Biophysical Journal Volume 100 June 2011 2633–2641
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Sterols Have Higher Affinity for Sphingomyelin than for Phosphatidylcholine Bilayers even at Equal Acyl-Chain Order Max Lo¨nnfors,† Jacques P. F. Doux,‡ J. Antoinette Killian,‡ Thomas K. M. Nyholm,† and J. Peter Slotte†* †
˚ bo Akademi University, Turku, Finland; and ‡Biochemistry of Membranes, Bijvoet Center for Biochemistry, Department of Biosciences, A Biomolecular Research, Utrecht University, Utrecht, The Netherlands
ABSTRACT The interaction between cholesterol and phospholipids in bilayer membranes is important for the formation and maintenance of membrane structure and function. However, cholesterol does not interact favorably with all types of phospholipids and, for example, prefers more ordered sphingomyelins (SMs) over phosphatidylcholines (PCs). The reason for this preference is not clear. Here we have studied whether acyl-chain order could be responsible for the preferred sterol interaction with SMs. Acyl-chain order was deduced from diphenylhexatriene anisotropy and from the deuterium order parameter obtained by 2H-NMR on bilayers made from either 14:0/14:0(d27)-PC, or 14:0(d27)-SM. Sterol/phospholipid interaction was determined from sterol bilayer partitioning. Cholestatrienol (CTL) was used as a fluorescence probe for cholesterol, because its relative membrane partitioning is similar to cholesterol. When CTL was allowed to reach equilibrium partitioning between cyclodextrins and unilamellar vesicles made from either 14:0/14:0-PC or 14:0-SM, the molar-fraction partitioning coefficient (Kx) was approximately twofold higher for SM bilayers than for PC bilayers. This was even the case when the temperature in the SM samples was raised to achieve equal acyl-chain order, as determined from 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy and the deuterium order parameter. Although the Kx did increase with acyl-chain order, the higher Kx for SM bilayers was always evident. At equal acyl-chain order parameter (DPH anisotropy), the Kx was also higher for 14:0-SM bilayers than for bilayers made from either 14:0/15:0-PC or 15:0-/14:0-PC, suggesting that minor differences in chain length or molecular asymmetry are not responsible for the difference in Kx. We conclude that acyl-chain order affects the bilayer affinity of CTL (and thus cholesterol), but that it is not the cause for the preferred affinity of sterols for SMs over matched PCs. Instead, it is likely that the interfacial properties of SMs influence and stabilize interactions with sterols in bilayer membranes.
INTRODUCTION Cholesterol is an important fluidity and permeability modulator in cell membranes and also helps to form and maintain a specific lateral organization in such membranes as well as in multicomponent model membrane systems (1–4). The interaction of cholesterol with phospholipids plays a major role in the regulation of membrane fluidity and permeability (4), and for the creation of lateral structure (5–7). However, cholesterol does not appear to interact with all types of phospholipids equally. Whereas cholesterol is able to interact with, e.g., monounsaturated phosphatidylcholines (PCs) which are abundant in cell membranes, when given a choice it prefers to associate with saturated phospholipids (8,9). The most common saturated phospholipids in cell membranes are the sphingomyelins (SMs). They have the same headgroup as PCs but in this case, it is attached to a long-chain base (usually 2-amino-4-octadecene-1,3-diol) to which saturated or monounsaturated C16-C24 acylchains are N-linked (10). If cholesterol can choose to interact with either acylmatched PCs or SMs, cholesterol prefers to interact with SMs (8,11,12). Some studies have suggested that cholesterol/SM interactions could be stabilized by hydrogen bonding (13,14). However, both NMR studies (15) and
Submitted February 24, 2011, and accepted for publication March 25, 2011.
