Anomeric Effects on the Stability of Bilayers of ... - ACS Publications

Ibaraki 305-8565, Japan, and Pharmaceutical Development Laboratory, Kirin Brewery Co., Ltd., 1-2-2 Souja-Machi Maebashi, Gunma 371-0853, Japan...
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Anomeric Effects on the Stability of Bilayers of Galactosylphytoceramides and on the Interaction with Phospholipids Minoru Nakano,† Rui Inoue,† Motoko Koda,† Teruhiko Baba,‡ Hiroki Matsunaga,§ Takenori Natori,§ and Tetsurou Handa*,† Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan, Department of Polymer Physics, National Institute of Material and Chemical Research, 1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan, and Pharmaceutical Development Laboratory, Kirin Brewery Co., Ltd., 1-2-2 Souja-Machi Maebashi, Gunma 371-0853, Japan Received March 1, 2000. In Final Form: June 1, 2000 The thermotropic behavior of the hydrated bilayers formed by novel glycosylceramides containing an anomeric galactose, a saturated long acyl chain (cerotoyl, C26), and a phytosphingosine base, R- and β-GalpCer, was investigated by means of differential scanning calorimetry (DSC) and X-ray diffraction measurements. DSC curves for hydrated R-GalpCer showed an endothermic transition from metastable gel to liquid crystalline states at 72.8 °C (∆H ) 12.0 kcal/mol) and exothermic transition of the reverse change at 66.3 °C (∆H ) -10.9 kcal/mol) at the heating/cooling rate of 1 °C/min. The metastable gel of R-GalpCer was remarkably stable in contrast with β-GalpCer or the typical animal β-galactosylceramide containing a long acyl chain. Annealing at 37 °C for 48 h completely converted the metastable gel of R-GalpCer into a stable gel, which was directly transformed to a liquid crystal (LC) at 74.9 °C with a 2-fold larger endothermic peak (∆H ) 23.8 kcal/mol). Cooling of the LC state brought about the transition into the metastable gel. X-ray diffraction measurements showed that both metastable and stable gels were lamellar structures. The diffraction patterns also suggested that the stable gel had a bilayer arrangement of higher crystallinity than the metastable gel. However, the metastable gel of β-GalpCer was rapidly converted to the stable gel: Upon heating, the stable gel was directly transformed to the LC state at 75.8 °C (∆H ) 19.0 kcal/mol), and cooling of the LC state led to successive transitions of the LC to the metastable gel at 71.0 °C and of the metastable gel to the stable gel at 64.8 °C. The stable gel gave sharp reflections at 1/4.66 and 1/4.06 Å-1. DSC findings also showed that at least 30 mol % of R-GalpCer was intermixing and the lower fraction of β-GalpCer was miscible with DPPC in the gel state. The less ordered metastable gel of R-GalpCer was assumed to be involved in the better miscibility with DPPC.

Carbohydrate-bearing lipids β-galactosyl-N-acylceramides (β-GalCers) are involved as recognition sites and structural elements of the plasma membrane of higher animal cells. It was suggested that the β-GalCer concentration, arrangement, and cluster formation within the membrane may be important modulators of cell contact, cell growth and regulation, and immunoresponse as a result of change in the expression of the glycolipid.1-4 It has been well established that hydrated β-GalCer forms limited major supermolecular structures: the lamellar liquid crystalline (LC) state; the metastable gel state obtained by cooling from temperature above the gel-LC transition; the stable gel state obtained by relaxation of the metastable state.5-7 Differential scanning calorimetry * To whom correspondence should be addressed. Fax: +81 75 753 4601. E-mail: [email protected]. † Kyoto University. ‡ National Institute of Material and Chemical Research. § Kirin Brewery Co., Ltd. (1) Kishimoto, Y.; Moser, H. W.; Suzuki, K. In Handbook of Neurochemistry; Lajtha, A., Ed.; Plenum Press: New York, 1985; Vol. 10, pp 125-151. (2) Lu, D.; Singh, D.; Morrow, M. R.; Grant, C. W. M. Biochemistry 1993, 32, 290-297. (3) Stewart, R. J.; Boggs, K. M. Biochemistry 1993, 32, 5605-5614. (4) Brown, R. E. J. Cell Sci. 1988, 111, 1-9. (5) Thompson, T. E.; Allietta, M.; Brown, R. E.; Johnson, M. L.; Tillack, T. W. Biochim. Biophys. Acta 1985, 817, 229-237. (6) Thompson, T. E.; Barenholz, Y.; Brown, R. E.; Correa-Freire, M.; Young, W. W.; Tillack, T. W. In Enzymes of Lipid Metabolism ll; Freysz, L., Dreyfus, H., Massarelli, R., Gatt, S., Eds.; Plenum Pub. Corp.: New York, 1986; pp 387-396.

