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-oleoyl-phosphatidylcholine/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.

Fig. 1. Solution spectra of GalCer

A, ¹³C NMR of [6-¹³C]GalCer. B, natural abundance ¹³C NMR of bovine brain GalCer (without hydroxy fatty acyl chains). Both spectra were acquired in CDCl₃: CD₃OD:D₂O (50:50:15 v/v/v). C1 and C6 indicate resonances of galactose.

Fig. 2. ¹³C NMR spectrum of [6-¹³C]-GalCer-POPC vesicles

Inset B shows an expanded δ region between 50 and 73 ppm. C_A, C_B, and C_C indicate the resonances of the glycerol carbons, and Cα and Cβ indicate the phosphocholine head-group resonances. The POPC vesicles contained 1 mol % [6-¹³C]GalCer.

Fig. 3. Transbilayer distribution of GalCer in POPC vesicles determined by ¹³C NMR

Left panel, ¹³C NMR spectra (55–75-ppm region) of POPC vesicles containing 1 mol % [6-¹³C]GalCer, acquired in the absence (lower spectrum), and in the presence of 5 mM Mn²⁺(upper spectrum). Right panel, transbilayer distribution of GalCer in POPC vesicles containing either 1 or 2 mol % [6-¹³C]GalCer.

Fig. 4. Transbilayer distribution of POPC and SPM determined by ³¹P NMR

Left panel, ³¹P NMR spectra of vesicles comprised of equimolar POPC and SPM and containing 1 mol % [6-¹³C]GalCer, acquired in the absence (upper spectrum), and in the presence of 1 mM Pr³⁺ (lower spectrum). The lower spectrum shows well resolved resonances derived from POPC (a) and SPM (b) of the inner leaflet and from POPC (c) and SPM (d) of the outer leaflet. Right panel, transbilayer distribution of POPC (A) and SPM (B) measured from ³¹P NMR data for vesicles comprised of POPC/SPM at the molar ratios of 2:1, 1:1, and 1:2 and containing 1 mol % [6-¹³C]GalCer.

Fig. 5. The effect of SPM on the transbilayer distribution of GalCer determined by ¹³C NMR

Left panel, ¹³C NMR spectra (50 –80-ppm region) of vesicles comprised of equimolar POPC and SPM and containing 1 mol % [6-¹³C]GalCer, acquired in the absence (lower spectrum) and in the presence of 5 mM Mn²⁺(upper spectrum). Right panel, transbilayer distribution of GalCer in vesicles composed of POPC/SPM at the molar ratios of 2:1, 1:1, and 1:2 and containing 1 mol % [6-¹³C]GalCer.