CHOLESTEROL-INDUCED INTERFACIAL AREA CONDENSATIONS OF GALACTOSYLCERAMIDES AND SPHINGOMYELINS WITH IDENTICAL ACYL CHAINS

Interest in the in-plane interactions that occur between cholesterol and simple sphingolipids has been stimulated by recent discoveries in virology and cell biology. Semliki Forest virus requires cholesterol in target membranes for binding, but the fusion event is dependent upon simple sphingolipids such as sphingomyelin, ceramide, or galactosylceramide being present in target membranes (Nieva et al., 1994; Wilschut et al., 1995). Other envelope viruses, such as HIV,1 are enriched in sphingomyelin and possess high cholesterol:phospholipid ratios compared to host cell plasma membranes (Aloia et al., 1993). With regard to the sorting of integral membrane proteins in the Golgi apparatus, the cholesterol–sphingolipid concentration is hypothesized to play a critical role in formation of membrane microdomains that differ in thickness from other regions. During sorting, Golgi proteins, but not plasma membrane proteins, may be excluded from the thicker, cholesterol-rich microdomains due to dimensional and compositional differences in their transmembrane helices (Bretscher & Munro, 1993). The trafficking, sorting, and localization of proteins bearing covalently attached glycosylphosphatidylinositol (GPI) lipid anchors also has been linked to specialized membrane regions enriched in cholesterol, glycosylceramides, and sphingomyelin [ for recent review, see Parton and Simons (1995)]. Determining and verifying the molecular details of these interesting cell biological processes requires that the nature of the in-plane interactions between cholesterol and simple sphingolipids be clearly defined. An effective way to investigate the in-plane interactions between cholesterol and other lipids is by Langmuir film balance approaches [e.g. Phillips (1972)]. An important advantage of studying lipid–lipid interactions using the monolayer approach is that the range of molecular areas known to occur in membrane systems can be investigated systematically while avoiding the mesomorphic changes that often occur in bulk hydrated dispersions (e.g. bilayer vesicles) as lipid composition is varied. Also, the in-plane lipid–lipid interactions can be isolated from the influences of changing transbilayer compositional distributions that may occur in bilayer systems when cholesterol mole fractions are varied [e.g. Sankaram and Thompson (1991) and Harris et al. (1995)]. For these reasons, monolayer investigations of sphingolipid–cholesterol interactions have proven useful (Lund-Katz et al., 1988; Johnston & Chapman, 1988; Gronberg et al., 1991; Slotte et al., 1993; Ali et al., 1994; Smaby et al., 1994; Bittman et al., 1994). In these earlier studies, interactions between cholesterol and different sphingolipids were evaluated by directly measuring the average cross-sectional molecular areas at various mixing ratios and surface pressures. The extent to which the average molecular area in a mixed composition film decreased below that predicted by summing the known molecular areas of the pure lipids (apportioned by mole fraction) provided a measure of the area condensation induced by cholesterol. This classic monolayer approach originally was used to show the marked condensing effect that cholesterol has on fluid-phase phosphoglycerides [e.g. Phillips, (1972) and references therein]. As a result, the extent of lipid monolayer condensation induced by cholesterol sometimes has been used as an indicator of affinity or interaction strength that cholesterol has for various phosphoglycerides or sphingolipids [e.g. de Kruyff et al., (1973), Lund-Katz et al. (1988), Gronberg et al. (1991), and Slotte et al. (1993)]. Using the area condensation approach, Johnston and Chapman (1988) and Slotte et al. (1993) investigated the mixing behavior of cholesterol with galactosylceramides (GalCer) containing their naturally occurring, mixed complement of acyl chains. Slotte et al. (1993) concluded that GalCer does not interact with cholesterol because no area condensation was observed. Subsequently, we showed that significant molecular area condensation does occur when cholesterol is mixed with GalCer if the acyl composition imparts fluid-phase behavior to this sphingolipid prior to mixing with cholesterol (Ali et al., 1994). Even when the acyl structure of GalCer was modified so as to maximize area condensation upon mixing with cholesterol, larger effects were observed with egg or bovine brain sphingomyelin (SM) than with GalCer when these sphingolipids were in comparable phase states prior to mixing with cholesterol (Ali et al., 1994). Recently, we noted that the acyl chain heterogeneity of egg and bovine brain SM significantly broadened the surface pressure range of their phase transitions compared to those of their predominant molecular species (e.g. egg SM = 86% 16:0 SM; bovine brain SM = 63% 18:0 SM) (Smaby et al., 1996). An examination of earlier studies, in which the cholesterol-induced condensations of SMs and other lipids had been compared, revealed that phase transitional broadening was probably an unrecognized contributor that enhanced the area condensations of SM. Here, we have synthesized different GalCer and SM species containing identical acyl chains (e.g. oleate, nervonate, and linoleate) as well as the predominant acyl residues in egg SM (palmitate) and in bovine SM (stearate). We have investigated the mixing behavior of these sphingolipids and cholesterol with two aims in mind: (1) to assess the contribution of phase transitional broadening to the cholesterol-induced condensations of naturally occurring SMs and (2) to carefully compare not only the changes in SM and GalCer cross-sectional area induced by cholesterol but also the effect that the sterol has on the molecular interfacial-packing density of GalCer relative to that of SM.