Effect of sterol carrier protein-2 expression on sphingolipid distribution in plasma membrane lipid rafts/caveolae.
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.
Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 77843-4466, USA.
Barbara P
BP
Atshaves
John R
JR
Jefferson
Avery L
AL
McIntosh
Adalberto
A
Gallegos
Bonnie M
BM
McCann
Kerstin K
KK
Landrock
Ann B
AB
Kier
Friedhelm
F
Schroeder
D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D021941:Caveolae / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007739:L Cells; D021962:Membrane Microdomains / Q000378:metabolism*; D051379:Mice; D020383:Neutral Glycosphingolipids / Q000378:metabolism; D010767:Phosphorylcholine / Q000494:pharmacology; D013107:Sphingolipids / Q000378:metabolism*; D013109:Sphingomyelins / Q000378:metabolism
D002352:Carrier Proteins; D020383:Neutral Glycosphingolipids; D013107:Sphingolipids; D013109:Sphingomyelins; C072087:sterol carrier proteins; D010767:Phosphorylcholine