Identification of a homology-independent linchpin domain controlling mouse and bank vole prion protein conversion

Abstract

Prions are unorthodox pathogens that cause fatal neurodegenerative diseases in humans and other mammals. Prion propagation occurs through the self-templating of the pathogenic conformer PrPSc, onto the cell-expressed conformer, PrPC. Here we study the conversion of PrPC to PrPSc using a recombinant mouse PrPSc conformer (mouse protein-only recPrPSc) as a unique tool that can convert bank vole but not mouse PrPC substrates in vitro. Thus, its templating ability is not dependent on sequence homology with the substrate. In the present study, we used chimeric bank vole/mouse PrPC substrates to systematically determine the domain that allows for conversion by Mo protein-only recPrPSc. Our results show that that either the presence of the bank vole amino acid residues E227 and S230 or the absence of the second N-linked glycan are sufficient to allow PrPC substrates to be converted by Mo protein-only recPrPSc and several native infectious prion strains. We propose that residues 227 and 230 and the second glycan are part of a C-terminal domain that acts as a linchpin for bank vole and mouse prion conversion.

Fig 1. Susceptibility of various rodent species in BH sPMCA.

(A) Amino acid comparison of the processed regions of Mo PrP, Ha PrP, and BV PrP. The sequence bar highlights the regions of BV PrP homologous to Mo PrP (orange) or Ha PrP (green), or are unique to BV PrP (blue). Black arrowheads denote the location of N-linked glycans. The locations of various structural domains are shown in black. OR = octapeptide repeat; PB = polybasic domain; GPI = glycophosphaditylinositol. Sequence alignments were performed using Multalin[25]. (B) Western blots showing three-round sPMCA reactions using normal brain homogenates (BH) from the species used in (A) as the substrates and initially seeded on day 0 with various seeds, as indicated. Day 0 samples are seeded reactions not subject to sonication. -PK = samples not subjected to proteinase K digestion; all other samples were proteolyzed.

Fig 2. Susceptibility of BV PrP^C and Mo PrP^C substrates in reconstituted sPMCA.

Western blots showing three-round reconstituted sPMCA reactions using either BV or Mo partially purified PrP^C substrates supplemented with PrP^0/0 BH and seeded with various seeds, as indicated. -PK = samples not subjected to proteinase K digestion; all other samples were proteolyzed.

Fig 3. Determining the minimum PrP sequence required for propagation of Mo protein-only recPrP^Sc seed.

(A) Table summarizing the ability of various BV/Mo PrP^C chimeras to propagate Mo protein-only recPrP^Sc. All constructs in this series are based on the BV PrP^C backbone and become progressively substituted with Mo residues from the C-terminus towards the N-terminus, except for the last two constructs which contain different combinations of 3 Mo substitutions. (B) Western blots of experiments summarized in (A), showing three-round reconstituted sPMCA reactions using partially purified PrP^C substrates supplemented with PrP^0/0 BH and seeded with various seeds, as indicated. -PK = samples not subjected to proteinase K digestion; all other samples were proteolyzed.

Fig 4. Determining the effect of single Mo amino acid mutations on the propagation of Mo protein-only recPrP^Sc.

(A) Table summarizing the effect of single Mo amino acid substitutions on the ability of BV/Mo PrP^C chimeras to propagate Mo protein-only recPrP^Sc. All constructs are based on the BV PrP^C backbone. (B) Western blots of experiments summarized in (A), showing three-round reconstituted sPMCA reactions using partially purified PrP^C substrates supplemented with PrP^0/0 BH and seeded with various seeds, as indicated. -PK = samples not subjected to proteinase K digestion; all other samples were proteolyzed.

Fig 5. Determining the BV amino acids that are sufficient to propagate protein-only recPrP^Sc.

(A) Table summarizing the ability of BV/Mo PrP^C chimeras to propagate Mo protein-only recPrP^Sc. All constructs are based on the Mo PrP^C backbone and become progressively substituted with BV residues from the C-terminus towards the N-terminus. (B) Western blots of experiments summarized in (A), showing three-round reconstituted sPMCA reactions using partially purified PrP^C substrates supplemented with PrP^0/0 BH and initially seeded with various seeds, as indicated. -PK = samples not subjected to proteinase K digestion; all other samples were proteolyzed.

Fig 6. Determining the effect of single BV amino acid mutations on the propagation of Mo protein-only recPrP^Sc.

(A) Table summarizing the effect of single BV amino acid substitutions on the ability of BV/Mo PrP^C chimeras to propagate Mo protein-only recPrP^Sc. All constructs are based on the Mo PrP^C backbone. (B) Western blots of experiments summarized in (A), showing three-round reconstituted sPMCA reactions using partially purified PrP^C substrates supplemented with PrP^0/0 BH and initially seeded with various seeds, as indicated. -PK = samples not subjected to proteinase K digestion; all other samples were proteolyzed.

Fig 7. Determining the effect of specific BV and Mo amino acids on the species barrier.

Western blots of experiments showing three-round reconstituted sPMCA reactions using partially purified PrP^C substrates supplemented with PrP^0/0 BH and initially seeded on day 0 with various seeds, as indicated. Day 0 samples are a seeded reaction not subject to sonication. -PK = samples not subjected to proteinase K digestion; all other samples were proteolyzed.

Fig 8. Propagation of Mo protein-only recPrP^Sc in sPMCA reactions with glycosylation-deficient PrP^C substrates.

Western blots of sPMCA reactions using brain homogenates from glycosylation mutants G1, G2, or G3 as substrates, as indicated. Reactions were seeded with Mo protein-only recPrP^Sc or RML, as indicated. -PK = samples not subjected to proteinase K digestion; all other samples were proteolyzed. Mouse amino acid numbering scheme is used to show locations of N-linked glycans on Mo PrP.