recent molecular dynamics (MD) calculations have suggested that hydrogen bonding, although important for stabilizing interlipid interactions among sphingolipids, is not responsible for cholesterol/SM association (16). Alternatively, it is possible that cholesterol prefers to interact with SM over an acyl-matched PC due to differences in acyl-chain order. Cholesterol is known to preferentially interact with ordered phospholipids (17,18), and indeed, the affinity of cholesterol for binary phospholipid bilayers appears to correlate with acyl-chain order in the bilayer, as measured from 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy (8,19). Cholesterol’s interaction with palmitoyl SM has often been compared with its interactions with dipalmitoyl PC (20–22), because the N- and sn-2-linked acyl-chains are identical, and because the two lipids have almost an identical gel-to-liquid crystalline phase transition temperature (~41 C (23–25)). However, in the fluid phase the deuterium order parameter for the palmitoyl chain in the N-linked (SM) or the sn-2-linked (PC) position is very different. According to atomistic MD simulation and deuterium NMR experiments, the SM acyl-chain is much more ordered than the corresponding chain in PC (26,27). Because it is not fully established how much the difference in acyl-chain order can influence the interaction of cholesterol with, e.g., SM or PC, a systematic study to resolve this question is warranted.
*Correspondence:
[email protected] Editor: Heiko H. Heerklotz. Ó 2011 by the Biophysical Society 0006-3495/11/06/2633/9 $2.00
doi: 10.1016/j.bpj.2011.03.066
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Thus, we have compared the affinity of cholestatrienol (cholesta-5,7,9-trien-3-b-ol; CTL) for membrane bilayers which are fluid, and contain either myristoyl SM or chainmatched PCs. The myristoyl chain length for SM was selected to yield bilayers which are fluid at experimentally feasible temperatures. Affinity was deduced from the equilibrium-partitioning coefficient (Kx) of CTL, and was determined under conditions where the bilayer acyl-chain order was as identical as possible. Acyl-chain order was deduced from DPH steady-state anisotropy measurements, and from the deuterium order parameters of the N-linked or the sn-2-linked tetradecanoate(d27) chains. Our results show clearly that CTL has a significantly higher affinity to SM bilayers when compared to PC bilayers at identical acyl-chain order. These results show that acyl-chain order, per se, does not explain the preferential interaction between CTL and SM.
Determination of steady-state fluorescence anisotropy The steady-state fluorescence anisotropy of DPH or CTL was measured on a T-format Quanta-Master spectrofluorometer (Photon Technology International, Birmingham, NJ), essentially following the procedure described in Jaikishan et al. (32). The multilamellar vesicles had the indicated lipid compositions, with 1 mol % DPH or 2 mol % CTL. The wavelengths of excitation and emission were 368/430 nm and 324/390 nm for DPH and CTL, respectively. The steady-state anisotropy was calculated as described in Lakowicz (33).
Sterol partitioning into unilamellar vesicles The distribution of CTL between methyl-b-cyclodextrin (Sigma-Aldrich) and extruded large unilamellar phospholipid vesicles (200-nm average pore diameter) was determined as described previously (19,34). The assay yields the molar fraction partition coefficient Kx, for CTL. The CTL and thus sterol concentration was 2 mol % in all partitioning experiments. A high Kx indicates a higher affinity of CTL for the bilayer as compared with methyl-b-cyclodextrin.
MATERIALS AND METHODS Highly pure 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), sphingosylphosphorylcholine, 1-tetradecanoyl-2-hydroxy-sn-glycero-3phosphocholine, and 1-pentadecanoyl-2-hydroxy-sn-glycero-3-phosphocholine were purchased from Avanti Polar Lipids (Alabaster, AL). Tetradecanoyl anhydride and tetradecanoyl(d27) acid were from SigmaAldrich (St. Louis, MO). Pentadecanoyl acid was from Larodan Fine Chemicals (Malmo¨, Sweden). Sphingosylphosphorylcholine and lysophosphatidylcholines were acylated as described previously (11,28). The synthesized phospholipids were purified by preparative high-performance liquid chromatography (RP C18, elution with methanol) and their identities were verified by electrospray ionization mass spectrometry (purity >99%). Stock solutions of lipids were prepared in hexane/2-propanol (3/2, by vol). All phospholipid solutions were taken to ambient temperature before use. The concentration of all phospholipid solutions was determined by phosphate assay subsequent to total digestion by perchloric acid (29). Stock solutions of the phospholipids were stored in the dark at 20 C. CTL was synthesized and purified as described previously (30,31). The identity of CTL was positively verified by mass spectrometry. DPH was purchased from Molecular Probes (Leiden, the Netherlands). Probes were stored under argon at 87 C until dissolved in argon-purged ethanol (CTL) or methanol (DPH). The concentration of stock solutions of CTL and DPH was determined by spectrophotometry using their molar absorption coefficients (3) values: 11,250 M1 cm1 at 324 nm for CTL, and 88,000 M1 cm1 at 350 nm for DPH. Stock solutions of fluorescent reporter molecules were stored in the dark at 20 C and used within a week. Deuterium-depleted water (2–3 ppm deuterium) was bought from Cambridge Isotope Laboratories (Andover, MA). All other inorganic and organic chemicals used were of the highest purity available. The solvents used were of spectroscopic grade. Water was purified by reverse osmosis followed by passage through a UF Plus water purification system (Millipore, Billerica, MA) having final resistivity of 18.2 MU cm.
