NR AWJG
AU Lund,C.; Olsen,C.M.; Tveit,H.; Skogtvedt,S.; Tranulis,M.A.
TI Cleavage and subcellular localisation of the ovine prion protein in transfected cell lines
QU International Conference - Prion 2006: Strategies, advances and trends towards protection of society - 3.10.-6.10.2006, Torino, Italy, Lingotto Conference Centre - Poster sessions CE-25
PT Konferenz-Poster
AB In normal brain and cell-culture, PrPc is cleaved near amino acid 113 (ovine numbering), separating the unstructured N-terminal tail from the globular C-terminal two-thirds of the molecule. This processing is found in a wide variety of species indicating an important event in PrP metabolism. The cellular site and the functional purpose of the processing are still unsettled. Whether the processing is of relevance to the development and/or sensitivity to prion disease, analogous to the processing of the Alzheimer peptide amyloid beta, also remains to be investigated. We have used several approaches to study the localisation of the major prion protein cleavage fragments: The murine (N2a) and humane (SH-SY5Y) neuroblastoma cells were transiently and stably transfected with the different ovine PrP-constructs. Constructs of the ovine PrP, with and without tags and GPI-anchor were made. To facilitate the studies of the short N-terminal cleavage product (9-10 kDa), a construct of PrP with a green fluorescent protein (GFP) in the N-terminal part was made. Other constructs had red fluorescent protein C-terminally with or without GFP N-terminally. By use of live imaging of fluorescent "tags" and immunohistochemistry with different monoclonal antibodies (Mabs), the full-size molecule and its two main cleavage products could be traced in the cell lines. To test whether structural alterations in the conserved hydrophobic domain of PrP could influence the processing, we introduced a number of amino acid substitutions and deletion mutants by site directed mutagenesis.
AD C. Lund, C.M. Olsen, S. Skogtvedt, M.A. Tranulis: Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, Oslo; H. Tveit: Department of Molecular Biosciences, University of Oslo
SP englisch
PO Italien