Design, synthesis of ceramide 1-phosphate analogs and their affinity for cytosolic phospholipase A2 as evidenced by surface plasmon resonance.

Ceramide 1-phosphate (C1P) is a lipid mediator that specifically binds and activates cytosolic phospholipase A2α (cPLA2α). To elucidate the structure-activity relationship of the affinity of C1P for cPLA2α in lipid environments, we prepared a series of C1P analogs containing structural modifications in the hydrophilic parts and subjected them to surface plasmon resonance (SPR). The results suggested the presence of a specific binding site for cPLA2α on the amide, 3-OH and phosphate groups in C1P structure. Especially, dihydro-C1P exhibited enhanced affinity for cPLA2α, suggesting the hydrogen bonding ability of 3-hydroxy group is important for interactions with cPLA2α. This study helps to understand the influence of specific structural moieties of C1P on the interaction with cPLA2α at the atomistic level and may lead to the design of drugs that regulate cPLA2α activation.

Passive Translocation of Phospholipids in Asymmetric Model Membranes: Solid-State 1H NMR Characterization of Flip-Flop Kinetics Using Deuterated Sphingomyelin and Phosphatidylcholine.

Although lateral and inter-leaflet lipid-lipid interactions in cell membranes play roles in maintaining asymmetric lipid bilayers, the molecular basis of these interactions is largely unknown. Here, we established a method to determine the distribution ratio of phospholipids between the outer and inner leaflets of asymmetric large unilamellar vesicles (aLUVs). The trimethylammonium group, (CH3)3N+, in the choline headgroup of N-palmitoyl-sphingomyelin (PSM) and 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) gave rise to a relatively sharp signal in magic-angle spinning solid-state 1H NMR (MAS-ss-1H NMR). PSM and DOPC have the same headgroup structure, but one phospholipid was selectively observed by deuterating the trimethylammonium group of the other phospholipid. The addition of Pr3+ to the medium surrounding aLUVs selectively shifted the chemical shift of the (CH3)3N+ group in the outer leaflet from that in the inner leaflet, which allowed estimation of the inter-leaflet distribution ratio of the unlabeled lipid in aLUVs. Using this method, we evaluated the translocation of PSM and DOPC between the outer and inner leaflets of the cholesterol-containing aLUVs, with PSM and DOPC mostly distributed in the outer and inner leaflets, respectively, immediately after aLUV preparation; their flip and flop rates were approximately 2.7 and 6.4 × 10-6 s-1, respectively. During the passive symmetrization of aLUVs, the lipid translocation rate was decreased due to changes in the membrane order, probably through the formation of the registered liquid-ordered domains. Comparison of the result with that of symmetric LUVs revealed that lipid asymmetry may not significantly affect the lipid translocation rates, while the lateral lipid-lipid interaction may be a dominant factor in lipid translocation under these conditions. These findings highlight the importance of considering the effects of lateral lipid interactions within the same leaflet on lipid flip-flop rates when evaluating the asymmetry of phospholipids in the cell membrane.

Urine protein quantification in human urine on boron-doped diamond electrodes based on the electrochemical reaction of Coomassie brilliant blue.

Urinalysis is attracting interest in personal healthcare management as part of a general move to improve quality of life. Urine contains various metabolites and the protein level in urine is an indicator of kidney function. In this study, a novel electrochemical sensing system based on boron-doped diamond (BDD) electrodes was developed for the detection of protein concentrations in human urine. BDD electrodes have the advantages of a wide electrochemical potential window and low non-specific adsorption, making them ideal for simple, rapid, and compact devices for home detection of bio-relevant substances. Coomassie brilliant blue (CBB), a dye that selectively and strongly binds to urine proteins, was found to be a redox-active indicator to show a decrease in its redox currents in relation to the concentration of protein in urine samples. Our detailed studies of BDD electrodes showed their limit of detection to be 2.57 µg mL-1 and that they have a linear response that ranges from 0 to 400 µg mL-1 in urine samples. We also investigated the detection of urine protein in different urine samples. Our results agreed with those obtained using conventional colorimetric analysis. We believe this to be the first study of electrochemical detection of urine protein in urine samples on BDD electrodes, which is of great significance to be able to obtain results with electrical signals rapidly compared to conventional colorimetric analysis. This CBB-BDD technique has the potential to assist healthcare management in the form of a rapid daily diagnostic test to judge whether a more detailed examination is needed.

