NR AXKN
AU Connolly,J.G.; Tate,R.J.; McLoughlin,V.; Norrby,K.; Jones,M.; MacGregor,I.R.; Baybutt,H.; Farquhar,C.F.; Head,M.W.
TI Comparison of Glycosylated Hamster, Mouse and Human Prion Protein Expressed in Xenopus Oocytes
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Protein Misfolding P01.69
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Poster
AB The starting point for understanding prion protein misfolding is the characterisation of normal genotypes in their native state. We have therefore expressed wild type mouse, hamster and human prion proteins, as well as 3F4-tagged mouse prion protein, in Xenopus oocytes. Oocytes were separately injected with RNAs encoding the various prion proteins and crude membrane fractions of the expressed proteins were extracted and assayed by Western Blotting using 3F4 and 6H4 monoclonal antibodies. All expressed proteins were recognised by both antibodies, except for wild type mouse prion protein which as expected was not recognised by 3F4. The expressed prion proteins were similar in size and abundance (>100ng/oocyte) to prion protein extracted from human brain. Non-glycosylated, mono-glycosylated, di-glycosylated and additional, higher molecular glycoforms were expressed. The non-glycosylated forms were similar in size to recombinant human and hamster prion protein and all forms were proteinase K sensitive (50 µg/ml, 1 hour, 37 °). Following treatment with PNGase F, the glycoforms reduced to a full length, non-glycosylated band although truncated forms of prion protein were also detected in some extracts. PIPLC treatment of intact oocytes expressing 3F4-tagged mouse prion protein reduced the amount of protein detectable by Western blotting compared to a matched set of controls. This suggests that prion protein was present on the cell surface and had been liberated by cleavage at the GPI anchor. Supporting this interpretation, prion protein could be detected on the surface of intact live oocytes by placing them directly upon wetted PVDF membrane and then immunoblotting the imprint. Thus oocytes offer a very versatile system for producing large amounts of wild type and mutant glycosylated prion proteins from several species and for studying their folding and biochemical properties under native conditions.
AD J.G. Connolly, R.J. Tate, V. McLoughlin, University of Strathclyde, S. I. Pharmacy & Biomedical Sciences, UK; K. Norrby, M. Jones, University of Edinburgh, Western General Hospital, National CJD Surveillance Unit, UK; I.R. MacGregor, Scottish Blood Transfusion Service, National Science Laboratory, UK; H. Baybutt, C.F. Farquhar, Neuropathogenesis Unit, UK; M.W. Head, University of Edinburgh, Western General Hospital, National CJD Surveillance Unit, UK
SP englisch
PO Schottland
EA pdf-Datei und Poster (geänderte Reihenfolge in der Autorenliste)