Abstract
In prion disease, direct interaction between the cellular prion protein (PrP(C)) and its misfolded disease-associated conformer PrP(Sc) is a crucial, although poorly understood step promoting the formation of nascent PrP(Sc) and prion infectivity. Recently, we hypothesized that three regions of PrP (corresponding to amino acid residues 23-33, 98-110, and 136-158) interacting specifically and robustly with PrP(Sc), likely represent peptidic components of one flank of the prion replicative interface. In this study, we created epitope-tagged mouse PrP(C) molecules in which the PrP sequences 23-33, 98-110, and 136-158 were modified. These novel PrP molecules were individually expressed in the prion-infected neuroblastoma cell line (ScN2a) and the conversion of each mutated mouse PrP(C) substrate to PrP(Sc) compared with that of the epitope-tagged wild-type mouse PrP(C). Mutations within PrP 98-110, substituting all 4 wild-type lysine residues with alanine residues, prevented conversion to PrP(Sc). Furthermore, when residues within PrP 136-140 were collectively scrambled, changed to alanines, or amino acids at positions 136, 137, and 139 individually replaced by alanine, conversion to PrP(Sc) was similarly halted. However, other PrP molecules containing mutations within regions 23-33 and 101-104 were able to readily convert to PrP(Sc). These results suggest that PrP sequence comprising residues 98-110 and 136-140 not only participates in the specific binding interaction between PrP(C) and PrP(Sc), but also in the process leading to conversion of PrP(Sc)-sequestered PrP(C) into its disease-associated form.
Highlights
Prion diseases such as Creutzfeldt-Jakob disease (CJD) in human, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, and scrapie in sheep, are a group of closely related fatal neurodegenerative conditions characterized by the change in the conformation of normal cellular prion protein (PrPC)4 into a pathogenic conformer (PrPSc) [1, 2]
Generation of Mouse PrP Mutants—Recently, we have identified three regions of the prion protein that interact with PrPSc
FIGURE 4. 136 –140 single PrP mutants and the 136 –140 scrambled PrP mutant show normal reactivity to the PrP-specific antibody 6H4, whereas the 136 –140A/PrP mutant, in which the original residues are all changed to alanine, shows reduced reactivity, possibly indicating an alteration of the helix-1 of PrP [37]
Summary
PrPC, cellular prion protein; PrPSc, scrapie prion protein; wt, wild-type; ScN2a, prion infected neuroblastoma cell line; PK, proteinase K; FACS, fluorescent-activated cell sorting. Within PrP 136 –158, mutants were prepared in which the native sequence within segments 136 –140 and 149 –158 was altered. Each of these novel full-length PrP molecules was individually expressed in the prion-infected neuroblastoma cell line (ScN2a). Conversion of the various mutated mouse PrPC substrates to PrPSc were measured and compared with the conversion of equivalently expressed epitope tagged wild-type (wt) mouse PrPC. When residues in position 136 –140 were individually substituted with alanine, mutants (Arg to Ala), (Pro to Ala), and 139 (Ile to Ala) did not convert to PrPSc
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