Effect of cholesterol on the lactosylceramide domains in phospholipid bilayers.

Lactosylceramide (LacCer) in the plasma membranes of immune cells is an important lipid for signaling in innate immunity through the formation of LacCer-rich domains together with cholesterol (Cho). However, the properties of the LacCer domains formed in multicomponent membranes remain unclear. In this study, we examined the properties of the LacCer domains formed in Cho-containing 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC) membranes by deuterium solid-state NMR and fluorescence lifetimes. The potent affinity of LacCer-LacCer (homophilic interaction) is known to induce a thermally stable gel phase in the unitary LacCer bilayer. In LacCer/Cho binary membranes, Cho gradually destabilized the LacCer gel phase to form the liquid-ordered phase by its potent order effect. In the LacCer/POPC binary systems without Cho, the 2H NMR spectra of 10',10'-d2-LacCer and 18',18',18'-d3-LacCer probes revealed that LacCer was poorly miscible with POPC in the membranes and formed stable gel phases without being distributed in the liquid crystalline domain. The lamellar structure of the LacCer/POPC membrane was gradually disrupted at around 60°C, whereas the addition of Cho increased the thermal stability of the lamellarity. Furthermore, the area of the LacCer gel phase and its chain order were decreased in the LacCer/POPC/Cho ternary membranes, whereas the liquid-ordered domain, which was observed in the LacCer/Cho binary membrane, was not observed. Cho surrounding the LacCer gel domain liberated LacCer and facilitated forming the submicron to nano-scale small domains in the liquid crystalline domain of the LacCer/POPC/Cho membranes, as revealed by the fluorescence lifetimes of trans-parinaric acid and trans-parinaric acid-LacCer. Our findings on the membrane properties of the LacCer domains, particularly in the presence of Cho, would help elucidate the properties of the LacCer domains in biological membranes.

Boron-Doped Diamond as a Quasi-Reference Electrode.

As a working electrode, boron-doped diamond (BDD) has been studied in detail in electrochemical processes because of its superior electrochemical properties. However, these characteristics have rarely been mentioned when BDD is used as a quasi-reference electrode (QRE). Herein, we conducted a systematic investigation on BDD electrodes, with different boron-doping levels (1 and 0.1%) and different surface terminations (hydrogen and oxygen) for their application as a QRE. A BDD electrode with 1% boron and a hydrogen-terminated surface achieved the best stability. Its open-circuit potential (OCP) exhibited less than 100 mV of potential drift over 6000 s and showed a minuscule half-wave potential difference (E1/2) of 0.0037 V in 0.1 mM K3[Fe(CN)6]/1 M KCl solution before and after the OCP measurement. Based on these observations, anions are found to contribute to the potential, which we preliminarily speculate as related to the capacitance caused by electrostatic adsorption on the positively charged hydrogen-terminated surface. The repeatability of measurement was verified through continuous cyclic voltammetry tests in 0.1 mM K3[Fe(CN)6]/1 M KCl, showing a maximum E1/2 difference of 0.042 V. The contribution of the redox couples was excluded, and the repeatability was considered to originate from its surface stability. Finally, a linear response of the optimized BDD as a QRE was validated (R2 > 0.99) by determination of free chlorine and dopamine concentrations, respectively. These results consolidate the existing fundamental research on BDD electrodes and promote the possibility of its application as a QRE in harsh environments or in vivo biological monitoring.