Preparation of vesicles Multilamellar vesicles at a final lipid concentration of ~50 mM were prepared by mixing the used lipids and probes (DPH or CTL) from organic solvent, after which the lipids were dried under nitrogen at ~37 C, followed by exposure to high vacuum for 45 min. The dry lipids were hydrated for 20 min above the Tm for the highest melting lipid with repeated shaking. Biophysical Journal 100(11) 2633–2641
Deuterium NMR measurement of acyl-chain order Two to three milligrams of lipid powder was hydrated with 50 ml of deuterium-depleted water at 40 C for 3 h and subjected to three freeze-thaw cycles. The samples were then transferred by centrifugation into 4 mm o.d. ZrO2 NMR-tubes. These were closed using Kel-F (3M Company, St. Paul, MN) inserts and caps. 2 H-NMR measurements were carried out on a wide-bore Ultrashield magnet (Bruker BioSpin, Billerica, MA) of 11.7 Twith an Avance III console (Bruker BioSpin). A single-channel goniometer probe was used, tuned at 76.783 MHz. The pulse sequence was a solid echo p/2-t -p/2-t- acquisition as described elsewhere (35), using 600-ms recycling delay, 50-ms echo delay, and 2.5-ms pulse duration. Sweep width was 200 kHz, and acquisition time was 4 ms. The spectra were collected with TopSpin 3 (Bruker BioSpin) using a digitization mode which optimizes the baseline in solid experiments. The spectra composed of 20 k scans were multiplied with a cosine function and zero-filled to 8192 points before Fourier transformation. The measurement were done at several temperatures with a precision of 0.1 C and an accuracy of ~1 C. The 2H spectra were dePaked using the NMR-DePaker software available from the Launchpad website (https://launchpad.net/). The Peak-Fitting Module in the Origin 7.5 software (OriginLab, Northampton, MA) was then used to deconvolute the dePaked spectra and to assign the peaks to carbon atoms along acyl-chains. The quadrupolar splittings determined from the dePaked spectra were used to calculate the order parameter, SCD(i), according to
DyQðiÞ ¼
3 e2 qQ 3cos2 q 1 SCDðiÞ ; 4 h
where e2qQ/h ¼ 167 kHz and q ¼ 0 .
RESULTS Acyl-chain order versus temperature The objective of this study was to compare CTL equilibrium partitioning into fluid unilamellar PC or SM bilayers at equal bilayer acyl-chain order. Because CTL fluorescence is very temperature-sensitive (water and oxygen-induced quenching becomes severe above 40 C and signal intensity
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FIGURE 1 DPH anisotropy versus temperature in pure phospholipid bilayers. Steady-state DPH anisotropy was recorded as a temperature function from bilayers prepared from the indicated phospholipids (see Materials and Methods). Each value is the average from two-to-three different measurements (mean 5 SE). The lines shown are polynomial fits to the data, and are given to guide the eye.
