Assembly of natural and recombinant prion protein into fibrils

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Abstract
The conversion of the a-helical, cellular isoform of the prion protein (PrPC) to the insoluble, p-sheet-rich, infectious, disease-causing isoform (PrPSc) is the fundamental event in the prion diseases. The C-terminal fragment of PrPSc (PrP 27-30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrPSc, PrP 27-30 polymerizes into rod-shaped structures with the ultrastructural and tinctorial properties of amyloid. To study the folding of PrP, both with respect to the formation of PrPSc from PrPC and the assembly of rods from PrP 27-30, we solubilized Syrian hamster (sol SHa) PrP 27-30 in low concentrations (0.2%) of sodium dodecyl sulfate (SDS) under conditions previously used to study the structural transitions of this protein. Sol SHaPrP 27-30 adopted a beta-sheet-rich structure at SIDS concentrations between 0.02% and 0.04% and remained soluble. Here we report that NaCl stabilizes SHaPrP 27-30 in a soluble, beta-sheet-rich state that allows fibril assembly to proceed over several weeks. Under these conditions, fibril formation occurred not only with sol PrP 27-30, but also with native SHaPrP(C). Addition of sphingolipids seems to increase fibril growth. When recombinant (rec) SHaPrP(90-231) was exposed to low concentrations of SIDS, similar to those used to polymerize sol SHaPrP 27-30 in the presence of 250 mm NaCl, fibril formation occurred regularly. When fibrils formed from PrP 27-30 or PrPC were bioassayed in transgenic mice overexpressing full-length SHaPrP, no infectivity was obtained, whereas annyloid fibrils formed of rec mouse PrP(89-230) were infectious. At present, it cannot be determined whether the lack of infectivity is caused by a difference in the structure of the fibrils or in the bioassay conditions.