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. 2018 Sep 7;115(8):1530–1540. doi: 10.1016/j.bpj.2018.08.042

Sphingomyelin Stereoisomers Reveal That Homophilic Interactions Cause Nanodomain Formation

Yo Yano 1,2, Shinya Hanashima 1, Tomokazu Yasuda 1, Hiroshi Tsuchikawa 1, Nobuaki Matsumori 3, Masanao Kinoshita 3, Md Abdullah Al Sazzad 4, J Peter Slotte 4,∗, Michio Murata 1,2,∗∗
PMCID: PMC6260218  PMID: 30274830

Abstract

Sphingomyelin is an abundant lipid in some cellular membrane domains, such as lipid rafts. Hydrogen bonding and hydrophobic interactions of the lipid with surrounding components such as neighboring sphingomyelin and cholesterol (Cho) are widely considered to stabilize the raft-like liquid-ordered (Lo) domains in membrane bilayers. However, details of their interactions responsible for the formation of Lo domains remain largely unknown. In this study, the enantiomer of stearoyl sphingomyelin (ent-SSM) was prepared, and its physicochemical properties were compared with the natural SSM and the diastereomer of SSM to examine possible stereoselective lipid-lipid interactions. Interestingly, differential scanning calorimetry experiments demonstrated that palmitoyl sphingomyelin, with natural stereochemistry, exhibited higher miscibility with SSM bilayers than with ent-SSM bilayers, indicating that the homophilic sphingomyelin interactions occurred in a stereoselective manner. Solid-state 2H NMR revealed that Cho elicited its ordering effect very similarly on SSM and ent-SSM (and even on the diastereomer of SSM), suggesting that SSM-Cho interactions are not significantly affected by stereospecific hydrogen bonding. SSM and ent-SSM formed gel-like domains with very similar lateral packing in SSM/Cho/palmitoyloleoyl phosphatidylcholine membranes, as shown by fluorescence lifetime experiments. This observation can be explained by a homophilic hydrogen-bond network, which was largely responsible for the formation of gel-like nanodomains of SSMs (or ent-SSM). Our previous study revealed that Cho-poor gel-like domains contributed significantly to the formation of an Lo phase in sphingomyelin/Cho membranes. The results of the study presented here further show that SSM-SSM interactions occur near the headgroup region, whereas hydrophobic SSM-Cho interactions appeared important in the bilayer interior for Lo domain formation. The homophilic interactions of sphingomyelins could be mainly responsible for the formation of the domains of nanometer size, which may correspond to the small sphingomyelin/Cho-based rafts that temporally occur in biological membranes.

Introduction

Sphingomyelin acts as a key lipid molecule in eukaryotic cell membranes by forming functional domains called lipid rafts (1). Specific proteins and lipids are considered to reside in the rafts, forming dynamic-ordered domains of submicrometer size (2). Diverse cellular events such as cell adhesion, signal transductions, and microbial invasion can be mediated by the lipid rafts, in which the dynamic assembly/disassembly equilibrium is regarded as a key factor of proper cellular functions (3). Sphingomyelin and cholesterol (Cho) are known to be the typical lipid constituents in the lipid rafts (4). Sphingomyelin is the most abundant sphingolipid species in eukaryotic cell membranes, constituting 2–15% of total lipid components (5, 6). Intermolecular sphingomyelin-sphingomyelin and/or sphingomyelin-Cho interplay has been thought to be responsible for the dynamic molecular assemblies of lipid rafts. In particular, Cho has been a key lipid in forming rafts, because it has high miscibility with sphingomyelin and other phospholipids in bilayers (7); their interactions with Cho then may transform a rigid gel phase to a liquid-ordered (Lo) phase (8). Previous studies using covalent conjugates with Cho (9) and molecular dynamics simulations (10) suggested that sphingomyelin-Cho interactions are induced by the combination of intermolecular hydrogen-bonds between the functional groups of sphingomyelin and van der Waals interactions of their hydrocarbon portions (11).

The structure of D-erythro-sphingomyelin with the natural stereochemistry contains two neighboring chiral centers (2S, 3R) in the membrane surface portion of the sphingosine backbone, namely the amide-bearing and hydroxy-substituted carbon atoms (Fig. 1) (12). The chirality is strictly controlled in the biosynthetic pathway, where the stereochemistry of the C2 position originates from the α-carbon of L-serine, followed by coupling with palmitoyl-CoA (coenzyme A) to form 3-keto-dihydrosphingosine. The hydroxy-bearing chiral center is introduced by subsequent enzymatic reductions to produce D-erythro-sphingomyelin (2S, 3R) (13). These chiral centers likely contribute to the formation of intermolecular hydrogen-bonding, which has long been considered important for lipid-raft formation (14). A previous study using chemically modified sphingomyelins such as 3-O-methylated and 2-N-methylated analogs demonstrated that O- or N-methylation disrupts the intermolecular hydrogen-bonding network, which results in decreased gel formation and reduces the ordering effects of Cho (15).

Figure 1.

Figure 1

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Chemical structures of sphingomyelin (D-erythro-SSM, SSM), SSM enantiomer (L-erythro-SSM, ent-SSM), SSM diastereomer (L-threo-SSM, threo-SSM), and their deuterated analogs.

