The influence of ceramide and its dihydro analog on the physico-chemical properties of sphingomyelin bilayers

Journal Pre-proof The influence of ceramide and its dihydro analog on the physico-chemical properties of sphingomyelin bilayers Masanao Kinoshita, Kaoru Tanaka, Nobuaki Matsumori

PII:

S0009-3084(19)30172-0

DOI:

https://doi.org/10.1016/j.chemphyslip.2019.104835

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CPL 104835

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Chemistry and Physics of Lipids

Received Date:

24 July 2019

Revised Date:

12 October 2019

Accepted Date:

15 October 2019

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The influence of ceramide and its dihydro analog on the physico-chemical properties of sphingomyelin bilayers

Masanao Kinoshita*, Kaoru Tanaka, Nobuaki Matsumori*

Department of Chemistry, Graduate School of Science, Kyushu University, Motooka 744, Nishi-ku,

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Fukuoka 819-0395, Japan

*Corresponding authors

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Masanao Kinoshita (Assistant Professor)

Department of Chemistry, Graduate School of Science, Kyushu University

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Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan

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Tel/fax: +81-92-802-4148 e-mail: [email protected]

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Nobuaki Matsumori (Professor)

Department of Chemistry, Graduate School of Science, Kyushu University

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Motooka 744, Nishi-ku, Fukuoka, 819-0395, Japan

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Tel/fax: +81-92-802-4153

e-mail: [email protected]

Graphical abstract

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Highlights 

We examined impact of the ceramide (Cer) and its dihydo-analog (DHCer) on the sphingomyelin bilayers.

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Both of Cer and DHCer form the Cer-rich and DHCer-rich domains, respectively,

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in sphingomyelin bilayers.

DHCer are more condensed in the DHCer-rich domains than Cer in the Cer-rich domains.

DHCer forms larger crystallites than Cer due to stronger intermolecular

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interaction of DHCer than that of Cer.

Abstract

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The influence of ceramide and its dihydro analog (Cer and DHCer, respectively; inclusively termed Cers) on sphingomyelin (SM) bilayers was examined. Fluorescent microscopy showed that

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SM/Cers binary bilayers undergo phase separation between Cers-rich and Cers-poor phases. Based on calorimetry, the content of Cers in the Cers-rich phase was estimated and the results show that

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DHCer in the DHCer-rich phase (17.5 mol%) is more condensed than Cer in the Cer-rich phase (15 mol%), likely due to a stronger intermolecular interaction of DHCer than that of Cer. Furthermore, the Cers-poor phase consists of almost pure SM. X-ray diffraction and water permeability measurements disclosed that the size of crystallites—lipid nano-clusters formed inside the Cers-rich phase—are larger for the DHCer-rich phase than those for the Cer-rich phase. Due to its stronger intermolecular interactions, DHCer could effectively suppress the inter-crystallite packing gaps to 2

avoid the energetic disadvantage, resulting in formation of larger crystallites.

Keywords: fluorescent microscopy, artificial rafts, phase separation, giant unilamellar vesicle, x-ray diffraction, calorimetry

1. Introduction

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Ceramide (Cer), which is the simplest precursor of the sphingolipids (SLs), consists of a sphingosine backbone and an N-linked acyl chain. Rigid and highly hydrophobic Cer molecule is widely known to be the main constituent of the outermost layer of skin (Berkers et al., 2018;

Chermprapai et al., 2018; Moore and Rawlings, 2017). While Cer is a minor component of the

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plasma-membrane (PM), it has attracted interest because metabolic conversion from SLs to Cer

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regulates various cellular processes including apoptosis (Haimovitz-friedman et al., 1994; Pettus et al., 2002), growth suppression (Obeid and Hannun 1995), and differentiation and senescence

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(Okazaki et al., 1990; Venable et al., 1995). Previously, Obeid et al. reported that a short chain Cer (C2:0) causes DNA fragmentation, which leads to programmed cell death (Obeid et al., 1993). On

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the other hand, ceramide-1-phosphate, an antagonist of Cer, attenuates the activity of the acid SMase, which is responsible for breaking down SM into phosphocholine and Cer. The reduction in the

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generation of Cer, due to the inactivity of SMase, results in the inhibition of cell death (Gómez-Muñoz, 2006; Gómez-Muñoz et al., 2004). These results indicate that the quantitative

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balance between SLs and Cer works as a signal switch for cell death and survival. With regard to PM-based signal transductions, the membrane sub-micro domains, called lipid rafts,

are also of interest because they work as a platform for important signal transductions. The putative rafts are rigid and ordered membrane domains, and mainly consist of SLs represented by sphingomyelin (SM; Fig. 1a) (Lingwood and Simons, 2010; Simons and Toomre, 2000). Due to their strong detergent resistance, lipid rafts are extracted as detergent resistant membrane (DRM) fractions 3

from PMs. Recently, it was found that SMases are recruited in DRMs in response to external stimuli (Cremesti et al., 2002; Gulbins et al., 2004). Therefore, it is not farfetched to say that lipid rafts provide favorable environments for the generation of Cer (Cremesti et al., 2002; Grassme et al., 2001; Gulbins et al., 2004; Johnston, 2008). From a structural aspect, hydrophobic Cer works as a lipid packing modulator and alters membrane properties (Leung et al., 2012). For example, phenylhexatriene (DHP) polarization and H-NMR measurements revealed that natural Cers and synthetic palmitoyl-Cer (C16:0-Cer) increase

