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Different Phase Behavior and Packing of Ceramides with Long (C16) and Very Long (C24) Acyls in Model Membranes: Infrared Spectroscopy Using Deuterated Lipids Barbora Školová,†,‡ Klára Hudská,† Petra Pullmannová,† Andrej Kovácǐ k,† Karel Palát,† Jaroslav Roh,† Jana Fleddermann,‡ Irina Estrela-Lopis,‡ and Kateřina Vávrová*,† †

Skin Barrier Research Group, Faculty of Pharmacy in Hradec Králové, Charles University in Prague, Heyrovského 1203, 50005 Hradec Králové, Czech Republic ‡ Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstrasse 16-18, 04275 Leipzig, Germany S Supporting Information *

ABSTRACT: Ceramides (Cer) are the central molecules in sphingolipid metabolism that participate in cellular signaling and also prevent excessive water loss by the skin. Previous studies showed that sphingosine-based Cer with a long 16C chain (CerNS16) and very long 24C-chain ceramides (CerNS24) differ in their biological actions. Increased levels of long CerNS16 at the expense of the very long CerNS24 have been found in atopic dermatitis patients, and this change correlated with the skin barrier properties. To probe the membrane behavior of the long CerNS16 and the very long chain CerNS24, we studied their interactions with fatty acids and cholesterol in model stratum corneum membranes using infrared spectroscopy. Using Cer with deuterated acyls and/or deuterated fatty acids, we showed differences in lipid mixing, packing, and thermotropic phase behavior between long and very long Cer. These differences were observed in the presence of lignoceric acid or a heterogeneous fatty acid mixture (C16−C24), in the presence or absence of cholesterol sulfate, and at 5−95% humidity. In these membranes, very long CerNS24 prefers an extended (splayed-chain) conformation in which the fatty acid is associated with the very long Cer chain. In contrast, the shorter CerNS16 and fatty acids are mostly phase separated. linoleic acid attached to the ω-hydroxyl, is essential for mammalian survival (reviewed in ref 12). Apart from these ultralong Cer, the other Cer classes in the uppermost epidermal layer (i.e., the stratum corneum (SC)) contain very long FFA. This high carbon content seems to be essential for proper skin barrier function. This assumption is supported by recent studies that showed increased levels of long CerNS16 at the expense of the very long CerNS24 in atopic dermatitis patients and that this change correlated with their impaired skin barrier properties.13,14 This Cer chain length shift is most likely caused by a down-regulation of elongases that create FFA longer than C16, as found in a murine atopic dermatitis model;15 this down-regulation may be a consequence of increased interferon gamma.16 At the model SC membrane level, studies on very short CerNS2−12 showed that such Cer form continuous short Cer-enriched domains with shorter lamellar periodicity as compared to those of the native, very long Cer.17,18 However, the reason the long CerNS16 cannot substitute for the very long CerNS24 at the membrane level is unknown. We hypothesized that a different interaction of long CerNS16 and very long CerNS24 with the other SC lipids,

1. INTRODUCTION Ceramides (Cer) are the central molecules in sphingolipid metabolism. These lipids are crucial for the life of the cell, where they regulate several phenomena, including cell growth, metabolism, development, differentiation, aging, and death.1,2 Cer are equally important outside of the cell in the extracellular space of the skin barrier because they prevent water loss in land-dwelling mammals.3−7 Cer are composed of a sphingoid base (sphingosine, dihydrosphingosine, phytosphingosine, or 6hydroxysphingosine8) and a fatty acid (FFA, either nonhydroxylated, α-hydroxylated, or ω-hydroxylated) that acylates the amino group of the sphingoid base. During Cer formation, a family of 6 Cer synthases can attach various FFAs to a sphingoid base, resulting in Cer with acyls varying from long (13−19 carbons) to very long (20−26 carbons) and ultralong (>28 carbons).7 These diverse chain lengths significantly influence Cer properties (reviewed in ref 9): Cer with long C16 acyl (CerNS16 = N-palmitoyl sphingosine, Figure 1) induce apoptosis, while Cer with very long C24 chain (CerNS24 = N-lignoceroyl sphingosine, Figure 1) have protective effects, suggesting that the equilibrium between long and very long chain Cer determines the fate of the cell.10,11 In the skin-barrier Cer, the chain length is important as well. In particular, the presence of ω-hydroxylated ultralong Cer, either covalently bound to the corneocyte envelope or free with © 2014 American Chemical Society