The influence of two neighboring chiral centers on membrane properties has been investigated using L-threo-sphingomyelin (2S, 3S), a diastereomer of the natural homolog (16, 17, 18). A differential scanning calorimetry (DSC) study showed that the phase transition temperature of pure threo-stearoyl-sphingomyelin (threo-SSM) was significantly lower than that of sphingomyelin (16, 18). In addition, the cooperativity of threo-SSM was higher than that of SSM, which was rather similar to the properties of saturated phosphatidylcholines (18). Thus, we adopted L-erythro-SSM, the enantiomer of SSM (ent-SSM; 2R, 3S), to investigate the role of stereochemistry in lipid-lipid interactions that lead to lateral domain formation. Although a racemic mixture of D/L-erythro-palmitoyl-sphingomyelin (PSM), was used in a previous study (19) to examine the basic physicochemical properties of its bilayers, those of membranes containing the pure enantiomer are not examined in detail.

In this study, we synthesized ent-SSM 3 (Fig. 1) and compared its physicochemical properties and lipid-lipid interactions with SSM 1 and threo-SSM 5 using DSC, solid-state 2H NMR, and fluorescence spectroscopy. The DSC thermograms indicated immiscibility of ent-SSM with SSM or PSM, and solid-state 2H NMR results showed a similar ordering effect of Cho on the two SSM bilayers. In ternary lipid bilayers (ent-SSM or SSM/POPC/Cho), these SSMs showed a similar membrane-ordering profile. Our results consistently demonstrate that the chirality of sphingomyelin directly contributed to the homophilic interactions through intermolecular stereochemical matching, whereas stereochemical effects were weak or nonexistent in sphingomyelin-Cho interactions.

Materials and Methods

General

Texas-Red 1,2-palmitoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-phospatidyl choline (POPC) were purchased from Avanti Polar Lipid (Alabaster, AL). SSM was purified from bovine brain sphingomyelin (Avanti Polar Lipids) by reverse-phase high-performance liquid chromatography (14). Synthesis of ent-SSM (compounds 3 and 4 in Fig. 1) is described in the Supporting Materials and Methods. Threo-SSM was synthesized following published procedures (18).

Cho and other chemicals for organic synthesis were purchased from Nacalai Tesque (Kyoto, Japan). Dry organic solvents and silica gel were obtained from Kanto Chemical (Tokyo, Japan). Deuterated solvents and deuterium-depleted water for NMR experiments were purchased from Cambridge Isotope Laboratories (Tewksbury, MA) and ISOTEC (St. Louis, MO). Solution NMR was collected using ECS-400 and ECA-500 spectrometers (JEOL, Tokyo, Japan). Electrospray ionization mass spectrometry spectra were collected using Linear Ion Trap Orbitrap (Thermo Fisher Scientific, Waltham, MA).

DSC

DSC was performed using a Nano-DSC (TA Instruments, New Castle, DE). Vesicles were prepared according to published procedures (20). Briefly, an appropriate molar ratio of SSM and its analogs in chloroform/methanol (4:1) was prepared and mixed, and the solvent was evaporated under a nitrogen stream. The samples were kept in high vacuum for 24 h. The residual lipid film was dispersed into Milli-Q (Merck Millipore, Billerica, MA) water and incubated for 30 min at 55°C with intermittent vortex mixing. Then, 500 μL of the bilayer sample was injected into the DSC sample cell (full cell). The heating-cooling scans were performed between 20 and 55°C with a linear temperature gradient of 0.5°C/min.

Solid-state 2H NMR

Multilamellar vesicles (MLVs) were prepared in a conventional manner. Deuterated lipids (10.5 millimoles (mmol)) were dissolved in methanol/chloroform (4:1), and an appropriate amount of Cho was added. After mixing, the organic solvent was removed, and the sample was dried for 12 h in high vacuum. The resulting lipid film was hydrated with water (1 mL) and vortexed at 65°C. The sample was then freeze-thawed several times, and the suspension was lyophilized. The obtained lipid film was rehydrated with deuterium-depleted water to 50% moisture (w/w) and freeze-thawed several times. Samples were then transferred into 5-mm glass tubes and sealed with epoxy glue.

Solid-state 2H NMR spectra were collected with a 300-MHz spectrometer (CMX-300; Varian, Palo Alto, CA) equipped with a 5-mm 2H static probe (Otsuka Electronics, Osaka, Japan). To observe the 2H quadrupole splitting pattern under static conditions, a solid-echo pulse sequence was used with a 90° pulse width set in 3 μs; the solid-echo delays were set in 12 and 11 μs, respectively, and the initial delay was set in 0.5 s. The sweep width was set to 200 kHz with 8 k data points, and the number of scans was ∼100,000. Free induction decay data were Fourier transformed upon exponential multiplication.