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the acyl-chain order of diacyl-phosphatidylcholine (diacyl-PC) bilayers in the fluid phase (Massey,

2001; Hsueh et al., 2002). Differential scanning calorimetry (DSC) demonstrated that the addition of

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Cer into dimyristoyl-PC (DMPC) and dipalmitoyl-PC (DPPC) bilayers induces phase segregation of the Cer-rich domains whose main transition temperature is higher than that of the host PC

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membranes (Holopainen et al., 1997; Morrow et al., 2009). X-ray diffraction experiments

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represented that the Cer-rich domains form the gel phase (Holopainen et al., 2000; Boulgaropoulos et al. 2012). These results indicate the excellent potency of Cer for the membrane ordering and the domain formation.

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It has been suggested that the lipidome of any mammalian cell contains two types of SLs (Kuikka et al., 2001; Ramstedt and Slotte, 2002; Slotte, 2016). One is the most abundant SL and the other is

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its dihydro-analog, which bears a single bond linkage at C4–C5 in the sphingosine base. This minor

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structural difference imposes different impacts on the physical properties of membranes. For example, dihydro-SM (DHSM, C16:0) bilayers undergo the main transition at 46.8 °C, which is much higher than that for normal SM (C16:0) bilayers (Tm = 40.8 °C) (Nyholm et al., 2003). The main transition enthalpy of the DHSM bilayer (∆H = 35.4 kJ/mol) is also higher than that of the SM bilayer (∆H = 33.1 kJ/mol). Furthermore, some fluorescent measurements revealed a higher immiscible-to-miscible transition temperature of DHSM (C18:0) than SM (C18:0) in

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di-oleoyl-phosphatidylcholine (DOPC) bilayers (Kinoshita et al., 2014; Slotte, 2016). These results suggest that the minor structural difference at C4−C5 markedly affects membrane properties and causes DHSM bilayers to form the thermally stable gel phase more than SM bilayers. Although it has been suggested that Cers are generated in lipid rafts, the difference in the influence of Cer (Fig. 1b) and its dihydo-analog (DHCer; Fig. 1c) (Fabrias et al., 2012; Kok et al., 1997) on the physico-chemical properties of lipid rafts is unknown. Specifically, basic information

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using model membranes has been lacking. In the present study, we compared the influence of Cer and DHCer (inclusively termed Cers) on the physico-chemical properties of SM bilayers using

fluorescent microscopy, differential scanning calorimetry (DSC), detergent solubility measurements.

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Moreover, structural difference in the Cer-rich and DHCer-rich phases was examined by wide angle

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x-ray diffraction (WAXD) and water permeability measurements.

Fig. 1. Chemical structures of (a) SM, (b) Cer, and (c) DHCer. The Cer bears a trans-double bond linkage at C4−C5 of the sphingosine base while DHCer bears a single bond (indicated by the red color).

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2. Materials & Methods 2.1. Materials Egg-yolk sphingomyelin, palmitoyl-Cer (C16:0 Cer) and palmitoyl-DHCer (C16:0 DHCer) were purchased from Avanti Polar Lipids (Alabaster AL). Palmitoyl-sphingomyelin (C16:0 SM) was extracted from the egg-yolk SM using HPLC and the purity was checked by thin layer chromatography, showing a single spot (Jungalwala et al., 1979). Here, we denote

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palmitoyl-sphingomyelin, palmitoyl-Cer and palmitoyl-DHCer as SM, Cer, and DHCer, respectively. The SM, Cer and DHCer were dissolved in chloroform/methanol (4:1 v/v) and stored at −30 ºC until use.

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2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glyce ro-3-phosphocholine (Bodipy-PC) was purchased from Thermo Fisher Scientific (Waltham, MA). It

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was dissolved in chloroform/methanol (4:1 v/v) at a concentration of 50 μg/mL and stored in a dark

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place at −30 ºC until use.

