The most infectious prion protein particles

Abstract

Neurodegenerative diseases such as Alzheimer's, Parkinson's and the transmissible spongiform encephalopathies (TSEs) are characterized by abnormal protein deposits, often with large amyloid fibrils. However, questions have arisen as to whether such fibrils or smaller subfibrillar oligomers are the prime causes of disease. Abnormal deposits in TSEs are rich in PrP(res), a protease-resistant form of the PrP protein with the ability to convert the normal, protease-sensitive form of the protein (PrP(sen)) into PrP(res) (ref. 3). TSEs can be transmitted between organisms by an enigmatic agent (prion) that contains PrP(res) (refs 4 and 5). To evaluate systematically the relationship between infectivity, converting activity and the size of various PrP(res)-containing aggregates, PrP(res) was partially disaggregated, fractionated by size and analysed by light scattering and non-denaturing gel electrophoresis. Our analyses revealed that with respect to PrP content, infectivity and converting activity peaked markedly in 17-27-nm (300-600 kDa) particles, whereas these activities were substantially lower in large fibrils and virtually absent in oligomers of < or =5 PrP molecules. These results suggest that non-fibrillar particles, with masses equivalent to 14-28 PrP molecules, are the most efficient initiators of TSE disease.

Figure 1

Analysis of fractionated PrP^res. a, Quantification of PrP (per 1-ml fractions) by dot immunoblotting with anti-PrP monoclonal antibody 3F4 (n ranged from 3 to 16 from four FlFFF runs), analysis of converting activity by solid-phase conversion assay (n = 9 from four FlFFF runs) and scrapie infectivity analysis by incubation time assays in hamsters (n = 4 animals from one FlFFF run). Mean incubation periods (days) are indicated by red numbers in the plot. b, Calculated specific converting activity for the individual FlFFF run assessed for infectivity (n = 4 converting activity analyses), the consensus of all four FlFFF runs (n = 9 converting activity analyses) and specific infectivity (n = 4 animals). c, In-line light scattering analyses of FlFFF runs indicating M_W (n = 3), r_h (n = 4) and r_g (n=4).The specific infectivity trace from b is superimposed. d, Expansion of a region of the plot shown in c. The solid red line indicates the centre of fraction 12, and the dashed red lines lead to arrowheads, which indicate the mass and radii of particles at this point in the elution. Mean values are shown; error bars represent ±standard error except for incubation time of disease, where they represent ±s.d.

Figure 2

PAGE analyses of detergent-treated PrP^res. a, b, Samples from FlFFF fractions were subjected to PAGE on 3-8% Tris-acetate gels and analysed by either silver stain (a) or immunoblotting with anti-PrP monoclonal antibody 3F4 after transfer to PVDF (b). c, d, Additional samples of purified PrP^res were boiled in SDS-PAGE buffer without sonication, subjected to PAGE on 10% Bis-Tris gels, and analysed in duplicate by either immunoblotting (c) or solid-phase conversion (d). Fraction numbers and types of PrP^res used (PK PrP^res, proteinase K-digested PrP^res) are shown at the top. PrP glycoforms and oligomers are indicated on the left, and molecular weight standards (kDa) are shown on the right.

Figure 3

Transmission electron microscopy analyses of fractionated PrP^res. Fraction numbers are indicated in the upper left of each panel. No particles were visible on control grids loaded with buffer alone. Scale bars, 100 nm.