Figure 1.
Western blot analysis of Nor98 isolates.
A: Representative western blots of PrPres from Nor98 isolates (Table S1). Lanes 1–2, Norwegian sheep Nor98 (AFRQ/AFRQ); lanes 3–5, Italian goat Nor98 (ALRQ/ALHQ); lanes 6–7, Italian sheep Nor98 (AFRQ/AFRQ and ALRR/ALHQ, respectively); lane 8, Italian goat scrapie (ALRQ/ALRQ); lane 9, Italian sheep scrapie (ALRQ/AFRQ). All samples were digested at 200 µg/ml PK. Membranes were probed with P4 (top) and SAF84 (bottom). B: Effect of PK digestion on Nor98 PrPSc. WB analysis of healthy, Nor98 (Italian sheep, AFRQ/AFRQ) and scrapie (Italian sheep, ALRQ/ALRQ) sheep brain homogenate treated with 10, 50 and 200 µg/ml PK. Replica blots were probed with P4 (top) and SAF84 (bottom). MW marker was loaded as indicated (M). C: Epitope mapping of Nor98 (Norwegian sheep AFRQ/AFRQ) PrPres produced after 200 µg/ml PK treatment. Membranes were probed with different mAbs, as indicated on the top of each blot. MW markers were loaded into the last lane of each blot. A-C: MW marker was loaded as indicated (M).
Figure 2.
Titration of proteinase K digestion.
Representative western blots of brain homogenates from Nor98 (#2), VPSPr (#7), GSS F198S (#11), A117V (#13), P102L (#15) and sCJD treated with increasing concentrations of PK (0–1000 µg/ml). Given the various PK-cleavage sites of the PrPSc types under investigation which affects mAbs binding to PrPres (see Fig. 3), membranes stained with different antibodies were selected in order to show the PK resistance of strong PrPres in each sample. Membranes were probed with 12B2 for Nor98 and GSS P102L, 9A2 for GSS A117V, 1E4 for VPSPr, and 3F4 for GSS F198S and sCJD samples.
Table 1.
Human and ovine cases used for comparative analyses.
Figure 3.
Epitope mapping of PrPSc treated with different PK concentrations.
PK digestion curves of VPSPr (#7), GSS A117V (#13) and GSS F198S (#11) were probed with 12B2, 9A2 and L42.
Table 2.
Monoclonal antibodies used for epitope mapping.
Figure 4.
A: Representative blots of PrPres from Nor98, GSS and VPSPr. Brain homogenates were treated with 100 µg/ml PK. Replica blots were probed with SAF32, 12B2, 9A2, 8G8, L42 mAbs. Case numbers are shown on the bottom of blots according to Table 1. B: Samples (ID #11, #1, #7, #13) were re-analysed at a lower PK concentration (50 µg/ml) to investigate partially cleaved epitopes. Replica blots were probed with 12B2, 9A2 and L42 mAbs. In these blots Nor98 is an internal control as it is recognised equally well by the 3 mAbs (see Fig. 4A). By comparison with Nor98, it is shown than i) in GSS A117V the L42 epitope is cleaved, the 12B2 is partially preserved and the 9A2 was fully preserved; ii) in VPSPr the 12B2 epitope is cleaved and the 9A2 partially preserved; iii) in GSS F198S the L42 epitope is partially preserved. C: Samples (ID #11, #1, #7, #13) were treated with 100 µg/ml PK and probed with 6D11, 8G8 and 12F10 mAbs. 6D11 and 8G8 bind to epitopes within the “core” of PrPres fragments and reveal the actual quantity of PrPres fragments in the samples analysed. By comparison, 12F10, which recognizes an epitope at the C-terminus of PrPres fragments, only detected VPSPr, suggesting that its epitope is cleaved in all other samples.
Figure 5.
Comparison of PrPres fragments in Nor98, GSS and VPSPr.
The aligned amino acid sequences at the N and C termini of all PrPres fragments, derived from epitope mapping as described in SI 5, are depicted. Amino acid numbering refers to sheep PrP for Nor98 and to human PrP for all other fragments. Coloured letters highlight the relevant epitopes as follows: orange for SAF32; blue for 12B2; green for 9A2; pink for 8G8; red for 12F10 and violet for L42. Note that when epitopes of mAbs partially overlap, only the aa differentiating the epitopes were coloured. On the right of each PrPres fragment is reported the predicted MW estimated by using the ProtParam (ExPASy) software.
Figure 6.
Conformational stability of insoluble PrPSc.
A: Western blot analysis of VPSPr 129MV (#7) and 129VV (#3) and GSS P102L (#15 cerebellum) cases, showing separation of insoluble PrPSc by a solubility assay. Samples were centrifuged at 20000 g for 1 h in presence of 1% sarkosyl. Supernatant (S) and pellet (P) fractions were analysed with (+) or without (-) PK treatment (50 µg/ml), along with aliquots of samples before centrifugation (Tot). Note that PrPres segregated into the insoluble fraction (compare lanes S+ and P+ in each blot). In each lane 0,7 mg TE were loaded. Membranes were probed with L42 mAb. B: Representative western blots of CSSA experiments in Nor98 (#1), VPSPr (#9), GSS F198S (#11), GSS A117V (#14) and GSS P102L (#16) cases. Lanes were loaded with insoluble PrP obtained as shown in Fig. 6A, with or without previous denaturation with increasing concentrations of GdnHCl, as shown on the top of each lane. Membranes were probed with L42. Molecular size markers are shown in kilodaltons on the right of each blot. C: Dose-response curves obtained by plotting the fraction of PrPSc remaining in the pellet as a function of GdnHCl concentration and best-fitted and using a four parameter logistic equation. Individual [GdnHCl]1/2 values are shown in Table 1.