title: In vitro conversion and seeded fibrillization of posttranslationally modified prion protein.
abstract: The conversion of the cellular isoform of the prion protein (PrP(C)) into the pathologic isoform (PrP(Sc)) is the key event in prion diseases. To study the conversion process, an in vitro system based on varying the concentration of low amounts of sodium dodecyl sulfate (SDS) has been employed. In the present study, the conversion of full-length PrP(C) isolated from Chinese hamster ovary cells (CHO-PrP(C)) was examined. CHO-PrP(C) harbors native, posttranslational modifications, including the GPI anchor and two N-linked glyco-sylation sites. The properties of CHO-PrP(C) were compared with those of full-length and N-terminally truncated recombinant PrP. As shown earlier with recombinant PrP (recPrP90-231), transition from a soluble α-helical state as known for native PrP(C) into an aggregated, β-sheet-rich PrP(Sc)-like state could be induced by dilution of SDS. The aggregated state is partially proteinase K (PK)-resistant, exhibiting a cleavage site similar to that found with PrP(Sc). Compared to recPrP (90-231), fibril formation with CHO-PrP(C) requires lower SDS concentrations (0.0075%), and can be drastically accelerated by seeding with PrP(Sc) purified from brain homogenates of terminally sick hamsters. Our results show that recPrP 90-231 and CHO-PrPC behave qualitatively similar but quantitatively different. The in vivo situation can be simulated closer with CHO-PrP(C) because the specific PK cleave site could be shown and the seed-assisted fibrillization was much more efficient.
authors.affiliation: Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Germany.
authors.forename: Jan
authors.initials: J
authors.lastname: Stöhr
authors.forename: Kerstin
authors.initials: K
authors.lastname: Elfrink
authors.forename: Nicole
authors.initials: N
authors.lastname: Weinmann
authors.forename: Holger
authors.initials: H
authors.lastname: Wille
authors.forename: Dieter
authors.initials: D
authors.lastname: Willbold
authors.forename: Eva
authors.initials: E
authors.lastname: Birkmann
authors.forename: Detlev
authors.initials: D
authors.lastname: Riesner
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002942:Circular Dichroism; D006224:Cricetinae; D003412:Cricetulus; D004591:Electrophoresis, Polyacrylamide Gel; D008854:Microscopy, Electron; D018621:PrPC Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D018620:PrPSc Proteins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
chemical_list: D018621:PrPC Proteins; D018620:PrPSc Proteins; D012967:Sodium Dodecyl Sulfate