Time-resolved fluorescence measurements

MLVs were prepared to a molar composition of 30:60:10 SSM/POPC/Cho, ent-SSM/POPC/Cho, and threo-SSM/POPC/Cho, respectively, following a published method (21). The lipid solutions were dried under a stream of nitrogen gas, and the lipids were hydrated with argon-purged water for 30 min at 65°C, followed by sonication at 65°C for 5 min. trans-parinaric acid (tPA) in methanol (1 mol %) was added to the vesicle suspensions (the final methanol concentration was less than 1 vol %), and the mixture was vortexed and sonicated at 65°C for 5 min. tPA in MLVs were excited at 298 nm, and the emission was measured at 405 nm. The tPA fluorescence lifetime was obtained using a FluoTime 200 spectrometer (PicoQuant, Berlin, Germany), with a PicoHarp 300E time-correlated single photon-counting module. Data acquisition and analyses were performed with FluoFit Pro software (PicoQuant). The obtained emission decay curves were resolved into three different lifetime components (τ1, τ2, and τ3) by best fitting of the exponential decays (22).

Results

DSC analysis

The phase transition temperature is a key indicator of the stability of lipid packing in the gel phase of bilayers. SSM, ent-SSM, and threo-SSM membranes were subjected to DSC measurements to collect their endothermic profiles (Fig. 2; Table 1). The main endothermic transitions of SSM and ent-SSM membranes were observed at 44.9 ± 0.2 and 45.0 ± 0.1°C, respectively, with asymmetrically broad peaks (T1/2 = 0.70 ± 0.15 and 0.70 ± 0.09; Fig. 2, a and b). These values agreed well with the previously reported result for the pure SSM membrane (44.5–44.8°C) (19, 23). As expected, the transition enthalpies of the SSM membrane (3.0 ± 0.4 and 9.4 ± 0.4 kcal/mol) and the ent-SSM membrane (2.6 ± 0.5 and 9.6 ± 0.5 kcal/mol) were indistinguishable (Table 1). On the other hand, threo-SSM showed a distinct profile from that of both SSM and ent-SSM, with a sharp main transition (T1/2 = 0.35 ± 0.05) at 44.1 ± 0.1°C, characteristic pretransition at 43.1 ± 0.2°C (Fig. 2 c), and with reduced transition enthalpies of 2.0 ± 0.3 and 7.0 ± 0.4 kcal/mol, respectively (Table 1). These DSC data indicate that the gel-to-fluid phase transition of the SSM membrane is identical to that of the ent-SSM membrane within experimental errors. In contrast, the distinguishable profile of threo-SSM indicates that molecular packing in the solid phase differed from that of the erythro-SSMs because of differences in intermolecular interactions.

Figure 2.

Figure 2

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DSC phase transition thermograms of MLVs consisting of SSM (a), ent-SSM (b), and threo-SSM (c). Thermograms were collected from three samples of each composition, and the thermograms exhibited good reproducibility. SDs are shown in Table 1.

Table 1.

Summary of the DSC Data of SSM, ent-SSM, threo-SSM, and Racemic SSM

Tp (°C) ΔHp (kcal/mol) Tm (°C) ΔHm (kcal/mol) T1/2 (°C)
SSM 33.2 ± 0.7 3.0 ± 0.4 44.9 ± 0.2 9.4 ± 0.4 0.70 ± 0.15
Ent-SSM 34.4 ± 0.7 2.6 ± 0.5 45.0 ± 0.1 9.6 ± 0.5 0.70 ± 0.09
Threo-SSM 43.1 ± 0.2 2.0 ± 0.3 44.1 ± 0.1 7.0 ± 0.4 0.35 ± 0.05
Racemic SSM – – 44.5 ± 0.3 8.2 ± 0.5 0.98 ± 0.08
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The average temperature of three measurements ± SD are shown and the SSM/ent-SSM ratio is 1:1. ΔH, mole transition enthalpy; T1/2, fullwidth half-maximum; Tp, pretransition temperature.

We examined the miscibility of ent-SSM with SSM using DSC. The phase transition of SSM membranes was observed in bilayers containing gradually increasing amounts of ent-SSM (from 0 to 100 mol %) in SSM membranes (Fig. 3). As a result, the pretransition disappeared with increasing content of ent-SSM, whereas the main transition temperature was not significantly altered but showed small fluctuations depending on the SSM/ent-SSM ratios (Fig. 3 a). The endothermic profile of the “racemic” (1:1 SSM/ent-SSM) condition still provided a clear transition peak. This profile has two potential explanations: one is that the phase separation (or immiscibility) occurs between SSM and ent-SSM domains, and the other is that the gel phase consisting of molecularly mixed SSM and ent-SSM happens to have a melting temperature very close to that of SSM. The small fluctuations in the melting temperatures of the SSM/ent-SSM bilayers (Fig. 3 b) were not found in the mixtures of D- dipalmitoylphosphatidylcholine (DPPC) (2S) and L-DPPC (2R) bilayers (Fig. S1). A previous study (24) has suggested that D-DPPC and L-DPPC are mixed well with each other. Thus, this fluctuation for the SSM mixtures in Fig. 3 may imply that SSM and ent-SSM are not perfectly mixed probably because of the formation of small domains for each sphingomyelin. In addition, line broadening observed for some of the mixtures may be accounted for by a loss of cooperativity due to coexisting SSM and ent-SSM domains.

Figure 3.

