NR AOVU
AU Levin,J.; Giese,A.; Bieschke,J.G.; Weber,P.; Bertsch,U.; Kretzschmar,H.A.
TI Probing the molecular composition of prion protein aggregates by dual-colour SIFT
QU International Conference - Prion diseases: from basic research to intervention concepts - TSE-Forum, 08.10.-10.10.2003, Gasteig, München - Poster session - DG-55
PT Konferenz-Poster
AB
PrPsc exists in different conformations or types, which are believed to encode different prion strains. These subtypes are showing different glycosylation patterns and different sizes of the PrP-fragment after PK-digestion. Different clinico-pathological phenotypes of sporadic CJD can be grouped into six variants reflecting the combination of two types of PrPsc and three genetic subtypes due to the M/V polymorphism at amino acid 129 of the human prion protein.
We have used a novel approach based on fluorescence correlation spectroscopy to develop a highly sensitive detection method for protein aggregates termed SIFT (Scanning for Intensely Fluorescent Targets). In addition to high sensitivity that can be exploited in diagnostic applications, dual-colour SIFT allows characterization of the labelling ratio for two separate probe molecules on a single particle level. Thus, this technique can be used to probe the composition of prion protein aggregates and to study the co-aggregation of different prion protein variants and the relative binding affinity of prion protein variants to purified PrPsc aggregates.
In a de novo aggregation assay, we could show that recombinant human PrP with methionine at codon 129 and recombinant human PrP with valine at codon 129 are capable of forming co-aggregates. In further experiments, we investigated the binding of these variants of human prion protein and other probe molecules such as monoclonal antibodies to purified prion rods from different molecular variants of CJD. We could show that different PrPsc preparations could be discriminated by different labelling ratios for certain probe molecules. This technique might be a future tool for efficient and fast prion detection and typing as well as for analysis of the molecular basis of species and strain barriers for prion transmission.
AD Johannes Levin, Armin Giese, Jan Bieschke, Petra Weber, Uwe Bertsch, Hans Kretzschmar, LMU Munich, Germany
SP englisch
PO Deutschland