Effect of Glycans and the Glycophosphatidylinositol Anchor on Strain Dependent Conformations of Scrapie Prion Protein: Improved Purifications and Infrared Spectra

Mammalian prion diseases involve conversion of normal prion protein, PrPC, to a pathological aggregated state (PrPres). The three-dimensional structure of PrPres is not known, but infrared (IR) spectroscopy has indicated high, strain-dependent beta-sheet content. PrPres 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 PrPres with GPI- and glycan-deficient PrPres produced in GPI-anchorless PrP transgenic mice. These analyses required the development of substantially improved purification protocols. Spectra of both types of PrPres revealed conformational differences between the 22L, ME7, and Chandler (RML) murine scrapie strains, most notably in bands attributed to beta-sheets. These PrPres spectra were also distinct from those of the hamster 263K scrapie strain. Spectra of wild-type and anchorless 22L PrPres were nearly indistinguishable. With ME7 PrPres, modest differences between the wild-type and anchorless spectra were detected, notably an similar to 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 PrPres, 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 PrPres preparations strongly suggested the presence of strain-dependent, solvent-inaccessible beta-core structures throughout most of the C-terminal half of PrPres molecules, with no remaining a-helix. Our IR data provide evidence that similar core structures also comprise wild-type PrPres.