Strain-specified relative conformational stability of the scrapie prion protein

Abstract

Studies of prion biology and diseases have elucidated several new concepts, but none was more heretical than the proposal that the biological properties that distinguish different prion strains are enciphered in the disease-causing prion protein (PrP(Sc)). To explore this postulate, we examined the properties of PrP(Sc) from eight prion isolates that propagate in Syrian hamster (SHa). Using resistance to protease digestion as a marker for the undenatured protein, we examined the conformational stabilities of these PrP(Sc) molecules. All eight isolates showed sigmoidal patterns of transition from native to denatured PrP(Sc) as a function of increasing guanidine hydrochloride (GdnHCl) concentration. Half-maximal denaturation occurred at a mean value of 1.48 M GdnHCl for the Sc237, HY, SHa(Me7), and MT-C5 isolates, all of which have approximately 75-d incubation periods; a concentration of 1.08 M was found for the DY strain with a approximately 170-d incubation period and approximately 1.25 M for the SHa(RML) and 139H isolates with approximately 180-d incubation periods. A mean value of 1.39 M GdnHCl for the Me7-H strain with a approximately 320-d incubation period was found. Based on these results, the eight prion strains segregated into four distinct groups. Our results support the unorthodox proposal that distinct PrP(Sc) conformers encipher the biological properties of prion strains.

Fig. 1.

Western blot analysis of denaturation transitions for the Sc237 and DY strains. Ten microliters of denatured and proteinase K-treated protein samples used in ELISA has been analyzed by Western blotting using anti-PrP mAb 3F4, as described in Materials and Methods (Kascsak et al. 1987). Brain homogenates (10 mg/mL) were prepared from Syrian hamsters infected with Sc237 (S) and DY (D) prion isolates.

Fig. 2.

ELISA of denaturation transitions for the Sc237 and DY strains. (A) P2 fractions prepared from brains of normal Syrian hamster (black bars), Sc237 (white bars), and DY (striped bars) were treated with GdnHCl and precipitated with methanol/chloroform, and ELISA wells were coated with 50 μL of ∼10 μg/mL of proteins. Proteins were denatured in situ with 3 M GdnSCN, and PrP was detected with mAb D18 (1 μg/mL). (B, C) After 1 h of denaturation, the samples were diluted and treated with PK for 1 h at 37°C. ELISA wells were coated with 50 μL of 3 (diamonds), 6 (circles), 12 (triangles), and 25 (squares) μg/mL of denatured proteins. PrP was detected with D18 Fab, which recognizes the epitope within residues 133–157 (Williamson et al. 1998). The sigmoidal patterns of Sc237 PrP (filled symbols in B) and DY PrP (open symbols in C) were plotted with a four-parameter algorithm by using a nonlinear least-squares fit and correlation coefficient >0.97. (D) The apparent fractional appearance (fapp) of Sc237 (filled symbols) and DY PrP (open symbols) were calculated for each O.D. and fitted for each antigen concentration, as described in Materials and Methods. Each data point shown is the mean of duplicate readings and error bars represent SEM.

Fig. 3.

ELISA of denaturation transitions for six prion strains. P2 fractions prepared from a pool of five brains were treated with GdnHCl, PK digested, and precipitated with methanol/chloroform as previously described. ELISA wells were coated with 50 μL of ∼10 μg/mL of proteins, and PrP was detected with anti-PrP D18 Fab. Sigmoidal patterns of PrP^Sc strains were plotted with correlation coefficient >0.97, and Fapp values were calculated for each O.D. Each symbol represents a separate experiment.

Fig. 4.

Incubation period and GdnHCl_1/2 values for eight SHa strains. ELISA wells were coated with ∼0.5 μg of P2 proteins and GdnHCl_1/2 values were interpolated from at least four denaturation curves. Each data point shown is the mean, and error bars represent standard deviation.