2.2. Giant unilamellar vesicle (GUV) preparation and fluorescent observation

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GUVs were prepared using the electroformation method originally developed by Angelova and Dimitrov (Angelova and Dimitrov, 1986). Briefly, 10 μL of SM/5 mol% Cers mixed solution (1

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mg/mL) in the presence of 0.2 mol% Bodipy-PC was spread on the surface of electrodes; platinum

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wires (100 μm diameter), and dried under vacuum for more than 12 h. Then, parallel aligned electrodes were put into Milli-Q water sandwiched between two cover glasses (24 mm × 60 mm, 0.12–0.17 mm thickness) using an open-square shaped rubber spacer. This chamber was fixed on a temperature-controlled aluminum block kept at 70 ºC (Sahara 310, Rocker Scientific Co., Ltd., Taipei, Taiwan). We incubated the sample for 50 min, applying a low frequency alternating current (AC) field (sinusoidal wave function, 10 Vpp, 10 Hz) by a function generator (20 MHz

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function/arbitrary waveform function generator, Agilent, Santa Clara CA). The GUVs were then cooled down to 25 ºC and equilibrated for 60 min. Fluorescent observation was carried out using the BZ-X700 (Keyence, Osaka, Japan) with an air objective lens (Plan Apoλ, 60x, N.A. 0.95, Nikon, Tokyo, Japan). The excitation (470 nm) and detection (525 nm) wavelengths were selected by dichroic mirrors OP-87763 (Keyence, Osaka, Japan).

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2.3. Differential scanning calorimetry (DSC)

The thermal phase behavior of SM bilayers in the absence and presence of Cers was examined by

X-DSC7000 (Hitachi High-Tech Science Corp., Tokyo, Japan). Bilayer samples were prepared using

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a conventional method. Briefly, 2 mg of SM and appropriate amounts of Cers dissolved in

chloroform/methanol (4:1) were mixed in a glass vial. The solution was dried under a flow of

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nitrogen and then under reduced pressure for at least 24 h. The resulting lipid film was dispersed into 20 μL of Milli-Q water (Simplicity UV, MerckMillipore, Billerica, MA) and incubated for

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approximately 30 min at 80–90 °C with intermittent vortexing. Then, 15 μL of the aliquots were enclosed in an aluminum cell for the subsequent DSC heating scan. A scanning rate of 1.0 °C/min

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was used for all DSC measurements. Obtained DSC thermograms were analyzed by Origin Pro

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2015J (Origin Lab. Corp., Northampton, MA).

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2.4. Temperature resolved turbidity measurements Bilayer samples were prepared using a conventional method. Briefly, appropriate amounts of SM

and SM/10 mol% Cers dissolved in chloroform/methanol (4:1) were mixed in a glass vial. The solution was dried under a flow of nitrogen and then under reduced pressure for at least 24 h. The resulting lipid film was dispersed into 2420 μL of Milli-Q water (Simplicity UV, Merck Millipore, Billerica, MA) and incubated for approximately 30 min at 70 °C with intermittent vortexing.

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Following the incubation of the sample for 30 min at 4 ºC, 580 µL of cold Triton X-100 was exogenously added to the lipid suspension. The final concentration of lipids was 0.473 mM and the molar ratio of Triton X-100 to total lipids was 0.7. The turbidity of the lipid/Triton X-100 suspension was measured using a spectrophotometer V−730 Bio (JASCO, Tokyo, Japan). A scanning rate of 1.0 °C/min and a detection wavelength of 400 nm were used for all turbidity measurements.

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2.5. Wide-angle x-ray diffraction (WAXD)

Lipid suspensions were prepared using a conventional method. Ten milligrams of SM/Cers in

chloroform/methanol (4:1 v/v) were dried under a flow of nitrogen, and the samples were stored in

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vacuo for one day to remove the organic solvent completely. The dried lipid films were then

suspended in 24 μL of Milli-Q water at 80–90 °C for 10 min with intermittent vortexing to form

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fully hydrated multilamellar vesicles (MLVs). The final concentration of these samples was 30 wt %.

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The sample solution was sandwiched with kapton polyimide films (963153, The Nilaco Corp., Tokyo, Japan) with an aluminum washer as a spacer. The sample was placed on a differential scanning calorimetry apparatus (FP 84, Mettler-Toledo, UT), which was used as a temperature

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controller. The temperature scanning rate of 1 ºC/min was used for all x-ray experiments. WAXD experiments were conducted at the Photon Factory Beamlines (BL-6A) in the High Energy

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Accelerator Research Organization (Tsukuba, Japan). A wavelength (λ) and sample-to-detector

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length were 0.10 nm, which is calibrated with silver behenate and, sample-to-detector length of 540 mm.

All diffraction patterns were recorded using an x-ray photon counting two-dimensional pixel array

detector PILATUS (DECTRIS Ltd., Baden-Daettwil, Switzerland) with a pixel size of 172 μm × 172 μm. The two-dimensional WAXD patterns were linearized by integrating the intensity along the Debye–Scherrer rings and converted to one-dimensional profiles using Fit2d. Then, the background

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was subtracted using an appropriate polynomial function and the WAXD peak of the Cers-rich phase was obtained. Diffraction peaks given by the kapton polyimide film is separately subtracted using blank sample. The fitting of the WAXD profiles were conducted by OriginPro 2015. To our best knowledge, Gauss function, Lorentz function (Young et al., 1997) and their weighted sum (pseudo-Voigt function) (Wertheim et al., 1974) have been widely used for the WAXD fit. As a trial, we fitted our WAXD profiles to the Lorentz and Gauss functions and found that they were fitted

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much better to the Lorentz function than to the Gauss function (Fig. S1). This result manifests that, even in the case of the pseudo-Voigt fit, the Lorentzian component dominantly contributes to the

peak fit rather than the Gaussian component. In fact, the Lorentz function was previously used for

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the fitting WAXD profiles of SM/Cer bilayers in the gel phase (Boulgaropoulos et al. 2012).

