Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane

TLDR

Sphingolipid microdomains form liquid‑ordered, cholesterol‑rich membrane domains enriched in GPI‑anchored proteins and can be isolated as detergent‑resistant, buoyant complexes. The study examined how methyl‑β‑cyclodextrin, a cholesterol‑binding agent that does not insert into membranes, affects these microdomains in lymphocytes. MBCD preferentially extracts cholesterol from the outer membrane, releasing GPI‑anchored proteins (Thy‑1, GM1, CD45, Lck, Fyn, CD59, CD44) and solubilizing transmembrane proteins while sparing caveolin, producing both vesicular and non‑vesicular microdomains distinct from those released by Triton X‑100.

Abstract

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-β-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.