Identifying key components of the PrPC-PrPSc replicative interface

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.

FIGURE 1.

Modifications of PrP regions 98–110 and 136–140 impair conversion of PrP^C into PrP^Sc in ScN2a cells. Western blots of PK-treated and non-treated lysates prepared from ScN2a cells transiently transfected with 3F4- or FLAG-tagged wt-PrP and PrP mutants and probed with antibody D13 (panel A) or D18 (panels C and E) for detection of total PrP, antibody 3F4 (panels B and D), or anti-FLAG antibody (panel F) for detection of transfected PrP. Compared with wt-PrP (panel B/lanes 1 and 2), 23–27/PrP mutant (panel B/lanes 3 and 4) and 101–104/PrP mutant (panel D/lanes 3 and 4), which robustly convert to protease-resistant PrP^Sc, no protease-resistant 101–110/PrP mutant (panel F/lanes 3 and 4), 136–140A/(panel B/lanes 5 and 6), and 136–140scrambled/PrP mutant (panel B/lanes 7 and 8) are detectable. As expected, the 101–110 mutant is not detected by antibody 3F4 (panel D/lanes 5 and 6). Samples 136–140 scrambled (panel B) and wt FLAG, 101–110 FLAG (panel F), in the figure appear separated, because even though processed at the same time, they were loaded in different gels or in the same gel but not adjacent to each other.

FIGURE 2.

Residues 136 (arginine), 137 (proline), and 139 (isoleucine), are individually crucial for conversion of PrP^C into PrP^Sc in ScN2a cells. Western blot of PK-treated and non-treated lysate prepared from ScN2a cells transiently transfected with 3F4-tagged 136–140/PrP mutants and probed with antibody D13 (panel A) or antibody 3F4 (panel B). PrP^C conversion into PrP^Sc is significantly reduced or impaired when the original PrP residues in position 136 (panel B/lanes 1 and 2), 137 (panel B/lanes 3 and 4), or 139 (panel B/lanes 7 and 8) are individually modified to an alanine residue. Individual substitutions of the original PrP residues in position 138 (methionine) (panel B/lanes 5 and 6) and 140 (histidine) (panel B/lanes 9 and 10) to alanine residues do not interfere with prion conversion.

FIGURE 3.

136–140/PrP mutants that do not convert into PrP^Sc and PrP with the FLAG peptide between β2 and α2, are present on the cells surface of N2a cells. Single cell suspensions of N2a cells transiently transfected with 3F4-tagged wt-PrP and 136–140/PrP mutants were analyzed by flow cytometry for presence of PrP on the cell surface. Profiles shown and the histogram indicate that all the 136–140 mutants are present on the cell surface at similar levels of the wt-protein (panel A). Single cell suspensions of N2a cells transiently transfected with 3F4-tagged wt- and 3F4+FLAG-tagged wt-PrP were analyzed by flow cytometry for the presence of PrP on the cell surface. Profiles shown and the histogram indicate the presence of PrP on the cell surface for both samples (panel B).

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). PK-treated and non-treated lysates prepared from ScN2a cells transiently transfected with 3F4-tagged wt-PrP and 136–140/PrP mutants were incubated with the antibody 6H4, recognizing the helix 1 of the prion protein, residues 144–154. The complex 6H4-PrP was captured onto paramagnetic beads coupled to an anti-mouse IgG reagent, and any precipitated wt-PrP or PrP mutants were detected via Western blot by the PrP-specific antibody D13 (panels B and E/2) or 3F4 (panels D and F/2). Total and transfected PrP were as well detected by antibody D13 (panels A and E/1) and antibody 3F4 (panels C–F/1), employing Western blots. The reactivity of antibody 6H4 was reduced only for the 136–140A PrP mutant (panel D, lane 2, panel F/2 lane 3), whereas regular reactivity for the other 136–140/PrP mutants (panel D, lanes 3–7, panel F/2, lane 4) is shown.