Abstract
The transmissible spongiform encephalopathies or prion diseases are a group of uniformly fatal neurodegenerative diseases that affect both humans and animals. The agent of these diseases is proposed to consist solely of PrPsc, the abnormal form of a host-encoded protein, the prion protein. Despite the lack of a nucleic acid component for the prion agent, strain diversity is observed among these diseases. The conformation of PrPsc is proposed to encode prion strain diversity. Prion strains can be characterized based on phenotypic properties and also on molecular properties of PrP^. This study investigated the hypothesis that characterization of prion strains using molecular properties of PrPsc is more discriminatory when more than one molecular property of PrPsc is examined. Two previously defined hamster adapted transmissible mink encephalopathy (TME) strains, hyper (HY) and drowsy (DY) TME, were utilized in this study. The molecular properties of PrPsc examined were the extent of PrPsc glycosylation, the molecular weight of PrPsc following PK treatment and the number of immunoreactive polypeptides of PrPsc following PK treatment. When multiple anti-PrP antibodies directed to different regions of the prion protein were used in this analysis, additional differences between the molecular properties of HY and DY PrPsc were observed. The choice of sample preparation was also important, with a greater number of differences observed between the immunoreactive patterns of HY and DY PrPsc when a PK treated homogenate was used as compared with a preparation that enriched for PrP^'. These additional differences were only observed when analysis was performed using the NuPAGE^ method of polyacrylamide gel electrophoresis and were not revealed when the Laemmli method was employed. Using more than one molecular property of PrPsc to characterize HY and DY TME analyzed by anti-PrP antibodies to different regions of the prion protein revealed differences between the two prion strains that were not observed when only one molecular property of PrPsc was determined with one anti-PrP antibody.|The influence of the host prion protein genotype on the strain-specific molecular properties of PrP^' was also investigated. Passage of DY TME from the Syrian hamster (SHa) into the Armenian hamster (AHa) alters the PK cleavage sites on DY PrPsc, which is suggestive of an altered conformation of DY PrPsc in the AHa. It was hypothesized that differences in the prion protein genotypes of the SHa and the AHa result in the altered conformation of DY PrPsc in the AHa. The PrP cell-free conversion reaction (CFCR) was performed with AHaPrPsc and DY PrPsc to investigate this hypothesis. The molecular weight of the PK treated conversion products did not mimic those of PK treated DY PrPsc isolated from the AHa. Instead they resembled those of DY PrPsc isolated from the SHa. These results suggest that some factor(s) present in vivo in the AHa but not in the CFCR was involved in determining the conformation of DY PrPsc following passage of DY TME into the AHa. To investigate this, the CFCR was performed in the presence of uninfected AHa brain homogenate or the chaperone protein GroEL. These modifications of the CFCR did not alter the molecular weights of the PK treated conversion products. This does not rule out that a factor present i?? vivo is involved in determining the conformation of DY PrPsc in the AHa, as the concentrations of the factor(s) present in the AHa brain homogenate may have been too low to exert an effect. The results of this study suggest that some factor(s) other than the conformation of PrPsc or the amino acid sequence of the host prion protein is involved in determining strain-specific properties of PrPsc.