Abstract
Aggregated prion protein (PrPSc), which is detergent-insoluble and partially proteinase K (PK)-resistant, constitutes the major component of infectious prions that cause a group of transmissible spongiform encephalopathies in animals and humans. PrPSc derives from a detergent-soluble and PK-sensitive cellular prion protein (PrPC) through an alpha-helix to beta-sheet transition. This transition confers on the PrPSc molecule unique physicochemical and biological properties, including insolubility in nondenaturing detergents, an enhanced tendency to form aggregates, resistance to PK digestion, and infectivity, which together are regarded as the basis for distinguishing PrPSc from PrPC. Here we demonstrate, using sedimentation and size exclusion chromatography, that small amounts of detergent-insoluble PrP aggregates are present in uninfected human brains. Moreover, PK-resistant PrP core fragments are detectable following PK treatment. This is the first study that provides experimental evidence supporting the hypothesis that there might be silent prions lying dormant in normal human brains.
Highlights
Sensitive to proteinase K (PK) digestion, PrPSc forms detergent-insoluble aggregates and is partially resistant to PK [3,4,5,6,7]
One is the presence of a small amount of endogenous PrPSc or PrP* in the uninfected brain and the second is the formation of PrPSc-derived oligomers
Detergent-insoluble PrP Species Are Present in Uninfected Human Brains—It has been shown that PrPC is recovered in a soluble fraction (S2), following ultracentrifugation in nondenaturing detergents at 100,000 ϫ g for 1 h at 4 °C, whereas PrPSc is recovered in an insoluble fraction (P2) [6, 7, 23]
Summary
Reagents and Antibodies—NaPTA, PK, and phenylmethylsulfonyl fluoride were purchased from Sigma. The molecular weight (Mr) of the various PrP species recovered in different fast protein liquid chromatography fractions was evaluated according to a calibration curve generated with the gel filtration of molecular mass markers (Sigma) including dextran blue (2,000 kDa), thyroglobulin (669 kDa), apoferittin (443 kDa), -amylase (200 kDa), alcohol dehydrogenase (150 kDa), albumin (66 kDa), and carbonic anhydrase (29 kDa). These standards were loaded independently at the concentrations recommended by Sigma in 200-l sample volumes. Following incubation with horseradish peroxidase-conjugated sheep anti-mouse IgG or donkey anti-rabbit IgG at 1:3,000, the PrP or caveolin-1 bands or spots were visualized on Kodak film by ECL Plus as described by the manufacturer
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