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2.6. Large unilamellar vesicles (LUVs) preparation and membrane permeability measurements

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LUV samples were prepared in order to measure the membrane permeability. One hundred micrograms of SM in the presence of 15 mol% pCer and 17.5 mol% DHCer were dissolved in chloroform/methanol (4:1 v/v) and dried under the flow of nitrogen gas. The remaining organic

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solvent was completely removed under reduced pressure for more than 24 h. The resulting lipid film was suspended in 1 mL of Milli-Q water at 70 °C with intermittent vortexing to form multilamellar

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vesicles (MLVs). The MLVs were extracted through a two-ply membrane filter with a pore size of

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Φ = 200 nm (GE Healthcare UK Ltd., Buckinghamshire, UK) 11 times using an Avanti mini-extruder (Avanti Polar Lipid Inc., Alabaster, AL, USA) to form LUVs (Patil & Jadhav 2014.; Kinoshita et al., 2017). Then, 500 μL of the LUV suspension was mixed with the appropriate concentration of sucrose solution, so as to apply osmotic pressure (500 mOsm/L and 730 mOsm/L) to LUVs. Following the exogenous addition of sucrose solutions, we measured the particle size distribution of the LUVs by dynamic light scattering (DLS) (BL-550, Horiba Osaka, Japan). All

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DLS measurements were conducted at 25 °C. In the DLS measurements, 1.590 mPa・s and 2.153 mPa・s were used as a viscosity of 500 mM and 730 mM sucrose solutions, respectively (Zhao & Li, 2005). The reflective index of 500 mM and 730 mM sucrose solutions were set to be 1.360 and 1.375, respectively, which were estimated by fitting the reflective index v.s. sucrose concentration plot to a linear function (Yunus & Rahman 1988).

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3.1. Observation of the Cer-rich/Cer-poor phase separation

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3. Results

Fig. 2. Fluorescent micrographs at the edges of (a) SM/5 mol% Cer and (b) SM/5 mol% DHCer GUVs in the

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presence of 0.2 mol% Bodipy-PC at 25 ºC. The Cers-poor phases were labelled by Bodipy-PC.

First, we observed Cers-induced phase separation in SM bilayers using fluorescent microscopy.

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Fig. 2a shows SM/5 mol% Cer binary GUVs in the presence of 0.2 mol% Bodipy-PC at 25 ºC, and indicates the phase separation between the Cer-rich and Cer-poor phases. This result is in line with a

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previous report (Sot et al., 2006). As Bodipy-PC is preferentially incorporated into the Cer-poor phase in SM/Cer GUVs (Matsufuji et al., 2019), the darker and brighter regions correspond to the Cer-rich and Cer-poor phases, respectively. In addition, we found that DHCer also induces phase separation between DHCer-rich and DHCer-poor phases in binary GUVs (Fig. 2b).

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3.2. Thermal phase behavior of SM bilayers in the presence of Cer and DHCer In order to examine the difference in the membrane properties between the Cer-rich and DHCer-rich phases, inclusively termed the Cers-rich phase, we examined the thermal phase behavior of SM/Cers binary bilayers using differential scanning calorimetry (DSC) (Fig. 3). The DSC heating thermogram of pure SM bilayers shows a sharp endothermic peak at Tsharp = 40.9 °C, corresponding to the gel-to-fluid phase transition (main transition) (Maulik and Shipley, 1996). The addition of Cer

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gives rise to the appearance of a broad shoulder at the higher temperature region of the sharp transition (indicated by arrows in Fig. 3a). Because the transition enthalpy of the broad peak

increases as the concentration of Cer increases, the broad shoulder corresponds to the main transition

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of the Cer-rich phase. This thermal phase behavior of SM/Cer bilayers is in line with a previous report (Sot et al., 2006). Similar results were obtained in the SM/DHCer mixtures; addition of

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DHCer also gives rise to a broad peak component, which corresponds to main transition of the

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DHCer-rich phase (indicated by arrows in Fig. 3b). It is noted that the transition temperature of the DHCer-rich phase is significantly higher than that of the Cer-rich phase under the same compositions (Fig. 3 and crosses in Fig. 4a). Thus, the DHCer-rich gel phase possesses higher

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thermal stability than the Cer-rich gel phase. To the best of our knowledge, this is the first

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comparison of the thermal phase behavior between SM/Cer and SM/DHCer binary bilayers.

