Species-independent inhibition of abnormal prion protein (PrP) formation by a peptide containing a conserved PrP sequence

Abstract

Conversion of the normal protease-sensitive prion protein (PrP) to its abnormal protease-resistant isoform (PrP-res) is a major feature of the pathogenesis associated with transmissible spongiform encephalopathy (TSE) diseases. In previous experiments, PrP conversion was inhibited by a peptide composed of hamster PrP residues 109 to 141, suggesting that this region of the PrP molecule plays a crucial role in the conversion process. In this study, we used PrP-res derived from animals infected with two different mouse scrapie strains and one hamster scrapie strain to investigate the species specificity of these conversion reactions. Conversion of PrP was found to be completely species specific; however, despite having three amino acid differences, peptides corresponding to the hamster and mouse PrP sequences from residues 109 to 141 inhibited both the mouse and hamster PrP conversion systems equally. Furthermore, a peptide corresponding to hamster PrP residues 119 to 136, which was identical in both mouse and hamster PrP, was able to inhibit PrP-res formation in both the mouse and hamster cell-free systems as well as in scrapie-infected mouse neuroblastoma cell cultures. Because the PrP region from 119 to 136 is very conserved in most species, this peptide may have inhibitory effects on PrP conversion in a wide variety of TSE diseases.

FIG. 1

Species specificity of the cell-free conversion reaction. (A) Immunopurified mouse (Mo) and hamster (Ha) ³⁵S-PrP-sen samples were incubated in the presence of hamster 263K (Ha 263K), mouse Obihiro (Mo Obi), or mouse 87V (Mo 87V) PrP-res. At the end of the incubation time, samples were treated (+) or not (−) with PK as described in Materials and Methods followed by SDS-PAGE analysis. Parallel experiments done using PrP-res from mouse Chandler scrapie gave results identical to those previously published (20). (B) Histogram representation of the cell-free conversion reactions induced by hamster 263K, mouse Obihiro, and mouse 87V PrP-res in the presence of immunopurified mouse or hamster ³⁵S-Prp-sen. Only the PK-resistant bands (19 to 24 kDa) showing the 6- to 8-kDa downward size shift relative to the untreated ³⁵S-PrP-sen precursor were quantified by PhosphorImager autoradiography. PK-resistant bands showing a downward size shift greater than 6 to 8 kDa (<19 kDa) likely represent partial conversion products and were not quantified. The results are expressed as the percent conversion of ³⁵S-PrP-sen to 19- to 24-kDa PK-resistant forms, and each histogram represents the means of five independent experiments ± standard deviations (bars).

FIG. 2

Kinetics of in vitro conversion reaction. The cell-free conversion reactions were performed by incubating hamster 263K (A) or mouse Obihiro (B) PrP-res with immunopurified mouse ³⁵S-PrP-sen (open circles) or hamster ³⁵S-PrP-sen (closed circles) at 37°C. At the indicated incubation times, the conversion reaction samples were digested with PK and then analyzed by SDS-PAGE and PhosphorImage autoradiography as described in the legend to Fig. 1. Percent conversion was plotted as a function of the incubation time. Each point represents the mean of two independent experiments.

FIG. 3

Dose response of the inhibition of conversion by hamster and mouse PrP peptides. The cell-free conversion reactions were performed by mixing PrP-res (200 ng) with immunopurified ³⁵S-PrP-sen (∼1 ng) as described in Materials and Methods in the presence of various concentrations of HaP109-141 or MoP108-140. At the end of the incubation time, the samples were digested by PK and analyzed as described in Materials and Methods. (A) Hamster 263K strain PrP-res plus hamster PrP-sen. (B) Mouse Obihiro strain PrP-res plus mouse PrP-sen; (C) mouse 87V strain PrP-res plus mouse PrP-sen. Each point represents the mean of four independent experiments ± standard deviation (bars). The data were plotted as relative percent conversion in the presence of peptide compared to the control reaction in the absence of peptide.

FIG. 4

Dose response of the inhibition of cell-free conversion by PrP peptides P119-136 and P119-128. (A to C) Representative results after PK digestion (+) of the samples. The first lane of each panel represents the PK-untreated sample (−). PrP-res was incubated with immunopurified ³⁵S-PrP-sen in the absence (control) or presence of the indicated concentrations of PrP peptides P119-136, P119-128, and P121-141 and the Alzheimer’s protein peptide Aβ1-40. (A and D) Hamster 263K PrP-res incubated with hamster ³⁵S-PrP-sen; (B and E) mouse Obihiro PrP-res incubated with mouse ³⁵S-PrP-sen; (C and F) mouse 87V PrP-res incubated with mouse ³⁵S-PrP-sen. Molecular mass markers are indicated on the left. (D to F) Dose-response curves of the inhibition of the cell-free conversion reactions induced by P119-136 (GAVVGGLGGYMLGSAMSR) and P119-128 (GAVVGGLGGY). The data represent the means of four independent experiments ± standard deviations (bars) and were plotted as relative percent conversion in the presence of peptide compared to the control reaction in the absence of peptide.

FIG. 5

Effect of PrP peptides P119-136 and P119-128 on the accumulation of PrP-res in Sc⁺MNB. (A) SDS-PAGE PhosphorImage of a representative experiment performed by treating Sc⁺MNB cells in the absence (control) or presence of indicated concentrations of Congo red, P119-136, P119-128, P121-141, or Aβ1-40. PrP-res was detected with a polyclonal rabbit anti-PrP serum and developed with the Amersham Vistra ECF system. (B) Dose-response curve of the inhibition of PrP-res accumulation induced by P119-136. The data represent the means of five independent experiments ± standard deviations (bars) and were plotted as relative percent conversion in the presence of peptide compared to the control culture in the absence of peptide.