PrP(c) molecule, should play a role in promoting the conversion into PrP(Sc). By using persistently scrapie-infected mouse neuroblastoma (Sc(+)-MNB) cells as a model system for prion replication, we set out to design dominant-negative mutants of PrP(c) that are capable of blocking the conversion of endogenous, wild-type PrP(c) into PrP(Sc). We constructed a deletion mutant (PrP(c)Δ114–121) lacking eight codons that span most of the highly amyloidogenic part, AGAAAAGA, of PrP(109–122). Transient transfections of mammalian expression vectors encoding either wild-type PrP(c) or PrP(c)Δ114–121 into uninfected mouse neuroblastoma cells (Neuro2a) led to overexpression of the respective PrP(c) versions, which proved to be correctly localized on the extracellular face of the plasma membrane. Transfection of Sc(+)-MNB cells revealed that PrP(c)Δ114–121 was not a substrate for conversion into a proteinase K-resistant isoform. Furthermore, its presence led to a significant reduction in the steady-state levels of PrP(Sc) derived from endogenous PrP(c). Thus, we showed that the presence of amino acids 114 to 121 of mouse PrP(c) plays an important role in the conversion process of PrP(c) into PrP(Sc) and that a deletion mutant lacking these codons indeed behaves as a dominant-negative mutant with respect to PrP(Sc) accumulation. This mechanism could form a basis for a new gene therapy and/or a prevention concept for prion diseases.