Figure 3

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DSC phase transition thermograms of the vesicles composed of SSM/ent-SSM (100−x:x) (a) and main transition temperatures of the corresponding mol % fractions (x) of ent-SSM in SSM/ent-SSM (solid black circle) and L-DPPC in D-DPPC/L-DPPC (100−x:x) (open circle) (b). DSC thermograms of D-DPPC/L-DPPC are shown in Fig. S1. The Tm in (b) are shown with the SDs (n = 3), many of which are smaller than the symbols.

Because the DSC experiments for a series of SSM/ent-SSM mixed membranes did not show significant changes in their melting temperatures, we adopted PSM (Tm, 41°C) instead to examine the miscibility of SSM/PSM and ent-SSM/PSM (Fig. 4) (25). The addition of PSM to the SSM membrane caused the complete coalescence of the two main transitions, which resulted in one broader main peak (Fig. 4 a). This indicates that PSM has high miscibility with SSM to form homogenous SSM/PSM gel phase. The peak broadening could have resulted from the two carbon differences between PSM and SSM, which likely destabilized the lipid packing in the gel domain. On the other hand, the main transitions of ent-SSM in the presence of PSM did not exhibit clear coalescence (Fig. 4 b). For the binary bilayers containing ent-SSM/PSM at the ratios of 1:1, 2:1, and 3:1, peak deconvolution indicated the presence of relatively small peaks at 41.9 ± 0.2, 42.6 ± 0.2, and 43.5 ± 0.2°C, whereas larger peaks were observed at 42.4 ± 0.1, 43.3 ± 0.0, and 44.3 ± 0.1°C, respectively (Fig. S2). These DSC results imply the presence of phase separation, with two gel phases consisting mainly of PSM and ent-SSM.

Figure 4.

Figure 4

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DSC phase transition thermograms in the main transition of SSM/PSM (a) and ent-SSM/PSM (b). From the top to the bottom of the thermograms, the molar ratios of SSM/PSM and ent-SSM /PSM are 1:0, 3:1, 2:1, 1:1, 1:2, 1:3, and 0:1. Reproducibility of the thermal behavior of the ent-SSM/PSM preparations was confirmed by three separate experiments (see Fig. S2).

2H NMR of SSM in Cho-containing membranes

We next evaluated the order of the lipid chains of SSM, ent-SSM, and threo-SSM based on the 2H NMR quadrupolar coupling (kHz) of selectively 2H-labeled lipids. In particular, a 10′,10′-d2-stearoyl moiety was used to label the SSMs to examine the ordering effect of Cho, because the 10 position of the acyl chain is known to exhibit the highest order in the presence of Cho (26, 27). 2H NMR measurements were carried out with MLVs consisting of SSM/Cho and ent-SSM/Cho (1:1) (Fig. 5 a). The quadrupole splitting values thus obtained were almost equivalent between the two MLV preparations for all the temperatures tested, indicating that like SSM, ent-SSM is ordered by Cho. It is suggested that the stereochemistry of the 2,3-positions of SSM-bearing amide and hydroxy groups did not significantly affect interactions with Cho as reported previously for enantiomeric Cho (28). Interestingly, d2-threo-SSM showed only marginal difference from SSM in the splitting width except for 25°C (Fig. 5 a), indicating that the stereochemical alteration near the headgroup portion did not markedly influence Cho-induced ordering of the stearoyl chain, particularly for 40 and 50°C. Further 2H NMR experiments to elucidate Cho-ordering effects were carried out using racemic-SSM/Cho bilayers. The quadrupolar splitting obtained from d2-SSM in an SSM/ent-SSM/Cho (1:1:2) system was quite similar to those observed with the SSM/Cho (1:1) system (Fig. 5 a); d2-ent-SSM in the same racemic mixture revealed the same splitting widths (Fig. S7; Table S1). The results indicate that SSM and ent-SSM are similarly ordered by Cho in the racemic membrane. In comparison with SSM, the temperature-dependent splitting of d2- palmitoylstearoylphosphatidylcholine (PSPC) (Fig. 5 c) in PSPC/Cho (1:1) membranes was also examined. The results show a close similarity to the SSM/Cho systems up to 40°C, but the splitting width became significantly smaller at 45 and 50°C. This observation indicates that SSM/Cho bilayers are more thermally stable than PSPC/Cho bilayers, which is likely due to the difference between ceramide and glycerol backbones (27). Unlike PSPC, SSM is known to form intermolecular hydrogen bonds with their amide groups (14). Cho effectively enhances orders in the middle regions of SSM acyl chains (26). It is likely that Cho’s ordering effect on the SSM hydrocarbon chains and the intermolecular hydrogen bond effectively strengthen ordered domains in SSM-Chol bilayers to enhance the thermal stability as compared with those of PSPC-Chol domains.

Figure 5.

Figure 5

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Cho-induced ordering effects on the acyl chain of SSM, ent-SSM, and threo-SSM in binary systems as determined by solid-state 2H NMR in the temperature range of 25–50°C. (a) Quadrupolar coupling Δν of d2-SSM/Cho (1:1), d2-ent-SSM/Cho (1:1), racemic SSM/Cho (d2-SSM/ent-SSM/Cho, 1:1:2), and d2-threo-SSM/Cho (1:1) is shown. (b) d2-SSM/Cho (1:1) (the same as shown in (a)), Δν of d2-SSM/CME (1:1), and d2-ent-SSM/CME (1:1) are shown. (c) Δν of d2-PSPC/Cho (1:1) and d2-PSPC/CME (1:1) is shown. Corresponding 2H NMR spectra and splitting width of Pake doublets are shown in Figs. S3–S11 and Table S1, respectively.

