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. 2005 May;14(5):1222-32.
doi: 10.1110/ps.041186605. Epub 2005 Mar 31.

Synthetic prions generated in vitro are similar to a newly identified subpopulation of PrPSc from sporadic Creutzfeldt-Jakob Disease

Olga V Bocharova  1 , Leonid Breydo, Vadim V Salnikov, Andrew C Gill, Ilia V Baskakov
Affiliations

Synthetic prions generated in vitro are similar to a newly identified subpopulation of PrPSc from sporadic Creutzfeldt-Jakob Disease

Olga V Bocharova et al. Protein Sci. 2005 May.

Abstract

In recent studies, the amyloid form of recombinant prion protein (PrP) encompassing residues 89-230 (rPrP 89-230) produced in vitro induced transmissible prion disease in mice. These studies showed that unlike "classical" PrP(Sc) produced in vivo, the amyloid fibrils generated in vitro were more proteinase-K sensitive. Here we demonstrate that the amyloid form contains a proteinase K-resistant core composed only of residues 152/153-230 and 162-230. The PK-resistant fragments of the amyloid form are similar to those observed upon PK digestion of a minor subpopulation of PrP(Sc) recently identified in patients with sporadic Creutzfeldt-Jakob disease (CJD). Remarkably, this core is sufficient for self-propagating activity in vitro and preserves a beta-sheet-rich fibrillar structure. Full-length recombinant PrP 23-230, however, generates two subpopulations of amyloid in vitro: One is similar to the minor subpopulation of PrP(Sc), and the other to classical PrP(Sc). Since no cellular factors or templates were used for generation of the amyloid fibrils in vitro, we speculate that formation of the subpopulation of PrP(Sc) with a short PK-resistant C-terminal region reflects an intrinsic property of PrP rather than the influence of cellular environments and/or cofactors. Our work significantly increases our understanding of the biochemical nature of prion infectious agents and provides a fundamental insight into the mechanisms of prions biogenesis.

