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

• Published in: PLoS pathogens 2018-01, Vol.14 (1), p.e1006797-e1006797
• 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: Amyloid; Animals; Arvicolinae; Biochemical characteristics; Biochemistry; Biology and Life Sciences; Biomedical research; Brain; Brain - metabolism; Brain architecture; Brain research; Disease; Endopeptidase K; Epitopes; Female; Fragmentation; Fragments; Gel electrophoresis; Infectivity; Laboratories; Mass spectrometry; Mass spectroscopy; Medicine and Health Sciences; Mice; Mice, Transgenic; NMR; Nuclear magnetic resonance; Pathogens; Prion protein; Prions; Prions - chemistry; Prions - metabolism; Protein Structure, Secondary; Proteinase; Proteins; Proteolysis; PrPSc Proteins - chemistry; PrPSc Proteins - metabolism; Recombinant Proteins - chemistry; Research and Analysis Methods; Scientific imaging; Sodium lauryl sulfate; Software; Strands; Supervision
• ISSN: 1553-7374; 1553-7366; 1553-7374
• DOI: 10.1371/journal.ppat.1006797

Very solid evidence suggests that the core of full length PrPSc is a 4-rung β-solenoid, and that individual PrPSc subunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-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 ~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 β-strands, helping us to predict the threading of the β-solenoid. We have now extended this approach to recombinant PrPSc (recPrPSc). The term recPrPSc refers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPSc 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 ~89/97-152, were detected in some recPrPSc preparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPSc 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 recPrPSc. Furthermore, recombinant PrPSc offers exciting opportunities for structural studies unachievable with brain-derived PrPSc.