Recombinant PrP.sup.Sc shares structural features with brain-derived PrP.sup.Sc: Insights from limited proteolysis

• Published in: PLoS pathogens 2018-01, Vol.14 (1)
• Main Authors: Sevillano, Alejandro M, Fernández-Borges, Natalia, Younas, Neelam, Wang, Fei, R. Elezgarai, Saioa, Bravo, Susana, Vázquez-Fernández, Ester, Rosa, Isaac, Eraña, Hasier, Gil, David, Veiga, Sonia, Vidal, Enric, Erickson-Beltran, Melissa L, Guitián, Esteban, Silva, Christopher J, Nonno, Romolo, Ma, Jiyan, Castilla, Joaquín, R. Requena, Jesús
• Format: Article
• Language: English
• Subjects: Analysis; Brain-derived neurotrophic factor; Care and treatment; Diagnosis; Prion diseases; Proteolysis; Solenoids
• ISSN: 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1006797

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