Purification and Properties of the Cellular and Scrapie Hamster Prion Proteins

Eur J Biochem. 1988 Sep 1;176(1):21-30. doi: 10.1111/j.1432-1033.1988.tb14246.x.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Brain Chemistry
  • Cricetinae
  • Cross Reactions
  • Disulfides / analysis
  • Electrophoresis, Polyacrylamide Gel
  • Immunologic Techniques
  • Mesocricetus
  • Prions / analysis*
  • Protein Processing, Post-Translational
  • Scrapie / metabolism*
  • Transcription, Genetic
  • Viral Proteins / genetics
  • Viral Proteins / isolation & purification*

Substances

  • Disulfides
  • Prions
  • Viral Proteins