Previous studies have often suggested that intermolecular hydrogen-bonding between sphingomyelin and Cho plays an important role in the formation of Lo domains (10, 29). To further investigate this hypothesis, we performed 2H NMR experiments using Cho methyl ether (CME), in which the 3-β-hydroxy moiety is blocked with a methyl group, thus greatly attenuating the hydrogen-bond-donating activity of Cho. In d2-SSM/CME and d2-ent-SSM/CME membranes, the quadrupolar splitting widths were comparable to that of the d2-SSM/Cho membrane (Fig. 5 b) for all the temperatures, including 50°C (higher than SSM’s Tm of 45°C). These results imply that a possible intermolecular hydrogen-bond, originating from the three-hydroxy group of Cho (as an hydrogen-bond donor), was not essential to enhance acyl-chain ordering by Cho. In contrast, in the PSPC/CME membrane, d2-PSPC was less susceptible to the CME-induced ordering effect above the main PSPC’s Tm of 49°C, which was significantly smaller than that of the PSPC/Cho membrane. These results indicate that the 3-β-hydroxy group of Cho was not essential for the intermolecular interactions between SSM and Cho but significantly contributed to the thermal stability of PSPC-Cho interactions, particularly at higher temperatures.

Fluorescence lifetime analysis

To further investigate the stereochemical effects of SSM on lateral lipid packing in biologically relevant bilayers, we measured fluorescence lifetimes of a fluorescent probe (tPA) incorporated into SSM-, ent-SSM-, and threo-SSM-containing ternary systems. We adopted SSM/POPC/Cho systems, which have often been used as a model membrane in biophysical studies of lipid-raft formation (30, 31, 32). According to a phase diagram of similar ternary bilayers, the lipid composition of SSM/POPC/Cho (30:60:10) that we adopted induced very little macroscopic phase separation (Fig. S12), such as coexisting Lo/liquid-disordered (Ld) and Lβ/Ld phases (33, 34, 35), whereas the formation of nanodomains having Lo or So characteristics was suggested as expected at 30°C (see Fig. S13 and Table S2 for details). In addition, these nanodomains have also been reported for monolayers by using x-ray scattering (36, 37, 38). Macroscopic domain segregation, particularly gel-phase formation, often hampers the precise evaluation of intermolecular interactions at nanosecond and/or nanometer scales. We used tPA as the fluorescence probe for the following two reasons: tPA favorably partitions into gel phase (So) or Lo domains, and its fluorescence lifetime is markedly affected by the mobility of surrounding lipid molecules at the nanosecond timescale (21, 39, 40, 41).

The results shown in Fig. 6 clearly demonstrate that the stereochemical difference between SSM and ent-SSM has a negligible effect on the lipid-packing properties of the ternary bilayers; the temperature-dependent changes in average lifetime (Fig. 6 a) and the three components of lifetime τ1, τ2, and τ3 (Fig. 6 b) were virtually identical between the SSM- and ent-SSM-containing membranes. In contrast, the bilayers containing threo-SSM exhibited a significant difference in both cases. Moreover, as seen in Fig. 6 a, the racemic SSMs showed a shorter averaged lifetime, indicating that a small but significant difference between optically pure SSM and racemic SSM, which was not apparent in the DSC and 2H NMR experiments (Figs. 2 and 5), could be detected in this experiment. These results will be interpreted in the following section.

Figure 6.

Figure 6

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Lifetime analysis of tPA in SSM, ent-SSM, and threo-SSM membranes containing ternary systems. The lipid ratio of SSMs/POPC/Cho was 30:60:10. (a) Intensity-weighted average lifetimes are shown by symbols with error bars, many of which are smaller than the symbols. SSM (circle with dotted line), ent-SSM (triangle with broken line), threo-SSM (square with two-dot chain line), and racemic SSM (solid line) are shown. (b) Fitted lifetime of τ1 (gel-like domain; black), τ2 (intermediately ordered domain; gray), and τ3 (fluidic domain; stripe), which are fluorescence lifetime components of tPA in SSM, ent-SSM, threo-SSM, and racemic SSM, are shown. The experiments were performed at 30°C. The values are given as the mean ± SD from at least three independent experiments (n), n ≥ 3; ∗p < 0.05 in t-test. NS, not significant.

Discussion

In the formation of Lo domains in membranes, sphingomyelin hydrocarbon chains play a major role through attractive van der Waals forces. Moreover, hydrogen-bond-donating functionalities such as amide and hydroxy groups certainly contribute to the interaction with neighboring lipid molecules. The balance of the intermolecular attractions, particularly homophilic and heterophilic interactions, could be a determining factor of the size and lifetime of sphingomyelin-rich-ordered domains. For this purpose, we adopted ent-SSM bearing the mirror-image configuration as a key probe molecule, particularly to discriminate SSM-SSM interactions from SSM-Cho interactions. SSM-SSM interaction is identical to that between ent-SSM and ent-SSM, whereas an SSM-Cho pairing should be typically different from the diastereomeric pairing of ent-SSM and Cho.

