title: Non-glycosylphosphatidylinositol (GPI)-anchored recombinant prion protein with dominant-negative mutation inhibits PrPSc replication in vitro.
abstract: Dominant-negative mouse prion protein (PrP) with a lysine mutation at codon 218 (Q218K) is known to inhibit prion replication. In order to gain further mechanistic insight into such dominant negative inhibition, non-glycosylphosphatidylinositol (GPI)-anchored recombinant PrP with Q218K (rPrP-Q218K) was investigated. When applied into scrapie-infected mouse neuroblastoma (ScN2a) cells, rPrP-Q218K but not wild-type rPrP (rPrP-WT) exclusively inhibited abnormal protease-resistant pathogenic isoform (PrPSc) replication without reducing the viability of the cells. It was even more efficient than quinacrine, which has already been prescribed for sporadic Creutzfeldt-Jakob disease (CJD) patients; 50% effective concentration (EC50) = 0.20 microM, 99% effective concentration (EC99) = 0.86 microM vs. EC50 = 0.45 microM, EC99 = 1.5 microM. Besides, no apparent cell damage was observed at the concentration of up to 4.3 microM (100 micrograms/ml). In combination treatment with 0.43 microM (10 micrograms/ml) of rPrP-Q218K, EC99 of quinacrine was decreased from 1.5 microM to 0.5 microM, and the cell viability was recovered from 50% to over 90% as inversely proportional to the concentration of quinacrine. Such combination could alleviate the side effects of quinacrine by reducing its effective concentration without changing or even acceleration the inhibition efficacy. Since homogeneous, high-quality rPrPs could be easily prepared from Escherichia coli in large quantities, rPrP-Q218K is a good candidate for a prion replication antagonist.
authors.affiliation: Department of Cortical Function Disorders, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan.
authors.forename: Hitaru
authors.initials: H
authors.lastname: Kishida
authors.forename: Yuji
authors.initials: Y
authors.lastname: Sakasegawa
authors.forename: Kota
authors.initials: K
authors.lastname: Watanabe
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Yamakawa
authors.forename: Masahiro
authors.initials: M
authors.lastname: Nishijima
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Kuroiwa
authors.forename: Naomi S
authors.initials: NS
authors.lastname: Hachiya
authors.forename: Kiyotoshi
authors.initials: K
authors.lastname: Kaneko
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D045744:Cell Line, Tumor; D002470:Cell Survival; D003062:Codon / Q000235:genetics; D007562:Creutzfeldt-Jakob Syndrome / Q000378:metabolism; D005799:Genes, Dominant; D017261:Glycosylphosphatidylinositols / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D018620:PrPSc Proteins / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D020836:Protein Structure, Quaternary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D003062:Codon; D017261:Glycosylphosphatidylinositols; D018620:PrPSc Proteins; D011994:Recombinant Proteins