title: Mechanism of scrapie prion precipitation with phosphotungstate anions.
abstract: The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrP(Sc)) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrP(Sc) under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40](3-) ion decomposes to give a lacunary [PW11O39](7-) (PW11) complex and a single orthotungstate anion [WO4](2-) (WO4). To measure the efficacy of each component of PTA, increasing concentrations of PW11, WO4, and mixtures thereof were used to precipitate PrP(Sc) from brain homogenates of scrapie prion-infected mice. The amount of PrP(Sc) isolated, quantified by ELISA and immunoblotting, revealed that both PW11 and WO4 contribute to PrP(Sc) precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrP(Sc) were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrP(Sc) in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrP(Sc) aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrP(Sc). We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrP(Sc) by PTA from tissue and might be optimized further for the detection of prions.
authors.affiliation: †Department of Chemistry, University of California, Berkeley, 211 Lewis Hall, Berkeley, California 94720, United States.|‡Institute for Neurodegenerative Diseases, University of California, San Francisco, 675 Nelson Rising Lane, San Francisco, California 94143, United States.
authors.forename: Dana J
authors.initials: DJ
authors.lastname: Levine
authors.affiliation: ‡Institute for Neurodegenerative Diseases, University of California, San Francisco, 675 Nelson Rising Lane, San Francisco, California 94143, United States.|§Department of Neurology, University of California, San Francisco, 675 Nelson Rising Lane, San Francisco, California 94143, United States.
authors.forename: Jan
authors.initials: J
authors.lastname: Stöhr
authors.affiliation: ‡Institute for Neurodegenerative Diseases, University of California, San Francisco, 675 Nelson Rising Lane, San Francisco, California 94143, United States.
authors.forename: Lillian E
authors.initials: LE
authors.lastname: Falese
authors.affiliation: ‡Institute for Neurodegenerative Diseases, University of California, San Francisco, 675 Nelson Rising Lane, San Francisco, California 94143, United States.
authors.forename: Julian
authors.initials: J
authors.lastname: Ollesch
authors.affiliation: ‡Institute for Neurodegenerative Diseases, University of California, San Francisco, 675 Nelson Rising Lane, San Francisco, California 94143, United States.|§Department of Neurology, University of California, San Francisco, 675 Nelson Rising Lane, San Francisco, California 94143, United States.
authors.forename: Holger
authors.initials: H
authors.lastname: Wille
authors.affiliation: ‡Institute for Neurodegenerative Diseases, University of California, San Francisco, 675 Nelson Rising Lane, San Francisco, California 94143, United States.|§Department of Neurology, University of California, San Francisco, 675 Nelson Rising Lane, San Francisco, California 94143, United States.
authors.forename: Stanley B
authors.initials: SB
authors.lastname: Prusiner
authors.affiliation: †Department of Chemistry, University of California, Berkeley, 211 Lewis Hall, Berkeley, California 94720, United States.
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Long
mesh_terms: D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000473:pathology; D004797:Enzyme-Linked Immunosorbent Assay; D051379:Mice; D010772:Phosphotungstic Acid / Q000737:chemistry*; D011328:Prions / Q000737:chemistry*; D012608:Scrapie / Q000473:pathology*
chemical_list: D011328:Prions; D010772:Phosphotungstic Acid