In DSC measurements, the Tm temperature of SSM/ent-SSM racemic membranes (44.5°C) was very close to that of SSM (44.9°C), and it exhibited slightly broad peaks (T1/2; 0.70 vs. 0.98°C), suggesting that the cooperativity in SSM/ent-SSM gel melting was minimally affected by the presence of 50 mol % of the antipode (Fig. 3; Table 1). Similarity among the Tm temperatures of PSM, ent-PSM, and PSM/ent-PSM racemic membranes have been reported using synthetic PSMs (19). In contrast, threo-SSM, a diastereomer of SSM (or ent-SSM), showed much narrower peak widths, although its Tm was not much different from that of SSM (Fig. 2; Table 1) (16, 18). This observation raises two possibilities: SSM and ent-SSM are mixed very well to form a homogeneous gel phase giving a single endothermic peak, or their miscibility is low enough to give rise to two overlapping endothermic peaks. To verify these two assumptions, the miscibility of SSM and ent-SSM with PSM (having lower Tm) was examined (Fig. 4). PSM was readily mixed with SSM to form a homogeneous gel phase, probably because of their stereochemical matching (Fig. 4), whereas the mixing of PSM with ent-SSM was limited, resulting in segregation into PSM-rich and ent-SSM-rich gel phases. These data strongly suggest that homophilic lipid interactions in PSMs (and ent-SSMs) are preferable over heterophilic PSM-ent-SSM interactions. This observation can be accounted for by a significant difference in stereospecific homophilicity, which is probably due to appropriate intermolecular hydrogen-bonds.

We next compared the ordering effects of Cho on SSM and ent-SSM using 2H NMR in the homogeneous Lo phase (SSM/Cho 1:1 mixtures; Fig. 5). We found very close doublet widths between d2-SSM/Cho and d2-ent-SSM/Cho bilayers, indicating that Cho exerts the same ordering effects on SSM and ent-SSM, regardless of their different stereochemical matching (Fig. 5 a). Cho elicited the ordering of threo-SSM to a similar extent, suggesting that SSM stereochemistry does not significantly influence SSM-Cho interactions. At 25°C (and 30°C), however, there is a certain difference in the ordering of acyl chains between threo-SSM and SSM. We deduce that this is caused by weaker intermolecular hydrogen-bonds of threo-SSM than those of SSM, and the difference is apparent at low temperature, because the packing of acyl chains becomes loose at higher temperature, which increases the intermolecular distance and weakens the hydrogen-bonding between the headgroup regions for both SSM and threo-SSM. These results further indicate that hydrogen-bonding is not significantly involved in Cho-sphingomyelin interactions, whereas their hydrophobic interactions are a main driving force to enhance membrane order.

The splitting widths of deuterated SSM and ent-SSM were unaffected when the 3β-OH group of Cho was blocked with an O-methyl group in CME (Fig. 5 b). This finding implies that hydrogen-bonding with 3β-OH of Cho is not essential for acyl-chain ordering. As a hydrogen-bond acceptor, an ether is generally known to be a weaker or hindered Lewis base than a hydroxy group. Thus, O-methylation could have affected the chain packing of SSM even if CME played a role as an acceptor in hydrogen-bond formation with SSM. In contrast, PSPC exhibited a significant reduction in the Pake doublet widths by CME at 50°C as compared with Cho (Fig. 5 c). This result suggests that the 3β-OH of Cho is important for stabilizing the Lo phase of PSPC at higher temperatures. These observations partially support our previous finding that Cho is located in the deeper (away from the membrane surface) interior of SSM membranes than in polycarbonate membranes (26, 27). The hydroxy group of Cho in SSM membranes, therefore, probably resides beyond the hydrogen-bond distance from the amide group of SSM (14, 26).

