title: Enzymatic degradation of PrPSc by a protease secreted from Aeropyrum pernix K1.
abstract: BACKGROUND
  An R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrP(Sc)) from different species (human, bovine, deer and mouse).
  
  
  RESULTS
  Degradation of the PrP(Sc) isoform by the R30 fraction and the purified protease was evaluated using the 6H4 anti-PrP monoclonal antibody. Fragments from the N-terminal and C-terminal of PrP(Sc) were also monitored by Western blotting using the EB8 anti-PrP monoclonal antibody, and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrP(Sc), the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 23 kDa. The purified pernisine was active between 58 °C and 99 °C, and between pH 3.5 and 8.0. The temperature and pH optima of the enzymatic activity of the purified pernisine in the presence of 1 mM CaCl(2) were 105 °C ± 0.5 °C and pH 6.5 ± 0.2, respectively.
  
  
  CONCLUSIONS
  Our study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest the pathological prion protein PrP(Sc).
authors: Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
authors: Marko
authors: M
authors: Snajder
authors: Tanja
authors: T
authors: Vilfan
authors: Maja
authors: M
authors: Cernilec
authors: Ruth
authors: R
authors: Rupreht
authors: Mara
authors: M
authors: Popović
authors: Polona
authors: P
authors: Juntes
authors: Vladka Čurin
authors: VČ
authors: Serbec
authors: Nataša Poklar
authors: NP
authors: Ulrih
mesh_terms: D041522:Aeropyrum / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D045726:Metalloproteases / Q000032:analysis* / Q000378:metabolism; D051379:Mice; D018620:PrPSc Proteins / Q000378:metabolism*; D020033:Protein Isoforms / Q000378:metabolism
chemical_list: D018620:PrPSc Proteins; D020033:Protein Isoforms; D045726:Metalloproteases