(DSC) and X-ray diffraction of hydrated β-GalCer with N-palmitoyl (C16) and N-lignoceroyl (C24) chains clearly show the rapid conversion of the metastable to stable state.8,9 DPPC and β-GalCer are completely miscible in the LC state, but in the gel state, both β-GalCers with N-palmitoyl (C16) and N-lignoceroyl (C24) chains show limited miscibility with 1,2-dipalmitoyl-L-phosphatidylcholine (DPPC).10,11 The solubility of the longer acyl chainβ-GalCer is less than 10 mol % in the DPPC-rich gel phase.11 Agelasphins, R-galactosylphytoceramides with a 2-hydroxy fatty acid, have been isolated from an extract of the amarine sponge (Agelas mauritianus) and shown to have antitumoral activity against several murine tumors in vivo with weak acute toxicity.12,13 The structure-antitumoral activity relationship shows an R-galactosylphytoceramide with a long saturated fatty acyl chain with 26 carbons, (R-D-galactopyranosyl)-N-cerotoyl-2-amino-1,3,4(7) Curatolo, W. Biochim. Biophys. Acta 1987, 906, 111-136. (8) Ruocco, M. J.; Atkinson, D.; Small, D. M.; Skarjune, R. P.; Oldfield, E.; Shipley, G. G. Biochemistry 1981, 20, 5957-5966. (9) Reed, R. A.; Shipley, G. G. Biochim. Biophys. Acta 1987, 896, 153-164. (10) Ruocco, M. J.; Shipley, G. G. Biophys. J. 1983, 43, 91-101. (11) Gardam, M.; Silvius, J. R. Biochim. Biophys. Acta 1989, 980, 319-325. (12) Kobayashi, E.; Motoki, K.; Uchida, T.; Fukushima, H.; Koezuka, Y. Oncology Res. 1995, 7, 529-534. (13) Morita, M.; Sawa, E.; Yamaji, K.; Sakai, T.; Natori, T.; Koezuka, Y.; Fukushima, H.; Akimoto, K. Biosci. Biotech. Biochem. 1996, 60, 288-292.

10.1021/la0003084 CCC: $19.00 © 2000 American Chemical Society Published on Web 07/29/2000

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β- anomeric galactose, a long fatty acyl chain (C26), and a phytosphingosine base, R- or β-GalpCer, and their interactions with phospholipids on the basis of DSC and X-ray diffraction measurements, as a first example of the glycolipids with a double hydrocarbon chain. Materials and Methods

Figure 1. Structures of galactosylceramides: (A) R-galactosylN-cerotoylphytoceramide (R-GalpCer); (B) β-galactosyl-N-cerotoylphytoceramide (β-GalpCer); (C) β-galactosyl-N-acylceramide (β-GalCer (C24)).