Depth-Dependent Segmental Melting of the Sphingomyelin Alkyl Chain in Lipid Bilayers.

The chain melting of lipid bilayers has often been investigated in detail using calorimetric methods, such as differential scanning calorimetry (DSC), and the resultant main transition temperature is regarded as one of the most important parameters in model membrane experiments. However, it is not always clear whether the hydrocarbon chains of lipids are gradually melting along the depth of the lipid bilayer or whether they all melt concurrently in a very narrow temperature range, as implied by DSC. In this study, we focused on stearoyl-d-sphingomyelin (SSM) as an example of raft-forming lipids. We synthesized deuterium-labeled SSMs at the 4', 10', and 16' positions, and their depth-dependent melting was measured using solid-state deuterium NMR by changing the temperature by 1.0 °C, and comparing with that observed from a saturated lipid, palmitoylstearoylphosphatidylcholine (PSPC). The results showed that SSM exhibited a characteristic depth-dependent melting, which was not observed for PSPC. The strong intermolecular hydrogen bonds between the sphingomyelin amide moiety probably caused the chain melting to start from the chain terminus through the middle part and end in the upper part. This depth-dependent melting implies that the small gel-like domains of SSM remain at temperatures slightly above the main transition temperature. These sphingomyelin features may be responsible for the biological properties of SM-based lipid rafts.

Behavior of Triterpenoid Saponin Ginsenoside Rh2 in Ordered and Disordered Phases in Model Membranes Consisting of Sphingomyelin, Phosphatidylcholine, and Cholesterol.

The ginsenoside Rh2 (Rh2) is a saponin of medicinal ginseng, and it has attracted much attention for its pharmacological activities. In this study, we investigated the interaction of Rh2 with biological membranes using model membranes. We examined the effects of various lipids on the membrane-disrupting activity of Rh2 and found that cholesterol and sphingomyelin (SM) had no significant effect. Furthermore, the effects of Rh2 on acyl chain packing (DPH anisotropy) and water molecule permeability (GP340 values) did not differ significantly between bilayers containing SM and saturated phosphatidylcholine. These results suggest that the formation of the liquid-ordered (Lo) phase affects the behavior of Rh2 in the membrane rather than a specific interaction of Rh2 with a particular lipid. We investigated the effects of Rh2 on the Lo and liquid-disordered (Ld) phases using surface tension measurements and fluorescence experiments. In the surface tension-area isotherms, we compared the monolayers of the Ld and Lo lipid compositions and found that Rh2 is abundantly bound to both monolayers, with the amount being greater in the Ld phase than in the Lo phase. In addition, the hydration state of the bilayers, mainly consisting of the Lo or Ld phase, showed that Rh2 tends to bind to the surface of the bilayer in both phases. At higher concentrations, Rh2 tends to bind more abundantly to the relatively shallow interior of the Ld phase than the Lo phase. The phase-dependent membrane behavior of Rh2 is probably due to the phase-selective affinity and binding mode of Rh2.

Simultaneous electrochemical detection of ozone and free chlorine with a boron-doped diamond electrode.

O3 and free chlorine play significant roles in disinfection and organic degradation. There are numerous reports about their mixed-use, yet detection of the residual concentrations is not easily accomplished, whilst the interactions between them remain unclear. Herein, we develop a detection method using a boron-doped diamond (BDD) electrode to achieve the simultaneous determination of O3 and free chlorine, which benefits from the unique property of the wide potential window of BDD electrodes. It is indicated that O3 can always be accurately determined at 0.35 V vs. Ag/AgCl in an acidic solution (pH = 4-5), whether or not free chlorine is present in the solution, whereas free chlorine can be precisely monitored at -0.78 V vs. Ag/AgCl only after the O3 is completely depleted. Furthermore, in a basic solution (pH = 9-10), the reduction peak of O3 at 0.57 V vs. Ag/AgCl promptly disappears accompanied by a decrease in the peak current of free chlorine at 1.41 V. All the phenomena observed in the acidic and basic solutions are concurrently confirmed in a quasi-neutral solution. Based on these complementary measurements, a mechanism is proposed, in which the O3 reduction results in partial oxidation of the BDD surface, hindering the reduction of free chlorine in the acidic mixture; whereas O3 reacts quickly with free chlorine in the basic solution, which causes the co-consumption of both of them. It is hoped these results will give us a guide as to how better utilize mixtures and more precisely simultaneously determine O3 and free chlorine in the mixture.

