PrPSc-Specific Antibody Reveals C-Terminal Conformational Differences between Prion Strains

Abstract

Understanding the structure of PrP(Sc) and its strain variation has been one of the major challenges in prion disease biology. To study the strain-dependent conformations of PrP(Sc), we purified proteinase-resistant PrP(Sc) (PrP(RES)) from mouse brains with three different murine-adapted scrapie strains (Chandler, 22L, and Me7) and systematically tested the accessibility of epitopes of a wide range of anti-PrP and anti-PrP(Sc) specific antibodies by indirect enzyme-linked immunosorbent assay (ELISA). We found that epitopes of most anti-PrP antibodies were hidden in the folded structure of PrP(RES), even though these epitopes are revealed with guanidine denaturation. However, reactivities to a PrP(Sc)-specific conformational C-terminal antibody showed significant differences among the three different prion strains. Our results provide evidence for strain-dependent conformational variation near the C termini of molecules within PrP(Sc) multimers.

Importance: It has long been apparent that prion strains can have different conformations near the N terminus of the PrP(Sc) protease-resistant core. Here, we show that a C-terminal conformational PrP(Sc)-specific antibody reacts differently to three murine-adapted scrapie strains. These results suggest, in turn, that conformational differences in the C terminus of PrP(Sc) also contribute to the phenotypic distinction between prion strains.

FIG 1

Epitopes of anti-PrP antibodies used in this study. The schematic representation of PrP primary structure (mouse residues 81 to 231) shows epitopes of anti-PrP antibodies. CHO indicate the N-linked glycosylation sites at residues 180 and 196 in PrP. A disulfide bond is indicated by S-S. Two β-sheets (β1 and β2) and three α-helices (α1, α2, and α3) exist in PrP^C; however, the secondary structure of PrP^C is not maintained in PrP^Sc. The C-terminal region is recognized by the PrP^Sc-specific MAb 6H10 (215 to 216, 221 to 223, and 228 [dark-blue arrows]) in PrP^Sc and the human Fab anti-PrP antibody R1 (224 to 230 [red arrow]) in both PrP^C and PrP^Sc.

FIG 2

Epitopes of PrP antibodies with more N-terminal linear epitopes were largely hidden in the folded conformation of PrP^RES. Purified PrP^RES from wild-type mice inoculated with the Chandler strain and with the 22L strain were used to examine the epitope accessibilities of the indicated more N-terminal PrP antibodies using indirect ELISA. The linear epitopes are shown in parentheses. Each of the epitopes was much less accessible in the folded conformation than after treatments with chaotropic 3 or 8 M GdnHCl solutions. OD₄₅₀ readings with background subtracted are indicated as the means and SD from triplicate wells. Similar results were obtained in at least three independent experiments.

FIG 3

Epitopes of PrP antibodies with linear epitopes near glycosylation sites were largely hidden in the folded conformation of PrP^RES. Preparations of wild-type murine Chandler (dark blue) and 22L (light blue) PrP^RES were either folded (untreated) or treated with 3 M or 8 M GdnHCl prior to indirect-ELISA measurements using the indicated primary anti-PrP antibodies, with epitopes shown in parentheses. OD₄₅₀ readings with background subtracted are indicated as the means and SD from triplicate wells. Similar results were obtained in at least three independent experiments.

FIG 4

The PrP^Sc-specific C-terminal antibody 6H10 recognized the abnormal conformations of Me7 and 22L, but not Chandler, PrP^RES. The OD₄₅₀ readings of MAbs 132, SAF-70, 8H4, and 94B4 after 8 M GdnHCl treatment showed that approximately equivalent amounts of PrP^RES were loaded into each well in ELISA plates. (A) MAb 132 was used to normalize the OD₄₅₀ reading. (B) The same data were normalized to MAb 8H4 readings. Twofold dilutions of PrP^RES were used to show that the OD₄₅₀ readings were in a responsive range (the nine bars on the right in panels A and B). (C) Because PrP^RES was treated with PK and several possible PK cleavage sites are at the C terminus of PrP, the C-terminal linear-epitope antibody R1 was used to examine whether the C-terminal epitopes were still present in PrP^RES. OD₄₅₀ readings with background subtracted are indicated as the means and SD from triplicate wells. *, P < 0.05; **, P < 0.01; ***, P < 0.005; one-way ANOVA followed by the FDR (Benjamini-Hochberg) test. Similar results were obtained in at least three independent experiments.

FIG 5

MAb 6H10 differed in its binding to the 22L and Chandler PrP^RES lacking GPI anchors and N-linked glycosylations. Purified GPI^neg Chandler and 22L PrP^RES were tested for epitope exposure of 6H10 using indirect ELISA. The OD₄₅₀ readings of MAbs 132, SAF-70, 8H4, and 94B4 after 8 M GdnHCl treatment showed that equivalent amounts of PrP^RES were loaded in each well in the ELISA plates, and MAb 132 was used to normalize the OD₄₅₀ readings. Twofold dilutions of PrP^RES were used to show that the OD₄₅₀ readings were in a responsive range. OD₄₅₀ readings with background subtracted are indicated as the means and SD from triplicate wells. *, P < 0.05; **, P < 0.01; ***, P < 0.005; unpaired two-tailed Student's t test. Similar results were obtained in at least three independent experiments.