octadecanetriol (R-GalpCer, in Figure 1), as the most promising candidate for clinical development.14 The antitumoral and immunostimulatory activities parallel the enhancing proliferation of natural killer T lymphocytes (NKT cells). Such bioactivities and proliferation effects were not observed for the glycosylphytoceramide-containing β-anomeric galactose, β-GalpCer (Figure 1).15-18 Although the two anomers R and β have the same molecular weight and hydrophilic-lipophilic balances, they show physicochemical distinction in addition to the physiological discrimination due to the difference of configuration at the anomeric center. For example, micellization behavior of R- and β-alkyl glycosides has been thoroughly investigated. Surface tension measurements of aqueous solutions of decyl glucosides have revealed that the β-anomer forms micelles but the R-anomer reaches a solubility limit before micellization can occur at increasing concentrations.19 Light scattering experiments for octyl glucosides have shown that the R-anomer has less solubility and forms larger assemblies than the β-anomer.20 However, X-ray and neutron scattering of dodecyl maltosides have indicated the formation of spherical micelles for the R-anomer and oblate ellipsoidal micelles for the β-anomer.21 These findings suggest that the anomeric configuration influences the intermolecular interaction and hence the solubility or association behavior. This can also be applied to the glycolipids with double hydrocarbon chain, although it has not been experimentally demonstrated. In the present study, we evaluated the polymorphic behavior of hydrated glycosyl ceramides containing R- or (14) Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa, E.; Yamaji, K.; Koezuka, Y.; Kobayashi, E.; Fukushima, H. J. Med. Chem. 1995, 38, 2176-2187. (15) Kobayashi, E.; Motoki, K.; Yamaguchi, Y.; Uchida, T.; Fukushima, H.; Koezuka, Y. Bioorg. Med. Chem. 1996, 4, 615-619. (16) Uchimura, A.; Shimizu, T.; Nakajima, M.; Ueno, H.; Motoki, K.; Fukushima, H.; Natori, T.; Koezuka, Y. Bioorg. Med. Chem. 1997, 4, 1447-1452. (17) Kawano, T.; Cui. J.; Koezuka, Y.; Toura, I.; Kaneko, Y.; Motoki, K.; Ueno, H.; Nakagawa, R.; Sato, H.; Kondo, E.; Koseki, H.; Taniguchi, M. Science 1997, 278, 1626-1629. (18) Spada, F. M.; Koezuka, Y.; Porcelli, S. A. J. Exp. Med. 1998, 188, 1529-1534. (19) Aveyard, R.; Binks, B. P.; Chen, J.; Esquena, J.; Fletcher, P. D. I. Langmuir 1998, 14, 4699-4709. (20) Focher, B.; Savelli, G.; Torri, G.; Vecchio, G.; McKenzie, D. C.; Nicoli, D. F.; Bunton, C. A. Chem. Phys. Lett. 1989, 158, 491-494. (21) Dupuy, C.; Auvray, X.; Petipas, C. Langmuir 1997, 13, 39653967.

R- and β-GalpCers were synthesized as described previously.14,22 The purity (greater than 98%) was determined by thinlayer chromatography. DPPC was purchased from Sigma-Aldrich Co. (Tokyo) and used without further purification. Differential Scanning Calorimetry (DSC). Calorimetry experiments were performed on a DSC3200 system (Mac Science Co., Yokohama, Japan) over the temperature range between 10 and 85 °C at a heating/cooling rate of 1 or 5 °C/min. R- or β-GalpCer or glycolipid-DPPC mixtures of ca. 3 mg were hydrated above 80 °C with excess Tris-HCl buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7) in the DSC sample cell. The temperature at the onset of the heat capacity change was taken as the transition temperature. Transition enthalpies were calculated from the area under the peak using gallium as a standard. The baseline was corrected with a buffer blank. Small-Angle and Wide-Angle X-ray Diffraction. The hydrated samples were flame-sealed into a quartz glass capillary (W. Mu¨ller, Berlin; 1.5 mm in outer diameter, 1/100 mm in wall thickness). X-ray diffraction experiments were performed with Ni-filtered Cu KR radiation (wavelength: 1.5418 Å) generated by a Rigaku RU-200 X-ray generator (Rigaku Co., Tokyo; 40 kV, 100 mA) with a double pinhole collimator (0.3 mm φ × 0.3 mm φ). The diffraction pattern was recorded with an imaging plate (Fuji Film Ltd., HR-III N) in a flat camera. The camera length was 270 mm for the small-angle and 50 mm for the wide-angle diffraction measurements. The recorded images were read on a Rigaku imaging plate reader, and the digitized data were analyzed on a Rigaku RINT 2000 system. Exposure time was 120 and 10 min for the small- and wide-angle diffraction measurements, respectively. The specimen temperature was controlled at 20 °C.