decreases markedly), we had to limit the phospholipid selection to species with acyl-chains that are fluid between room temperature and ~45 C. We chose to study 14:0/14:0-PC (Tm 24 C (36), and our data not shown) and 14:0-SM (Tm ~ 27 C, data not shown). In addition, we prepared 14:0/15:0-PC and 15:0/14:0-PC (Tm 32 and 31 C, respectively; our data not shown, but see also (37)) to allow for comparison of the effect of moderate changes in chain length and molecular asymmetry in PC on CTL partitioning. Measuring DPH steady-state anisotropy above the transition temperature for vesicles prepared from each of the phospholipids, we obtained information about relative acyl-chain order in the fluid phase and plotted it against temperature (Fig. 1). It can be seen that 14:0-SM was the most ordered phospholipid of the ones tested, whereas 14:0/14:0-PC was the most disordered (at equal temperature). 14:0/15:0-PC and 15:0/14:0-PC were intermediate in acyl-chain order. This trend is wholly expected based on the acyl-chain composition and the respective Tm for each
A
fully hydrated phospholipid. It is likely that the extensive hydrogen-bonding network present at the SM bilayer interface contribute to the stabilization of intermolecular interaction, thus leading to more ordered SM bilayers (13,14). PC bilayers do not have similar interlipid hydrogen-bonding networks. To get more-detailed direct information about acyl-chain order at different membrane depths, we also measured the 2 H-NMR spectra of perdeuterated 14:0 fatty acid linked to the sn-2 position of 14:0/14:0-PC, or the N-linked position of 14:0-SM. This is a more direct determination of acylchain order compared to the DPH anisotropy measurements. The other benefit of deuterium measurements is that information is obtained for selected segments of the acyl-chain, whereas DPH anisotropy only yields average information from the hydrophobic part of the bilayer membrane. Fig. 2 A shows representative 2H spectra of multilamellar liposomes of either 14:0/14:0(d27)-PC or 14:0(d27)-SM. To be able to determine order parameter profiles for the SM and PC bilayers the 2H spectra were deconvoluted into q ¼ 0 spectra through dePaking (35,38,39). The deuterium order profile for the perdeuterated acyl-chain was calculated from the dePaked spectra at each measured temperature, and data for 14:0/14:0(d27)-PC is shown in Fig. 3 A. It can be seen that the order parameter of carbons 2–8 are undistinguishable from the spectra, only more distal carbons experienced increased disorder. This is consistent with previous studies on 14:0/14:0(d27)-PC (40,41). The order parameter profiles of 14:0(d27)-SM at different temperatures are shown in Fig. 3 B. The assignment of the peaks in the 14:0(d27)-SM spectra was based on published results with 16:0(d31)-SM (27), assuming that shortening the acyl-chain by two carbons does not affect the interfacial orientation of the acyl-chains. Except at 34 C, no clear plateau region was obtained in the order parameter profiles of 14:0(d27)-SM. Instead, the acyl-chain order appeared to decrease with carbon distance from the interface, as previously observed for 16:0(d31)-SM (27). The chain order at
B
FIGURE 2 2H-NMR spectrum of unaligned 14:0/ 14:0(d27)-PC or 14:0(d27)-SM liposomes. (A) Original measurements for PC and SM. (B) dePaked spectra for the same samples.
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FIGURE 4 Average deuterium order parameter (SCD) versus temperature for 14:0/14:0(d27)-PC and 14:0(d27)-SM bilayers. The average order parameter was calculated for carbons 2–8 in PC and 3–8 in SM. The lines shown are polynomial fits to the data, and are given to guide the eye.
CTL orientation in bilayers is much more restricted than DPH orientation, because the sterol is adjacent to phospholipid acyl-chains, and because the b-hydroxyl will be positioned close to the membrane/water interface. DPH is dominantly hydrophobic and is not expected to have the
FIGURE 3 Deuterium order parameter (SCD) in 14:0/14:0(d27)-PC or 14:0(d27)-SM. The calculated SCD values are given for PC (upper panel) and SM (lower panel) as a function of carbon number.
