Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane.
Sphingolipid microdomains are thought to result from the organization of plasma membrane sphingolipids and cholesterol into a liquid ordered phase, wherein the glycosylphosphatidylinositol (GPI)-anchored proteins are enriched. These domains, resistant to extraction by cold Triton X-100, can be isolated as buoyant membrane complexes (detergent-resistant membranes) in isopycnic density gradients. Here the effects of methyl-beta-cyclodextrin (MBCD), a specific cholesterol-binding agent that neither binds nor inserts into the plasma membrane, were investigated on the sphingolipid microdomains of lymphocytes. MBCD released substantial quantities of GPI-anchored Thy-1 and glycosphingolipid GM1, and also other surface proteins including CD45, and intracellular Lck and Fyn kinases. From endothelial cells, MBCD released GPI-anchored CD59, and CD44, but only a negligible amount of caveolin. Most MBCD-released Thy-1 and CD59 were not sedimentable and thus differed from Thy-1 released by membrane-active cholesterol-binding agents such as saponin and streptolysin O, or Triton X-100. Unlike that released by Triton X-100, only part of the Thy-1 molecules released by MBCD was buoyant in density gradients and co-isolated with GM1. Finally, treatment of Triton X-100-isolated detergent-resistant membranes with MBCD extracted most of the cholesterol without affecting the buoyant properties of Thy-1 or GM1. We suggest that (1) MBCD preferentially extracts cholesterol from outside, rather than within the sphingolipid microdomains and (2) this partly solubilizes GPI-anchored and transmembrane proteins from the glycerophospholipid-rich membrane and releases sphingolipid microdomains in both vesicular and non-vesicular form.
Ilangumaran
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Department of Pathology, Centre Médical Universitaire, 1 rue Michel Servet, 1211 Geneva 4, Switzerland.
Hoessli
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DC
D000818:Animals; D018957:CD59 Antigens / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002784:Cholesterol / Q000378:metabolism*; D003505:Cyclodextrins / Q000378:metabolism / Q000494:pharmacology*; D003902:Detergents / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects; D005677:G(M1) Ganglioside / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism; D017493:Leukocyte Common Antigens / Q000378:metabolism; D008214:Lymphocytes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D011505:Protein-Tyrosine Kinases / Q000187:drug effects / Q000378:metabolism; D013107:Sphingolipids / Q000737:chemistry / Q000378:metabolism*; D018800:Thy-1 Antigens / Q000378:metabolism; D047392:beta-Cyclodextrins
C495540:CAV1 protein, human; D018957:CD59 Antigens; C495541:Cav1 protein, mouse; D051242:Caveolin 1; D022461:Caveolins; D003505:Cyclodextrins; D003902:Detergents; D017261:Glycosylphosphatidylinositols; D018960:Hyaluronan Receptors; D008565:Membrane Proteins; D013107:Sphingolipids; D018800:Thy-1 Antigens; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D005677:G(M1) Ganglioside; D002784:Cholesterol; D011505:Protein-Tyrosine Kinases; D017493:Leukocyte Common Antigens