Abstract
The scrapie isoform of the prion protein, PrP(Sc), is the only identified component of the infectious prion, an agent causing neurodegenerative diseases such as Creutzfeldt-Jakob disease and bovine spongiform encephalopathy. Following proteolysis, PrP(Sc) is trimmed to a fragment designated PrP 27-30. Both PrP(Sc) and PrP 27-30 molecules tend to aggregate and precipitate as amyloid rods when membranes from prion-infected brain are extracted with detergents. Although prion rods were also shown to contain lipids and sugar polymers, no physiological role has yet been attributed to these molecules. In this work, we show that prion infectivity can be reconstituted by combining Me(2)SO-solubilized PrP 27-30, which at best contained low prion infectivity, with nonprotein components of prion rods (heavy fraction after deproteination, originating from a scrapie-infected hamster brain), which did not present any infectivity. Whereas heparanase digestion of the heavy fraction after deproteination (originating from a scrapie-infected hamster brain), before its combination with solubilized PrP 27-30, considerably reduced the reconstitution of infectivity, preliminary results suggest that infectivity can be greatly increased by combining nonaggregated protease-resistant PrP with heparan sulfate, a known component of amyloid plaques in the brain. We submit that whereas PrP 27-30 is probably the obligatory template for the conversion of PrP(C) to PrP(Sc), sulfated sugar polymers may play an important role in the pathogenesis of prion diseases.
Highlights
PrPSc, the abnormal isoform of PrPC, is the only known component of the prion, an agent causing fatal neurodegenerative disorders such as bovine spongiform encephalopathy and CreutzfeldtJakob disease [1]
We show that prion infectivity can be reconstituted by combining Me2SO-solubilized PrP 27–30, which at best contained low prion infectivity, with nonprotein components of prion rods, which did not present any infectivity
Disruption of prion rods into detergent protein lipid complexes resulted in the retention of their protease resistance property concomitantly with an increase in their prion infectivity, suggesting that solubilized PrPSc is more infectious than the aggregated prion protein [9]
Summary
Sucrose Gradients—Three hundred l of 10, 15, 20, 25, 30, and 60% sucrose in phosphate-buffered saline were loaded into centrifuge tubes adapted for the TLS-55 rotor of the TL 100 ultracentrifuge (Beckman Instruments) to form a zonal gradient. The three pooled bottom fractions (H, heavy) were tested either directly or after a deproteination treatment that included the following: 1) denaturation with 4.5 M guanidium thiocyanate (GndSCN) (final concentration) for 15 min, 2) precipitation with methanol to wash out the GndSCN, 3) resuspension in 2% sarkosyl STE buffer before digestion with PK (100 g/ml for 60 min at 37 °C) to form NPH (non protein heavy) fractions. To form the combined fractions, original samples (L, NPH, or HS (heparan sulfate)) were mixed at equal volumes and incubated for 16 h at 4 °C. All volumes of original samples were adjusted before inoculation to contain the same concentration of L or NPH samples present in the mixtures. Samples containing only L or H fractions were supplemented with 2% sarkosyl to retain similar concentrations of the components in the infectivity assay
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