Molecular characteristics of the scrapie agent
The prion diseases of humans and animals, including Creutzfeld-Jakob disease (CJD) and Kuru of humans, scrapie of sheep and Bovine Spongiform Encephalopothy (BSE), are a family of closely linked neurodegenerative disorders that have proved to be both heritable and transmittable. The aetiology of these disorders has been shown to be intrinsically linked to a novel infectious particle made up either solely or overwhelmingly of protein, the major component of which is the prion precursor protein (PrP). PrP is a 253 amino acid protein modified during synthesis by the addition of a glycophosphatidinol (GPI) anchor and either one or two N-linked sugar chains. PrP normally exists within the cell in a predominantly a-helical conformation (PrPc), but can adopt an alternative, predominantly p-sheet structure closely linked to the disease state. In this thesis, investigations into two of the molecular characteristics of the scrapie agent are described: the impact of the GPI anchor on infectivity and the biochemical effect of the codon 129 M/V polymorphism. The M/V polymorphism plays a major role in the disease process---with the codon 129 status of an individual modifying both susceptibility to disease and disease course. No alterations were observed in the stability, structure, protease resistance or copper binding properties of the a-helical form of PrP due to this polymorphism. Both forms had a similar propensity to form the P-rich isoform of PrP and, once in the P-rich conformation, no differences in structure, protease resistance or stability were observed between the two polymorphic variants. The codon 129 polymorphism was found, however, to have a dramatic impact on the ability of the prion protein to form ordered aggregates. To investigate the contribution of the GPI anchor to the infectivity of PrPSc, a novel proteolytic treatment was used to remove the C-terminal and GPI from the prion protein. The infectious properties of PrP80 lacking the GPI were examined using in vitro, cell based and in vivo models of prion replication. No alteration in the ability of C-terminally truncated PrP80 to propagate was observed.