title: GPI-anchorless human prion protein is secreted and glycosylated but lacks superoxide dismutase activity.
abstract: Prion protein (PrP) is a glycosylphosphatidylinositol (GPI)-anchored membrane protein that is thought to play a role in anti-oxidative stress. It remains controversial whether PrP elicits superoxide dismutase (SOD) activity itself or indirectly by activating cellular SOD. Our previous studies showed that soluble PrP produced by a baculovirus expression system did not exhibit any SOD activity in a marginally glycosylated form. In the present study, we developed a mammalian expression system for a truncated soluble form of human prion protein with the native signal peptide but without a GPI-anchor site, driven by the peptide chain elongation factor 1alpha promoter in stably transfected rabbit-kidney epithelial RK13 cells, to investigate the SOD activity of mammalian PrP. This recombinant product, denoted sPrP, is secreted in large quantities in medium and can be isolated in very high purity and yield (more than 1 mg sPrP per 2 litres medium). Characterization by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tunicamycin treatment revealed that a fully glycosylated form of sPrP was secreted from the cells. SOD activity in cell lysate showed a decrease in sPrP-expressing RK13 cells and an increase in wild-type PrP-expressing RK13 cells compared to empty vector-transfected RK13 cells or parent cells. Purified or immunoprecipitated sPrP did not show any SOD activity. In conclusion, the GPI-anchor site, but not glycosylation, appears to be essential for the secretion of PrP. In addition, mammalian PrP itself does not act as a functional SOD enzyme.
authors.affiliation: Department of Virology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan. sakudo@biken.osaka-u.ac.jp
authors.forename: Akikazu
authors.initials: A
authors.lastname: Sakudo
authors.forename: Izuru
authors.initials: I
authors.lastname: Nakamura
authors.forename: Shotaro
authors.initials: S
authors.lastname: Tsuji
authors.forename: Kazuyoshi
authors.initials: K
authors.lastname: Ikuta
mesh_terms: D000818:Animals; D002460:Cell Line; D003470:Culture Media; D006031:Glycosylation; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006801:Humans; D011328:Prions / Q000302:isolation & purification / Q000378:metabolism*; D021381:Protein Transport; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013482:Superoxide Dismutase / Q000172:deficiency*; D014162:Transfection
chemical_list: D003470:Culture Media; D017261:Glycosylphosphatidylinositols; D011328:Prions; D011994:Recombinant Proteins; D013482:Superoxide Dismutase