NR AXJG
AU Bellon,A.; Solforosi,L.; Schaller,M.; Abalos,G.C.; Cruite,J.; Laude,H.; Williamson,R.A.
TI Cellular Prion Protein Overexpression Promotes Prion-like Conformations in Transgenic Mice and Transfected Cells
QU International Conference - Prion 2007 (26.-28.9.2007) Edinburgh International Conference Centre, Edinburgh, Scotland, UK - Book of Abstracts: Oral Abstracts FC4.2
IA http://www.prion2007.com/pdf/Prion Book of Abstracts.pdf
PT Konferenz-Vortrag
AB PrPsc, an abnormal misfolded isoform of the cellular prion protein, PrPc, is a pathognomonic marker of prion disease. Diagnosis of prion infections is based largely on the detection of PrPsc, which is typically partially resistant to proteinase K (PK) digestion, but may also be present to varying degrees in a PK sensitive form. Our laboratory has developed three different PrPsc-reactive engineered antibodies, displaying PrP sequence grafts composed of amino acids 19-33, 89-112 and 136-158. These IgG reagents have been shown to bind robustly to PrPsc before or after PK treatment, but do not recognize PrPc. In this study, we have sought to evaluate the effect of PrP overexpression upon its conformation. PrP conformation in the brains of transgenic mice, and in transfected cultured cells, has been examined both immunologically, using the grafted IgGs 19-33, 89-112 and 136-158, and with the use of sucrose velocity gradients. Firstly, four transgenic mouse lines overexpressing mouse (tga20), hamster (tg7), human (tg650) and ovine (tg338) PrP between 4-fold to 8-fold over wild-type levels, were studied using an immunoprecipitation assay. PK sensitive forms of PrP reacting with all three of the motif-grafted antibodies were clearly immunoprecipitated from brain homogenates prepared from each of these mouselines, but not from wild type mice. This phenomenon was found to be independent of the age of the animals (within a 3-month to 15-month age range). Similarly, a significant quantity of PrPsc-like molecules was also recovered from lysates of transfected RK13 cells overexpressing mouse PrP following immunoprecipitation with IgGs 19-33, 89-112 and 136-158. Subsequently, PrP from the brains of tga20 mice was allowed to sediment through a sucrose gradient. These experiments indicated that, in contrast to equivalently prepared wild type brain samples, a subpopulation of PrP in the tga20 tissues can be observed in higher sucrose fractions, partially overlapping those from which PrPsc can be recovered from prion-infected brains. These findings indicate that a proportion of the PrP population encountered in tga20 brain tissue likely exists in a polymeric state. These data collectively indicate that PrPsc-like molecular forms, as characterized by antibody reactivity and separation profiles, can be detected in healthy non-prion infected transgenic mice overexpressing wild-type PrPc as well as in transfected cells also overexpressing PrP.
AD A. Bellon, L. Solforosi, M. Schaller, G. Abalos, J. Cruite, R.A. Williamson, The Scripps Research Institute, Immunology, USA; H. Laude, INRA, Virologie Immunologie Moleculaires, France
SP englisch
PO Schottland