Purification and properties of the cellular and scrapie hamster prion proteins

Abstract

During scrapie infection an abnormal isoform of the prion protein (PrP), designated PrPSc, accumulates and is found to copurify with infectivity; to date, no nucleic acid has been found which is scrapie-specific. Both uninfected and scrapie-infected cells synthesize a PrP isoform, denoted PrPC, which exhibits physical properties that differentiate it from PrPSc. PrPC was purified by immunoaffinity chromatography using a PrP-specific monoclonal antibody cross-linked to protein-A--Avidgel. PrPSc was purified by detergent extraction, poly(ethylene glycol) precipitation and repeated differential centrifugation of PrPSc polymers. Both PrP isoforms were found to have the same N-terminal amino acid sequence which begins at a predicted signal peptide cleavage site. The first 8 residues of PrPC were found to be KKXPKPGG and the first 29 residues of PrPSc were found to be KKXPKPGGWNTGGSXYPGQGSPGGNRYPP. Arg residues 3 and 15 in PrPSc and 3 in PrPC appear to be modified since no detectable signals (denoted X) were found at these positions during gas-phase sequencing. Both PrP isoforms were found to contain an intramolecular disulfide bond, linking Cys 179 and 214, which creates a loop of 36 amino acids containing the two N-linked glycosylation sites. Development of a purification protocol for PrPC should facilitate comparisons of the two PrP isoforms and lead to an understanding of how PrPSc is synthesized either from PrPC or a precursor.

Detection of the Disease-Associated Isoform of the Prion Protein in Formalin-Fixed Tissues by Western Blot

“…The 3 bands characteristic of un-, mono-, and di-glycosylated PrP Sc between 20 and 30 kD were clearly present in all positive samples. 13 As expected, proteinase K digested all detectable PrP C under conditions of the experiment and the lane containing a negative sample was observed as an empty lane. Shown in Fig.…”

Dynamics of a truncated prion protein, PrP(113–231), from 15N NMR relaxation: Order parameters calculated and slow conformational fluctuations localized to a distinct region

“…It is yet to be established whether PrP Sc retains any of the structural elements found in PrP C , although recent data suggests that residues 160-220 undergo significant conformation change to form amyloid fibers. 13,24 In contrast, modeling based on 2D crystals, 46 and epitope mapping studies 47 of PrP C and PrP Sc , as well as the presence of the intra-molecular disulphide in PrP Sc , 48 suggests that much of helix B and C may be retained in the scrapie form. At face value, the J(0) values hint at the possibility that helix A of PrP C has relatively more flexibility than helix B and C. However, the effect of anisotropic tumbling needs to be taken into account.…”

Cell-free formation of protease-resistant prion protein

“…The first 22 amino acids are a signal peptide that is cleaved during biosynthesis. 29,30 After the signal peptide is a region that contains 5 octarepeats of sequence: PHGGGWGQ.…”