In SSM/POPC/Cho systems, we further examined the stereospecific interactions between SSM and Cho by evaluating the membrane order based on tPA lifetime (Fig. 6) in comparison with other experimental results. The intensity-weighted average lifetimes of ent-SSM/POPC/Cho bilayers, which exhibited the formation of highly ordered domains at 30°C or lower temperature, were strikingly similar to those of SSM/POPC/Cho bilayers for all the temperatures tested (Fig. 6 a). The results clearly indicate that the membrane order of ent-SSM-containing bilayers is almost identical to that of SSM, although their heterophilic interactions such as SSM/Cho and SSM/POPC should significantly differ with respect to diastereomeric recognition. On the other hand, the average lifetimes of tPA in the threo-SSM membrane below 30°C were significantly shorter than those of the SSM membrane (Fig. 6 a). This fluidity change likely originates from the different molecular order between SSM and threo-SSM domains. Racemic SSM-containing membranes (SSM/ent-SSM/POPC/Cho 15/15/60:10) exhibited lifetimes similar to those of threo-SSM; a possible explanation for racemic membrane will be discussed below. In the 2H NMR spectra of a homogeneous Lo phase, the Cho-induced ordering effect on threo-SSM was comparable to that on SSM (Fig. 5 a) at 40 and 50°C. However, the DSC analysis (Fig. 2) indicated that the gel/liquid-crystalline phase transition profile was clearly different between threo-SSM and SSM. Therefore, the gel component in the ternary system may play a key role for the difference in tPA lifetimes. The average lifetimes of threo-SSM were further examined with the three lifetime components τ1, τ2, and τ3, which are thought to correspond to gel-like, intermediate-ordered, and fluid phases (41), respectively. In addition, the lifetime component τ1 has been used as a marker of a gel phase in lipid bilayers (21, 22). In the ternary bilayers at 30°C, the longest τ1 components of SSM, ent-SSM, racemic SSM, and threo-SSM were 40.1 ± 3.0, 42.2 ± 4.0, 37.5 ± 1.3, and 31.2 ± 2.7 ns, respectively (Fig. 6 b). These lifetime data suggest that SSM and ent-SSM form gel-like domains with very similar ordering profiles because the homophilic lipid interaction of SSMs and that of ent-SSMs are theoretically identical (as also evidenced by DSC). In contrast, threo-SSM apparently exhibited lower ordering of gel-like domains at 30°C, probably because of its weaker homophilic interaction. The racemic SSM showed a significantly longer lifetime in the τ1 component than threo-SSM (Fig. 6 b), probably indicating that both SSM and ent-SSM form their own gel-like domains, even under the racemic conditions. The data also point to the importance of SSM stereochemistry in their homophilic interactions. A difference in acyl-chain packing between SSM (ent-SSM) and threo-SSM was also observed in the DSC experiments; threo-SSM revealed a lower enthalpy value at the main transition (Table 1). The second lifetime component τ2, which is known to be derived largely from the intermediately ordered phase, was almost identical for all the SSM bilayer systems as shown in Fig. 6 a (21, 22). Taken together, the results consistently indicate that the gel-like domains are more ordered in the presence of SSM (or ent-SSM) and less ordered in the threo-SSM-containing bilayers.

Because the DSC results (Fig. 4) showed the immiscibility of PSM and ent-SSM, SSM and ent-SSM probably segregate from each other in the ternary lipid system, resulting in the separate formation of gel-like domains. Thus, the τ1 component of the racemic bilayers reflecting the properties of the gel phase was not greatly influenced by the formation of segregated SSM and ent-SSM domains (Fig. 6 b). On the other hand, the average lifetime of the racemic system was significantly shorter than that of the SSM system in lower temperatures (Fig. 6 a). A possible explanation for this difference is illustrated in Fig. 7. The average lifetime is markedly affected by a fractional ratio of the phase contributing to the longest lifetime component τ1. Rearrangement of nanodomains occurs at the nanosecond timescale by their fusion and fission in a stereospecific manner. For the racemic bilayers, this dynamic process leads to reductions in the time-averaged size and fractional area of nanodomains with respect to tPA partition. A decrease in the average size of nanodomains due to a lower fusion rate shortens the residence time of tPA in the highly ordered phase, thus resulting in shorter average lifetimes; a low fusion rate has been shown to significantly decrease the size of oil drops in emulsion (42), and the domain size effect on the fractional area has been discussed in our previous study (22). This hypothetical image of nanodomains consisting mostly of SM, as shown in Fig. 7 a, could be one of the possible explanations for SM-induced Lo domains.

Figure 7.

Figure 7

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Conceptual illustrations for explaining a difference in size of nanodomains between enantiomeric-pure (a) and racemic (b) sphingomyelins. The domains are temporally drawn as circles; the black circles consist mostly of natural sphingomyelin, and the white circles consist mostly of ent-sphingomyelin. The probability of fusion between two neighboring domains should be higher for the enantiomeric-pure aggregates (a) than the racemic mixture (b), whereas the fission probability of domains with a similar size should be the same between them. This situation results in the presence of larger domains in the pure sphingomyelin membrane than in the racemic one (see the text for details). Interdomain areas consist of Cho, other colipid (e.g., unsaturated PC), and residual sphingomyelin. Asterisks indicate intermediate states in the process of nanodomain fission or fusion.

As discussed above, homophilic SSM-SSM interactions through hydrogen-bonding in the headgroup region of membranes are thought to play a key role in lipid ordering, particularly in forming gel-like domains. Regarding the mechanism of lipid-raft formation, we assume that sphingomyelin molecules tend to associate with each other, resulting in the formation of a stereospecific hydrogen-bonding network. On the other hand, Cho preferably enhances the acyl-chain ordering of sphingomyelin in the interior of membranes (43) and promotes lipid-raft formation together with hydrogen-bond network between their headgroups (14, 43). Therefore, the use of ent-SSM instead of SSM does not lead to a significant difference in membrane-ordering effects induced by Cho, because the Cho-interaction site is quite distant from the headgroup region of membranes. The 2H NMR results in Fig. 5 a also indicate weak interactions between SSM and ent-SSM, in which diastereomeric interactions between their headgroups, if any, have little effect on the chain packing of these lipids. Further 2H NMR experiments in Fig. 5, b and c showed that the O-methylation of Cho does not affect SSM-Cho interactions but significantly influences PSPC-Cho interactions. Taken together, these results consistently suggest that the homophilic hydrogen-bonding network of SSMs plays a crucial role in the formation of highly ordered gel-like domains in SSM/POPC/Cho bilayer systems.