Lipid chain-driven interaction of a lipidated Src-family kinase Lyn with the bilayer membrane.

N-Myristoylation is a process of ubiquitous protein modification, which promotes the interaction of lipidated proteins on cell surfaces, in conjunction with reversible S-palmitoylation. We report the cooperative lipid-lipid interaction of two acyl chains of proteins, which increases the protein-membrane interaction and facilitates selective targeting of membranes containing anionic lipids. Lyn is a member of the Src family kinases distributed on the membrane surface by N-myristoyl and neighbouring S-palmitoyl chain anchors at the unique N-terminus domain. We prepared N-terminal short segments of lipidated Lyn to investigate the behaviour of each acyl chain in the lipid composition-dependent membrane interaction by solid-state nuclear magnetic resonance (NMR) analysis. Solid-state 31P-NMR studies revealed that S-palmitoylation of N-myristoylated Lyn peptides increased the interaction between peptides and phospholipid head groups, particularly with the anionic phosphatidylserine-containing bilayers. The solid-state 2H-NMR of Lyn peptides with a perdeutero N-myristoyl chain indicated an increase (0.6-0.8 Å) in the extent of the N-myristoyl chain in the presence of nearby S-palmitoyl chains, probably through the interaction via the acyl chains. The cooperative hydrocarbon chain interaction of the two acyl chains of Lyn increased membrane binding by extending the hydrocarbon chains deeper into the membrane interior, thereby promoting the peptide-membrane surface interaction between the cationic peptide side chains and the anionic lipid head groups. This lipid-driven mechanism by S-palmitoylation promotes the partition of the lipidated proteins to the cytoplasmic surface of the cell membranes and may be involved in recruiting Lyn at the signalling domains rich in anionic lipids.

LnDOTA-d8 , a versatile chemical-shift thermometer for 2 H solid-state NMR.

2 H solid-state nuclear magnetic resonance (NMR) is a method for examining the mobility and orientation of molecules in the field of biophysics. In studies on lipid bilayer membranes, 2 H NMR is often adopted to detect a phase transition from the gel to the liquid-crystal phase, which is observed as a change in spectral shape, and to evaluate the ordering of lipid alkyl chains using quadrupole coupling values. Because the mobility of membrane lipids is highly temperature dependent, precise temperature control is a prerequisite for evaluating the physical properties of membranes. Generally, NMR instruments monitor the temperature of the variable temperature (VT) gas. The temperature inside the sample tube and the VT gas match only when the heat generated by the radio frequency (rf) pulse emitted from the coil or magic angle spinning is significantly lower than the cooling capacity of the VT gas. In other words, the sample temperature inside the tube depends on the measurement method. Therefore, in this study, we took advantage of temperature-dependent changes in the chemical shift of a paramagnetic metal-ligand complex. We designed and synthesized a deuterated ligand complex and evaluated its temperature dependence as a thermometer for 2 H solid-state NMR spectroscopy. We chose Tb, Dy, Ho, and Er as the paramagnetic central metals. We then measured the 2 H NMR spectrum of each metal complex and confirmed the 2 H chemical shift to be temperature dependent. Furthermore, with the use of the thermometer molecule with Er, we succeeded in accurately evaluating the segmental melting of an alkyl chain in lipid bilayers with 0.1°C accuracy.

Diosgenin-induced physicochemical effects on phospholipid bilayers in comparison with cholesterol.