Results Calorimetric Behavior of Hydrated r- and βGalpCer. Figure 2A shows the polymorphic DSC behavior of R-GalpCer. The initial heating at the rate of 1 °C/min gave an endothermic transition at 72.8 °C with ∆H of 12.0 kcal/mol, following a pretransition at 25.7 °C (∆H ) 0.45 kcal/mol) (Figure 2A, a). In the subsequent cooling, an exothermic transition appeared at 66.3 °C with ∆H of -10.9 kcal/mol (Figure 2A, b). An identical heatingcooling cycle of hydrated R-GalpCer between 10 and 85 °C was reproduced at the temperature scanning rates of 1 and 5 °C/min. When the hydrated sample was annealed at 37 °C for more than 48 h, a distinct calorimetric behavior was observed in the heating run: An endothermic transition with 2-fold larger enthalpy (∆H ) 23.8 kcal/ mol) was observed at a higher temperature (74.9 °C), and the pretransition disappeared (Figure 2A, c). The following cooling run presented an exothermic transition with the same enthalpy (∆H of -10.9 kcal/mol) and at the same temperature (66.3 °C) as the initial cooling run (Figure 2A, b). The hydrated sample annealed for less than 48 h showed coexistence of the lower- and higher-temperature endothermic peaks (Figure 2B). These findings indicate the following: (1) The hydrated R-GalpCer phase which underwent the endothermic transition to LC with the higher-temperature and higher enthalpy was the stable gel or crystalline form. (2) The phase with the lowertemperature transition was the metastable gel which was formed directly by cooling the LC. Cooling at a rather slow rate (1 °C/min) did not convert the LC or metastable (22) Morita, M.; Natori, T.; Akimoto, K.; Osawa, T.; Fukushima, H.; Koezuka, Y. Bioorganic Med. Chem. Lett. 1995, 5, 699-704.

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Figure 2. Polymorphic DSC behaviors of hydrated galactosylphytoceramides (heating or cooling rate at 1 °C/min): (A) R-GalpCer, (a) heating scan, (b) cooling scan, (c) heating scan after annealing at 37 °C for 48 h; (B) R-GalpCer, progressive conversion of the metastable to the stable gel with annealing at 37 °C for 0 h (1), 3 h (2), 6 h (3), 12 h (4), 24 h (5), and 48 h (6); (C) β-GalpCer, (a) heating scan, (b) cooling scan.

gel states into the stable gel. (3) The relative stability of the metastable gel to the stable gel was considerably high, and the complete conversion from the former to the latter required extensive annealing. The thermotropic findings presented here are distinct from those of typical animal β-GalCer (in Figure 1) with a long acyl chain which show a low relative stability of the metastable gel and a rather faster relaxation of the metastable to stable gels.8,9 Figure 2C shows the calorimetric behavior of β-GalpCer. The initial heating scan at the rate of 1 °C/min gave an endothermic transition at 75.8 °C with ∆H of 19.0 kcal/ mol, a similar value for the stable gel-LC transition of R-GalpCer. Subsequent cooling exhibited successive exothermic transitions at 71.0 °C (∆H ) -11.5 kcal/mol) and 64.8 °C (∆H ) -5.1 kcal/mol). Identical cycles were reproduced at the heating-cooling rate of 1 and 5 °C/min, indicating the fast relaxation of the middle (metastable) phase of the β-anomeric species X-ray Diffraction of Stable and Metastable Hydrated r- and β-GalpCer. The X-ray diffraction patterns were recorded at 20 °C for the stable and metastable gels of hydrated R-GalpCer. The former was produced by 48 h annealing of the metastable gel at 37 °C, and the latter was prepared by a rapid cooling of the LC state. Figure 3A,B shows the diffraction patterns of the small- and wideangle regions for the metastable gel, respectively. Lamellar reflections (h ) 1 and weakly 2) were observed, corresponding to a bilayer periodicity of 66.0 Å (Figure 3A). The fluctuating baseline and the low intensity of reflections indicated the formation of less ordered lamellar structures. In the wide-angle region, reflections appeared at 2θ ) 21.2 and 23.0° corresponding to 1/4.20 and 1/3.86 Å-1, respectively (Figure 3B). Figure 4A,B presents the smalland wide-angle diffraction patterns for the stable gel, respectively. Lamellar reflections (h ) 1 and 2, in Figure 4A) indicate a bilayer periodicity of 66.2 Å, a value close to that of the metastable one. A similar minor change in the position of the small-angle reflection of the bilayer

Figure 3. X-ray diffraction of the metastable gel of hydrated R-GalpCer (20 °C): (A) small-angle region; (B) wide-angle region.

periodicity is known between the metastable and stable gels of typical β-GalCer.9 In the wide-angle region, strong reflections appeared at 20.3, 21.2, 23.0, and 24.5° (1/4.37, 1/4.19, 1/3.87, and 1/3.64 Å-1, respectively) for the stable gel (Figure 4B), showing a highly ordered chain-packing state. Figure 5A,B presents the small- and wide-angle diffraction patterns for the stable gel of β-GalpCer, respectively. Lamellar reflections (h ) 1 and 2, in Figure 5A) indicate a bilayer periodicity of 70.5 Å, which is 4-5 Å larger than that of R-GalpCer. In the wide-angle region, two strong reflections appeared at 19.0° (1/4.66 Å-1) and 21.9° (1/4.06 Å-1), which indicated close packing of the hydrocarbon chains (Figure 5B).