Next, we consider the lateral segregation profile of SSM-rich domains in the SSM/POPC/Chol ternary system. In line with previous reports (32, 44, 45, 46), our present results indicate that SSM domains have highly ordered hydrocarbon chains. Because the stereospecific SSM-SSM recognition is thought to be responsible for the intermolecular interactions in the headgroup region of membranes, the hydrophobic interactions in the interior of bilayers needs to be further examined to explain lateral segregation. The alicyclic structure of Cho, which is mainly responsible for the ordering of lipid chains, greatly facilitates Lo phase formation by interacting with SSM chains in the membrane interior, whereas 3β-OH of Cho plays a negligible role in hydrogen-bonding with SSM. Moreover, the 2H NMR data in Fig. 5 b show that the ordering effect of CME on SSM chains is not distinguishable from that of Cho for all the temperatures tested, indicating that the size and polarity of Cho’s headgroup do not significantly influence the packing properties of SSM chains. These observations imply that molecular-level mixing of SSM and Cho rarely occurs and that a large portion of SSM forms relatively pure homophilic aggregates. As we reported previously (22), gel-like domains consisting mostly of SSM occur in SSM/Cho binary bilayers. In the conditions of the ternary systems in Fig. 6, the gel phase should similarly occur at 30°C according to previous DSC results (30). Thus, the dominant domains present in these bilayers are likely to consist mostly of SSM but to contain little Cho. If the size of these domains were large enough, this membrane profile could be regarded as the gel (or So) phase. However, their size should be very small, because microscopic observation showed no phase separation at 30°C, and Pake double signals of 2H NMR rule out the presence of the macroscopic gel phase but, judging from their splitting width, reveal the formation of intermediate phase between Lo and Ld (Figs. S12 and S13) phases. Based on these observations, we propose that the Lo phase in Cho-containing bilayers consists largely of nanodomains of sphingomyelin that are not mixed well with Cho. To exert the ordering effect on the acyl chains, Cho interacts with the nanodomain, in which the domain size needs to be small enough for Cho to elicit its ordering effects on all the sphingomyelin molecules inside the domain.

Conclusions

The ent-SSM was synthesized to investigate the role of the amide and hydroxyl functionalities in homophilic interactions of sphingomyelins responsible for domain formation. DSC experiments showed the immiscibility of ent-SSM with PSM, suggesting the copresence of two gel phases consisting of PSM and of ent-SSM because of their stereoselective homophilic interactions. Solid-state 2H NMR revealed that Cho enhances the ordering of SSM and ent-SSM to the same extent, indicating that the stereospecific SSM-Cho interaction with its polar functionality, if any, could not significantly influence the packing of SSM molecules. Fluorescence lifetime experiments further demonstrated that in SSM/POPC/Cho (30:60:10) ternary bilayers, SSM and ent-SSM formed gel-like domains in a very similar manner. These results indicate that the homophilic interaction of SSM through the amide (or hydroxyl) group enhanced the formation of nanodomains, which consisted mostly of SSM (or ent-SSM). This nanodomain could be stabilized by the hydrogen-bonding network among SSM molecules, whereas hydrophobic interactions with Cho enhanced the ordering of their hydrocarbon chains. This homophilic nanodomain of SSM, which can be regarded as a “sphingomyelin cluster,” possibly accounts for the highly ordered lipid chains in the sphingomyelin/Cho-induced Lo domains in model membranes and possibly in lipid rafts of cell membranes.

Author Contributions

Y.Y., T.Y., M.A.A.S., and M.K. performed experiments. All authors interpreted data. Y.Y., S. H., J.P.S., and M.M. wrote the manuscript with input from all authors. All authors have approved the final version of the manuscript.

Acknowledgments

The authors thank Drs. K. Kabayama, N. Inazumi, and Y. Todokoro (Osaka University) for their help for confocal microscopic observations and NMR measurements.

This work was supported by Grants-in-Aid for Scientific Research (S) 16H06315 and by (C) 15K01801 from Japan Society for the Promotion of Science. Y.Y. was supported by Japan Science and Technology Agency Exploratory Research for Advanced Technology Lipid Active Structure Project. (JPMJER1005). The Slotte laboratory was supported by grants from the Sigrid Juselius Foundation, the Jane and Aatos Erkko Foundation, and the Magnus Ehrnrooth Foundation.

Editor: Arne Gericke.

Footnotes

Supporting Materials and Methods, thirteen figures, and two tables are available at http://www.biophysj.org/biophysj/supplemental/S0006-3495(18)31020-8.

Contributor Information

J. Peter Slotte, Email: jpslotte@abo.fi.

Michio Murata, Email: murata@chem.sci.osaka-u.ac.jp.

Supporting Material

Document S1. Supporting Materials and Methods, Figs. S1–S13, and Tables S1 and S2
mmc1.pdf (513.7KB, pdf)
Document S2. Article plus Supporting Material
mmc2.pdf (1.6MB, pdf)

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Associated Data

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Supplementary Materials

Document S1. Supporting Materials and Methods, Figs. S1–S13, and Tables S1 and S2
mmc1.pdf (513.7KB, pdf)
Document S2. Article plus Supporting Material
mmc2.pdf (1.6MB, pdf)

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