Sphingomyelin–cholesterol interactions in biological and model membranes

Chemistry and Physics of Lipids 102 (1999) 13 – 27 www.elsevier.com/locate/chemphyslip

Sphingomyelin–cholesterol interactions in biological and model membranes J. Peter Slotte * Department of Biochemistry and Pharmacy, A, bo Akademi Uni6ersity, PO Box 66, FIN 20521 Turku, Finland

Abstract Cholesterol and sphingomyelin are both important plasma membrane constituents in cells. It is now becoming evident that these two lipid classes affect each other’s metabolism in the cell to an extent that was not previously appreciated. It is the aim of this review to present recent data in the literature concerning both molecular and membrane properties of the two lipid classes, how they interact in membranes (both biological and model), and the consequences their mutual interaction have on different functional and metabolic processes in cells and lipoproteins. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Sphingolipids; Sterols; Lipid interaction; Biological membranes; Cell metabolism; Lipoproteins; Vesicles; Monolayers

1. Introduction Both cholesterol and sphingomyelin are important constituents of cellular plasma membranes. These molecules are chemically as well as functionally different, and still they appear to co-localize in the same membrane compartment, and even to be attracted to each other. The objective of this review is to present new data in the literature, which relate to the effects of and reasons for cholesterol–sphingomyelin interactions in membranes. The reader may want to consult other recent reviews which deal with closely related topics, such as the cellular distribution and transport of cholesterol (Liscum and Dahl, 1992; Lis-

* Tel.: +358-2-2154689; fax: + 358-2-2154745. E-mail address: [email protected] (J.P. Slotte)

cum and Faust, 1994; Liscum and Underwood, 1995), cellular lipid traffic (Van Meer, 1989; Pagano, 1990; Voelker, 1991; Allan and Kallen, 1993), transport and metabolism of sphingomyelin (Koval and Pagano, 1991), the role of sphingolipids in cell signaling (Kolesnick, 1991, 1994; Hannun and Bell, 1993; Hannun, 1994), sphingolipid organization in biomembranes (Brown, 1998), sphingolipid–cholesterol rafts (Harder and Simons, 1997; Simons and Ikonen, 1997) and detergent-resistant membrane domains (Brown and London, 1997).

2. The molecular composition of sphingomyelin The sphingomyelin molecule is composed of a polar phosphorylcholine head group, an amidelinked acyl chain, and a long chain sphingoid base

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(Fig. 1). The polar head group of sphingomyelin is similar to the phosphorylcholine head group of phosphatidylcholines. The amide-linked acyl chains of sphingomyelin are usually saturated or monounsaturated and contain 14 – 24 carbons (O’Brien and Rouser, 1964; Kishimoto et al., 1969; White, 1973; Barenholz and Thompson, 1980). It is known that the acyl chain composition of sphingomyelins in the amide-linked position varies from tissue to tissue, so that e.g. brain white matter contains sphingomyelin with nervonic acid (24:1D15c), whereas brain grey matter consists of sphingomyelins with predominantly stearic acid (18:0) in the amide-linked position (O’Brien and Sampson, 1965). Brain-derived sphingomyelin may also contain a-hydroxylated acyl chains (Kishimoto et al., 1969). Sphingomyelins isolated from cultured cells (e.g. human skin fibroblasts, human erythro leukemia cells, bovine aortic smooth muscle cells, baby hamster kidney cells) is usually a mixed population, containing both long (22:0, 24:0, 24:1D15c) and intermediate length (16:0 and 18:0) acyl chains (Kronqvist, Leppima¨ki and Slotte, unpublished observations). In HL-60 cells, the predominant

sphingomyelin species were reported to contain palmitic and nervonic acids (Fitzgerald et al., 1995). There appears to be a plethora of different long chain sphingoid bases in naturally occurring sphingomyelins (see Merrill and Sweeley (1996), and references therein). Sphingosine (D-erythro-2amino-trans-4-octadecene-1,3-diol) is the prevalent backbone in most mammalian sphingolipids, including sphingomyelin. All naturally occurring sphingomyelins (and sphingolipids in general) have the D-erythro configuration (Sarmientos et al., 1985). However, the erythro configuration of the sphingoid base is somewhat labile, especially under acidic conditions. Consequently, acid hydrolysis of e.g. sphingomyelin often yield lysosphingomyelin (or sphingosine-1-phosphorylcholine) with altered stereochemical configuration, so that both D-erythro and L-threo isomers are obtained (Sripada et al., 1987). The stereoconfiguration of sphingomyelins can directly be determined by NMR, as shown by Sarmientos et al. (1985). An alternative (and simple) method to determine the extent of epimerization in semi-synthetic and acyl chain defined sphingomyelins (synthesized from lysosphingomyelin prepared by acid hydrolysis of e.g. bovine brain sphingomyelin) is to perform silica gel 60 high-performance thinlayer chromatography (elution with chloroform/ methanol/acetic acid/water 25:15:4:2 by volume), in which system L-threo-SM migrates ahead of the acyl-matched D-erythro-SM species (Ramstedt and Slotte, 1999). For a thorough review of the properties of sphingomyelin, see Barenholz and Thompson (1980) and Barenholz (1984).