the interface was higher in 14:0-SM bilayers than in 14:0/ 14:0-PC bilayers at selected temperatures. This is quantified in Fig. 4. For both lipids the chain order was lowered with increasing temperature. We can conclude that lipid order determined by either DPH anisotropy or deuterium solidstate NMR yield the same results in our system. A
CTL equilibrium partitioning To determine how CTL equilibrium partitioning was affected by bilayer acyl-chain order and phospholipid type, we measured CTL partitioning between cyclodextrin and different unilamellar bilayers at different temperatures, and correlated the Kx with bilayer acyl-chain order. Based on the relative bilayer acyl-chain order as reported by DPH anisotropy, it can be seen in Fig. 5 A that the affinity of CTL to bilayers was lower for PC-containing membranes as compared with bilayers prepared from 14:0-SM at comparable bilayer acyl-chain order. This difference in Kx was clearly seen at different degrees of bilayer acyl-chain order, and appeared to become more accentuated as the bilayer acyl-chain order increased (lower temperature). Changing the length of the sn-1 or the sn-2 acyl-chain in the PC did not alleviate the difference in affinity seen between SM and PC, suggesting that acyl-chain match (or mismatch) was not significantly responsible for the difference in bilayer affinity of CTL. Biophysical Journal 100(11) 2633–2641
B
FIGURE 5 CTL partitioning into phospholipid bilayers as a function of DPH (A) or CTL anisotropy (B). The relative and average acyl-chain order was determined from DPH anisotropy at different temperatures (see Fig. 1), or from CTL anisotropy (see Materials and Methods). CTL partitioning into phospholipid bilayers was determined at the indicated temperatures. The Kx of CTL is plotted against DPH or CTL anisotropy. Each value is the average from two-to-three determinations 5 SE. The lines shown are linear regression fits to the data, and are given to guide the eye.
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same orientation in the bilayer as CTL (42). We therefore also determined CTL anisotropy at different temperatures and for different bilayer systems, and correlated this with CTL partitioning. When we plotted the CTL anisotropy values against the corresponding CTL partitioning coefficient, we could clearly see that even at identical CTL anisotropy values, the CTL Kx was higher in 14:0-SM-containing bilayers as compared to 14:0/14:0-PC-containing bilayers (Fig. 5 B). Finally, when CTL Kx was plotted against the deuterium order parameter obtained from 2H-NMR measurements, it again became clear that at comparable acyl-chain order, the CTL Kx was markedly higher with 14:0-SM bilayers as compared to 14:0/14:0-PC bilayers (Fig. 6). CTL Kx was plotted against the average deuterium order parameter obtained from carbon/deuterium pairs 2–8 for the PC and 3–8 for the SM, because this segment is most likely involved in interactions with adjacent sterol. Also, the difference in CTL Kx for 14:0/14:0-PC and 14:0-SM bilayers become markedly more prominent as the deuterium order parameter increased. To further analyze the binding of CTL to 14:0-SM and 14:0/14:0-PC bilayers, van’ t Hoff plots were made from the Kx determined at different temperatures. From the plots shown in Fig. 7 one can see that the binding of CTL to both phospholipid bilayers was an exothermic process (as ln K is rising with 1/T). This was expected as binding of cholesterol to both POPC and PSM bilayers has been shown to be exothermic (43). From the slope of the plots one can obtain DHo(SM) ¼ 43.5 kJ/mol and DHo(PC) ¼ 35.2 kJ/mol. From the intercepts, one obtains DSo(SM) ¼ 110.4 J/mol and DSo(PC) ¼ 93.5 J/mol. This indicates that the binding of CTL to the SM bilayers is enthalpically more favorable and entropically less favorable than CTL binding to the PC
FIGURE 6 CTL partitioning into phospholipid bilayers as a function of bilayer acyl-chain order parameter (SCD). CTL partitioning into phospholipid bilayers was determined at the indicated temperatures. The Kx of CTL is plotted against the average SCD order parameter for carbons 2–8 (PC) or 3–8 (SM). Each value is the average from two-to-three determinations 5 SE. The lines shown are linear regression fits to the data, and are given to guide the eye.
