engleski

Lipid raft isolation from mouse brain tissue under conditions that retain submembrane distribution of gangliosides and proteins

Cell membranes are not uniform in their protein and lipid composition but organized in microdomains termed lipid rafts with higher concentration of (glyco)sphingolipids, cholesterol, and specific membrane proteins. The investigation of lipid rafts is thriving since the disturbed lipid microenvironment is recognized as highly important in pathogenesis of numerous human disorders. It is challenging to isolate lipid rafts from the bulk membrane in a way that would accurately reflect their composition and organization in living cells. Most often, detergent-resistant membranes enriched in cholesterol and sphingomyelin are extracted using the non-ionic detergent Triton X-100 (Tx-100). However, Tx-100 causes a redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins, which makes it an unacceptable choice for investigating the exact relationship between membrane gangliosides and proteins. The aim of this work was to develop a method for lipid raft isolation that would retain ganglioside and protein distribution for the purposes of a larger study investigating the crosstalk between gangliosides and specific membrane proteins. Since no-detergent methods of lipid raft isolation were not sufficiently effective or reproducible in our hands, the detergent Brij O20 was used in extraction followed by optimized sucrose density ultracentrifugation. Successful raft isolation was confirmed by Western blotting of non-lipid raft membrane protein transferrin receptor and lipid raft markers protein flotilin and ganglioside GM1 detected by cholera toxin subunit B immuno-overlay. The distribution of these markers, as well as gangliosides GT1b, GD1a, and specific membrane proteins, was compared in lipid rafts and the bulk membrane isolated with Tx-100 and Brij O20. Ganglioside distribution was found to be different as a result of Tx-100 vs. Brij020 isolation. Using Tx-100, the majority of ganglioside GD1a appears in raft fractions with almost no staining in bulk membrane, while Brij O20 isolation reveals a higher proportion of GD1a in the bulk membrane. Striking evidence for protein redistribution in Tx-100 isolation was found for transmembrane protein neuroplastin (Np) which is known to be affected by ganglioside environment. In Tx-100 isolation, the vast majority of Np is segregated in non- raft fractions (90% of all Np), compared to Brij O20 isolation where around 70% of Np is distributed in non-raft fractions. We show that isolating lipid rafts using Tx-100 leads to redistribution of gangliosides and specific proteins. This work will enable more accurate lipid raft analysis in respect to ganglioside and membrane proteins composition and lead to improved resolution of lipid- protein relations within lipid rafts.

Lipid rafts, gangliosides, neuroplastin, Brij O20, Triton X-100

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