3. Biosynthesis of sphingomyelin

Fig. 1. Structure of cholesterol.

D-erythro-N-stearoyl-sphingomyelin

and

Sphingomyelin appears to be synthesized by the enzymatic transfer of the phosphorylcholine moiety of phosphatidylcholine to ceramide (catalyzed by sphingomyelin synthase), thus yielding sphingomyelin and diacylglycerol (Kishimoto et al., 1969; Voelker and Kennedy, 1982; Spence et al., 1983; Marggraf and Kanfer, 1984). The direct transfer of CDP-choline to ceramide is apparently a pathway of minor significance in most cells

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(Voelker and Kennedy, 1982). There is currently a dispute regarding the major site of sphingomyelin synthesis, since synthetic activities have been demonstrated in several different cellular compartments (Futerman et al., 1990; Jeckel et al., 1990; van Echten et al., 1990; Kallen et al., 1993, 1994). In studies using subcellular fractionation techniques (rat liver), it was observed that cis Golgi fractions accounted for the largest activity of sphingomyelin synthesis, whereas only a small fraction of the synthetic activity could be attributed to the plasma membrane fraction (Futerman et al., 1990; Jeckel et al., 1990). In baby hamster kidney cells, however, it was reported that a substantial synthesis of sphingomyelin occurred in a compartment distinct from the cis/medial Golgi. This compartment was presumed to include the endocytic recycling pathway (Kallen et al., 1993, 1994). In a more recent work, Miro Obradors et al. (1997) observed that a substantial amount of sphingomyelin synthase activity in baby hamster kidney cells co-sedimented with plasma membrane and did not co-sediment with cis/medial Golgi. However, van Helvoort and colleagues reported that, according to their studies with human liver-derived HepG2 cells, and baby hamster kidney BHK-21 cells, little if any SMsynthase was localized to the endocytic pathway (van Helvoort et al., 1997). These authors concluded that most of the sphingomyelin synthase activity was present in the Golgi, and to a small extent at the cell surface (van Helvoort et al., 1997). Before this issue is settled more definitely, it can be assumed that some sphingomyelin synthase activity may occur in different compartments within the cell, and this assumption also implies that there may be different sphingomyelin pools within the cell. On the other hand, it is also likely that the predominant site of sphingomyelin synthesis may differ from one cell type to another, as a different synthetic topology may be needed for functionally specialized cells. This is indeed shown in a recent report, where it was demonstrated that the plasma and myelin membrane compartment accounted for at least half of the sphingomyelin synthase activity in oligodendrocytes (Vos et al., 1995).

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4. Subcellular distribution of sphingomyelin The distribution of sphingomyelin mass among intracellular organelles has been examined using cell fractionation studies, and by sphingomyelinase degradation experiments. Results with both techniques suggest that more than half of the cellular sphingomyelin mass is confined to the plasma membrane (with most of it being in the exoleaflet), for a review see Koval and Pagano (1991). With fibroblasts it was reported that as much as 90% of the total cell sphingomyelin mass was in the plasma membrane compartment (Lange et al., 1989). Again, as was the case with cholesterol distribution, it appears that cells with an extensive recycling of plasma membranes through the endocytic system also have a larger fraction of sphingomyelin in intracellular compartments (inaccessible to exogenously applied sphingomyelinase) (Allan and Kallen, 1993). It also appears that at least in BHK cells a substantial fraction of the internal pool of sphingomyelin does not normally reach the surface very efficiently (Quinn and Allan, 1992). The predominant exoleaflet orientation of plasma membrane sphingomyelin apparently results from the topological orientation of sphingomyelin synthase, since sphingomyelin flip-flop from one leaflet to the other is very limited, and since a membrane translocase acting on sphingomyelin has not yet been observed. Endogenous neutral sphingomyelinases, the activity of which can be stimulated with various cytokines (Brindley et al., 1996; Spiegel et al., 1996; Testi, 1996; Ghosh et al., 1997), appear to utilize sphingomyelin present on the endoleaflet of the plasma membrane. It has been estimated that only 10–20% of cellular sphingomyelin is involved in cytokine-induced turnover of cell sphingomyelin (Linardic and Hannun, 1994). Since the sphingomyelin cycle involves a cytosolic sphingomyelinase and the intracellular release of choline phosphate, this pool of sphingomyelin appears to localize to the inner leaflet of the plasma membrane (or to a closely related compartment) (Linardic and Hannun, 1994). Studies on TNFa-induced turnover of sphingomyelin in human skin fibroblasts indicated that only sphin-

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gomyelin in the endoleaflet was degraded, since sphingomyelin introduced into the exoleaflet was resistant to hydrolysis (Andrieu et al., 1996).

physical studies of cholesterol–phospholipid interactions, see McMullen and McElhaney (1996).

