Effect of Sterol Carrier Protein‐2 Expression on Sphingolipid Distribution in Plasma Membrane Lipid Rafts/Caveolae

Abstract

Although sphingolipids are highly important signaling molecules enriched in lipid rafts/caveolae, relatively little is known regarding factors such as sphingolipid binding proteins that may regulate the distribution of sphingolipids to lipid rafts/caveolae of living cells. Since early work demonstrated that sterol carrier protein-2 (SCP-2) enhanced glycosphingolipid transfer from membranes in vitro, the effect of SCP-2 expression on sphingolipid distribution to lipid rafts/caveolae in living cells was examined. Using a non-detergent affinity chromatography method to isolate lipid rafts/caveolae and non-rafts from purified L-cell plasma membranes, it was shown that lipid rafts/caveolae were highly enriched in multiple sphingolipid species including ceramides, acidic glycosphingolipids (ganglioside GM1); neutral glycosphingolipids (monohexosides, dihexosides, globosides), and sphingomyelin as compared to non-raft domains. SCP-2 overexpression further enriched the content of total sphingolipids and select sphingolipid species in the lipid rafts/caveolae domains. Analysis of fluorescence binding and displacement data revealed that purified human recombinant SCP-2 exhibited high binding affinity (nanomolar range) for all sphingolipid classes tested. The binding affinity decreased in the following order: ceramides > acidic glycosphingolipid (ganglioside GM1) > neutral glycosphingolipid (monohexosides, hexosides, globosides) > sphingomyelin. Enrichment of individual sphingolipid classes to lipid rafts/caveolae versus non-rafts in SCP-2 expressing plasma membranes followed closely with those classes most strongly bound to SCP-2 (ceramides, GM1 > the neutral glycosphingolipids (monohexosides, dihexosides, and globosides) > sphingomyelin). Taken together these data suggested that SCP-2 acts to selectively regulate sphingolipid distribution to lipid rafts/caveolae in living cells.