Sphingomyelin Modulates the Transbilayer Distribution of Galactosylceramide in Phospholipid Membranes

Abstract

The interrelationships among sphingolipid structure, membrane curvature, and glycosphingolipid transmembrane distribution remain poorly defined despite the emerging importance of sphingolipids in curved regions and vesicle buds of biomembranes. Here, we describe a novel approach to investigate the transmembrane distribution of galactosylceramide in phospholipid small unilamellar vesicles by 13 C NMR spectroscopy. Quantitation of the transbilayer distribution of [6-13 C]galactosylceramide (99.8% isotopic enrichment) was achieved by exposure of vesicles to the paramagnetic ion, Mn 2؉ . The data show that [6-13 C]galactosylceramide prefers (70%) the inner leaflet of phosphatidylcholine vesicles. Increasing the sphingomyelin content of the 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles shifted galactosylceramide from the inner to the outer leaflet. The amount of galactosylceramide localized in the inner leaflet decreased from 70% in pure 1-palmitoyl-2-oleoyl-phosphatidylcholine vesicles to only 40% in 1-palmitoyl-2-oleoyl-phosphatidylcholine/ sphingomyelin (1:2) vesicles. The present study demonstrates that sphingomyelin can dramatically alter the transbilayer distribution of a monohexosylceramide, such as galactosylceramide, in 1-palmitoyl-2-oleoylphosphatidylcholine/sphingomyelin vesicles. The results suggest that sphingolipid-sphingolipid interactions that occur even in the absence of cholesterol play a role in controlling the transmembrane distributions of cerebrosides.Sphingolipids participate in a number of important cellular processes that require membrane budding, fission, or vesiculation (1, 2). Examples include infectious processes involving bacterial toxin and envelope virus entry into cells (3, 4), exosomal antigen presentation (5), and processes related to the terminal stages of apoptosis (6). Many recent investigations, by this laboratory and others, have focused on the in-plane lateral interactions among sphingolipids, cholesterol, and other membrane lipids (7-10). As a result, significant new insights into sphingolipid organization in membranes have emerged, including the identification and characterization of sphingolipid-enriched, liquid-ordered microdomains, often referred to as rafts (11-13). With so much emphasis on lipid lateral interactions, studies of sphingolipid transmembrane distribution have been relatively few (14), and the interrelationships among sphingolipid structure, membrane curvature, and glycosphingolipid transmembrane distribution remain poorly understood (15).Much of what is currently known about the mechanical forces affecting membrane curvature has been achieved by investigations of phosphoglyceride model membranes (16). The elastic constants associated with a fluid membrane are the bending elastic modulus and the spontaneous curvature (17, 18). The bending elastic modulus is the resistance of membranes to curvature or the bending rigidity, whereas the spontaneous curvature is the inherent curvature of an unconstrained membrane section and changes with lipid structure. Bec...

Phosphatidylserine Stimulates Ceramide 1-Phosphate (C1P) Intermembrane Transfer by C1P Transfer Proteins

“…When the sphingolipid source vesicles are POPC, the observed 35% C1P transfer equilibrium value suggests that 35% of C1P localizes in the vesicle outer leaflet. This value is similar to that of GalCer (39,56). When anionic phosphoglycerides are added to the mix, their influence also needs consideration.…”

Nonadditive Compositional Curvature Energetics of Lipid Bilayers

“…Nuclear magnetic resonance experiments on small lipid vesicles indicate sphingomyelin prefers the positively curved outer leaflet [45, 46], consistent with a positive value of c 0 . In contrast, x-ray crystallography of mixtures of sphingomyelin and DOPE in the inverse hexagonal phase at low sphingomyelin concentration (10% or less) show that sphingomyelin has a weak effect on the negative $c_0^{\text{mix}}$ of the mixture [43], indicating that it also has a negative curvature preference (though somewhat weaker than a PE lipid).…”

Glycolipid transfer proteins

“…GalCer, at concentrations below 30 mole%, is miscible in both liquid-crystalline and gel-phase SM [35], whereas GalCer mixing in PC becomes less favorable with increasing unsaturation of the fatty acyl chains in PC. NMR studies of GalCer transbilayer distributions in PCs and SMs suggest preferential lateral interactions between GalCer and SM [36,37]. Interestingly, the GLTP-mediated transfer of AV-GalCer seems to be affected by its miscibility state in bilayer membranes composed of SM and PC.…”