6. Subcellular distribution of cholesterol 5. Molecular and membrane properties of cholesterol Cholesterol is an alicyclic lipid molecule, the structure of which contains four fused rings (all trans-anti ) (Duax et al., 1976), a single hydroxyl group at carbon 3, a double bond between carbons 5 and 6, and an iso-octyl side chain at carbon 17. The molecule is practically insoluble in aqueous media, with the critical aggregation concentration estimated to be 30 nM (Haberland and Reynolds, 1973). The 3b-OH function of cholesterol is important, since a fatty acid can be esterified to this position, either by acyl-CoA:cholesterol acyl transferase within the cell (Billheimer and Gillies, 1989), or by lecithin:cholesterol acyl transferase (Fielding, 1989) in the plasma compartment. Since membrane cholesterol is unesterified, the free 3b-OH group of cholesterol may engage in hydrogen bonding with water (Boggs, 1987) and possibly with co-lipids in the membrane. Cholesterol is an essential component of cellular plasma membranes in higher organisms. Cholesterol interacts with membrane phospholipids and influences their physico-chemical properties. The important membrane properties that are directly or indirectly influenced by membrane levels of cholesterol include solute permeability in bilayer membranes (for a review, see Yeagle, 1985), phospholipid acyl chain mobility and orientational order in bilayer membranes (Gally et al., 1976; Stockton and Smith, 1976; Yeagle, 1985) and lateral packing density of phospholipids in monolayer membranes (Chapman et al., 1969; Smaby et al., 1994). Cholesterol also has a marked influence on lateral phase separations (Smutzer and Yeagle, 1985), and on the effective free volume of membranes (Straume and Litman, 1987), two parameters which are directly related to the flexibility of membrane proteins (e.g. ion channels, enzymes) and hence to their function in membranes. For an excellent recent review on

Most of the cellular unesterified cholesterol can be found in the plasma membrane compartment. Depending on the cell type examined, and the assay method used, plasma membranes have been reported to contain from about half to as much as 90% of the total cellular unesterified cholesterol (Lange et al., 1989; Warnock et al., 1993). It appears that cells with an extensive recycling of plasma membranes through the endocytic system have a larger fraction of intracellular cholesterol (Allan and Kallen, 1993). It is not surprising that cellular plasma membranes contain such a high concentration of cholesterol, since several of the cellular cholesterol transport pathways are directed toward the cell surface. Newly synthesized cholesterol is transported vectorially from the intracellular site of synthesis to the cell surface, where it appears with a transfer half-time of 10– 18 min (De Grella and Simoni, 1982; Kaplan and Simoni, 1985; Urbani and Simoni, 1990; Lange et al., 1991). Exogenously derived cholesterol, entering the cell with LDL particles, is thought to be quantitatively transported from lysosomes to the cell surface, where it accumulates (Tabas et al., 1988). However, with Chinese hamster ovary (CHO) cells, Underwood et al. (1998) recently demonstrated that there exists an alternative pathway that directs some of the exogenously derived lysosomal cholesterol directly to the endoplasmic reticulum, where it can be esterified by acylCoA:cholesterol acyltransferase. In the CHO cells, the bulk of lysosomal cholesterol still was delivered to the plasma membrane. According to results obtained by the Tabas laboratory for macrophage cells, excess cholesterol does not appear to move to the endoplasmic reticulum for esterification by ACAT until the solubilizing capacity of the plasma membrane compartment is saturated (Xu and Tabas, 1991). It is suggested that the capacity of plasma membranes to solubilize cholesterol is largely a function of its sphingomyelin content (Okwu et al., 1994).

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7. Effect of sphingomyelin on cell cholesterol homeostasis Since Patton (1970) made the observation that there was a positive correlation between the contents of cholesterol and sphingomyelin mass in the membranes of rat liver hepatocytes, many other studies in recent years have strengthened the view that these two lipid classes co-localize to a significant extent in biological membranes, and affect each others homeostasis. The incorporation of sphingomyelin mass from liposomes into fibroblast membranes was observed to result in a reduced esterification of cell cholesterol, and to an increased formation of newly synthesized cholesterol (Gatt and Bierman, 1980; Kudchodkar et al., 1983). Since both the ACAT-catalyzed esterification reaction, and the biosynthesis of cholesterol are acutely regulated by the cellular level of unesterified cholesterol, the sphingomyelin loading experiments suggested that sphingomyelin incorporation directly led to a net flow of cholesterol from intracellular sites to the plasma membrane compartment. Later, Slotte and Bierman (1988) were able to show that degradation of sphingomyelin mass (with the aid of exogenous sphingomyelinase) in cultured fibroblasts led to a dramatic activation of the endogenous esterification of cholesterol. Since the ACAT-reaction in the endoplasmic reticulum is sensitive to the flow of substrate cholesterol, its activation strongly suggested that sphingomyelin degradation resulted in a flow of cholesterol from the cell surface into the substrate pool of ACAT. This study was later reproduced by others in a variety of cell types (Gupta and Rudney, 1991; Stein et al., 1992a). It has also been reported that activation of sphingomyelin degradation in fibroblasts with TNFa, via an endogenous sphingomyelinase, leads to the activation of cholesterol esterification in the treated cells (Chatterjee, 1994). In addition to affecting the rate of endogenous cholesterol esterification, the sphingomyelinase-induced cholesterol translocation also influenced the rate of cholesterol biosynthesis. Slotte and Bierman (1988) originally demonstrated that the incorporation of sodium [14C] acetate into de novo sterols was reduced markedly in cells treated with