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region of 0 e X e 0.3. At X ) 0.5 (1/1 mixture), a broad, but single, endothermic peak was observed at the heating rate of 5 °C/min (Figure 6B, a); however, two separated peaks appeared at the rate of 1 °C/min (Figure 6B, b). In addition, annealing the mixtures at 37 °C for 48 h changed the position of the two peaks (Figure 6B, c). These findings suggested progressive phase separation of the mixed bilayer into the DPPC-rich (onset of transition at 45 °C) and R-GalpCer-rich (onset at 67 °C) bilayer phases. However, in the mixed lamellar phase of β-GalpCer and DPPC, separate endothermic peaks appeared even when X was 0.3 (Figure 6C, a). Annealing the mixtures at 37 °C showed little change in the DSC thermograph (Figure 6C, b). These findings indicated lower miscibility of the β- than R-GalpCer with DPPC. Discussion

Figure 4. X-ray diffraction of the stable gel of hydrated R-GalpCer (20 °C): (A) small-angle region; (B) wide-angle region.

Figure 5. X-ray diffraction of the stable gel of hydrated β-GalpCer (20 °C): (A) small-angle region; (B) wide-angle region.

Miscibility of r- or β-GalpCer with Phospholipid. Exothermic transitions were recorded on DSC for hydrated mixtures of R- or β-GalpCer/DPPC when cooling from 85 °C (both components in the LC state) to 10 °C (both in the gel state). The transition onset temperature was continuously changed as a function of the glycolipid mole fraction of mixed bilayers (data not shown), indicating homogeneous mixing in the LC phase. If a phase separation occurs in the LC, the degree of thermodynamic freedom is zero under atmospheric pressure, leading to an invariant region in the transition temperature. Endothermic transitions of the lipid mixtures in the heating run were complicated. For the mixed lamellar phase (bilayers) of R-GalpCer and DPPC, single endothermic peaks appeared when the mole fraction of the glycolipid, X, was smaller than 0.3. Annealing these mixtures at 37 °C did not change the endothermic transition (Figure 6A, a, b). These findings indicated a homogeneous mixing in the gel phase in the

Thermotropic Properties of r- and β-GalpCer Bilayers. Figure 7A shows the thermotropic conversion of hydrated R-GalpCer. The metastable gel was obtained by cooling the LC state, and the stable gel was obtained only by extensive annealing of the metastable gel. The endothermic transition from the metastable gel to the LC states and the exothermic transition of the reverse change gave similar absolute values of enthalpy (12.0 and 10.9 kcal/mol, respectively). The metastable gel of R-GalpCer was remarkably stable under the experimental conditions in contrast with β-galactosylceramide, β-GalCer, and β-galactosylphytoceramide, β-GalpCer, containing a long acyl chain. Extensive annealing was required to convert the metastable gel of R-GalpCer into the stable gel. During the annealing, the metastable and stable gels coexisted. The latter was directly transformed to the LC state with a 2-fold larger endothermic peak. Cooling the LC state from 85 °C brought about a transition into the metastable gel. For β-GalpCer, however, cooling of the LC state completely converted it to the stable gel via the metastable gel state, and the stable gel was directly transformed to the LC state (Figure 7B). These findings suggest that the nucleation and/or nuclear growth in conversion from the LC or metastable gel states to the stable gel of R-GalpCer are more retarded than those of β-GalCer and β-GalpCer. Two factors are responsible for this retardation: the existence of an R-anomeric galactose bond and/or phytosphingosine base. The findings of this study demonstrated that the anomeric galactose appears to play a primary role in the stability of the metastable gel of galactosylphytoceramides. However, when the metastable gel of a typical animal β-galactosylceramide (β-GalCer) is heated, the endothermic transition from the metastable gel to LC states is often followed by the immediate exothermic transition from the LC to the stable gel state, and when there is cooling, the exothermic transition from the LC to the metastable gel state is followed by a second exothermic transition from the metastable to the stable gel.5-9 This behavior is different from that of β-GalpCer, which showed complete conversion of the LC to the stable gel and direct transition of the stable gel to the LC state on cooling and heating, respectively. Therefore, sphingosine and phytosphingosine bases in β-GalCer and β-GalpCer, respectively, also play roles in determining the relative stability of the stable and metastable states. The X-ray diffraction of the small-angle region of hydrated R-GalpCer demonstrated that both metastable and stable gels were lamellar structures with similar bilayer periodicities. For the wide-angle diffraction of the stable gel, a number of strong reflections were observed which indicated a crystalline chain-packing arrangement

