Regular Article
The Metabolism and Imaging in Live Cells of the Bovine Prion Protein in Its Native Form or Carrying Single Amino Acid Substitutions

https://doi.org/10.1006/mcne.2000.0953Get rights and content

Abstract

Prion diseases are probably caused by an abnormal form of a cellular glycoprotein, the prion protein. Recent evidence suggests that the prion strain causing BSE has been transmitted to humans, thereby provoking a variant form of Creutzfeldt–Jacob disease. In this work, we analyzed the behavior of normal and malformed isoforms of the bovine PrP in transfected mammalian cell lines. Biochemical and immunocytochemical assays were complimented with imaging of live cells expressing fusion constructs between PrP and GFP. Bovine homologues of human E200K and D178N (129M) mutations were used as models of pathogenic isoforms. We show that the GFP does not impair the metabolism of native and mutant bPrPs and is thus a valid marker of PrP cellular distribution. We also show that each amino acid replacement provokes alterations in the cell sorting and processing of bPrP. These are different from those ascribed to both murine mutant homologues. However, human and bovine PrPs carrying the D178N genotype had similar cellular behavior.

References (67)

  • J. Lippincott-Schwartz et al.

    Rapid redistribution of Golgi proteins into the ER in cells treated with brefeldin A: Evidence for membrane cycling from Golgi to ER

    Cell

    (1989)
  • A. Negro et al.

    Susceptibility of the prion protein to enzymic phosphorylation

    Biochem. Biophys. Res. Commun.

    (2000)
  • R.B. Petersen et al.

    Effect of the D178N mutation and the codon 129 polymorphism on the metabolism of the prion protein

    J. Biol. Chem.

    (1996)
  • S.B. Prusiner et al.

    Scrapie prions aggregate to form amyloid-like birefringent rods

    Cell

    (1983)
  • N. Singh et al.

    Prion protein aggregation reverted by low temperature in transfected cells carrying a prion protein gene mutation

    J. Biol. Chem.

    (1997)
  • N. Stahl et al.

    Scrapie prion protein contains a phosphatidylinositol glycolipid

    Cell

    (1987)
  • W. Swietnicki et al.

    Familial mutations and the thermodynamic stability of the recombinant human prion protein

    J. Biol. Chem.

    (1998)
  • R.G. Will et al.

    A new variant of Creutzfeldt-Jakob disease in the UK

    Lancet

    (1996)
  • D.C. Bolton et al.

    Identification of a protein that purifies with the scrapie prion

    Science

    (1982)
  • D.C. Bolton et al.

    Scrapie PrP 27–30 is a sialoglycoprotein

    J. Virol.

    (1985)
  • N. Bons et al.

    Natural and experimental oral infection of nonhuman primates by bovine spongiform encephalopathy agents

    Proc. Natl. Acad. Sci. USA

    (1999)
  • D.R. Brown et al.

    The cellular prion protein binds copper in vivo

    Nature

    (1997)
  • M.E. Bruce et al.

    Transmission to mice indicate that ‘new variant’ CJD is caused by the BSE agent

    Nature

    (1997)
  • B. Caughey et al.

    Prion protein biosynthesis in scrapie-infected and uninfected neuroblastoma cells

    J. Virol.

    (1989)
  • F.E. Cohen et al.

    Structural clues to prion replication

    Science

    (1994)
  • J. Collinge et al.

    Molecular analysis of prion strain variation and the aethiology of ‘new variant’ CJD

    Nature

    (1996)
  • D.G. Donne et al.

    Structure of the recombinant full-length hamster prion protein PrP(29-231): The N-terminus is highly flexible

    Proc. Natl. Acad. Sci. USA

    (1997)
  • L. Ellgaard et al.

    Setting the standards: quality control in the secretory pathway

    Science

    (1999)
  • P. Gambetti et al.

    Fatal familial insomnia and familial Creutzfeldt-Jakob disease: Clinical, pathological and molecular features

    Brain Pathol.

    (1995)
  • P. Gambetti et al.

    Insomnia in prion diseases: Sporadic and familial

    N. Engl. J. Med.

    (1999)
  • F.L. Garcia et al.

    NMR structure of the bovine prion protein

    Proc. Natl. Acad. Sci. USA

    (2000)
  • L.G. Goldfarb et al.

    Fatal familial insomnia and familial Creutzfeldt-Jakob disease: Disease phenotype determined by a DNA polymorphism

    Science

    (1992)
  • J.S. Griffith

    Self-replication and scrapie

    Nature

    (1967)
  • Cited by (62)

    • Interaction between the cellular prion (PrP<sup>C</sup>) and the 2P domain K<sup>+</sup> channel TREK-1 protein

      2006, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      To exclude interference of the fluorescent protein tags in the physiological post-translational modifications of PrPC and TREK-1, an immunoblot analysis of the PrPC/DsRed and TREK-1/EGFP proteins in transfected HeLa cells was performed. In particular, PrPC/DsRed revealed an hetero-disperse signal of 50–75 kDa, likely attributed to un-, mono-, and di-glycosylated isoforms, similar to the electrophoretic pattern recently described for the GFP-bPrP construct in mammalian transfected cell lines [34]. Furthermore, PrPC glycoforms heterogeneity has been previously described in cultured cell lines (reviewed in [5] and [35]).

    • GFP-tagged PrP supports compromised prion replication in transgenic mice

      2006, Biochemical and Biophysical Research Communications
      Citation Excerpt :

      In agreement with previous cell culture studies [4–7], Western blots of extracts from stably transfected SMB and SMB-PS cells demonstrated that EGFPrP-N existed predominantly as multiple glycoforms migrating at ∼60–72 kDa with an additional fragment migrating at ∼55 kDa in uninfected SMB-PS cells (Fig. 1A). As demonstrated previously [4,5], release of EGFPrP-N from the surface of stably transfected SMB and SMB-PS cells with phosphatidylinositol phospholipase C (PIPLC) indicated that the fusion protein was correctly targeted to the plasma membrane and anchored to the cell surface by a GPI lipid anchor (data not shown). The persistence of a 3F4-reactive EGFPrP-N protease-resistant core corresponding to PrP27–30 following PK treatment of extracts from SMB cells stably expressing EGFPrP-N (Fig. 1A, lane 8) demonstrated that EGFPrPC-N was converted to EGFPrPSc-N.

    View all citing articles on Scopus
    1

    To whom correspondence and reprint requests should be addressed. Fax: +39 049 807 3310. E-mail: [email protected].

    View full text