Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrP(C), into a plethora of assemblies, PrP(Sc), associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrP(Sc) structures. However, the degree to which intra- and inter-strain PrP(Sc) heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrP(Sc) subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrP(Sc) subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrP(Sc) particles. We found that each strain had a continuum of PrP(Sc) assemblies, with strong correlation between PrP(Sc) quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrP(Sc) aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrP(Sc) took place at a hydrodynamic radius (R(h)) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrP(Sc) subpopulations and demonstrate that while PrP(Sc) quaternary structure is a major contributor to PrP(Sc) structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrP(Sc) particles from maintaining proteinase K resistance below an R(h) of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.