Effect of ceramide N-acyl chain and polar headgroup structure on the properties of ordered lipid domains (lipid rafts)

Abstract

Ceramides are sphingolipids that greatly stabilize ordered membrane domains (lipid rafts), and displace cholesterol from them. Ceramide-rich rafts have been implicated in diverse biological processes. Because ceramide analogues have been useful for probing the biological function of ceramide, and may have biomedical applications, it is important to characterize how ceramide structure affects membrane properties, including lipid raft stability and composition. In this report, fluorescence quenching assays were used to evaluate the effect of analogues of ceramide with different N-acyl chains or different sphingoid backbones on raft stability and sterol content. The effect of replacing 18 mol% of sphingomyelin (SM) with ceramide in vesicles composed of a 1:1 (mol:mol) mixture of SM and dioleoylphosphatidylcholine (DOPC), with or without 25 mol% sterol, was examined. In the absence of sterol, the thermal stability of the SM-rich ordered domains increased with ceramide N-acyl chain length in the order C2:0 approximately C6:0 approximately C8:0<no ceramide<C12:0<C16:0. In vesicles containing 25 mol% cholesterol (1:1:0.66 sphingolipid:DOPC:cholesterol), the dependence of raft stability on ceramide N-acyl chain length increased in the order C8:0 approximately C6:0<C2:0<C12:0 approximately no ceramide<C16:0. We also studied the stability of lipid rafts in the presence of N-lauroyl- and N-palmitoylsphingosine analogues containing altered structures in or near the polar portion of the sphingoid base. In almost all cases, the analogues stabilized rafts to about the same degree as a normal ceramide containing the same acyl chain. The only exception was N-palmitoyl-4D-ribophytosphingosine, which was very strongly raft-stabilizing. We conclude that variations in sphingoid base structure induce only insignificant changes in raft properties. N-Lauroyl and N-palmitoylsphingosine and their analogues displaced sterol from rafts to a significant degree. Both C12:0 and C16:0 analogues of ceramide may be good mimics of natural ceramide, and useful for cellular studies in which maintenance of the normal physical properties of ceramide are important.

Sphingosine Kinase-1 Protects Multiple Myeloma from Apoptosis Driven by Cancer-Specific Inhibition of RTKs

“…1C) analyses revealed a dosedependent increase in cell surface ceramide levels in U266 cells following EGCG treatment. Displacement of cholesterol from the lipid raft on the plasma membrane occurs after generation of ceramide by ASM (17), and EGCG has been shown to induce disruption of cholesterol-rich lipid rafts in colon cancer cells (27). Therefore, we next investigated the involvement of ASM in EGCGinduced disruption of lipid raft domains by staining with the lipid-mimetic dialkyl-indocarbocyanine (DilC16) and using a cold Triton X-100 solubility assay ( Fig.…”

“…In addition, ASMinduced generation of ceramide leads to displacement of cholesterol from lipid rafts on the plasma membrane (17). However, whether ASM influences cholesterol-rich lipid raft formation, which is associated with receptor tyrosine kinase (RTK) activation (18), is unknown.…”

Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers

“…The first is ceramide chain heterogeneity (ESM is 84% C 16 and 16% longer saturated chains); the ability of ceramides to stabilize liquid-ordered phases and to form gel phase domains has been shown to depend on chain length (Chiantia et al, 2007;Megha et al, 2007;Fig. 7.…”

“…Ceramide has also been shown to displace cholesterol from raft domains in model and cellular membranes (Ali et al, 2006;Chiantia et al, 2006;Megha and London, 2004;Megha et al, 2006;Yu et al, 2005b). Several recent studies have concluded that the ability of ceramide to modulate the properties of liquid-ordered domains and form ceramide-enriched gel domains is affected by the chain length and that short-chain ceramides (<C 12 ) are not good models for studies of the biological activity of ceramide (Chiantia et al, 2007;Megha et al, 2007).…”

Clustering and Lateral Concentration of Raft Lipids by the MAL Protein

“…In general, liquid ordered domains are thicker than the surrounding liquiddisordered membrane. Moreover, long saturated fatty acyl tails have been shown to be a prerequisite for the association between sphingolipid rafts and glycophosphatidylinositol (GPI)-anchored proteins (Benting et al, 1999;Megha et al, 2007). A proposed offshoot of such protein-lipid mismatching interactions is that integral membrane proteins might be key determinants of membrane thickness.…”