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sphingomyelinase compared with control fibroblasts. Later, Gupta and Rudney (1991) demonstrated that sphingomyelin degradation in conjunction with the resulting cholesterol flow down-regulated the activity of HMG-CoA reductase, the key regulatory enzyme in cholesterol biosynthesis. It now appears that the sphingomyelinase-induced flow of cholesterol from the cell surface to the endoplasmic reticulum directly affects the proteolytic cleavage of sterol regulatory element binding protein-2 (SREBP-2), thereby preventing the release of the active aminoterminal fragment from membranes (Scheek et al., 1997). This amino-terminal fragment of SREBP-2 is active in regulating the nuclear transcription of several genes encoding for enzymes involved in the regulation of biosynthesis and uptake of cholesterol (Scheek et al., 1997). When the cellular distribution of cholesterol was determined in control and sphingomyelin-depleted fibroblast cells using filipin staining and fluorescence microscopy, it was observed that the filipin staining pattern was not significantly different in control and sphingomyelin-depleted cells (Po¨rn and Slotte, 1995). Assuming that filipin stained cholesterol equally well in the presence and absence of sphingomyelin in the plasma membranes, these results suggest that the extent of cholesterol translocation away from the cell surface is not very extensive in sphingomyelin-depleted cells. Another interesting effect of plasma membrane sphingomyelin on cell cholesterol metabolism was observed in the human intestinal CaCo2 cell line, in which the content of sphingomyelin in the apical membrane was found to regulate cholesterol uptake from bile salt micelles (Chen et al., 1992). A 60% degradation of cell sphingomyelin resulted in a 50% decrease of cholesterol absorption, as well as a down-regulation of endogenous cholesterol biosynthesis. In addition, the basolateral secretion of cholesterol was likewise reduced in sphingomyelin-depleted CaCo2 cells (Chen et al., 1992). These authors suggested that the sphingomyelin content of the intestinal cells directly affected cholesterol flux, and that the overall process may be regulated by endogenous sphingomyelinases present in pancreatic juice.

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8. Does cholesterol interact selectively with sphingomyelin in biological membranes? Whereas sphingomyelin degradation using either exogenous (Slotte and Bierman, 1988; Po¨rn and Slotte, 1990; Slotte et al., 1990; Gupta and Rudney, 1991; Stein et al., 1992a,b) or endogenous sphingomyelinase (Chatterjee, 1994) leads to increased cholesterol esterification activity in a variety of cells, degradation of cell surface phosphatidylcholine does not (Po¨rn et al., 1993). Consequently, it appears that under the conditions used, cholesterol associated preferentially with sphingomyelin. In a recent study where the rate of cholesterol efflux from cells to b-cyclodextrins was examined, it was found that sphingomyelin degradation dramatically increased cholesterol efflux from the cells — phosphatidylcholine degradation had no significant effect on cholesterol efflux (Ohvo et al., 1997). These results, together with the results of Po¨rn et al. (1993), suggest that at least in human skin fibroblasts, grown under normal cell culture conditions, hardly any of the cell unesterified cholesterol is influenced by a removal of phosphatidylcholine. Instead, cholesterol homeostasis is most dramatically influenced by changes in cell membrane sphingomyelin.

