Role of lipid rafts and GM1 in the segregation and processing of prion protein.

Laura Botto,Silvia Coco,Alessandro Negro,Roberto Chiesa,Paola Palestini,Emiliano Biasini,Silvia Sesana,Diana Cunati,Alessandra Bulbarelli,Laura Colombo,Massimo Masserini,Sophie Mouillet-Richard

doi:10.1371/journal.pone.0098344

Abstract

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.

Highlights

PrPC was first identified as a normal cellular protein almost 30 years ago [1], but its physiological function remains uncertain
Secondary antibodies for enhanced chemiluminescence (ECL) detection, anti-mouse and/or anti-rabbit-HRP conjugates were from Pierce; PNGasi F from New England Biolabs; Cholerae Toxin B subunit (CTB) conjugate Alexa Fluor 594 and secondary antibodies conjugate with Alexa Fluor 594 and Alexa Fluor 488 were from Invitrogen
PrPC distribution in cerebellar granule cells (CGCs) To monitor the enrichment of PrPC in lipid rafts, we fractionated cold-detergent lysates of CGCs by sucrose gradient and analyzed the fractions by immunoblotting with either 6H4Ab, that recognizes an epitope between residues 144–152, or SAF32Ab that recognizes residues 79–92 in the octapetide repeat region (Fig. 1A)

Summary

Introduction

PrPC was first identified as a normal cellular protein almost 30 years ago [1], but its physiological function remains uncertain. The proposed functions of PrPC are related to its localization on the cell surface. PrPC is synthesized in the secretory pathway and the mature form is N-glycosylated and anchored to the cell surface by means of a glycosylphosphatidylinositol (GPI)anchor. GPI-anchored PrPC is present in lipid rafts, microdomains enriched in cholesterol, gangliosides, sphingomyelin and acylated proteins, related to a wide range of biological processes, including intracellular trafficking, transmembrane signalling, lipid and protein sorting, viral uptake and regulated proteolysis [3,4]. PrPSc (scrapie prion protein), is the misfolded isoform of PrPC and is the main cause for a group of fatal neurodegenerative disorders known as prion diseases or transmissible spongiform encephalopathies, including Creutzfeldt–Jakob disease, Gerstmann–Straussler–Scheinker syndrome, fatal familial insomnia and kuru in humans, scrapie in sheep, bovine spongiform encephalopathy in cattle and chronic wasting disease in deer and elk [2].

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