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Molecular mass, biochemical composition, and physicochemical behavior of the infectious form of the scrapie precursor
1Laboratory of Central Nervous System Studies, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892.
Abstract:
A highly purified fraction obtained from scrapie (263-K strain)-infected hamsters' brains by an alternative procedure without proteinase K treatment contained a protease-resistant form of the scrapie precursor protein (PrPSc) and infectivity of 9.9 +/- 0.7 log LD50/ml. Polyclonal antibodies produced against hamster scrapie amyloid protein (PrP27-30) and used in a neutralization test diminished infectivity of the PrPSc preparations by 1.6 log after intracerebral inoculation and by 1 log after intraperitoneal inoculation. PrPSc was subjected to size-exclusion HPLC; greater than or equal to 60% of the eluted infectious units were recovered from the peak with an apparent mass of 30.4 +/- 0.6 kDa. Characterization by UV absorption spectra, SDS/PAGE, immunoblots, N-terminal amino acid sequence, and neutral sugar and amino sugar analyses demonstrated homogeneity of the infectious units. The neutral sugar and amino sugar compositional analyses revealed high mannose, glucosamine, fucose, and sialic acid content. This demonstrated an extensive posttranslational modification by the complex type of N-linked glycosylation and glycane core of C-terminal glycolipid of PrPSc. The results correspond to the predicted size, composition, and sequence of PrPSc and indicate that this protein may be the only component of scrapie infectious unit or the infectious form of scrapie precursor.
Insights
Researchers purified scrapie infectious units, identifying a protease-resistant form of the scrapie precursor protein (PrPSc). Antibodies reduced infectivity, and characterization revealed PrPSc
Area of Science:
- Neuroscience
- Biochemistry
- Infectious Diseases
Background:
- Scrapie is a fatal neurodegenerative disease caused by prions.
- The scrapie precursor protein (PrPSc) is central to prion infectivity.
- Previous purification methods often involved proteinase K, potentially altering PrPSc.
Purpose of the Study:
- To isolate and characterize the infectious agent of scrapie without proteinase K treatment.
- To determine the role of PrPSc in scrapie infectivity.
- To investigate the biochemical properties of the infectious PrPSc.
Main Methods:
- Purification of scrapie-infected hamster brain fractions using an alternative procedure.
- Antibody-mediated neutralization assays to assess infectivity reduction.
- Size-exclusion High-Performance Liquid Chromatography (HPLC) for mass determination.
- Biochemical analyses including SDS/PAGE, immunoblots, N-terminal sequencing, and sugar composition analysis.
Main Results:
- A highly purified fraction containing protease-resistant PrPSc and high infectivity (9.9 log LD50/ml) was obtained.
- Antibodies against PrP27-30 significantly reduced scrapie infectivity.
- HPLC analysis identified infectious units with an apparent mass of ~30.4 kDa.
- Detailed compositional analysis revealed extensive N-linked glycosylation and a C-terminal glycolipid.
Conclusions:
- The purified PrPSc fraction contains the scrapie infectious agent.
- PrPSc is likely the sole component of the scrapie infectious unit.
- Posttranslational modifications, including glycosylation, are significant features of infectious PrPSc.