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Fig. 3. DSC heating thermograms of SM bilayers in the presence of 0−12.5 mol% (a) Cer and (b) DHCer. The molar

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fractions of Cers, xcer and xDHCer, are described in the Figure. Blue and red arrows show the main transitions of the Cer-rich and DHCer-rich phases, respectively. Dashed lines indicate the main transition temperature of pure SM

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bilayers.

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3.2. Compositional analysis of the Cers-rich and Cers-poor phases In order to estimate the composition of the Cers-rich phase, we plotted the main transition

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enthalpy of the Cer-poor and DHCer-poor phases, inclusively termed the Cers-poor phase, (∆Hsharp) as a function of the Cers composition (xCers) (Fig. 4b). As a result, the ∆Hsharp decreases linearly as

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the xCers increases, showing that the Cers-poor phase transforms into the Cers-rich phase as the concentration of Cers increases. The extrapolation of the ∆Hsharp vs xCers plot to the ∆Hsharp = 0

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(dashed lines in Fig. 4b) allows us to determine the Cers composition at which transformation from the Cers-poor phase to the Cers-rich phases is completed. Consequently, it was found that the Cer-rich phase consists of SM/15±1.4 (S.D.) mol% Cer while the DHCer-rich phase consists of SM/17.5±0.4 (S.D.) mol% DHCer. On the other hand, we speculated that the Cers-poor phase consists of almost pure SM bilayers because their transition temperatures (Tsharp) are kept constant

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irrespective of the concentrations of Cers (circles in Fig. 4a).

Fig. 4. Dependence of the (a) main transition temperature and (b) transition enthalpy on the molar fraction of Cer and DHCer. The circles and crosses in panel (a) show the transition temperature (peak-top temperature) of the Cers-poor and Cers-rich phases, respectively. (b) shows the transition enthalpy of the Cers-poor phase (∆Hsharp). The results obtained for SM/Cer and SM/DHCer are indicated by blue and red symbols, respectively. The dashed lines in the panel show linear fitting for ΔHsharp vs xCers.

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Furthermore, these results are confirmed by temperature scanning detergent (Triton X-100) solubility measurements. Turbidity of pure SM bilayers monotonously decrease at ~25 ºC (black curve in Fig. 5). Because fluid bilayer membranes are easily dissolved in Triton X-100, this turbidity drop likely corresponds to the main transition of SM bilayers (Lichtenerg et al., 2013). The

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solubilization temperature of pure SM bilayers (~25 ºC; indicted by an arrow in Fig. 5) is lower than the main transition temperature obtained in the DSC measurements (circles in Fig. 4a). Probably, the

small amounts of Triton X-100 are incorporated into SM bilayers and destabilize the gel phase (Patra et al., 1998). On the other hand, SM/10 mol% Cers bilayers led to a stepwise decrease in turbidity

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(blue and red curves in Fig. 5); namely, the Cers-poor and Cers-rich phases are dissolved at the first

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and second drops of turbidity, respectively. Even after all membrane domains transformed into the fluid phase, the turbidity does not reach zero due to the cloud point of Triton X-100 near 55ºC (Sot et

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al. 2006).

Consequently, we found that the solubilization temperature of the Cers-poor phase is close to that

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of pure SM bilayers (indicated by an arrow in Fig. 5), demonstrating again that the Cers-poor phase consists of almost pure SM. Moreover, in order to estimate the areal ratio of the Cers-rich phase in

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SM/Cers bilayers, we compared turbidity of SM/10 mol% Cers and SM/10 mol% DHCer bilayers at 39ºC, where almost all Cers-poor domains are dissolved in Triton X-100 while the Cers-rich

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domains are not. In addition, the clouding of Triton X-100 is negligibly small at 39ºC (see black curve in Fig. 5). As a result, the Cers-rich phase accounts for 51±3 (S.D.) % and 39±5 (S.D.) % of SM/10 mol% Cer and SM/10 mol% DHCer bilayers, respectively. Besides, enthalpy analysis showed that the molar fraction of Cers is lower in the Cer-rich phase (15±1.4 mol%) than in the DHCer-rich phase (17.5±0.7 mol%) (Fig. 4b). In such a case, the Cer-rich phase (64±2 %) should be larger than that of the DHCer-rich phase (55±1 %) at the same content of Cers (xCers = 10 mol%), 14

being in line with the turbidity measurements. However, the ratio of the Cers-rich to all membrane domains estimated by turbidity measurements is 13%−16% smaller than that estimated by enthalpy analysis. This discrepancy is not surprising because the turbidity at the plateau regions shows the areal ratio of the Cers-rich phases while DSC analysis shows molar ratio of lipids incorporated in the Cer-rich phases. Previously, it was reported that the lateral occupied area of Cer (16:0) is smaller than that of SM (C16:0) (Wilke and Maggio, 2009). In addition, hydrophobic Cers are likely buried

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under the hydrophilic polar head of SM as depicted by umbrella model (Altetxe et al. 2013).