Diosgenin (DGN), which is a sterol occurring in plants of the Dioscorea family, has attracted increasing attention for its various pharmacological activities. DGN has a structural similarity to cholesterol (Cho). In this study we investigated the effects of the common tetracyclic cores and the different side chains on the physicochemical properties of lipid bilayer membranes. Differential scanning calorimetry showed that DGN and Cho reduce the phase transition enthalpy to a similar extent. In 2H NMR, deuterated-DGN/Cho and POPC showed similar ordering in POPC bilayers, which revealed that DGN is oriented parallel to the membrane normal like Cho. It was suggested that the affinity of DGN-Cho in membrane is stronger than that of DGN-DGN or Cho-Cho interaction. 31P NMR of POPC in bilayers revealed that, unlike Cho, DGN altered the interactions of POPC headgroups at 30 mol%. These results suggest that DGN below 30 mol% has similar effects with Cho on basic biomembrane properties.

Sphingomyelins and ent-Sphingomyelins Form Homophilic Nano-Subdomains within Liquid Ordered Domains.

Sphingomyelin (SM), a major component of small domains (or lipid rafts) in mammalian cell membranes, forms a liquid-ordered phase in the presence of cholesterol (Cho). However, the nature of molecular interactions within the ordered SM/Cho phase remains elusive. We previously revealed that stearoyl-SM (SSM) and its enantiomer (ent-SSM) separately form nano-subdomains within the liquid-ordered phase involving homophilic SSM-SSM and ent-SSM-ent-SSM interactions. In this study, the details of the subdomain formation by SSMs at the nanometer range were examined using Förster resonance energy transfer (FRET) measurements in lipid bilayers containing SSM and ent-SSM, dioleoyl-phosphatidylcholine and Cho. Although microscopy detected a stereochemical effect on partition coefficient favoring stereochemically homophilic interactions in the liquid-ordered state, it showed no significant difference in large-scale liquid-ordered domain formation by the two stereoisomers. In contrast to the uniform domains seen microscopy, FRET analysis using fluorescent donor- and acceptor-labeled SSM showed distinct differences in SM and ent-SM colocalization within nanoscale distances. Donor- and acceptor-labeled SSM showed significantly higher FRET efficiency than did donor-labeled SSM and acceptor-labeled ent-SSM in lipid vesicles composed of "racemic" (1:1) mixtures of SSM/ent-SSM with dioleoylphosphatidylcholine and Cho. The difference in FRET efficiency indicated that SSM and ent-SSM assemble to form separate nano-subdomains. The average size of the subdomains decreased as temperature increased, and at physiological temperatures, the subdomains were found to have a single-digit nanometer radius. These results suggest that (even in the absence of ent-SM) SM-SM interactions play a crucial role in forming nano-subdomains within liquid-ordered domains and may be a key feature of lipid microdomains (or rafts) in biological membranes.

Conformation and Orientation of Branched Acyl Chains Responsible for the Physical Stability of Diphytanoylphosphatidylcholine.

Diphytanoylphosphatidylcholine (DPhPC) is a synthetic phospholipid in which two methyl-branched acyl chains are introduced into the glycerol moiety, mimicking phospholipids of eukaryotic and eubacterial origins. The lipid bilayers of DPhPC reproduce the outstanding physical properties of methyl-branched lipids that occur in archaeal membranes. DPhPC is commonly used as the base lipid in biophysical experiments, particularly for recording ion-channel currents. However, the dynamics of lipid molecules that induces their useful physical properties is still unclear. In this study, we examined the conformation and orientation of the methyl-branched acyl chain of DPhPC in a membrane using 2H nuclear magnetic resonance (NMR) measurements of the synthetic lipid with a high stereochemical purity and molecular dynamics (MD) simulations. Deuterium-labeled 3',3'-CD3,D-DPhPC (2) and 7',7'-CD3,D-DPhPC (3) showed the characteristic quadrupole splitting width in the 2H NMR spectra, which corresponded to the bent orientation reported for the archaeal lipid PGP-Me [Yamagami, M., et al. (2019) Biochemistry 58, 3869-3879]. However, MD simulations, which reproduced the 2H NMR results well, unveiled the unknown features of DPhPC in the membrane; DPhPC has a chain-specific average orientation, where two bent orientations with upward and downward methyl groups occur at positions C3 and C7 of the sn-1 and sn-2 chains of DPhPC, respectively. These MD and NMR results reveal that these two bent orientations define the average orientation of DPhPC for the shallow part of the acyl chains, which is considered to be an important factor in the stability of DPhPC membranes.

