The subcellular trafficking of the prion protein: Characterisation of the function of the PrPc N-terminus

Transmissible spongiform encephalopathies (TSEs) are degenerative diseases of the central nervous system in humans and animals, and include Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep and bovine spongiform encephalopathy in cattle. These spongiform encephalopathies can manifest as sporadic, familial and acquired disorders and are caused by the conformational alteration of the non-pathogenic cellular prion protein (PrPc) into a infectious isoform denoted PrPSc. The latter therefore represents a pathogenic agent (prion) which does not contain nucleic acids. During biogenesis, PrPc undergoes posttranslational modifications with the addition of two N-linked carbohydrate chains and a glycosyl-phosphatidyl-inositol-(GPI)-anchor. Properly folded PrPc transits through the Golgi compartment and the secretory pathway and is attached to the outer leaflet of the plasma membrane by the GPI-anchor. The cellular function of the prion protein is still unknown, although binding of copper to the octapeptide repeat sequence located at its N-terminus suggests a role of PrPc related to this phenomenon. In the present work, the physiological function of the N-terminal part of PrPc in subcellular trafficking was analysed. Metabolic labelling and surface-biotinylation assays were performed in order to compare the intracellular trafficking and turnover of PrPc mutants showing specific deletions within their N-terminal sequence with those of wild type PrPc (wtPrPc). Upon transient expression of these constructs in murine neuroblastoma cells, these deletions, although not influencing the biochemical properties or the cell surface expression of these proteins, lead to a delay in their endocytosis. The prolongation of the internalisation kinetics was shown to be dependent on the length of the deletion: truncation of the complete N-terminus leads to the almost complete inhibition of internalisation. The analysis of the kinetics of degradation showed a similar correlation with the N-terminal part of PrPc, since the half-life of the PrP-mutants was significantly prolonged when compared to that of the wild type protein. Additionally performed detailed analysis of the secretory pathway with immunoprecipitation assays showed that N-terminally truncated PrP molecules reach the plasma membrane at a later time point, when compared with wtPrPc. A closer analysis of the processing of the sugar molecules linked to these proteins performing an Endo-H digestion revealed that this delay in the transport to the cell surface takes place in a cellular compartment following the mid-Golgi. The following studies were done with a chimeric protein consisting of the short N-terminal segment of Xenopus laevis, which does not contain the copper-binding octarepeat region, fused to the N-terminally truncated mouse PrPc. These studies showed that endocytosis of this protein and its transport through the secretory pathway were comparable to those of the mouse wtPrPc. It was therefore concluded that the N-terminus belonging to a phylogenetically remote species can rescue the wild type trafficking phenotype. These results indicate that the N-proximal domain of the prion protein functions as a targeting element and is essential for both transport to the plasma membrane and modulation of endocytosis. The data support a model in which the N-terminal part of PrPc represents an epitope for binding to a transmembrane receptor containing internalisation-promoting motifs or for inclusion of PrPc into the secretory raft-compartments. The present work also indicates for the first time that copper affinity of the octarepeats and subcellular trafficking represent separate aspects in the life-cycle of the prion protein.

Level thesis:degree_level

thesis.doctoral

Grantor dc:publisher

Ludwig-Maximilians-Universität

Year

2003

Author dc:creator

• Nunziante, Maximilian

Repository record source_url

https://edoc.ub.uni-muenchen.de/1418/

OAI identifier oai:identifier

oai:edoc.ub.uni-muenchen.de:1418