Received: June 27, 2014 Revised: August 13, 2014 Published: August 14, 2014 10460

dx.doi.org/10.1021/jp506407r | J. Phys. Chem. B 2014, 118, 10460−10470

The Journal of Physical Chemistry B

Article

Water was deionized, distilled, and filtered through a Milli-Q purification system (Millipore). 2.2. Synthesis of Deuterated Ceramides (d-CerNS24 and d-CerNS16). 2.2.1. General. Thin layer chromatography was performed on Merck (Darmstadt, Germany) aluminum plates with silica gel 60 F254. Merck Kieselgel 60 (0.040−0.063 mm) was used for column chromatography. Melting points were recorded on a Büchi B-545 apparatus (Büchi Labortechnik, Flawil, Switzerland) and are uncorrected. Infrared spectra were measured on a Nicolet 6700 (Thermo Scientific, Waltham, MA). 1H and 13C NMR spectra were recorded on a Varian Mercury Vx BB 300 or VNMR S500 NMR spectrometer (Varian, Palo Alto, CA). Chemical shifts are reported as δ values in parts per million (ppm) and were indirectly referenced to tetramethylsilane via the solvent signal. Electrospray ionization mass spectrometry (ESI MS) was measured using an Acquity UPLC with MS/MS Quattro Microdetection (Micromass+Waters, Milford, MA). 2.2.2. General Procedure for the Synthesis of d-Cer (Figure 1). Perdeuterated lignoceric or palmitic acid (0.14 mmol), 1ethyl-3-(3-(dimethylamino)propyl)carbodiimide (0.17 mmol), and N-hydroxybenzotriazole (0.16 mmol) were dissolved in dry CH2Cl2 under nitrogen, and then sphingosine (0.14 mmol) in CH2Cl2 was added at 0 °C. The reaction was allowed to warm to room temperature, and the mixture was stirred overnight. Next, the reaction mixture was washed with 0.1 M HCl and water. The organic phase was dried over Na2SO4, concentrated under reduced pressure, and purified by silica column chromatography using 50:1 chloroform/methanol as a mobile phase. 2.2.3. N-((2S,3R,4E)-1,3-Dihydroxyoctadec-4-en-2-yl)tetracosanamide-d47 (d-CerNS24). Yield = 83%, white crystals, mp = 89−92 °C, Rf = 0.44 (CHCl3/methanol 10:1). 1H NMR (300 MHz, CDCl3): δ = 6.24 (d, J = 7.4 Hz, 1H), 5.78 (dt, J = 14.4, 6.8 Hz, 1H), 5.53 (dd, J = 15.4, 6.3 Hz, 1H), 4.51−4.13 (m, 1H), 4.03−3.82 (m, 2H), 3.70 (apparent d, J = 10.6 Hz, 1H), 2.76 (br s, 2H), 2.15−1.93 (m, 2H), 1.45−1.10 (m, 22H), 0.88 (t, J = 6.5 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ = 174.0, 134.3, 128.8, 74.7, 62.5, 54.5, 32.3, 31.9, 29.7, 29.6, 29.5, 29.4, 29.2, 29.1, 28.6, 28.4, 28.1, 22.7, 14.1. IR (ATR): ν 3293, 2919, 2850, 2195, 2088, 1645, 1557, 1466, 1091, 1087, 721 cm−1. MS (ESI+) m/z 680.5 (M + H+ − H2O). 2.2.4. N-((2S,3R,4E)-1,3-Dihydroxyoctadec-4-en-2-yl)hexadecanamide-d31 (d-CerNS16). Yield = 82%, white crystals, mp = 92−95 °C, Rf = 0.39 (CHCl3/methanol 10:1). 1 H NMR (500 MHz, CDCl3): δ = 6.31 (d, J = 7.2 Hz, 1H), 5.78 (dt, J = 14.1, 6.8 Hz, 1H), 5.52 (dd, J = 15.5, 6.4 Hz, 1H), 4.31 (dd, J = 6.0, 3.8 Hz, 1H), 4.08−3.79 (m, 2H), 3.70 (dd, J = 11.2, 3.2 Hz, 1H), 2.67 (br s, 2H), 2.13−1.93 (m, 2H), 1.31− 1.20 (m, 22H), 0.88 (t, J = 6.8 Hz, 3H). 13C NMR (125 MHz, CDCl3): δ = 174.1, 134.3, 128.8, 74.5, 62.5, 54.5, 32.3, 31.9, 29.7, 29.6, 29.6, 29.5, 29.4, 29.2, 29.1, 28.3, 28.2, 22.7, 14.1. IR (ATR): 3297, 2917, 2850, 2193, 2089, 1645, 1554, 1468, 1090, 721 cm−1. MS (ESI+) m/z 551.9 (M + H+ − H2O), 591.9 (M + Na+). 2.3. Preparation of the Lipid Samples. 2.3.1. Samples A (Cer/LA/Chol/CholS). The lipid samples were prepared as equimolar mixtures of Cer (or d-Cer), Chol, and lignoceric acid (LA, or perdeuterated d-LA) along with 5 wt % CholS. The lipids were dissolved in 2:1 hexane/96% ethanol (v/v) and mixed in the appropriate ratios in glass vials. The solvents then were evaporated using a stream of nitrogen and overnight vacuuming. These lipid films were heated to 90 °C, which is