Because these factors likely reduce the surface occupied area of the Cers-rich phase and, thus,

turbidity measurements indicate the smaller ratio of the Cers-rich phase to all membrane domains

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than enthalpy analysis. Meanwhile, on the basis of the turbidity, it is reasonable for us to compare

the surface occupied areas of the Cer-rich and DHCer-rich phases because the lattice spacing of the

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those two phases (see Fig. 6 and text).

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carbon chains and, thus, the average molecular areas of the SM and Cers, are almost same between

Fig. 5. Temperature scanning turbidity measurements of (black) pure SM, (blue) SM/10 mol% Cer and (red) SM/10

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mol% DHCer bilayers in the presence of Triton X-100. An arrow indicates the first drop in turbidity, which corresponds to the solubilization of the Cers-poor phase. The molar ratio of Triton X-100 to lipids is 0.7. For easy comparison of the turbidity change, the initial turbidity is set to be 100%.

3.3. Structural analysis of the Cers-rich phase

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s (nm )

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2.38 2.36 2.34

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Peak position (nm-1)

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Intensity (a.u.)

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2.32 2.30 45

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Tempeature (℃)

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FWHM (nm-1)

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0.06 0.04 0.02 0.00

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Tempeature (℃)

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Fig. 6. (a) Linearized WAXD profiles of the (blue profile) Cer-rich and (red profile) DHCer-rich phases consisting of

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SM/15 mol% Cer and SM/17.5 mol% DHCer bilayers, respectively, at 44 ºC. Under these conditions almost all bilayer domains form the Cers-rich gel phase (Fig. 4). For easy comparison of full-width at half maximums (FWHMs), the peak heights were set to be equal between these two profiles. S is the absolute value of the scattering vector; s=2sinθ/λ. (b) Peak positions and (c) FWHMs of the (blue circles) Cer-rich and (red circles) DHCer-rich phases are plotted as a function of temperature. The peak positions and FWHMs are estimated by fitting the WAXD

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profiles shown in Fig. S2 to one or two Lorentz functions (see Fig. S3 for detail analysis).

Wide angle x-ray diffraction (WAXD) is a useful methodology to investigate the lateral packing structures of lipid carbon chains (Oka et al., 2017; Mills et al., 2008). Fig. 6a shows the WAXD

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profiles of SM/15 mol% Cer (Cer-rich phase) and SM/17.5 mol% DHCer (DHCer-rich phase)

bilayers at 44 ºC, where the Cers-rich phase forms the gel phase (crosses in Fig. 4a). Both of the

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Cer-rich and DHCer-rich phases give rise to a single WAXD peak at the similar position (2.38 nm−1 and 2.37 nm−1 for the Cer-rich and DHCer-rich phases, respectively), suggesting that the carbon

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chains form a hexagonal-like lattice with almost the same spacing of 0.42 nm−1. Furthermore, temperature scanning WAXD measurements disclosed that the peak positions are almost the same

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between the Cer-rich and DHCer-rich phases across experimental temperature range (Fig. 6b) Next, we compared crystallite sizes formed inside the Cer-rich and DHCer-rich phases based on

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the FWHMs of the WAXD peaks. The full-width at half maximums (FWHMs) of the x-ray diffractions depend largely on the size of crystallites, which are lipid nano-clusters formed inside the

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Cers-rich phase and/or, the packing order of the carbon chains. The influence of crystallite size and packing order on the FWHM is generally known as the particle broadening and the inhomogeneous strain broadening, respectively. Previously, de Keijser et al. (1981) reported that that the particle broadening is expressed by the Lorentz function while the strain broadening the Gauss function. Here, we noticed that our WAXD profiles show the Lorentzian line shape but not the Gaussian (see

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§2.5 and Fig. S1). This fact suggests that, in our case, FWHMs of the WAXD peaks depend dominantly on the crystallite size. Fig. 6a and c show that the FWHMs are smaller for the DHCer-rich phase than those for the Cer-rich phase across the experimental temperature range. In accordance with Scherrer’s equation (Patterson 1939), the FWHM of the WAXD peak is inversely related to the size of the crystallites. (Mills et al., 2008; Zhang et al., 2008). Therefore, we speculated that the crystallites formed in the DHCer-rich phase are larger than those formed in the Cer-rich

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phase (Fig. 7).

Fig. 7. Schematic illustrations of the (a) Cers-rich/Cers-poor micro-phase separation as observed in Fig.2. The green

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and red regions in panel (a) show the Cers-rich and Cers-poor phases, respectively. Panels (b) and (c) show crystallites formed inside the Cer-rich and DHCer-rich phases, respectively. In the Cers-rich phase, the carbon chains

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of lipids form a hexagonal-like lattice. A unit of a single crystallite formed inside the Cers-rich phase is indicated by

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different colors in panels (b) and (c).

