NR AORA
AU Cancellotti,E.; Tuzi,N.L.; Baybutt,H.N.; Aitchison,L.; Bradford,B.; Manson,J.
TI Molecular and intracellular characterisation of gene targeted mice with altered glycosylated PrP
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - BR-25
PT Konferenz-Poster
AB
The PrP protein has two consensus sites for N-linked glycosylation (residues 180 and 196 in mouse) that can be occupied or not with different sugars. Mature PrP molecules are characterised by di- or mono- or un- glycosylated forms. Cell culture experiments have shown that the glycosylation can influence the PrP trafficking and alter its presence on the cell membrane. Thus it is possible that the localisation in different cell compartments of modified host PrP may influence the course of disease. The prion hypothesis proposes that an altered form of PrP is the infectious agent, but it is difficult to see how this can accommodate the observation of numerous strains of agent, each possessing characteristic incubation time and pathology. However it has been suggested that a post-translational modification such as the glycosylation of PrP could provide the protein with strain specificity.
In order to study the role of PrP glycosylation in both the localisation and normal function of PrP and to address its role in TSE disease, we have produced gene targeted transgenic mice with mutations at first, second or both PrP glycosylation sites. We introduced mutations into the PrP gene using site directed mutagenesis, which results in the partial or complete absence of N-linked glycosylation. Three mutant PrP genes have been produced N180T, N196T and both N180T and N196T. Cell lines were produced in which the endogenous murine PrP gene was replaced by the mutated PrP genes and used to produce transgenic mice with the glycosylation mutant PrP genes. The mutated PrP genes are expressed under the same control elements as the wild type gene. The level of PrP mRNA and protein has been assessed in the transgenic mice and we have demonstrated that the mutations prevent glycosylation at the altered sites. The effect of this altered glycosylation on the cellular trafficking and localisation of PrP will be presented.
AD Enrico Cancellotti, Nadia L. Tuzi, Herbert Baybutt, Lorraine Aitchinson, Barry Bradford, Jean Manson, Neuropathogenesis Unit, Institute for Animal Health, Edinburgh, UK
SP englisch
PO Deutschland