Expression of unglycosylated mutated prion protein facilitates PrP(Sc) formation in neuroblastoma cells infected with different prion strains.
Prion replication involves conversion of the normal, host-encoded prion protein PrP(C), which is a sialoglycoprotein bound to the plasma membrane by a glycophosphatidylinositol anchor, into a pathogenic isoform, PrP(Sc). In earlier studies, tunicamycin prevented glycosylation of PrP(C) in scrapie-infected mouse neuroblastoma (ScN2a) cells but it was still expressed on the cell surface and converted into PrP(Sc); mutation of PrP(C) at glycosylation consensus sites (T182A, T198A) produced low steady-state levels of PrP that were insufficient to propagate prions in transgenic mice. By mutating asparagines to glutamines at the consensus sites, we obtained expression of unglycosylated, epitope-tagged MHM2PrP(N180Q,N196Q), which was converted into PrP(Sc) in ScN2a cells. Cultures of uninfected neuroblastoma (N2a) cells transiently expressing mutated PrP were exposed to brain homogenates prepared from mice infected with the RML, Me7 or 301V prion strains. In each case, mutated PrP was converted into PrP(Sc) as judged by Western blotting. These findings raise the possibility that the N2a cell line can support replication of different strains of prions.
Korth
Carsten
C
Institute for Neurodegenerative Diseases1 and Departments of Neurology2 and Biochemistry and Biophysics3, Box 0518, University of California, San Francisco, CA 94143-0518, USA.
Kaneko
Kiyotoshi
K
Institute for Neurodegenerative Diseases1 and Departments of Neurology2 and Biochemistry and Biophysics3, Box 0518, University of California, San Francisco, CA 94143-0518, USA.
Prusiner
Stanley B
SB
Institute for Neurodegenerative Diseases1 and Departments of Neurology2 and Biochemistry and Biophysics3, Box 0518, University of California, San Francisco, CA 94143-0518, USA.
D019943:Amino Acid Substitution; D000818:Animals; D016384:Consensus Sequence; D006031:Glycosylation; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009447:Neuroblastoma / Q000378:metabolism / Q000821:virology*; D018620:PrPSc Proteins / Q000096:biosynthesis* / Q000235:genetics; D011328:Prions / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology*; D021281:Quantitative Structure-Activity Relationship; D014162:Transfection; D014407:Tumor Cells, Cultured; D014779:Virus Replication
D018620:PrPSc Proteins; D011328:Prions