Moreover, in order to support our speculation, we conducted water permeability measurements. It

has been reported that water molecules pass through packing gaps formed in the lipid membranes (Cass & Finkelstein, 1967; Deamer & Bramhall 1986; Kinoshita et al., 2010). In such a case, water permeability should be inversely related to the crystallite size because the assembly of smaller

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crystallites induces larger amount of the inter-crystallite packing gaps. Here, water permeability through the Cers-rich membrane was measured by osmotic methods whereby an osmotic gradient was applied to the LUVs, and the resulting volume change due to the water efflux from LUVs was measured (Mathai et al., 2007; Mlekoday et al., 1983). As a result, the average particle size of the SM/15 mol% Cer LUVs decreased from 154 nm to 123 nm and 119 nm under the osmotic pressure of 500 mOsm/L and 730 mOsm/L, respectively (Fig. 8a). On the other hand, the particle size of

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SM/17.5 mol% DHCer LUVs (164 nm) did not decrease under the osmotic pressure of 500 mOsm/L (165 nm) and 730 mOsm/L (161 nm), respectively (Fig. 8b). Moreover, we measured time course of the volume change at 730 mOsm/L and found that the decrease in the particle size of SM/15 mol%

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Cer LUVs is saturated within ~10 min. On the other hand, significant change in the particle size was not detected for SM/17.5 mol% DHCer LUVs (Fig. 8c). These results indicate that the DHCer-rich

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phase contains less packing gaps than the Cer-rich phase and, thus, the crystallite size of the

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DHCer-rich phase is larger than that of the Cer-rich phase. On the other hand, it is known that water permeability depends not only on the amount of the packing gap but also the compositional ratio of lipids (de Gier et al., 1968; Mathai et al., 2007). Thus, we measured water permeability of SM/15

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mol% DHCer LUVs at 750 mOsm/L. As a result, their particle size gradually decreased up to ~30 min (Fig. S4), indicating that the permeability rate of SM/15 mol% DHCer LUVs is significantly

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lower than that of SM/15 mol% Cer LUVs. Furthermore, it has been reported that the structure of

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lipid carbon chains often affects the water permeability of membranes. For example, Mathai et al. (2007) disclosed that the presence of the cis-double bonds in carbon chains significantly increases the membrane permeability, using dilauroylphosphatidylcholine (DLPC) and dioleoylphosphatidylcholine (DOPC). However, in our case, Cer bears a trans-double bond at C4–C5 of sphingosine but not cis-double bond (Fig. 1), and the trans-double bond produces membrane permeability similar to that of saturated chains [Roach et al., 2004]. Taken together, we

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speculate that that the water permeability depends dominantly on the crystallite rather than the lipid

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compositions and/or structures.

Fig. 8. Particle size distribution of the LUVs consisting of (a) SM/15 mol% Cer (Cer-rich phase) and (b) SM/17.5

mol% DHCer (DHCer-rich phase) at 25 ºC under the osmotic pressures of (black curves) 0 mOsm/L, (green curves)

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500 mOsm/L and (pink curves) 730 mOsm/L. The data were obtained after the equilibration of the LUVs for 30 min

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under those osmotic pressures. (c) Time course of change in the particle sizes of SM/15 mol% Cer (blue circles) and SM/17.5 mol% DHCer (red circles) LUVs under the osmotic pressure of 730 mOsm/L. All data were normalized by

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LUV size at 0 mOsm/L. We repeated same measurements for three times and the average values were shown. In addition, the data were fitted to a single exponential function (blue curve) and a linear function (red line). Bars

4. Discussion

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indicate standard deviation.

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It has been reported that Cer works as a lipid packing modulator and induces the formation of

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Cer-rich gel phase in phospholipid membranes (Holopainen et al., 2000; Morrow et al., 2009; Boulgaropoulos et al. 2012). While the gel phase is structurally different from raft-like liquid ordered (Lo) phase, Cer has been widely employed for the raft-oriented study due to its excellent potency for the ordering of lipid carbon chains and the domain formation (Cremesti et al., 2002; Megha & London, 2004; Megha et al., 2007). However, influence of the physiologically-relevant two types of Cers on the SM bilayers has not been compared. In the present study, we investigated

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the influence of Cer and DHCer on the physico-chemical properties of SM bilayers (Fig. 1). Microscopic observation and DSC measurements revealed that these Cers give rise to the formation of the Cers-rich phase (Fig. 2), whose transition temperature is markedly higher than the main transition temperature of pure SM bilayers (arrows in Fig. 3). Enthalpy analysis revealed that the DHCers are more condensed in the DHCer-rich phase (17.5±0.7 mol%) than Cers, which are conserved in the Cer-rich phase (15±1.4 mol%) (Fig. 4b). This result is supported by detergent

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solubility measurements, in which the amount of the Cer-rich phase is larger than that of the

DHCer-rich phase at the same content of Cers (Fig. 5). Recently, Sazzad and Slotte demonstrated that DHCer has higher aggregation formability than Cer in unsaturated lipid bilayers such as

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palmitoyl-oleoyl-phosphatidylcholine (POPC) and DOPC. Thus, they suggest that DHCer is capable of stronger intermolecular interactions than Cer (Sazzad and Slotte, 2016). Probably, such strong