Figure 1. Synthesis of Cer with deuterated acyls (panel A) and list of the lipids used in this work and their abbreviations (panel B). EDC, 1ethyl-3-(3-(dimethylamino)propyl)carbodiimide; HOBt, N-hydroxybenzotriazole; rt, room temperature.

cholesterol (Chol), and FFA might provide an explanation. The model of the SC lipid organization recently proposed by Iwai et al. suggested that very long chain Cer adopt a fully extended conformation (i.e., with chains pointing in opposite directions); the model also indicated that Chol is associated with the Cer sphingoid chain and that FFA is associated with the very long acyl chain and enables segregation of the immiscible Chol and very long chain FFA.19 This result led us to the possibility that the proposed arrangement could be confirmed at the lipidmembrane level by infrared spectroscopy (FTIR). Using deuterated FFA (d-FFA) and Cer with deuterated acyl (dCerNS24 and d-CerNS16), we could determine whether FFA really associates with the very long CerNS24 acyl chain without being mixed with Chol or the sphingosine chain and whether the long CerNS16 adopts the same arrangement or a different one. In this work, we synthesized CerNS24 and CerNS16 (nonhydroxy acyl sphingosines) with C24 and C16 perdeuterated acyl chains. Next, we prepared membrane models based on major skin barrier lipids, either unlabeled or deuterated, to study their interactions using FTIR. The membranes were composed of an equimolar mixture of CerNS24 or CerNS16, FFA (either lignoceric acid, LA, or a mixture of FFA with C16− C24 chains similar to those in the SC), and Chol, either with or without 5% cholesterol sulfate (CholS), and were constructed at different hydration levels. The synthesis scheme and the studied lipids are given in Figure 1.

2. MATERIALS AND METHODS 2.1. Chemicals. Sphingosine (synthetic, >99% stereochemically pure, i.e., (2S,3R,4E)), unlabeled CerNS16, and unlabeled CerNS24 (synthetic, >99% stereochemically pure, i.e., (2S,3R,4E)) were purchased from Avanti Polar Lipids (Alabaster, AL), and deuterated fatty acids were from C/D/ N isotopes (Pointe-Claire, Canada). Deuterated Cer were synthesized as described below. All other chemicals and solvents were from Sigma-Aldrich (Schnelldorf, Germany). 10461