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Figure 6. DSC behaviors of the galactosylceramide-DPPC mixture: (A) heating at 1 °C/min of R-GalpCer-DPPC mixture with the mole fraction of the glycolipid of 0.3 after cooling from 85 to 10 °C (a) or after annealing at 37 °C for 48 h (b); (B) heating of the R-GalpCer-DPPC mixture with the mole fraction of the glycolipid of 0.5 at the rate of 5 °C/min (a), 1 °C/min (b) after cooling from 85 to 10 °C, or 1 °C/min after annealing at 37 °C for 48 h (c); (C) heating at 1 °C/min of β-GalpCer-DPPC mixture with the mole fraction of the glycolipid of 0.3 after cooling from 85 to 10 °C (a) or after annealing at 37 °C for 48 h (b).

Figure 7. Thermotropic conversion of hydrated R-GalpCer (A) and β-GalpCer (B).

of hydrocarbon chains. However, it is obvious from the wide-angle diffraction pattern that the metastable gel has less crystallinity of the hydrocarbon chains than the stable gel. For β-GalCer with the N-lignoceroyl chain, Reed and

Shipley9 referred to the stable gel as a crystal bilayer with a specific chain-packing arrangement and to the metastable gel as a simple bilayer gel (Lβ or Lβ′) with hexagonally packed chains. The diffraction measurements of R-GalpCer were quite similar to those of previous studies.9 The nucleation of the crystalline bilayer structure appears to be retarded in the LC state where the acyl chains are packed in a hexagonal subcell. The transition entropies of the metastable and stable gels to LC (35 and 68 cal mol-1 K-1, respectively) also suggested greater structural modification in the latter conversion. In hydrated DPPC bilayers, conversion from metastable gel (Lβ) to stable crystalline (LC) is, however, accompanied by the appreciable decrease in the bilayer periodicity because of dehydration,23 which was not observed for R-GalpCer. The X-ray diffraction of hydrated β-GalpCer showed that the stable gel was a lamellar structure with a marginally longer bilayer periodicity. The stable gel gave two sharp reflections, which indicated close packing of the acyl chains. The reason that the diffraction pattern of the stable gel of β-GalpCer differs from that of R-anomer providing a number of reflections is still unknown. It may reflect the difference of hydrocarbon chain-ordering; that is, as shown in Figure 7, the stable gel of β-GalpCer may have the bilayer phase with nontilted hydrocarbon chainpacking arrangement in the orthorhombic subcell, whereas the hydrocarbon chains of R-GalpCer are tilted in the stable bilayer gel, which induces a complex diffraction pattern in the wide-angle region and decreases the bilayer periodicity. For the crystal structure analysis of 2-D-hydroxystearoylgalactosyldihydrosphingosine (C18), the bilayer length was determined to be 61 Å.24 This suggests the bilayer molecular length of GalpCers to be 61 + 1.25 × (26 - 18) ) 71 Å, by assuming the water layer thickness (23) Ruocco, M. J.; Shipley, G. G. Biochim. Biophys. Acta 1982, 691, 309-320. (24) Pascher, I.; Sundell, S. Chem. Phys. Lipids 1977, 20, 175-191.