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FIGURE 7 van’ t Hoff plot for CTL association with 14:0-SM and 14:0/ 14:0-PC bilayers. Values shown are the mean 5 SE from two average values determined at each indicated temperature (1/T). The lines are linear regression fits to each set of data.
bilayers in the examined temperature range. Hence, the observed higher affinity of the sterol for the SM bilayers must be due to enthalpic contributions. DISCUSSION The affinity of cholesterol or other sterols for phospholipid bilayers is the net result of many competing influences. Cholesterol may prefer to interact with a specific phospholipid (e.g., a saturated one with a large headgroup) and these favorable interactions would be expected to result in an increased sterol affinity for bilayers made of such phospholipids (8,44,45). Cholesterol may also have an aversion for interacting with certain phospholipids (e.g., polyunsaturated phospholipids (46,47)), and would thus be forced to interact with phospholipids it might not otherwise prefer. We have in this study focused on the partitioning of CTL between cyclodextrins and vesicles prepared from one-component phospholipid bilayers. We wanted to determine whether bilayer acyl-chain order was the sole determinant of sterol affinity when sterol partitioning into PC or SM bilayers was compared. Before discussing the results obtained for PC and SM bilayers, let us first critically discuss the experimental conditions used in our assays. CTL was used as a cholesterol mimic in our study, because its fluorescence properties are useful and allow for experimental setups not possible with, e.g., radiolabeled cholesterol (19,34,48,49). CTL is not chemically identical to cholesterol, because its B- and C-rings contain additional double bonds, which render the sterol slightly more polar (30,31). The conjugated double bonds are also likely to affect the planarity of the sterol as compared to that of cholesterol (50). A change in the ring-system planarity and/or polarity is likely to affect sterol/acyl-chain interaction in bilayers (51). However, CTL is known to partition into liquid-order domains, to condense phospholipid packing, and increase acyl-chain order (19,21,34,53), thus Biophysical Journal 100(11) 2633–2641
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showing cholesterol-like membrane effects. Although CTL’s affinity for cyclodextrins and bilayer membranes is most likely slightly different from cholesterol’s (thus affecting absolute partitioning coefficients), it has been shown that CTL’s relative partitioning between cyclodextrins and bilayer membranes is comparable to that seen for radiolabeled cholesterol (19,34).We therefore conclude that results obtained for CTL are representative of the behavior of cholesterol. The types of phospholipids we chose for the study are common in cell membranes of most cells; however, the molecular species selected are only minor species (13,54,55). We chose molecular species with tetradecanoyl or pentadecanoyl acyl-chains in the sn-1or sn-2 positions (PCs), or with tetradecanoyl in the N-linked position of SM. These fairly short acyl-chains render the respective phospholipids fluid in a temperature window (25–45 C) which is suitable with regard to the fluorescence properties of CTL. Water and oxygen-induced quenching of CTL fluorescence emission becomes excessive at elevated temperatures. Furthermore, 14:0/14:0-PC and 14:0-SM are also fairly comparable molecules with regard to shape and hydrophobic length, because the 14:0 chain in sn-1 of PC is of comparable length with the long-chain base of SM, considering that the glycerol backbone in PC adds three carbons to its effective length. The molecules used are not highly asymmetric and are not expected to give rise to interdigitation (56). We can now consider how acyl-chain order in the bilayer membranes has been determined. The simple way was to measure DPH anisotropy in bilayer membranes as a temperature-function. The order of the acyl chains will affect the dynamic behavior of DPH and this is reported in the anisotropy function. DPH anisotropy measurements have successfully been used to demonstrate temperature and cholesterol-induced changes in bilayer acyl-chain order (57–61). A more direct way to determine acyl-chain order is to measure the deuterium order parameter (SCD) of the lipids under study (62). We did this for 14:0/14:0(d27)-PC and 14:0(d27)-SM at different temperatures. Looking at Fig. 4, we can, for instance, see that the 14:0(d27)-SM bilayer had to be at a 10–12 C higher temperature than the corresponding 14:0/14:0(d27)-PC bilayer, to yield equal SCD (comparisons made at SCD of 0.20 or 0.19). With DPH anisotropy, equal hri was obtained in 14:0/14:0-PC and 14:0-SM bilayers when the SM bilayer was at a 11–13 C higher temperature (comparisons made at hri of 0.12 and 0.10). These results suggest that both methods give qualitatively comparable acyl-chain order results. Thus, our results are consistent with the conclusion by Heyn (63) that the order parameters obtained from deuterium NMR and fluorescence depolarization (of DPH) show surprisingly good correlation. When the CTL partitioning was determined at different temperatures for bilayers containing either a PC with tetraBiophysical Journal 100(11) 2633–2641