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Figure 2. Infrared spectra of the model skin lipid membranes containing ceramides (Cer) with a very long C24 (A,B) or long C16 (C,D) acyl with the proposed lipid arrangement for model membranes with very long chain CerNS24 in the red rectangle. The lipid samples were composed of an equimolar mixture of the pertinent Cer (nonhydroxy acyl sphingosine class; CerNS24 or CerNS16), lignoceric acid (LA), cholesterol (Chol), and 5 wt % cholesteryl sulfate (CholS). Cer and LA were either unlabeled or deuterated (the latter indicated by the prefix d-). Samples without Cer (labeled as No Cer) are shown for comparison. Panels A and C show the CH2 scissoring vibrations of unlabeled lipid chains; panels B and D show the CD2 scissoring vibrations of deuterated lipid chains in the model membranes. For an easier orientation, the origin of the respective CH2 or CD2 vibration is indicated at the right. The doublet of the scissoring contour indicates packing of the lipids in an orthorhombic subcell, which only occurs between the same isotopes; therefore, it is indicative of mixing of the lipid chains. Sph, sphingosine chain. This figure shows representative spectra. For further information and the thermotropic behavior of these lipids, see Figure 3.

2.3.4. Samples D (Cer/LA/Chol, 5−95% Humidity). To probe the effects of hydration on the proposed CerNS24 arrangement, samples D were prepared. Cer, LA, and Chol were dissolved in 2:1 chloroform/methanol (v/v) and were mixed in an equimolar ratio in a glass vial. The solvent was evaporated, and the lipid samples were hydrated with 10 mM pyridine buffer at pH 5.5 (with NaCl to maintain the 154 mM ionic strength). The hydrated samples were heated to 95 °C and then slowly (approximately 4 h) cooled to room temperature. This heating−cooling cycle was repeated four times. 2.4. Fourier-Transform Infrared (FTIR) Spectroscopy. To study the lipid chain conformation and packing and the behavior of the polar headgroup regions at skin temperature and during the phase transitions, attenuated total reflectance FTIR spectroscopy was used. The lipid samples A, B, and C were placed on the ATR crystal, and the FTIR spectra were collected on a Nicolet IMPACT 400 or a Nicolet 6700 spectrometer (Thermo Scientific, Waltham, MA) equipped with a heated single-reflection MIRacle ATR ZnSe crystal (PIKE technologies, Madison, WI) or a Nicolet Smart iTR with ZnSe plate (Thermo Scientific, Waltham, MA). A clamping mechanism with constant pressure was used. The spectra were generated by the coaddition of 128 or 256 scans collected at 2 cm−1 resolution. The temperature dependence of the IR spectra was studied over the range of 28−100 °C with 2 °C steps using a temperature control module (PIKE technologies, Madison, WI). After each temperature increment, the sample was allowed to stabilize for 10 min before the spectrum was measured. The spectra were analyzed using Bruker OPUS software. The exact

well above the main phase transition temperature, equilibrated for 10 min, and slowly (approximately 3 h) cooled to room temperature. They then were incubated at 32 °C in the humidity of the ambient air for 24 h. 2.3.2. Samples B (Cer/FFA/Chol/CholS). To probe the role of the FFA heterogeneity, samples B were prepared as equimolar mixtures of the Cer, Chol, and FFA along with 5 wt % CholS. FFAs (or d-FFAs) were mixed in a molar % corresponding to the native composition of human skin: 1.8% hexadecanoic acid, 4.0% octadecanoic acid, 7.6% eicosanoic acid, 47.8% docosanoic acid, and 38.8% LA.20 All of these FFAs were either protonated or deuterated. This particular FFA composition was also chosen because the constituents are also commercially available in their perdeuterated forms. The lipids were dissolved in 2:1 hexane/96% ethanol (v/v) and mixed in the appropriate ratios. The solvents then were evaporated using a stream of nitrogen and overnight vacuuming. These lipid films were heated to 90 °C, which is well above the main phase transition temperature, equilibrated for 10 min, and slowly (approximately 3 h) cooled to room temperature. They then were incubated at 32 °C in the humidity of the ambient air for 24 h. 2.3.3. Samples C (Cer/LA/Chol). Samples C were prepared without CholS. Cer, LA, and Chol were dissolved in 2:1 chloroform/methanol (v/v) and were mixed in an equimolar ratio in a glass vial. The solvent was evaporated, and the lipid samples were heated to 95 °C and then slowly (approximately 4 h) cooled to room temperature. They then were incubated at 32 °C in the humidity of the ambient air for 24 h. 10462



