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

Abstract

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.