NR ATHZ
AU Lund,C.; Olsen,C.M.; Tveit,H.; Ersdal,C.; Prydz,K.; Harbitz,I.; Tranulis,M.A.
TI Proteolytic processing of the prion protein in cell culture
QU International Conference - Prion 2005: Between fundamentals and society's needs - 19.10.-21.10.2005, Congress Center Düsseldorf - Poster Session: Cell Biology of PrPc and PrPsc CELL-31
PT Konferenz-Poster
AB
In brain and other tissues, as well as in cell culture, the cellular prion protein (PrPc), undergoes a conserved proteolytic processing around amino acid 113 (ovine numbering), separating the unstructured N-terminal tail from the globular C-terminal two-thirds of the molecule. There are several hypotheses concerning the cellular site and purpose of this cleavage, but the debate is not yet settled.
Here we present data from comparative studies of the processing of ovine PrP in four cell lines; murine (N2a) and human (SH-SY5Y) neuroblastoma cells, human adenocarcinoma cells (LoVo) and canine epithelial kidney cells (MDCK).
We have established a well suited model for studying the processing of PrP by use of green fluorescent protein (GFP) as a molecular tag. This, combined with a panel of monoclonal antibodies (Mabs), made the study of full-size molecules and its two main cleavage products highly efficient. PrP appears to be processed similarly in all cell lines in an amazingly robust process. Interestingly, the major N-terminal cleavage product is retained within the cells in all assays. We also show that absence of GPI-anchor and N-glycan modifications do not affect the proteolytic processing of PrP. Such processing also appears to be unrelated to the protein convertase Furin or other Furin-dependent proteolytic enzymes, since PrP is effectively cleaved in the Furin deficit LoVo cells. Furthermore, inhibition of the processing by traditional proteinase inhibitors, such as leupeptin, aprotinin, galardin (Zn-metalloproteinase inhibitor) or proteasome inhibitors, such as lactacystin or by insertion of mutations affecting the putative cleavage site, appears very hard to achieve. Bafilomycin A1 which is an inhibitor of vacuolar proton pumps (H+/ATPases) does however reduce the proteolytic processing of PrP.
AD Christoffer Lund, Christel M. Olsen, Cecilie Ersdal, Ingrid Harbitz, Michael A. Tranulis, Norwegian School of Veterinary Science, Norway; Heidi Tveit, Kristian Prydz, University of Oslo, Norway
SP englisch
PO Deutschland