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Figures

Figure 1.
Figure 1.
GdnHCl-induced denaturation of the amyloid fibrils of rPrP 89-230. Amyloid fibrils were incubated for 30 min at 24°C (▿) or 57°C (•) in the presence of variable concentrations of GdnHCl. The GdnHCl concentration was then adjusted to 0.55 M, followed by a ThT-binding assay. The experiments were conducted at least in duplicate. The lines represent the results of the fitting to a two-state model (Santoro and Bolen 1988). The increase in ThT fluorescence observed at low concentrations of denaturant is due to GdnHCl-induced dissociation of coaggregated amyloid fibrils.
Figure 2.
Figure 2.
Limited PK digestion of the amyloid form of rPrP 89-230. (A) Amyloid fibrils or α-rPrP 89-230 (0.2 mg/mL) were treated with PK for 1 h at 37°C at the following PK:rPrP ratios (w/w): 1:2000 (lanes 2), 1:500 (lanes 3), 1:100 (lanes 4), 1:20 (lanes 5), no PK (lanes 1) and analyzed by SDS-PAGE followed by silver staining (amyloid fibrils, left panel; α-rPrP 89-230, middle panel) or by Western blotting using Fabs R1 and P (right panel). MW markers, lane 6. A 14-kDa fragment detectible by Fab R1 appeared as a result of partial degradation in the time-course of amyloid formation. (B) Online capillary HPLC-MS analysis of the amyloid form after limited digestion with PK. Electrospray mass spectrum and deconvoluted mass spectrum of species (inset) showing partial resistance to PK. Several PK-resistant species were identified by comparison of measured and theoretical masses, including polypeptides spanning residues 138–230 (measured mass 11,395.56 Da; theoretical mass 11,395.56 Da), residues 141–230 (measured mass 10,997.32 Da; theoretical mass 10,998.01 Da), residues 152–230 (measured mass 9,512.07 Da; theoretical mass 9,513.58 Da), residues 153–230 (measured mass 9,398.29 Da; theoretical mass 9,399.47 Da), and residues 162–230 (measured mass 8,182.63 Da; theoretical mass 8,184.07).
Figure 3.
Figure 3.
PK-resistant fragments. (A) PK-resistant core of the amyloid fibrils of Mo rPrP 89-231; epitopes for Fabs P and R1 are highlighted (present work). (B) PK-resistant core of the novel subpopulation of PrPSc found in spCJD; sites of PK digestion were identified by N-terminal sequencing using Edman degradation (Zou et al. 2003). (C) PK-resistant core of PrPSc generated in mice upon second passage of transmission of hamster PrPSc strain 263 K as determined by Western blotting using an antibody against the C terminus. Approximate sites of cleavage were identified by epitope mapping of PK-resistant fragments of Mo PrP generated in the cell-free conversion assay upon incubation with hamster PrPSc strain 263 K (Lawson et al. 2004). (D) PK-resistant core of hamster (Ha) PrPSc generated in the presence of 2.5 M GdnHCl. The approximate location of PK cleavage was found within residues 115–143 by epitope mapping, and is represented by the light gray area (Kocisko et al. 1996). PK-resistant regions are represented by the dark gray area, and partial PK-resistant regions by the light gray area.
Figure 4.
Figure 4.
PK-resistant core of the amyloid form displays seeding activity. (A) The kinetics of fibril formation for rPrP 89-230 (0.3 mg/mL; reaction volume 0.15 mL) seeded with 0.04% (w/w) (brown triangles), 0.2% (orange triangles), and 1% (yellow triangles) of preformed fibrils; with 2.5% fibrils treated with PK (green triangles); and in nonseeded reaction (blue triangles). Std. dev. for duplicate samples are represented by error bars. The kinetics were monitored using the automated format as described in Materials and Methods. (B) Dependence of the lag phase of fibrils formation on the amount of seed (circles); the solid line represents the result of the fitting to a linear function. The apparent seeding activity in the reactions seeded with 2.5% of fibrils pretreated with PK at PK/rPrP ratio of 1:20 (green square) was calculated using the linear dependence between the length of the lag phase and Lg[amount of seed].
Figure 5.
Figure 5.
PK-resistant core of the amyloid form maintains β-rich structure. (A) Electron micrographs of the amyloid fibrils of rPrP 89-230 without PK treatment (left panel), and treated with PK at 37°C for 1 h at the PK to rPrP ratio of 1:20 (right). Scale bar = 0.5 μm. (B) FTIR spectra of rPrP 89-230 in the α-monomeric form (dashed line), the intact amyloid form (solid line), and the amyloid form after treatment with PK (dotted line). (C) Second derivatives of FTIR spectra. The line definitions are the same as for panel B. Fibrils were incubated with PK for 1 h at 37°C at the PK:rPrP ratio 1:20.
Figure 6.
Figure 6.
In vitro conversion of rPrP 23-230 into the amyloid fibrils. (A) FTIR spectra of rPrP 23-230 in the amyloid form (solid line) and in α-monomeric form (dashed line). (B) Second derivatives of FTIR spectra. The line definitions are the same as for panel A.
Figure 7.
Figure 7.
Limited PK digestion of the amyloid form of rPrP 23-230 reveals two conformers. (A) SDS-PAGE followed by silver staining represents the relative amount of PK-resistant fragments. Amyloid fibrils of rPrP 23-230 (left panel) or α-rPrP 23-230 (0.2 mg/mL) (right) were treated with PK for 1 h at 37°C at the following PK:rPrP ratios (w/w): no PK (lanes 1), 1:10,000 (lanes 2), 1:5,000 (lanes 3), 1:1000 (lanes 4), 1:500 (lanes 5), 1:100 (lanes 6), and 1:50 (lanes 7). Apparent molecular masses of fragments are given in kDa at the left. (B) Western blot of the PK-resistant fragments treated with three Fabs: P (left panel), D18 (middle), and R1 (right). PK:rPrP ratios are the same as in panel A. The 9-kDa fragment containing epitope for Fab P appeared only at low PK:rPrP ratios (shown by an arrow). This fragment, however, was fully digested at high concentrations of PK. (C) Two patterns of PK digestion. Epitopes for Fab P are shown as light gray boxes, for D18 as dark gray boxes, and for R1 as black boxes; approximate locations of PK cleavage sites are shown by small arrows.
Figure 8.
Figure 8.
Two subpopulations of the amyloid fibrils of rPrP 23-230. (A) Fluorescence microscopy of the amyloid fibrils (top panel). The emission intensity (bottom panel) monitored across the image at the position shown by the red dashed line. The fibrils of minor subpopulations display emission intensities four-–eightfold higher than that of fibrils of the major subpopulation (scale bar = 2 μm). (B) Electron micrographs of negatively stained fibrils (scale bar = 0.5 μm). The minor subpopulation displayed much stronger propensity for staining with uranyl acetate and accounted for ~5%–10% of all fibrils.
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