9. Effect of cholesterol on sphingomyelin homeostasis in cells Since the membrane level of sphingomyelin, by affecting the subcellular distribution of cholesterol, can indirectly regulate the rate of cholesterol biosynthesis; it is of some interest to know whether changes in plasma membrane cholesterol levels, or changes in cell cholesterol homeostasis in general, also may affect rates of phospholipid biosynthesis. It has been shown that cholesterol surface transfer from liposomes to cultured rat aortic smooth muscle cells increased the incorporation of radio-labelled acetate into phosphatidylcholine (Slotte and Lundberg, 1983). Consequently, the cells responded to the increased flux of cholesterol into them by increasing the synthesis of phosphatidylcholine, apparently to help solubilize the excess free cholesterol. Later, it

was demonstrated that cholesterol loading into macrophages using b-VLDL as cholesterol carriers also stimulated the biosynthesis of phosphatidylcholine (Shiratori et al., 1994). With intestinal CaCo2 cells, it was observed that the regulation of HMG-CoA reductase (cholesterol biosynthesis) and serine palmitoyltransferase (sphingosine synthesis) were independent of each other (Chen et al., 1993). However, it was found that when the membrane mass of either cholesterol or sphingomyelin were altered, parallel changes occurred in the rate of synthesis of these two lipids. Cell cholesterol mass can easily, efficiently and rapidly be depleted or replenished using either extracellular cyclodextrin acceptors (cholesterol depletion) or pre-made cholesterol–cyclodextrin inclusion complexes (for cholesterol loading) (Awad et al., 1996; Ohvo and Slotte, 1996). Using this technique, Leppima¨ki et al. (1998) were able to demonstrate that cholesterol depletion (20% reduction in cell-free cholesterol) led to a markedly (70%) increased synthesis of sphingomyelin in cultured fibroblasts. With time (24 h) the cellular content of sphingomyelin mass also increased significantly in cholesterol-depleted cells. Careful examination of the cellular events indicated that the activity of serine palmitoyl transferase was increased in cholesterol-depleted cells, leading to the increased formation of sphingomyelin. The effect of cholesterol depletion of sphingomyelin synthesis was rather specific, since no changes were observed in the rate of [14C] palmitic acid incorporation into other sphingolipid classes. In this same study, cholesterol loading was shown to stimulate phosphatidylcholine synthesis (consistent with the findings of Slotte and Lundberg (1983) and Shiratori et al. (1994)) and to inhibit sphingomyelin synthesis. These results clearly show that rates of phosphatidylcholine and sphingomyelin synthesis are affected (although differently for each phospholipid class) by the level of cholesterol in the cells. In addition to cholesterol, it appears that oxidized sterols also may affect the regulation of sphingomyelin synthesis, as shown in Chinese hamster ovary cells, where 25-hydroxycholesterol was reported to stimulate sphingomyelin synthesis (Ridgway, 1995).

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10. Sphingolipid–cholesterol domains in cell membranes Cell membranes contain a complex mixture of glycosphingolipids, in addition to sphingomyelin, glycerophospholipids and cholesterol. It has been suggested that lateral assemblies of sphingolipids and cholesterol may function as platforms in membranes for different cellular processes (Harder and Simons, 1997; Simons and Ikonen, 1997). It is assumed that specific lateral interactions among the sphingolipids induce the formation of laterally organized microdomains or rafts, which directly participate in the lateral recruitment of certain proteins to these lateral domains, as well as in the association of GPI-anchored proteins to plasma membrane invaginations, the caveolaes (for reviews see Bretscher and Munro, 1993; Parton and Simons, 1995; Simons and Ikonen, 1997). Cholesterol is assumed to associate with the rafts either by functioning as a spacer within the sphingolipid domains, or by associating specifically with proteins (e.g. caveolin) in certain membrane domains (caveolae) (Murata et al., 1995). The molecular mechanisms involved in the spontaneous formation of sphingolipid microdomains in model and biological membranes were recently reviewed (Brown and London, 1997; Brown, 1998).

11. Sphingomyelin and cholesterol in lipoprotein structure and function Lipoproteins are spherical aggregates of lipids and (apo)proteins which in the circulation function to transport hydrophobic water-insoluble lipids (e.g. triacylglycerols and cholesteryl esters) from the intestine and the liver to peripheral tissues (for recent reviews on lipoprotein assembly and metabolism, see Davis and Vance, 1996; Fielding and Fielding, 1996; Schneider, 1996). The degradation of triacylglycerols in model lipid emulsions by lipoprotein lipase was recently shown to be greatly affected by the ratio of cholesterol to sphingomyelin in the emulsion surface (Lobo and Wilton, 1997). It was demonstrated that when only phosphatidylcholine was

