Characterization of prion protein-enriched domains, isolated from rat cerebellar granule cells in culture

The biological functions of (PrPC) and its possible interaction with other specific molecular membrane partners remain largely unknown. The aim of this study is to gain information on the molecular environment of PrPC by analyzing the lipid and protein composition of a PrPC-enriched membrane subfraction, called Prion Domain, PrD. This domain was obtained by immunoprecipitation of Detergent-Resistant Microdomains, DRM, of rat cerebellar granule cells under conditions designed to preserve lipid-mediated membrane organization. The electrophoretic pattern of PrD, after staining with Coomassie blue, showed the enrichment of some protein bands in comparison with DRM. μLC-ESI-MS/MS analysis showed that Thy-1 and different types of myosin were strongly enriched in PrD and, in a lesser extent, also OBCAM, LSAMP and tubulin, present altogether in a single band. Experiments using the chemical crosslinker BS3 suggested the existence of an interaction between PrPC and NCAM. Concerning lipids, the comparison between PrD and DRM showed a similar phospholipid/sphingolipid ratio, a phospholipid/cholesterol ratio doubled, and a strong decrease of plasmenilethanolamine (19,7 ± 3,5 % vs 38.3 ± 1.2 %). In conclusion, the peculiar lipid composition and in particular the presence of proteins involved in synaptic plasticity, cell adhesion, cytoskeleton regulation and signaling, suggest an important physiological role in neurons of Prion Domain.