Proteinase K-sensitive disease-associated ovine prion protein revealed by conformation-dependent immunoassay
Résumé
PrPSc accumulates in prion-affected individuals in the form of amorphous aggregates. Limited proteolysis of PrPSc results in a protease-resistant core of PrPSc of molecular mass of 27-30 kDa (PrP27-30). Aggregated forms of PrP co-purify with prion infectivity, although infectivity does not always correlate with the presence of PrP27-30. This suggests that discrimination between PrPC and PrPSc by proteolysis may underestimate the repertoire and quantity of PrPSc subtypes. We have developed a conformation-dependent immunoassay (CDI) utilizing time-resolved fluorescence to study the conformers of disease-associated PrP in natural cases of sheep scrapie, without using Proteinase K-treatment to discriminate between PrPC and PrPSc. The capture-detector CDI utilizes N-terminal- and C-terminal-specific anti-PrP monoclonal antibodies that recognize regions of the prion protein differentially buried or exposed depending upon the extent of denaturation of the molecule. PrPSc was precipitated from scrapie-infected brain stem and cerebellum tissue following sarkosyl extraction, with or without the use of sodium phosphotungstic acid, and native and denatured PrPSc detected by CDI. PrPSc was detectable in brain tissue from homozygous VRQ and ARQ scrapie-infected sheep brains. The highest levels of PrPSc were found in homozygous VRQ scrapie-infected brains. The quantity of PrPSc was significantly reduced, up to 90% in some cases, when samples were treated with Proteinase K prior to the CDI. Collectively, our data show that the level of PrPSc in brain samples from cases of natural scrapie display genotypic differences and that a significant amount of this material is Proteinase K-sensitive.
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