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present in the lipid emulsion surface, addition of cholesterol increased lipoprotein lipase activity. However, in the additional presence of physiological concentration of sphingomyelin, cholesterol no longer stimulated lipoprotein lipase activity. It was also shown that hydrolytic removal of sphingomyelin by sphingomyelinase in the absence of cholesterol increased lipoprotein lipase-mediated triacylglycerol hydrolysis. These findings suggest that the cholesterol–sphingomyelin balance in triacylglycerol-rich lipoproteins may affect or regulate their metabolism by lipoprotein lipase. Direct evidence for this suggestion was recently reported by Jeong et al. (1998), who showed that apoprotein E knockout mice generate sphingomyelinenriched lipoproteins, the fractional catabolic rate of which is markedly reduced because these particles are more resistant to intravascular enzymes. High density lipoproteins partake in cholesterol removal processes, in which cholesterol in peripheral tissue is transported to the liver, for ultimate excretion from the body. Nascent HDL is formed in the circulation from lipid-free apolipoprotein A-I and phospholipids. Whereas lipid-free A-I is a poor lipid acceptor, association of as little as 5 mol of phosphatidylcholine with apo A-I is sufficient to transform the lipid-free apo A-I into a distinct lipoprotein-like particle that is a significantly better acceptor of cellular cholesterol. Interestingly, inclusion of sphingomyelin into the phosphatidylcholine –A-I complex further stimulates cholesterol efflux significantly (Zhao et al., 1996a). It is plausible that sphingomyelin functions as a trap for cholesterol in the phospholipid–A-I complex, because of its high affinity for cholesterol (Clejan and Bittman, 1984). In another study with reconstituted discoidal HDL particles (containing apo A-I), it was observed that increasing the content of either phosphatidylinositol or sphingomyelin, up to 20 mol/particle, was associated with significantly increased abilities of the reconstituted lipoprotein particles to promote cholesterol efflux (Zhao et al., 1996b). This study suggested that in contrast to interlipoprotein cholesterol transfers, the efflux of cholesterol from cultured fibroblasts is less sensitive to factors that affect the frequency of molecular collisions and more dependent on the

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ability of an HDL particle to absorb and retain cholesterol molecules. Since sphingomyelin and phosphatidylinositol appear to modulate this adsorption/desorption of cholesterol to HDL, variations in the concentration of these lipids within HDL would be expected to affect plasma cholesterol homeostasis. Cholesterol which is removed from cell membranes by apo A-I containing lipoprotein particles eventually becomes esterified in the circulation by the action of lecithin – cholesterol acyltransferase (LCAT). It is known that the activity of LCAT toward HDLs is affected by the concentration of sphingomyelin in the lipoprotein surface monolayer. Cholesterol esterification in a proteoliposome model system was inhibited by up to 90% in the presence of sphingomyelin, and this inhibition was reversed by treatment with bacterial sphingomyelinase (Subbaiah and Liu, 1993). The inhibition induced by sphingomyelin could be overcome by increasing the concentration of phosphatidylcholine, but not unesterified cholesterol or apoprotein A-I, in the substrate. The ability of various lipoproteins to act as substrates for purified LCAT varied inversely with the sphingomyelin/phosphatidylcholine ratio. These results show that sphingomyelin is a physiological inhibitor of cholesterol esterification in the plasma, by virtue of its competition with phosphatidylcholine, the acyl donor for the reaction. In more recent studies on the effect of sphingomyelin on LCAT activity at the HDL surface, Bolin and Jonas (1996) could demonstrate that the dominant mechanism for the inhibition of LCAT activity by sphingomyelin was the impaired binding of the enzyme to the interface. It is plausible that the tight interaction between cholesterol and sphingomyelin in the surface monolayer of the HDL particle directly prevents the attachment of the enzyme to the lipoprotein surface monolayer. It has been reported that the phospholipid subclass distribution in apo A-I Seattle nascent HDL shows a significant enrichment in sphingomyelin and phosphatidylethanolamine compared with wild-type HDL (Lindholm et al., 1998). Studies with this mutated HDL lipoprotein showed that LCAT reactivity was impaired, in that cholesterol esterification was only half that of wild-type com-

plexes. This finding strongly implies a physiologically significant role for sphingomyelin in determining the further metabolism of HDL in the circulation.

12. Effect of sphingomyelin on cholesterol desorption from membranes The interaction of cholesterol with sphingomyelin or phosphatidylcholine in model membrane systems is known to differ markedly. This difference is best illustrated in cholesterol exchange or desorption studies, where exchange kinetics have been determined as a function of the donor membrane phospholipid composition. Clejan and Bittman (1984) showed that cholesterol exchange between Mycoplasma gallisepticum cells and acceptor phospholipid vesicles was markedly slower when the donor cell membranes were enriched in sphingomyelin. Later, several investigators have shown that cholesterol desorption from donor vesicles is markedly retarded by sphingomyelin, as compared with acyl chain-matched phosphatidylcholines (Fugler et al., 1985; Bar et al., 1987; Lund-Katz et al., 1988). For a thorough discussion of cholesterol exchange processes, see Phillips et al. (1987) and Bittman (1993). The efflux of cholesterol from red blood cells to acceptor vesicles is also known to be a function of the red blood cell membrane sphingomyelin content, with slower efflux rates being seen in cells with higher sphingomyelin/phosphatidylcholine ratios (Gold and Phillips, 1990). Even lipid transfer protein-mediated cholesterol exchange is influenced by the presence of sphingomyelin in the donor membrane. Billheimer and Gaylor (1990) observed that sphingomyelin dramatically retarded cholesterol transfer catalyzed by the nonspecific lipid transfer protein (or sterol carrier protein-2), when compared with a situation with phosphatidylcholines in the donor membranes instead. Recently a method was developed that allows for direct measurement of cholesterol desorption from phospholipid-containing monolayers (Ohvo and Slotte, 1996). b-Cyclodextrin can act as an acceptor of monolayer cholesterol without signifi-