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decanoyl or pentadecanoyl acyl-chains in the sn-1or sn-2 positions (PC), or an SM with tetradecanoyl in the N-linked, and the Kx was plotted against the DPH anisotropy function (Fig. 3), we clearly show that at equal DPH anisotropy the Kx of CTL was highest for SM bilayers and lowest for 14:0/14:0-PC bilayers. If a 15:0 fatty acid was linked to either sn-1 or sn-2, the measured acyl-chain order parameter was slightly higher (Fig. 1), and the Kx versus DPH anisotropy was intermediate between 14:0/14:0-PC and 14:0-SM. These results infer that: 1. CTL (and therefore cholesterol) interacts more favorably with SM than PC at equal acyl-chain order parameter. 2. Chain length (14:0 or 15:0 in PC) only had a moderate effect on CTL (and cholesterol) interaction and bilayer sterol affinity. Similar results were largely obtained when CTL Kx was plotted against the CTL anisotropy (Fig. 4) or the deuterium order parameter (Fig. 5 A).The SCD average values used in Fig. 5 A were taken from the order parameter of the acylchain segment most likely to interact with the sterol (carbons 2–8 in PC and 3–8 in SM). However, even if the order parameter of all carbons had been included in the average, a plot very similar to Fig. 5 A would still be obtained (data not shown). It can also be noted that the difference in Kx between PC and SM appeared to increase markedly when the bilayer acyl-chain order parameter increased (temperature decreased). This difference may result from temperature-related effects (stabilization at decreased temperatures) on the hydrogen-bonding network which is different in SM and PC bilayers and which is likely to affect the measured CTL partitioning coefficient. It is clear that the higher affinity that CTL/cholesterol shows for SM bilayers as compared to PC bilayers with similar degree of ordering must derive from the structural differences between the two phospholipids. Based on their functional groups, SM will have more possibilities to form hydrogen bonds than PC. Whereas PCs can only be acceptors for hydrogen bonding, SM can form both inter- and intramolecular hydrogen bonds with other phospholipids and with cholesterol, a fact that also shows up in MD simulations (26). Intermolecular SM-SM hydrogen bonds are expected to affect the dynamics in SM bilayers, decreasing both lateral diffusion rates and rotational motions, which in turn should indirectly also strengthen interactions between SM and cholesterol. Such differences in dynamics are probably also contributing to the observed difference in acyl-chain order between 14:0/14:0-PC and 14:0-SM. The importance of hydrogen bonding between cholesterol and SM for their interaction has been debated (15). However, several MD simulations suggest that hydrogen bonds form between cholesterol and SM, and they would therefore be expected to result in some stabilization of interaction not seen in a comparable cholesterol/PC system. It has also been observed that methylation of the OH-group
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or the NH-function in SM almost completely abolishes favorable interaction between sterol and SM (55). Hence, we find it likely that hydrogen bonding and interfacial interactions contribute to the stabilization of sterol/SM association. In recent studies it has been observed that the affinity of cholesterol/CTL for SM-containing bilayers would largely be determined by the higher chain order in these samples (8,19). In these studies the bilayers were composed of POPC and SM in addition to the sterol. It is possible that the hydrogen bonding between SM and cholesterol was less important in these samples because hydrogen bonding between PC and SM may have dominated, in line with what has been observed in MD simulations of POPC:PSM:cholesterol bilayers (16). In this study, where pure SM bilayers were compared to pure PC bilayers, the hydrogen bonds between SM and cholesterol can be expected to be more frequent. Although SM and PC share a common headgroup (phosphocholine), its