Prion strain-dependent differences in conversion of mutant prion proteins in cell culture

Abstract

Although the protein-only hypothesis proposes that it is the conformation of abnormal prion protein (PrP(Sc)) that determines strain diversity, the molecular basis of strains remains to be elucidated. In the present study, we generated a series of mutations in the normal prion protein (PrP(C)) in which a single glutamine residue was replaced with a basic amino acid and compared their abilities to convert to PrP(Sc) in cultured neuronal N2a58 cells infected with either the Chandler or 22L mouse-adapted scrapie strain. In mice, these strains generate PrP(Sc) of the same sequence but different conformations, as judged by infrared spectroscopy. Substitutions at codons 97, 167, 171, and 216 generated PrP(C) that resisted conversion and inhibited the conversion of coexpressed wild-type PrP in both Chandler-infected and 22L-infected cells. Interestingly, substitutions at codons 185 and 218 gave strain-dependent effects. The Q185R and Q185K PrP were efficiently converted to PrP(Sc) in Chandler-infected but not 22L-infected cells. Conversely, Q218R and Q218H PrP were converted only in 22L-infected cells. Moreover, the Q218K PrP exerted a potent inhibitory effect on the conversion of coexpressed wild-type PrP in Chandler-infected cells but had little effect on 22L-infected cells. These results show that two strains with the same PrP sequence but different conformations have differing abilities to convert the same mutated PrP(C).

FIG. 1.

Formation of Mo3F4-derived PrP^Sc in Ch-N2a58 and 22L-N2a58 cells. (A) Western blot using polyclonal anti-PrP antibody SS in N2a58, Ch-N2a58 (Ch), and 22L-N2a58 (22L) cells without (−) or with (+) PK treatment. (B) Expression levels of Mo3F4 PrP (left panel) and detection of Mo3F4-derived PrP^Sc (right panel) were measured by Western blot using monoclonal antibody 3F4. Mock, untransfected cells. (C) After consecutive treatments of PK and PNGase F, untransfected (left panel) and Mo3F4 PrP-transfected cells (right panel) were analyzed by Western blotting using SS and 3F4 antibodies, respectively. Molecular mass markers are indicated in kilodaltons on the left side of each panel.

FIG. 2.

Strain-dependent inhibitory effect of Q218K mutation on PrP^Sc formation of wild-type Mo3F4. (A) Conversion to 3F4-positive PrP^Sc (upper panels) and expression of Mo3F4, SHa, Hu, Q218K, and Q218E (lower panels) were measured by Western blot using 3F4 antibody. The 3F4 epitope was present in all these constructs. (B and C) The inhibitory effect of the constructs was determined by cotransfection with Mo3F4 in the DNA ratio of 1:1 or 1:2. The blots were probed with 3F4 antibody. Mock, untransfected cells; +vector(1:2), cotransfection of Mo3F4 and pcDNA3.1(+) at a 1:2 ratio.

FIG. 3.

Strain-specific effects of Q185R mutation on PrP^Sc formation. (A) Conversion to 3F4-positive PrP^Sc (upper panels) and expression of Mo3F4, Q90R, Q97R, Q159R, Q167R, Q171R, Q185R, Q211R, Q216R, and Q222R (lower panels) were measured by Western blotting using 3F4 antibody. The 3F4 epitope was present in all these constructs. (B) Inhibitory effects of constructs that did not convert were determined by cotransfection with Mo3F4 at a 1:1 or 1:2 DNA ratio. The blots were probed with 3F4 antibody.

FIG. 4.

Strain-specific PrP^Sc formation of Q185R, Q185K, Q218R, and Q218H mutated PrP. (A) Conversion to 3F4-positive PrP^Sc (upper panels) and expression of Mo3F4, Q185R, Q185K, Q185H, Q185E, and Q185L (lower panels) were measured by Western blot using 3F4 antibody. The 3F4 epitope was present in all these constructs. (B) Western blotting of Mo3F4, Q218R, Q218K, Q218H, Q218E, and Q218L was done as in A.

FIG. 5.

Strain-specific effects of L42-positive mutated PrPs on PrP^Sc formation. (A) Conversion to L42-positive PrP^Sc (upper panels) and expression of MoL42, Q185R, Q218H, Q218R, and Q218K (lower panels) were measured by Western blot using L42 antibody. The L42 epitope was present in all these constructs. (B) Inhibitory effects of SHa, Hu, and Q218K were determined by cotransfection with MoL42 at a 1:1 or 1:2 DNA ratio. The blots were probed with the L42 antibody. Molecular mass markers are indicated in kilodaltons on the left side of each panel.

FIG. 6.

Amino acid sequence of Mo3F4 and the position of mutations. The amino acid residue number is based on Mo3F4 PrP. The secondary structures in mouse PrP^C are indicated in white boxes at the top. Boxed residues indicate the representative mutations tested; + indicates that conversion occurred in the two cells; − indicates that conversion did not occur in the two cells; “C” indicates Chandler-specific conversion; “L” indicates 22L-specific conversion.

FIG. 7.

Comparison of 22L, Chandler, and 87V PrP^Sc by infrared spectroscopy. Second-derivative Fourier transform IR spectra are shown for PK-treated PrP^Sc samples isolated from the brains of mice affected by either 22L, Chandler, or 87V scrapie. Spectral differences, especially in the β-sheet region of the spectrum, provide evidence that PrP^Sc proteins associated with these murine-adapted scrapie strains have distinct conformations. For comparison, a highly α-helical protein, hemoglobin, gives strong absorbance (represented by a negative deflection) at ∼1,657 cm⁻¹, with only minor absorbance in the β-sheet region. Similar results were obtained from at least two independent preparations of each strain of PrP^Sc.