RESUMO
The prion protein (PrPC) is highly expressed within the nervous system. Similar to other GPI-anchored proteins, PrPC is found in lipid rafts, membrane domains enriched in cholesterol and sphingolipids. PrPC raft association, together with raft lipid composition, appears essential for the conversion of PrPC into the scrapie isoform PrPSc, and the development of prion disease. Controversial findings were reported on the nature of PrPC-containing rafts, as well as on the distribution of PrPC between rafts and non-raft membranes. We investigated PrPC/ganglioside relationships and their influence on PrPC localization in a neuronal cellular model, cerebellar granule cells. Our findings argue that in these cells at least two PrPC conformations coexist: in lipid rafts PrPC is present in the native folding (α-helical), stabilized by chemico-physical condition, while it is mainly present in other membrane compartments in a PrPSc-like conformation. We verified, by means of antibody reactivity and circular dichroism spectroscopy, that changes in lipid raft-ganglioside content alters PrPC conformation and interaction with lipid bilayers, without modifying PrPC distribution or cleavage. Our data provide new insights into the cellular mechanism of prion conversion and suggest that GM1-prion protein interaction at the cell surface could play a significant role in the mechanism predisposing to pathology.
Assuntos
Gangliosídeo G(M1) , Microdomínios da Membrana , Proteínas PrPC , Proteínas PrPSc , Proteólise , Animais , Gangliosídeo G(M1)/química , Gangliosídeo G(M1)/metabolismo , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/química , Microdomínios da Membrana/metabolismo , Neurônios/metabolismo , Proteínas PrPC/química , Proteínas PrPC/metabolismo , Proteínas PrPSc/química , Proteínas PrPSc/metabolismo , Estrutura Secundária de Proteína , Ratos , Ratos Sprague-DawleyRESUMO
The biological functions of prion protein (PrP(C)) 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 PrP(C) by analyzing the lipid and protein composition of a PrP(C)-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. microLiquid chromatography-electrospray ionization-mass spectrometry (microLC-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 cross-linker BS(3) suggested the existence of an interaction between PrP(C) and neural cell adhesion molecule (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 signalling, suggest an important physiological role in neurons of Prion Domain.