Interactions of OSW-1 with Lipid Bilayers in Comparison with Digitonin and Soyasaponin.

OSW-1, a unique steroidal saponin isolated from the bulbs of Ornithogalum saundersiae, has potent cell-growth inhibition activity. In this study, we conducted fluorescence measurements and microscopic observations using palmitoyloleoylphosphatidylcholine (POPC)-cholesterol (Chol) bilayers to evaluate the membrane-binding affinity of OSW-1 in comparison with another steroidal saponin, digitonin, and the triterpenoid saponin, soyasaponin Bb(I). The membrane activities of these saponins were evaluated using calcein leakage assays and fitted to the binding isotherm by changing the ratios of saponin-lipids. Digitonin showed the highest binding affinity for the POPC-Chol membrane (Kapp = 0.38 µM-1) and the strongest membrane disruptivity in the bound saponin-lipid ratio at the point of 50% calcein leakage (r50 = 0.47) occurrence. OSW-1 showed slightly lower activity (Kapp = 0.31 µM-1; r50 = 0.78), and the soyasaponin was the lowest in the membrane affinity and the calcein leakage activity (Kapp = 0.017 µM-1; r50 = 1.66). The effect of OSW-1 was further assessed using confocal microscopy in an experiment utilizing DiI and rhodamine 6G as the fluorescence probes. The addition of 30 µM OSW-1 induced inward membrane curvature in some giant unilamellar vesicles (GUVs). At the higher OSW-1 concentration (58 µM, r50 = 0.78) where the 50% calcein leakage was observed, the morphology of some GUVs became elongated. With digitonin at the corresponding concentration (35 µM, r50 = 0.47), membrane disruption and formation of large aggregates in aqueous solution were observed, probably due to a detergent-type mechanism. These saponins, including OSW-1, required Chol to exhibit their potent membrane activity although their mechanisms are thought to be different. At the effective concentration, OSW-1 preferably binds to the bilayers without prominent disruption of vesicles and exerts its activity through the formation of saponin-Chol complexes, probably resulting in membrane permeabilization.

Efficient preparation of human and mouse CD1d proteins using silkworm baculovirus expression system.

CD1d is a major histocompatibility complex (MHC) class I-like glycoprotein and binds to glycolipid antigens that are recognized by natural killer T (NKT) cells. To date, our understanding of the structural basis for glycolipid binding and receptor recognition of CD1d is still limited. Here, we established a preparation method for the ectodomain of human and mouse CD1d using a silkworm-baculovirus expression system. The co-expression of human and mouse CD1d and ß2-microglobulin (ß2m) in the silkworm-baculovirus system was successful, but the yield of human CD1d was low. A construct of human CD1d fused with ß2m via a flexible GS linker as a single polypeptide was prepared to improve protein yield. The production of this single-chained complex was higher (50 µg/larva) than that of the co-expression complex. Furthermore, differential scanning calorimetry revealed that the linker made the CD1d complex more stable and homogenous. These results suggest that the silkworm-baculovirus expression system is useful for structural and biophysical studies of CD1d in several aspects including low cost, easy handling, biohazard-free, rapid, and high yielding.

Efficient diversification of GM3 gangliosides via late-stage sialylation and dynamic glycan structural studies with 19F solid-state NMR.