dynamic and orientational properties may be different for SM and PC. Headgroup properties are known to markedly affect cholesterol/phospholipid interaction (20,44,64). In MD simulations of PSM and DPPC bilayers, small differences in headgroup orientation have been observed (26). It was observed that the SM headgroup had two orientational distributions of which the most common one is tilted 15 toward the interior of the bilayer. NMR studies of SM headgroups have also indicated that the conformation of the SM phosphocholine headgroup differs from that in PC (65–67). It has been suggested that this could lead to better shielding of sterols from water, thereby making sterol/SM interaction more favorable (16). The same MD simulation study also indicated that cholesterol have a higher tendency to form charge pairs with the phosphocholine headgroup of SM than with PC (16). Another possible contributing factor to the observed difference in partitioning at equal (bulk) acyl-chain order is the possibly different local ordering of acyl-chains by CTL. Suppose CTL orders its neighboring SM acyl-chains more than it orders the neighboring PC acyl-chains, then this local order would influence the partitioning process. Because it is impossible to measure the local order, we measured instead how the inclusion of 33 mol % cholesterol affected DPH anisotropy in 14:0/14:0-PC and 14:0-SM bilayers at different temperatures but at equal average order (see Fig. S1 in the Supporting Material). At temperatures where the acyl-chain order was equal in the absence of sterol, the ordering effect of cholesterol was slightly higher on SM bilayers than on PC bilayers. Hence, the higher affinity of CTL for SM bilayers could have been partially due to larger ordering effects of the sterol on the neighboring SM acyl-chains. However, as cholesterol displayed only a slightly larger ordering effect on SM acyl-chains, it is clear that other factors also must have contributed to the higher affinity. The thermodynamic parameters for CTL binding to 14:0/ 14:0-PC and 14:0-SM bilayers obtained from the van’ t Hoff
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plots show that insertion of CTL in the bilayers is entropically unfavorable. This can be explained by the increased order in the bilayers as a result of interactions with the sterol. The finding that CTL association with SM bilayers was more unfavorable than with PC bilayers can be rationalized by the observation that SM bilayers are slightly more ordered by sterol inclusion. The exothermic enthalpies obtained from the van’ t Hoff plots show that binding of CTL to phospholipid bilayers was enthalpically driven, as expected based on previously reported data (43). Isothermal titration calorimetry and molecular dynamics simulations have previously shown that the transfer of cholesterol from fluid POPC to fluid SM bilayers is enthalpically driven (43,68). The transfer of CTL from 14:0/14:0-PC to 14:0-SM bilayers would also be enthalpically driven, as the binding of CTL to 14:0-SM bilayers was more exothermic than binding to 14:/14:0-PC bilayers. The enthalpic gain of transferring of CTL from 14:0/14:0-PC to 14:0-SM bilayers could be due to a conformational enthalpy change but also to increased formation of hydrogen bonds when the sterol is moved to the SM bilayer. We conclude that sterol/phospholipid interaction is markedly influenced by bilayer acyl-chain order, and that CTL or cholesterol prefers to interact with ordered phospholipids. However, when the bilayer acyl-chain order is equal, CTL and cholesterol appears to prefer interacting with SM over PC. This seems to be in part due to a larger ordering effect of sterols on SM acyl-chains, an effect that may be brought about by stronger interactions between SM and sterols. Stronger interactions between SM and CTL could be due to better shielding from water by the SM headgroup and charge pairing between this and the sterol. Hydrogen bonding between the sterol and SM is also most likely to be an important stabilizing factor that is supported by enthalpically driven transfer of CTL from 14:0/14:0-PC to 14:0-SM bilayers and the fact that, in PC:SM mixtures, the affinity of sterols was influenced by the acyl-chain order.
SUPPORTING MATERIAL One figure is available at http://www.biophysj.org/biophysj/supplemental/ S0006-3495(11)00518-2. This study was supported by generous grants from the Sigrid Juselius foundation (to J.P.S.), Academy of Finland (to T.K.M.N.), the Foundation of ˚ bo Akademi University (to J.P.S.), and the Netherlands Organization for A Scientific Research (NWO-TOP grant No. 700-54-303 to J.P.F.D.).
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