Effect of Glycans and GPI Anchor on Strain Dependent Conformations of Scrapie Prion Protein: Improved Purifications

Mammalian prion diseases involve conversion of normal prion protein, PrP C , to a pathological aggregated state (PrP res ). The 3D structure of PrP res is not known but infrared (IR) spectroscopy has indicated high, strain-dependent beta sheet content. PrP res molecules usually contain a glycophosphatidylinositol (GPI) anchor and large Asn-linked glycans, which can also vary with strain. Using IR spectroscopy, we tested the conformational effects of these post-translational modifications by comparing wild type PrP res with GPI- and glycan-deficient PrP res produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrP res revealed conformational differences between the 22L, ME7 and Chandler (RML) murine scrapie strains, most notably in bands attributed to beta sheets. These PrP res spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild type and anchorless 22L PrP res were nearly indistinguishable. With ME7 PrP res , modest differences between the wild type and anchorless spectra were detected, notably a ~2-cm −1 shift in an apparent beta sheet band. Collectively, the data provide evidence that the glycans and anchor do not grossly affect the strain-specific secondary structures of PrP res , at least relative to the differences observed between strains, but can subtly affect turns and certain beta sheet components. Recently reported H/D exchange analyses of anchorless PrP res preparations strongly suggested the presence of strain-dependent, solvent-inaccessible beta core structures throughout most of the C-terminal half of PrP res molecules, with no remaining alpha helix. Our present IR data provide evidence that similar core structures also comprise wild type PrP res . The structure of the infectious, pathological form of prion protein (PrP res or PrP Sc ) 1 has been an enduring mystery for decades. During the course of transmissible spongiform encephalopathies (TSEs) or mammalian prion diseases, PrP res is formed from the host’s normal prion protein, PrP sen or PrP C , in a process that involves oligomerization and massive conformational change. PrP C is largely alpha helical and disordered (1-3), while PrP res is an