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interactions between DHCers promote condensation of DHCer into the DHCer-rich phase. Moreover,

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DHCer could also interact favorably with SMs. Previously, it was reported that trans-unsaturation in the vicinity of C4 of the sphingosine backbone facilitates intra-molecular hydrogen bonding in the polar region (Yappert and Borchman, 2004). In addition, the replacement of the C4−C5 double bond

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by the single bond augments the inter-molecular hydrogen bonding between Cers (Brockman et al., 2004). In such a case, rather than Cer, DHCer should effectively interact with SM, which possesses

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abundant moieties involved in hydrogen bonding (Slotte, 2016). Such strong hydrogen bond

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interactions of DHCer with DHCer and/or SM likely explain the higher phase transition temperature of the DHCer-rich phase than that of the Cer-rich phase (crosses in Fig. 4a). Although DHCer is capable of stronger intermolecular interactions than Cer, the lattice spacing of carbon chains is almost the same between the Cer-rich and DHCer-rich phases (Fig. 6a). This is not surprising because both of the Cer-rich and DHCer-rich phases form the tightly packed gel phase. Namely, in the gel phase, van der Waals contacts overcome intermolecular interactions induced by DHCer,

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hampering the further approach of adjacent lipid molecules. Finally, we compared structural differences between the Cer-rich and DHCer-rich phases using WAXD and water permeability measurements and found that the crystallite size of the DHCer-rich phase is larger than that of the Cer-rich phase (Figs. 6 and 8). Why does DHCer form larger crystallites than Cer? As we mentioned above, DHCer is capable of forming stronger intermolecular hydrogen bonds than Cer (Sazzad and Slotte, 2016). In such cases, DHCer should form larger

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crystallites to avoid packing gaps because the packing gaps cut the hydrogen bond network and

confer an energetic disadvantage to the DHCer-rich phase. One may argue that, rather than Cers, SM

should dominantly affect the size of crystallites as the main constituent of the Cers-rich phase is SM;

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85 mol% (= 100 − 15) and 82.5 mol% (= 100 – 17.5) SM for the Cer-rich and DHCer-rich phases, respectively. We speculate that compact Cers are suitable molecules to fill nano- or less-sized

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packing gaps. Thus, Cers could preferentially reside at the edge of crystallites so as to suppress the

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packing gaps. However, direct information on the nano- or less-scale distribution of Cers is not

5. Conclusion

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accessible and, thus, further nanoscopic studies are needed.

It has been reported that Cer is capable of ordering lipid carbon chains (Massey, 2001; Hsueh et

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al., 2002). Recently, it was suggested that Cer generation occurs in SM-rich membrane domains,

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called lipid rafts, because there are several lines of evidence that SMase is recruited into the rafts. Therefore, Cer likely affects the physicochemical properties of lipid rafts. In the present study, we examined the influence of two types of physiologically relevant Cers (Fig. 1), which have a small structural difference at C4−C5 of the sphingosine base, on the SM bilayer. SM/Cers bilayers undergo phase separation between the Cers-rich and Cers-poor phases. In addition, DHCer has higher condensability than Cer probably due to the stronger intermolecular

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interaction of DHCer than that of Cer. Furthermore, due to such strong interactions, DHCer favorably forms larger crystallites with less packing gaps inside the DHCer-rich phase. This result could give important biological implications. It is known that many myristoylated and palmitoylated proteins interact favorably with lipid rafts (Levental et al., 2010; Resh, 2004; Webb et al., 2000). When these proteins bind to lipid membranes, their acyl chain, called the lipid packing sensor, recognizes packing gaps and anchors the protein to the membranes (Hatzakis et al., 2009).

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Although the packing gaps are usually created in a membrane curvature dependent manner (Vanni et al., 2013), the rigid raft membranes might not alter their geometry. Thus, instead of the alteration of

membrane curvature, the qualitative balance of two Cers might control the amounts of packing gaps

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in the raft membranes and regulate affinity of acylated proteins to lipid rafts.

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Acknowledgements

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The synchrotron x-ray radiation experiments were conducted at 6A in the high energy accelerator laboratory (PF-KEK, Tsukuba, Ibaraki, Japan) under approval of the Photon Factory Program Advisory Committee (Proposal No. 2016G162 & No. 2018G135), supported by professor Nobutaka

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Shimizu (KEK-PF, Ibaraki, Japan), Hiroshi Takahashi (Gunma University, Gunma, Japan) and Yasuko Obata (Hoshi Medical University, Tokyo, Japan). DSC measurements were conducted at the

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center of advanced instrumental analysis of Kyushu University. We would like to thank Editage

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(www.editage.com) for English language editing.

Funding

This work was supported by Japan Society for the Promotion of Science KAKENHI (17K15107) and in part JST ERATO (the Lipid Active Structure Project) and Kyushu University Interdisciplinary Programs in Education and Projects in Research Development (P&P).

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