Generation of structurally distinct ovine prion protein soluble oligomers.
Thomas Haertlé, Human Rezaei, Jeanne Grosclaude

In pathologies due to protein misassembly, low oligomeric states of the misfolded proteins rather than large aggregates play an important biological role. In prion diseases the lethal evolution is associated with formation of PrPSc, a misfolded and amyloid form of the normal cellular prion protein PrP. Sheep is a unique example among mammalian species to present a strong correlation between genotype and prion disease susceptibility phenotype. Indeed a well-defined set of PrP polymorphisms at positions 136, 154 and 171define susceptibility to scrapie, ranging from very high susceptibility for V136-R154-Q171 variant (VRQ) to resistance for A136-R154-R171 variant (ARR). To get better insight into the molecular mechanisms of scrapie susceptibility/ resistance, the unfolding pathways of the different full-length recombinant sheep prion protein variants were analysed by differential scanning calorimetry in a wide range of pH. In the pH range 4.5–6.0, thermal unfolding occurs through a reversible one-step process while at pH 4.5 and 6.0 unfolding intermediates are formed, which are stable in the temperature range 65–80 8C. While these general behaviours are shared by all variants, VRQ and ARQ (susceptibility variants) show higher thermal stability than AHQ and ARR (resistance variants) and the formation of their unfolding intermediates requires higher activation energy than in the case of AHQ and ARR.
The heat-induced oligomerization pathway of the full length recombinant ARQ variant (A136, R154, Q171) at acidic pH gave additional insight into the molecular mechanisms of PrPC oligomerization,. This led to the irreversible formation of two well-identified soluble oligomers. Both oligomers displayed higher beta-sheet content when compared to the monomer. Small angle X-ray scattering allowed the determination of the molecular masses for each oligomer, 12mer and 36mer, as well as their distinct oblate shapes. The two species differed in accessibility of polypeptide chain epitopes and of pepsin-sensitive bonds, in a way suggesting distinct conformations for their monomeric unit. The conversion pathway leading to these novel oligomers, displaying contrasted biochemical reactivities, might be a clue to unravel their biological roles