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peak positions were determined from the second derivative spectra. Samples D were applied to the surface of a ZnSe ATR crystal (72 × 10 × 6 mm trapezoid, 45° face angle, six active reflections) in an Excalibur FTS3100 Fourier transform IR spectrometer (Varian, Darmstadt, Germany) at 32 °C. The hydration degree of the lipid film was adjusted with a moisture generator (HumiVar 5.0, Leipzig, Germany) to relative humidity values between 5% and 95% with an accuracy of 28C).4,7,12 The formation of very long Cer requires elongation of FFA by a family of enzymes called elongases (ELOVL1−7)33 and a specific Cer synthase that acylates the sphingoid base with this very long FFA (for a review, see Rabionet et al.7). Previous studies showed a deficiency in very long chain CerNS24 in atopic eczema and psoriasis that was compensated by increased levels of long chain CerNS16.13−16 Because such shortening of the Cer acyl chain by 8 carbons correlated with the impaired barrier function, it appears that the very long chains in skin Cer play a special role in the SC lipid organization and function. However, the molecular organization of these lipids has been a subject of debate.34 Cer, as a double-chain amphiphile, could adopt a hairpin conformation, with both chains pointing in the same direction, and an extended conformation (splayed-chain conformation), with its chains pointing in opposite directions. Although the latter conformation has rarely been considered as a possibility in lipid membranes, some authors have argued that in the SC barrier, which contains multilamellar lipid membranes rather than simple bilayers, this conformation may provide several advantages: extended Cer would increase the cohesion of the adjacent lipid lamellae, reduce the molecular packing strain associated with the unfavorable ratio of the hydrophobic/ hydrophilic cross-sectional area of Cer, and be devoid of swellable hydrophilic interfaces.35 This arrangement of Cer chains was actually proposed previously in 1989 by Swartzendruber et al.36 A counterargument would be that during the development of the skin lipid barrier, the Cer precursors, glucosylceramides and sphingomyelins, would most likely prefer the hairpin conformation due to their large and hydrophilic polar headgroups. Thus, the hydrolysis of these precursors would release Cer in the hairpin conformation. However, the chain flip in Cer from a hairpin into the extended conformation was found to occur relatively rapidly.37 Notably, Rabionet et al.38 recently found 1-O-acylCer in the human skin barrier; in such a triple-chain Cer, one chain will certainly point in the opposite direction, resembling the extended Cer conformation. The model proposed by Iwai et al.19 further refined the extended Cer concept: Chol is associated with the Cer sphingoid chain, and FFA is associated with its very long acyl chain, which enables segregation of the immiscible Chol and long-chain FFA. Our results indicate that the extended conformation of very long CerNS24 is indeed present in its mixture with Chol and FFA (either LA or heterogeneous FFA mixture). This CerNS24 conformation and interaction with Chol and FFA persist either with or without CholS and under hydration. The miscibility of CerNS24 with LA in these model membranes is consistent with X-ray results that show only a single lamellar phase with a repeat distance of 5.2−5.4 nm (short periodicity phase, SPP) and some separated Chol in CerNS24/LA/Chol membranes either with or without CholS.18,39 The separation of some Chol molecules from these membrane models as observed by X-ray diffraction is also consistent with the proposed extended Cer conformation because the cross-sectional area of Chol is 0.38 nm2, which would be occupied by approximately two FFA molecules or two extended Cer chains. This assumption seems to be supported by the observation that Chol fully incorporates in the lamella at a 1:0.5:1 molar ratio of Cer/Chol/FFA (E. H. Mojumdar, G. S. Gooris, J. A. Bouwstra, unpublished data, cited in Mojumdar et al.40). 10467



