Proteolytic processing of the ovine prion protein in cell cultures

Biochem Biophys Res Commun. 2005 Nov 11;337(1):232-40. doi: 10.1016/j.bbrc.2005.09.031.

Abstract

The cellular compartment and purpose of the proteolytic processing of the prion protein (PrP) are still under debate. We have studied ovine PrP constructs expressed in four cell lines; murine neuroblastoma cells (N2a), human neuroblastoma cells (SH-SY5Y), dog kidney epithelial cells (MDCK), and human furin-deficient colon cancer cells (LoVo). Cleavage of PrP in LoVo cells indicates that the processing is furin independent. Neither is it reduced by some inhibitors of lysosomal proteinases, proteasomes or zinc-metalloproteinases, but incubation with bafilomycin A1, an inhibitor of vacuolar H+/ATPases, increases the amount of uncleaved PrP in the apical medium of MDCK cells. Mutations affecting the putative cleavage site near amino acid 113 reveal that the cleavage is independent of primary structure at this site. Absence of glycosylphosphatidylinositol anchor and glycan modifications does not influence the proteolytic processing of PrP. Our data indicate that PrP is cleaved during transit to the cell membrane.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Cell Line
  • Cell Line, Tumor
  • Dogs
  • Enzyme Inhibitors / pharmacology
  • Green Fluorescent Proteins / genetics
  • Humans
  • Macrolides / pharmacology
  • Mice
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Prions / genetics
  • Prions / metabolism*
  • Protein Transport
  • Recombinant Fusion Proteins / metabolism
  • Sheep / metabolism
  • Vacuolar Proton-Translocating ATPases / antagonists & inhibitors

Substances

  • Enzyme Inhibitors
  • Macrolides
  • Prions
  • Recombinant Fusion Proteins
  • Green Fluorescent Proteins
  • bafilomycin A1
  • Vacuolar Proton-Translocating ATPases