Recombinant PrPSc shares structural features with brain-derived PrPSc: Insights from limited proteolysis.

Very solid evidence suggests that the core of full length PrPSc is a 4-rung beta-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the beta-strands and connecting loops that make up the PrPSc solenoid. Using high resolution SDS-PAGE followed by epitope analysis, and mass spectrometry, we identified positions similar to 116/118, 133-134, 141, 152-153, 162, 169 and 179 (murine numbering) as Proteinase K (PK) cleavage sites in PrPSc. Such sites likely define loops and/or borders of beta-strands, helping us to predict the threading of the beta-solenoid. We have now extended this approach to recombinant PrPSc (recPrP(Sc)). The term recPrP(Sc) refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of similar to 100%. Limited proteolysis of mouse and bank vole recPrP(Sc) species yielded N-terminally truncated PK-resistant fragments similar to those seen in brain-derived PrPSc, albeit with varying relative yields. Along with these fragments, doubly N- and C-terminally truncated fragments, in particular similar to 89/97-152, were detected in some recPrP(Sc) preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrP(Sc) and brain PrPSc prions. The observed differences, in particular the distinct yields of specific PK-resistant fragments, are likely due to differences in threading which result in the specific biochemical characteristics of recPrP(Sc). Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.