Sialic acid-containing glycoconjugates are involved in important biological processes such as immune response, cancer metastasis, and viral infection. However, their chemical syntheses have been challenging, mainly due to the difficulties in the α-sialylation of oligosaccharides. Very recently, we established a completely stereoselective sialidation method using a macrobicyclic sialyl donor. Herein, we describe a rational and efficient synthesis of sialoglycolipids via direct sialylation of a glycolipid at a late-stage, based on our novel sialidation method. The synthetic method enabled the development of GM3 ganglioside analogs with various C5-modifications of the sialosyl moiety. Furthermore, the synthesized analog was subjected to solid-state 19F NMR analysis on the model membranes and it revealed the influence of cholesterol on glycan dynamics.

Enantiomers of phospholipids and cholesterol: A key to decipher lipid-lipid interplay in membrane.

Most phospholipids constituting biological membranes are chiral molecules with a hydrophilic head group and hydrophobic alkyl chains, rendering biphasic property characteristic of membrane lipids. Some lipids assemble into small domains via chirality-dependent homophilic and heterophilic interactions, the latter of which sometimes include cholesterol to form lipid rafts and other microdomains. On the other hand, lipid mediators and hormones derived from chiral lipids are recognized by specific membrane or nuclear receptors to induce downstream signaling. It is crucial to clarify the physicochemical properties of the lipid self-assembly for the study of the functions and behavior of biological membranes, which often become elusive due to effects of membrane proteins and other biological events. Three major lipids with different skeletal structures were discussed: sphingolipids including ceramides, phosphoglycerolipids, and cholesterol. The physicochemical properties of membranes and physiological functions of lipid enantiomers and diastereomers were described in comparison to natural lipids. When each enantiomer formed a self-assembly or interacted with achiral lipids, both lipid enantiomers exhibited identical membrane physicochemical properties, while when the enantiomer interacted with chiral lipids or with the opposite enantiomer, mixed membranes exhibited different properties. For example, racemic membranes comprising native sphingomyelin and its antipode exhibited phase segregation due to their strong homophilic interactions. Therefore, lipid enantiomers and diastereomers can be good probes to investigate stereospecific lipid-lipid and lipid-protein interactions occurring in biological membranes.

Nonlamellar-Phase-Promoting Colipids Enhance Segregation of Palmitoyl Ceramide in Fluid Bilayers.

Ceramide is an important intermediate in sphingolipid homeostasis. We examined how colipids, with negative intrinsic curvature and which may induce curvature stress in the bilayers, affected the segregation of palmitoyl ceramide (PCer). Such colipids include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and tetra-linoleoyl cardiolipin (CL). In 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers, PCer formed ordered, gel-like domains at concentrations above 10 mol% at 23°C, as evidenced by the change in the average lifetime of the trans-parinaric acid emission. When POPE or DOPE were included in the DOPC bilayer (at 20:80 or 40:60 POPE or DOPE to DOPC, by mol), the lateral segregation of PCer was facilitated in a concentration-dependent manner, and less PCer was required for the formation of the ordered ceramide-rich domains. Inclusion of CL in the DOPE bilayer (at 10:90 or 20:80 CL to PC, by mol) also caused a similar facilitation of the lateral segregation of PCer. The PCer-rich domains formed in the presence of POPE, DOPE, or CL in DOPC bilayers were slightly more thermostable (by 2-10°C) when compared to PCer-rich domains in DOPC-only bilayers. Nonlamellar phases were not present in bilayers in which the effects of POPE or DOPE on PCer segregation were the largest, as verified by 31P NMR. When palmitoyl sphingomyelin was added to the different bilayer compositions at 5 mol%, relative to the phospholipids, PCer segregated into gel domains at lower concentrations (2-3 mol% PCer), and the effect of POPE on PCer segregation was eliminated. We suggest that the effects of POPE, DOPE, and CL on PCer segregation was in part influenced by their effects on membrane curvature stress and in part because of unfavorable interactions with PCer due to their unsaturated acyl chains. These lipids are abundant in mitochondrial membranes and are likely to affect functional properties of saturated ceramides in them.

