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

Alejandro M. Sevillano, Natalia Fernández-Borges, Neelam Younas, Fei Wang, Saioa R. Elezgarai, Susana Bravo, Ester Vázquez-Fernández, Isaac Rosa, Hasier Eraña, David Gil, Sonia Veiga, Enric Vidal, Melissa L. Erickson-Beltran, Esteban Guitián, Christopher J. Silva, Romolo Nonno, Jiyan Ma, Joaquín Castilla, Jesús R. Requena

Research output: Contribution to journalArticle

Abstract

Very solid evidence suggests that the core of full length PrPScis a 4-rung β-solenoid, and that individual PrPScsubunits stack to form amyloid fibers. We recently used limited proteolysis to map the β-strands and connecting loops that make up the PrPScsolenoid. 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 recPrPScrefers to bona fide recombinant prions prepared by PMCA, exhibiting infectivity with attack rates of ~100%. Limited proteolysis of mouse and bank vole recPrPScspecies 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 recPrPScpreparations; similar fragments are characteristic of atypical strains of brain-derived PrPSc. Our results suggest a shared architecture of recPrPScand brain PrPScprions. 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 PrPScoffers exciting opportunities for structural studies unachievable with brain-derived PrPSc.

Original languageEnglish
Article numbere1006797
JournalPLoS Pathogens
Volume14
Issue number1
DOIs
Publication statusPublished - Jan 1 2018

ASJC Scopus subject areas

  • Parasitology
  • Microbiology
  • Immunology
  • Molecular Biology
  • Genetics
  • Virology

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    Sevillano, A. M., Fernández-Borges, N., Younas, N., Wang, F., R. Elezgarai, S., Bravo, S., Vázquez-Fernández, E., Rosa, I., Eraña, H., Gil, D., Veiga, S., Vidal, E., Erickson-Beltran, M. L., Guitián, E., Silva, C. J., Nonno, R., Ma, J., Castilla, J., & R. Requena, J. (2018). Recombinant PrPScshares structural features with brain-derived PrPSc: Insights from limited proteolysis. PLoS Pathogens, 14(1), [e1006797]. https://doi.org/10.1371/journal.ppat.1006797