Recombinant human prion protein mutants huPrP D178N/M129 (FFI) and huPrP+9OR (fCJD) reveal proteinase K resistance

Abstract

The Semliki-Forest virus (SFV) system was used to overexpress human wild-type and mutant prion proteins as well as FLAG-tagged human and bovine PrP in mammalian cells. The application of recombinant SFV vectors allowed a high-level production of highly glycosylated prion proteins with a molecular weight ranging from 25 to 30 kDa for recombinant wild-type human PrP and from 26 to 32 kDa for wild-type bovine PrP. Further, we report here the generation of recombinant mutant prion proteins that are associated with inherited human prion diseases such as fatal familial insomnia (FFI) and Creutzfeldt-Jakob disease (CJD). Both mutated variants, the FFI-associated PrP carrying a mutation at amino acid position 178 and the CJD-linked form containing an insertion of nine additional octarepeats reveal proteinase K resistance, one of the typical biochemical properties of the infectious scrapie isoform of the prion protein. By contrast, recombinant wild-type PrP was completely proteinase K sensitive when expressed in SFV-transfected BHK cells. The subcellular location of both PrP mutants at the cell surface and in intracellular compartments of transfected BHK cells was similar to that of wild-type PrP. In order to purify recombinant human and bovine PrP from cell lysates, a FLAG-tag was introduced either at the N-terminus behind the signal peptide or at the C-terminus close to the adhesion site of the GPI anchor. N-terminal insertion did not extensively influence the trafficking of the FLAG-tagged protein to the cell surface, whereas insertion close to the GPI attachment site clearly affected the transport of the majority of PrP to the cell membrane, probably resulting in their retention within the secretory pathway. All FLAG-tagged prion proteins were expressed efficiently in BHK cells and showed a typical glycosylation pattern, allowing their rapid and simple purification via anti-FLAG antibody chromatography.

The 37‐kDa/67‐kDa Laminin Receptor Acts as a Receptor for Infectious Prions and Is Inhibited by Polysulfated Glycanes

“…Transcriptions were performed as described elsewhere [28]. The correct length of the transcripts was verified by agarose gel electrophoresis.…”

Prion Protein Promotes Growth Cone Development through Reggie/Flotillin-Dependent N-Cadherin Trafficking

“…Many different functions have, in fact, been attributed to PrP (Linden et al, 2008), and increasingly many PrP interacting proteins are being identified with positive and negative effects on axon growth (Gauczynski et al, 2002;Zanata et al, 2002;Santuccione et al, 2005;Parkyn et al, 2008;Devanathan et al, 2010), myelination (Bremer et al, 2010), synapse formation (Collinge et al, 1994), and long-termpotentiation (LTP) (Khosravani et al, 2008) (for review, see Linden et al, 2008). Connected to these functions are signal transduction pathways that PrP seems to trigger in cooperation with cis-interaction partners and/or lipid rafts (Simons and Ehehalt, 2002).…”

Glycosylation modification of human prion protein provokes apoptosis in HeLa cells in vitro

“…For example, one of the HuPrP mutants, HuPrP E200K h is closely related to familial Creutzfeldt-Jakob disease (fCJD) (15). A valine to isoleucine mutation at residue 180 was detected in a French patient with Creutzfeldt-Jakob disease (CJD) (16).…”