Glycan-controlled epitopes of prion protein include a major determinant of susceptibility to sheep scrapie.

A key feature of prion encephalopathies is the accumulation of a misfolded form of the host glycoprotein PrP. Cell-free and cell culture studies have shown that the efficiency of conversion of PrP into the disease-associated form is influenced by its amino acid sequence and also by its carbohydrate moiety. Here, we characterize four novel glycoform-dependent monoclonal antibodies raised against prokaryotic recombinant sheep PrP. We demonstrate that these antibodies discriminate the PrP monoglycosylated species, since two of them recognize molecules that have the first Asn glycosylation site occupied (mono1) while the other two recognize molecules glycosylated at the second site (mono2). Remarkably, the recognition of PrP by the anti-mono2 antibodies was strongly influenced by the amino acid present at position 171, i.e., either Gln or Arg. This polymorphism is known to be the main determinant of susceptibility and resistance to scrapie in sheep. Altogether, our findings lead us to propose that each glycan chain controls the accessibility of PrP determinants located close upstream from their attachment site. The monoglycoform-assigned and the allotype-restricted antibodies described here, the first to date, should provide further opportunities to investigate the involvement of each glycan chain in PrP conversion in relation to prion strain diversity and the basis of the resistance conferred by the Arg-171 amino acid.

Insight into the PrPC-->PrPSc conversion from the structures of antibody-bound ovine prion scrapie-susceptibility variants.

Prion diseases are associated with the conversion of the alpha-helix rich prion protein (PrPC) into a beta-structure-rich insoluble conformer (PrPSc) that is thought to be infectious. The mechanism for the PrPC-->PrPSc conversion and its relationship with the pathological effects of prion diseases are poorly understood, partly because of our limited knowledge of the structure of PrPSc. In particular, the way in which mutations in the PRNP gene yield variants that confer different susceptibilities to disease needs to be clarified. We report here the 2.5-A-resolution crystal structures of three scrapie-susceptibility ovine PrP variants complexed with an antibody that binds to PrPC and to PrPSc; they identify two important features of the PrPC-->PrPSc conversion. First, the epitope of the antibody mainly consists of the last two turns of ovine PrP second alpha-helix. We show that this is a structural invariant in the PrPC-->PrPSc conversion; taken together with biochemical data, this leads to a model of the conformational change in which the two PrPC C-terminal alpha-helices are conserved in PrPSc, whereas secondary structure changes are located in the N-terminal alpha-helix. Second, comparison of the structures of scrapie-sensitivity variants defines local changes in distant parts of the protein that account for the observed differences of PrPC stability, resistant variants being destabilized compared with sensitive ones. Additive contributions of these sensitivity-modulating mutations to resistance suggest a possible causal relationship between scrapie resistance and lowered stability of the PrP protein.

Amyloidogenic unfolding intermediates differentiate sheep prion protein variants.

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 171 (sheep numbering) govern scrapie susceptibility, 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 degrees C. 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. Furthermore, secondary structures of the unfolding intermediates differentiate variants: ARR unfolding intermediate exhibits random coil structure, contrasting with the beta-sheet structure of VRQ and ARQ unfolding intermediates. The rate of the unfolding intermediate formation allows us to classify genetic variants along increasing scrapie susceptibility at pH 4.0, VRQ and ARQ rates being the highest. Rather poor correlation is observed at pH 7.2. Upon cooling, these intermediates refold into stable species, which are rich in beta-type secondary structures and, as revealed by thioflavin T fluorescence and electron microscopy, share amyloid characteristics. These results highlight the prion protein plasticity genetically modulated in sheep, and might provide a molecular basis for sheep predisposition to scrapie taking into account both thermodynamic stability and transconformation rate of prion protein.