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cantly causing desorption of monolayer phospholipids. The desorption experiments are carried out at a constant lateral surface pressure with a zeroorder trough, which allow for determination of desorption kinetics. It was found that the desorption of cholesterol from N-palmitoylsphingomyelin monolayers was markedly slower than the comparable desorption from a dipalmitoylphosphatidylcholine mixed monolayer (Ohvo and Slotte, 1996). These results agree completely with previous findings concerning the effect of sphingomyelin on cholesterol desorption from bilayer membranes. In an effort to examine the molecular requirements for the high affinity association between cholesterol and sphingomyelin, synthetic analogues of sphingomyelin have been prepared in which different functional groups were altered systematically. These analogues were incorporated into model membrane systems together with cholesterol in order to examine how the functional alterations in the sphingomyelin molecule affected the strength of interaction with cholesterol. Kan et al. (1991) synthesized sphingomyelin analogues in which the hydroxy group at the 3 position of sphingomyelin was replaced with an O-alkyl group or with hydrogen, and measured the efflux of [14C]cholesterol from such donor vesicles. Vesicles prepared from 3-deoxy- and 3O-methyl-N-stearoyl-sphingomyelin had the same rate of [14C]cholesterol desorption, which was only about 1.5-fold faster than desorption from N-stearoyl-sphingomyelin vesicles. These data indicate that the hydroxy group of sphingomyelin was not critical for the strong interaction of cholesterol with sphingomyelin. However, if the 3-hydroxy group was replaced with a sterically bulky group (e.g. ethoxy or tetrahydropyranyloxy) this change interfered markedly with the molecular packing of cholesterol and sphingomyelin in bilayer membranes, and markedly increased cholesterol desorption rates (Kan et al., 1991). In monolayer membranes it was similarly found that the cholesterol oxidation susceptibility (by cholesterol oxidase) was not markedly different in 3-deoxy-sphingomyelin monolayers as compared with 3-OH sphingomyelin monolayers (Gronberg et al., 1991).

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It has been suggested that cholesterol–sphingomyelin interaction in membranes can be stabilized by hydrogen bonds between the 3b-OH group of cholesterol and the amide-linkage in sphingomyelin (Sankaram and Thompson, 1990). To further test this possibility, sphingomyelins were synthesized in which the amide-linked fatty acid was replaced by a carbonyl ester-linked acyl chain (Bittman et al., 1994). The rate of cholesterol oxidation (by cholesterol oxidase) was markedly higher in O-stearoyl-sphingomyelin monolayers (0.36× 1013 molecules oxidized/s) than it was in N-stearoyl-sphingomyelin monolayers (about 0.05× 1013 molecules/s — the conditions were: 50 mol% cholesterol, temperature 30°C). Similarly, the rate of cholesterol oxidation in small unilamellar vesicles prepared from either of these sphingomyelins (to 50 mol% cholesterol) was much higher in O-stearoyl-sphingomyelin vesicles (20.9 arbitrary units) than in N-stearoylsphingomyelin vesicles (4.4 arbitrary units) (Bittman et al., 1994). These findings imply that the amide-linked fatty acid function in sphingomyelin has a profound stabilizing effect on cholesterol–sphingomyelin interactions. It is possible that the amide group of sphingomyelin engages in hydrogen bonding with the hydroxy group of cholesterol, thereby stabilizing the interaction.

13. Hydrophobic mismatch effects Hydrophobic mismatch among the acyl chains of different phosphatidylcholine species has been shown to affect their mutual miscibility in model membrane systems (Lehtonen et al., 1996; Silvius et al., 1996). The interaction between cholesterol and phospholipids also appear to be influenced by the hydrophobic length of the two molecular species. Using high sensitivity DSC, McMullen et al. (1993) showed that cholesterol increased the bilayer transition temperature of the broad component of the DSC endotherms for saturated, equal-length phosphatidylcholines with less than 17 carbons per chain, whereas cholesterol decreased the transition temperature for phosphatidylcholines with acyl chains longer than 17.

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Fig. 2. Average oxidation rate of cholesterol in mixed monolayers containing disaturated phosphatidylcholines as a function of phospholipid acyl chain length. The monolayers contained 50 mol% cholesterol, were kept at 22°C and a lateral surface pressure of 20 mN/m. Oxidation of cholesterol was induced by cholesterol oxidase in the subphase. Values are averages9 SEM from three different monolayers of each composition. Adapted from Mattjus et al. (1994).

