Assembly of natural and recombinant prion protein into fibrils

Abstract

The conversion of the alpha-helical, cellular isoform of the prion protein (PrP C ) to the insoluble, beta-sheet-rich, infectious, disease-causing isoform (PrP Sc ) is the fundamental event in the prion diseases. The C-terminal fragment of PrP Sc (PrP 27-30) is formed by limited proteolysis and retains infectivity. Unlike full-length PrP Sc , PrP 27-30 polymerizes into rod-shaped structures with the ultra-structural and tinctorial properties of amyloid. To study the folding of PrP, both with respect to the formation of PrP Sc from PrP C 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 SDS 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 SDS, 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 PrP C were bioassayed in transgenic mice overexpressing full-length SHaPrP, no infectivity was obtained, whereas amyloid 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.

Mechanisms of prion protein assembly into amyloid

“…In the absence of PrP Sc seeds, 80 ng/ l recPrP incubated with 0.03% SDS and 250 mM NaCl formed fibrils within 35 days, as described in ref. 19. Increasing the recPrP concentration to 300 ng/ l accelerated the formation of amyloid fibrils: Fibrils formed after 7 days of incubation.…”

“…Previously, we described an in vitro conversion system, in which different conformational states of recPrP could be established by varying the concentration of SDS (18). In a follow-up study, this system was optimized to produce amyloid fibrils from recombinant and natural PrP (19). Low concentrations of SDS (0.02-0.03%), 250 mM NaCl, and a neutral buffer are essential for fibril formation.…”

“…We established conditions for amyloid formation [low concentration of SDS and 250 mM NaCl (19)]; recPrP in this so-called preamyloid state is soluble for at least 7 days before forming amyloid (Fig. 1).…”

“…The conditions were derived from our earlier studies of the conformational transitions of recPrP induced by varying the SDS concentration but in the absence of NaCl (19,23). A very similar SDS-conversion system was also used successfully by other groups (24).…”

Prion detection by an amyloid seeding assay

“…In prion diseases, amyloid deposition has been observed in both animals and humans (4-6) but is a nonobligatory feature of the disease (7,8). Recombinant prion proteins (recPrP) with sequences derived from various species and composed of full-length and truncated segments of the protein have been shown to form amyloid fibers (9)(10)(11).…”