Impact of Acyl Chain Mismatch on the Formation and Properties of Sphingomyelin-Cholesterol Domains.

Lateral segregation and the formation of lateral domains are well-known phenomena in ternary lipid bilayers composed of an unsaturated (low gel-to-liquid phase transition temperature (Tm)) phospholipid, a saturated (high-Tm) phospholipid, and cholesterol. The formation of lateral domains has been shown to be influenced by differences in phospholipid acyl chain unsaturation and length. Recently, we also showed that differential interactions of cholesterol with low- and high-Tm phospholipids in the bilayer can facilitate phospholipid segregation. Now, we have investigated phospholipid-cholesterol interactions and their role in lateral segregation in ternary bilayers composed of different unsaturated phosphatidylcholines (PCs) with varying acyl chain lengths, N-palmitoyl-D-erythro-sphingomyelin (PSM), and cholesterol. Using deuterium NMR spectroscopy, we determined how PSM was influenced by the acyl chain composition in surrounding PC environments and correlated this with the affinity of cholestatrienol (a fluorescent cholesterol analog) for PSM in the different PC environments. Results from a combination of time-resolved fluorescence measurements of trans-parinaric acid and Förster resonance energy transfer experiments showed that the relative affinity of cholesterol for phospholipids determined the degree to which the sterol promoted domain formation. From Förster resonance energy transfer, deuterium NMR, and differential scanning calorimetry results, it was clear that cholesterol also influenced both the thermostability of the domains and the degree of order in and outside the PSM-rich domains. The results of this study have shown that the affinity of cholesterol for both low-Tm and high-Tm phospholipids and the effects of low- and high-Tm phospholipids on each other influence both lateral structure and domain properties in complex bilayers. We envision that similar effects also contribute to lateral heterogeneity in even more complex biological membranes.

Cholesterol-Induced Conformational Change in the Sphingomyelin Headgroup.

Sphingomyelin (SM) and cholesterol (Cho) are the important lipids for the formation of biologically functional membrane domains, lipid rafts. However, the interaction between Cho and the headgroup of SM remains unclear. In this study, we performed solid-state NMR experiments to reveal the Cho effects on the headgroup conformation using 2H-labeled stearoyl-SM (SSM). Deuterated SSMs at the Cα, Cß, and Cγ positions of a choline moiety were separately prepared and subjected to NMR measurements to determine the quadrupolar splitting of 2H signals in hydrated SSM unitary and SSM/Cho (1:1) bilayers. Using 2H NMR and 13C-31P REDOR data, the conformation and orientation of the choline moiety were deduced and compared with those derived from molecular dynamics simulations. In SSM unitary bilayers, three torsional angles in the phosphocholine moiety, P-O-Cα-Cß, were found to be consecutive +gauche(g)/+g/+g or -g/-g/-g. The orientation and conformation of the SSM headgroup were consistent with the results of our molecular dynamics simulations and the previous results on phosphatidylcholines. The quadrupolar coupling at the α methylene group slightly increased in the presence of Cho, and those at the Cß and Cγ decreased more significantly, thus suggesting that Cho reduced the gauche conformation at the Cα-Cß torsion. The conformational ensemble in the presence of Cho may enhance the so-called umbrella effect of the SSM headgroup, resulting in the stabilization of Cho near the SM molecules by concealing the hydrophobic Cho core from interfacial water. We also examined the effect of the chiral centers at the sphingosine chain to the headgroup conformation by determining the enantiomeric excess between the diastereomeric +g/+g/+g and -g/-g/-g conformers using (S)-Cα-deuterated and (R)-Cα-deuterated SSMs. Their 2H NMR measurements showed that the chiral centers induced the slight diastereomeric excess in the SM headgroup conformation.