The hydrophobic length of cholesterol (17.5 A, ) was calculated to match the mean hydrophobic thickness of a 17:0 phosphatidylcholine bilayer (McMullen et al., 1993). When the interaction of cholesterol with phosphatidylcholines was measured in monolayer membranes using cholesterol oxidase as a probe, the oxidation susceptibility of cholesterol was observed to be lowest in phosphatidylcholine monolayers having acyl chain lengths between 14:0 and 17:0, and higher in membranes with phosphatidylcholines having more than 17 or less than 14 carbons per chain (see Fig. 2) (Mattjus et al., 1994). This finding suggested that cholesterol interacted more favorably with phosphatidylcholines having chain lengths between 14 and 17 carbons than with longer or shorter chain phosphatidylcholines. The above-mentioned study was, however, complicated by the fact the phosphatidylcholines used (di10:0–di20:0) displayed different phase states at the temperatures examined. To eliminate these complications, the study was repeated using shorter chain phosphatidylcholines (which all were liquid-expanded) and the shorter cholesterol analog, androsterol, which lacks the iso-octyl side chain (Ohvo et al., 1998). The rate of androsterol desorption from mixed monolayers to b-cyclodextrin in the subphase was a clear function of the host phosphatidylcholine acyl chain length (see Fig.

3). The slowest rate of androsterol desorption (i.e. best androsterol–phosphatidylcholine interaction) was seen from a di14 phosphatidylcholine monolayer, whereas the desorption rate increased when the host phosphatidylcholine had shorter or longer chains. Similar results were seen when the cholesterol oxidase susceptibility of androsterol was examined in small unilamellar vesicles (Ohvo et al., 1998). The results of this study agree qualitatively well with a calorimetry study in which the miscibility of androsterol in phosphatidylcholine was determined as a function of the phosphatidylcholine acyl chain length (McMullen et al., 1994). Since cell membranes contain sphingomyelins with e.g. 16:0, 18:0, 24:0 as well as 24:1 fatty acids in the N-linked position, albeit not necessarily within the same lateral domain, it is of

Fig. 3. Desorption of androsterol from mixed monolayers containing androsterol and disaturated phosphatidylcholines to b-cyclodextrin, as a function of the phosphatidylcholine acyl chain length. The monolayers contained 33 mol% androsterol and 67 mol% phospholipid (at 25°C). The monolayer lateral surface pressure was kept constant at 20 mN/m. The final concentration of b-cyclodextrin in the subphase was 0.33 mM. The desorption rate of androsterol from a monolayer of PC 10:0 was arbitrarily set to 100% (corresponding to an androsterol desorption rate of 2.8 nmol/cm2, min). Values are averages 9SEM from three to five separate experiments with each monolayer composition. Adapted from Ohvo et al. (1998).

J.P. Slotte / Chemistry and Physics of Lipids 102 (1999) 13–27

great interest to determine how the chain length affects cholesterol’s interactions with the sphingomyelins. Hydrophobic mismatch theory would predict that cholesterol would not associate favorably with very long chain (i.e. physiological) sphingomyelins. In order to study this problem, Ramstedt and Slotte (1998) synthesized different racemic sphingomyelins, to contain amide-linked acyl chains with lengths from 14 to 24 carbons. As controls, phosphatidylcholines were prepared which had palmitic in the sn-1 position, and matched chains in the sn-2 position. The relative interaction of cholesterol with these phospholipids was determined at 22°C by measuring the rate of cholesterol desorption from mixed monolayers (50 mol% cholesterol; at a lateral surface pressure of 20 mN/m) to b-cyclodextrin in the subphase (1.7 mM). The rate of cholesterol desorption was lower from saturated sphingomyelin monolayers than from chain-matched phosphatidylcholine monolayers. In sphingomyelin monolayers, the rate of cholesterol desorption decreased as the N-linked chain became longer, whereas the opposite was true for phosphatidylcholine monolayers (higher desorption rate from monolayers of longer phosphatidylcholines). These results show that cholesterol interacts favorably with long chain sphingomyelins (low rate of desorption) whereas its interaction (or miscibility) with long chain phosphatidylcholines is much weaker. The introduction of a single cis-unsaturation in the Nlinked acyl chain of sphingomyelins led to slightly faster rates of cholesterol desorption as compared with saturated sphingomyelins. The results of this study show that cholesterol is capable of interacting with all physiologically relevant (including long chain) sphingomyelins present in the plasma membrane bilayer of cells. These findings also imply that forces other than van der Waals attractive forces and hydrophobic interactions stabilize cholesterol’s interaction with sphingomyelins. It is possible that a hydrogen bond between the amidelinked acyl chain of sphingomyelin and the free 3b-OH group of cholesterol stabilizes this interaction (Sankaram and Thompson, 1990; Bittman et al., 1994).

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Acknowledgements I thank Dr Peter Mattjus for discussions and comments on the manuscript. The support from the Academy of Finland, the Sigrid Juselius Foundation, and the A, bo Akademi University is gratefully acknowledged.

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