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5. CONCLUSION Our results suggest that extended conformation of the very long chain ceramide NS24 is present in its mixture with fatty acids and cholesterol, either with or without cholesterol sulfate. Whether this specific ceramide arrangement occurs in more complex biological systems must be established. Nevertheless, we showed that the extended ceramide/fatty acid interaction requires a ceramide with a very long (e.g., 24 carbons) acyl chain and that this behavior is not shared by its long chain ceramide analogue with 16 carbons. These differences between very long and long ceramides are relevant to the biology of the skin lipid barrier because this tissue prefers ceramides with very long or ultralong acyl chains over ceramides with long chains. Inadequate elongation of the fatty acids used by ceramide synthases is most likely the reason for the increased ratio of long/very long ceramides that was found in atopic dermatitis,13−16 psoriasis,16 and also in reconstructed skin models;55 this increased ratio was also associated with higher permeability. We show here that an inadequate length of the ceramide acyl markedly affects its phase behavior and packing in model stratum corneum lipid membranes.

Figure 7. A proposed molecular model of the short periodicity phase (SPP) unit based on data from this work and from Iwai et al.19 Although this suggested arrangement would be locally asymmetric, the presence of domains of such asymmetric lamellae with an alternating direction in the asymmetry would be consistent also with the neutron scattering length density profiles described by Groen et al.39 and Mojumdar et al.40

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ASSOCIATED CONTENT

S Supporting Information *

Supporting Figures S1 (infrared spectra and thermotropic phase behavior of pure ceramides with deuterated acyls dCerNS24 and d-CerNS16), S2 (infrared spectra of the methylene scissoring regions in the Cer/FFA/Chol/CholS samples at 32 °C), S3 (infrared spectra of the methylene scissoring regions in the Cer/LA/Chol samples at 32 °C), and S4 (infrared spectra of the methylene scissoring regions in the Cer/LA/Chol samples at 32 °C and at 5−95% hydration). This material is available free of charge via the Internet at http:// pubs.acs.org.

mixtures as compared to that in its pure form can be explained by its miscibility with some Chol molecules. This hypothesis is in agreement with previous results from X-ray diffraction suggesting that CerNS16 forms two lamellar phases with Chol: one in an approximately 2:3 ratio, forming lamellar structures with 3.50 nm repeat distances, and another with an approximately 1:3 molar ratio with a repeat distance of 4.24 nm.49 The authors also noted that the behavior of a very long chain CerNS24 was very different from that of CerNS16. Similar results were also found for CerNS18: it was mixed with Chol already at physiological temperature.48 In addition, Langmuir monolayer studies showed that phytosphingosinebased CerNP16 mixed well with Chol while CerNP24 did not.50 Our results suggest that the sphingosine chain and acyl chain of CerNS16 share one domain. This may indicate a hairpin conformation or extended conformation of CerNS16 with a random distribution of its symmetric chains. The hairpin conformation of Cer would lead to a cross-sectional area very close to that of Chol that might be advantageous for their miscibility. This conformation could also explain the observed higher sensitivity of CerNS16 than of CerNS24 membranes to hydration. Previous work suggested low hydration of the intermembrane space of the SC lipid model membrane containing Cer6 (CerAP18, i.e., Cer with symmetric chains similar to CerNS16); the thickness of the water layer was only approximately 1 Å at full hydration.51 The authors also hypothesized that this is compatible with the extended conformation of CerAP18 and that a chain flip transition of CerAP18 from an extended to a hairpin conformation may occur upon hydration.51−53 It is also possible that both CerAP18 conformations coexist.54

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +420-495-067-497. Fax: +420-495-067-167. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was cofinanced by the European Social Fund, the state budget of the Czech Republic (project no. CZ.1.07/ 2.3.00/30.0061), the Czech Science Foundation (13-23891S), and Charles University in Prague (SVV 260 062, GAUK 652412). We thank Iva Vencovska for her technical assistance.

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REFERENCES

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