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is negligible and by taking the interdigitation of the hydrocarbon chains into account. This is in good agreement with β-GalpCer (70.5 Å). However, the lamellar periodicity of the stable gel of R-GalpCer (66.2 Å) is small, and therefore, both the interdigitation and tilting of the chains have to be considered. It has been reported that alterations in the chain length and the chain saturation affect the thermodynamic behavior of β-GalCer and the formation of the metastable and stable gels. Increased unsaturation in the N-acyl chain of β-GalCer results in inhibition of formation of the stable gel from the less stable or metastable gel phases.25,26 Introduction of a 2-hydoxy group into the N-acyl chain of β-GalCer leads to greater motional freedom and/or conformational disorder,27 a lower gel-liquid crystalline transition temperature, and higher kinetic barriers of the metastable gel to the stable gel state.28 The experimental findings of the present study indicated that, in addition to chain length and chain saturation, R- and β- anomeric galactose played an important role in the thermodynamic stability of the polymorphic states. Bruzik and Nyholm29 have strongly suggested that the LC-metastable gel transformation of β-GalCer is not associated with a conformation change of the headgroup, while the gel phase relaxation to the stable crystal involves a reorientation of the galactose moiety from the layerperpendicular to the layer-tilted position by a rotation of the C1-O1 bond of the sphingosine. The rapid cooling of the LC state to the metastable gel simply traps the layerperpendicular orientation. In the 13C NMR spectra, the most prominent difference between the metastable and stable gels is observed in the signal position of the anomeric carbon of the galactose moiety.29 This reorientation process of the galactose moiety could also be applicable to β-GalpCer. However, the 13C NMR measurements revealed a distinction between the headgroup orientations of R- and β-alkyl glycosides in the LC of the lipid bilayer; that is, β-alkyl glycosides have layer-perpendicular conformation of their headgroup, but the R linkage requires the sugar ring to be nearly parallel to the plane of the bilayer.30 If this observation is also valid for GalpCers,

the galactose moiety of R-GalpCer must be in a layerparallel orientation even in the LC or metastable gel. This suggests that the layer-parallel orientation in the metastable gel, unlike in the stable gel, provides greater motional freedom of the hydrocarbon chains, and the relaxation process to the stable gel needs the rearrangement of the headgroups with high kinetic barriers. Enhanced Miscibility of r-GalpCer with DPPC. β-GalCer with a long (C24) saturated acyl chain (Nlignoceroyl-β-GalCer) is completely miscible with DPPC in the LC state, while phase separation is detected over a wide range of compositions in the gel state of mixtures (from 0.1 to 0.9 of the glycolipid mole fraction, X).11 The same glycolipid is found to be miscible in the gel bilayers of 1-stearoyl-2-oleoylphosphatidylcholine up to X ) 0.2.31 However, freeze-fracture electron microscopy has demonstrated the separation of the glycolipid-rich domains in the 1-palmitoyl-2-oleoylphosphatidylcholine host matrixes of X ) 0.1.32 A similar limited miscibility of the β-anomer with phosphatidylcholine was also found in the present study for a galactosylphytoceramide, β-GalpCer. The hydrated R-GalpCer, however, showed higher miscibility with DPPC than β-GalpCer in the gel state. R-Hydroxylation of the N-acyl chain of β-GalCer molecules leads to greater motional freedom and/or conformational disorder in the gel states of the glycolipid and mixed bilayers with phosphatidylcholine.32,33 Johnston and Chapman34 have attributed the greater miscibility with the phospholipid to the reduced glycolipid-glycolipid attraction force due to the chemical modification. The better miscibility of R-GalpCer than β-GalpCer with DPPC in the bilayer gel state implied that the R- or β-anomeric galactose played a crucial role in the motional freedom of GalCer molecules, the stability of the relatively disordered (metastable) gel state, and the intermiscibility with phospholipid. Cluster formation of glucosylceramide within biomembranes has been assumed to be an important modulator for cellular functions as a result of the change in the expression of the glycosyl groups.1-4 The findings of the present study suggested that the anomeric sugar plays a role in the modulation of cellular functions.

(25) Reed, R. A.; Shipley, G. G. Biophys. J. 1989, 55, 281-292. (26) Haas, N. S.; Shipley, G. G. Biochim. Biophys. Acta 1995, 1240, 133-141. (27) Singh, D.; Jarrell, H. C.; Florio, E.; Fenske, D. B.; Grant, C. W. M. Biochim. Biophys. Acta 1992, 1103, 268-274. (28) Curatolo, W.; Jungalwala, F. B. Biochemistry 1985, 24, 66086613. (29) Bruzik, K. S.; Nyholm, P.-G. Biochemistry 1997, 36, 566-575. (30) Sanders, C. R.; Prestegard, J. H. J. Am. Chem. Soc. 1992, 114, 7096-7107.

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