Ceramide-1-phosphate transfer protein (CPTP) regulation by phosphoinositides.

Ceramide-1-phosphate transfer proteins (CPTPs) are members of the glycolipid transfer protein (GLTP) superfamily that shuttle ceramide-1-phosphate (C1P) between membranes. CPTPs regulate cellular sphingolipid homeostasis in ways that impact programmed cell death and inflammation. CPTP downregulation specifically alters C1P levels in the plasma and trans-Golgi membranes, stimulating proinflammatory eicosanoid production and autophagy-dependent inflammasome-mediated cytokine release. However, the mechanisms used by CPTP to target the trans-Golgi and plasma membrane are not well understood. Here, we monitored C1P intervesicular transfer using fluorescence energy transfer (FRET) and showed that certain phosphoinositides (phosphatidylinositol 4,5 bisphosphate (PI-(4,5)P2) and phosphatidylinositol 4-phosphate (PI-4P)) increased CPTP transfer activity, whereas others (phosphatidylinositol 3-phosphate (PI-3P) and PI) did not. PIPs that stimulated CPTP did not stimulate GLTP, another superfamily member. Short-chain PI-(4,5)P2, which is soluble and does not remain membrane-embedded, failed to activate CPTP. CPTP stimulation by physiologically relevant PI-(4,5)P2 levels surpassed that of phosphatidylserine (PS), the only known non-PIP stimulator of CPTP, despite PI-(4,5)P2 increasing membrane equilibrium binding affinity less effectively than PS. Functional mapping of mutations that led to altered FRET lipid transfer and assessment of CPTP membrane interaction by surface plasmon resonance indicated that di-arginine motifs located in the α-6 helix and the α3-α4 helix regulatory loop of the membrane-interaction region serve as PI-(4,5)P2 headgroup-specific interaction sites. Haddock modeling revealed specific interactions involving the PI-(4,5)P2 headgroup that left the acyl chains oriented favorably for membrane embedding. We propose that PI-(4,5)P2 interaction sites enhance CPTP activity by serving as preferred membrane targeting/docking sites that favorably orient the protein for function.

Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes.

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short "shelf life". Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long "shelf life" after production (≥6 days) and resilience to freeze-thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.

Structural analyses of 4-phosphate adaptor protein 2 yield mechanistic insights into sphingolipid recognition by the glycolipid transfer protein family.

The glycolipid transfer protein (GLTP) fold defines a superfamily of eukaryotic proteins that selectively transport sphingolipids (SLs) between membranes. However, the mechanisms determining the protein selectivity for specific glycosphingolipids (GSLs) are unclear. Here, we report the crystal structure of the GLTP homology (GLTPH) domain of human 4-phosphate adaptor protein 2 (FAPP2) bound with N-oleoyl-galactosylceramide. Using this domain, FAPP2 transports glucosylceramide from its cis-Golgi synthesis site to the trans-Golgi for conversion into complex GSLs. The FAPP2-GLTPH structure revealed an element, termed the ID loop, that controls specificity in the GLTP family. We found that, in accordance with FAPP2 preference for simple GSLs, the ID loop protrudes from behind the SL headgroup-recognition center to mitigate binding by complex GSLs. Mutational analyses including GLTP and FAPP2 chimeras with swapped ID loops supported the proposed restrictive role of the FAPP2 ID loop in GSL selectivity. Comparative analysis revealed distinctly designed ID loops in each GLTP family member. This analysis also disclosed a conserved H-bond triplet that "clasps" both ID-loop ends together to promote structural autonomy and rigidity. The findings indicated that various ID loops work in concert with conserved recognition centers to create different specificities among family members. We also observed four bulky, conserved hydrophobic residues involved in "sensor-like" interactions with lipid chains in protein hydrophobic pockets and FF motifs in GLTP and FAPP2, well-positioned to provide acyl chain-dependent SL selectivity for the hydrophobic pockets. In summary, our study provides mechanistic insights into sphingolipid recognition by the GLTP fold and uncovers the elements involved in this recognition.

Phosphatidylserine Stimulates Ceramide 1-Phosphate (C1P) Intermembrane Transfer by C1P Transfer Proteins.

Genetic models for studying localized cell suicide that halt the spread of pathogen infection and immune response activation in plants include Arabidopsis accelerated-cell-death 11 mutant (acd11). In this mutant, sphingolipid homeostasis is disrupted via depletion of ACD11, a lipid transfer protein that is specific for ceramide 1-phosphate (C1P) and phyto-C1P. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we investigated the functional regulation of ACD11 and CPTP by anionic phosphoglycerides and found that 1-palmitoyl-2-oleoyl-phosphatidic acid or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (≤15 mol %) in C1P source vesicles depressed C1P intermembrane transfer. By contrast, replacement with 1-palmitoyl-2-oleoyl-phosphatidylserine stimulated C1P transfer by ACD11 and CPTP. Notably, "soluble" phosphatidylserine (dihexanoyl-phosphatidylserine) failed to stimulate C1P transfer. Also, none of the anionic phosphoglycerides affected transfer action by human glycolipid lipid transfer protein (GLTP), which is glycolipid-specific and has few cationic residues near its glycolipid binding site. These findings provide the first evidence for a potential phosphoglyceride headgroup-specific regulatory interaction site(s) existing on the surface of any GLTP-fold and delineate new differences between GLTP superfamily members that are specific for C1P versus glycolipid.

Inhibition of tumor growth by a newly-identified activator for epidermal fatty acid binding protein.

Our previous studies have demonstrated that expression of epidermal fatty acid binding protein (E-FABP) in tumor associated macrophages (TAMs) promotes macrophage anti-tumor activity by enhancing IFNß responses in tumor models. Thus, E-FABP represents a new protective factor in enhancing tumor immune surveillance against tumor development. Herein, we report the compound 5-(benzylamino)-2-(3-methylphenyl)-1,3-oxazole-4-carbonitrile (designated EI-05) as a novel E-FABP activator for inhibition of mammary tumor growth. EI-05 was selected from the ZINC compound library using molecular docking analysis based on the crystal structure of E-FABP. Although EI-05 is unable to bind E-FABP directly, it significantly increases E-FABP expression in macrophages during inflammation. Stimulation of macrophages with EI-05 remarkably enhances lipid droplet formation and IFNß production, which further promotes the anti-tumor activity of macrophages. Importantly, administering EI-05 in vivo significantly inhibits mammary tumor growth in a syngeneic mouse model. Altogether, these results suggest that EI-05 may represent a promising drug candidate for anti-tumor treatment through enhancing E-FABP activity and IFNß responses in macrophages.

Sphingolipid transfer proteins defined by the GLTP-fold.

Glycolipid transfer proteins (GLTPs) originally were identified as small (~24 kDa), soluble, amphitropic proteins that specifically accelerate the intermembrane transfer of glycolipids. GLTPs and related homologs now are known to adopt a unique, helically dominated, two-layer 'sandwich' architecture defined as the GLTP-fold that provides the structural underpinning for the eukaryotic GLTP superfamily. Recent advances now provide exquisite insights into structural features responsible for lipid headgroup selectivity as well as the adaptability of the hydrophobic compartment for accommodating hydrocarbon chains of differing length and unsaturation. A new understanding of the structural versatility and evolutionary premium placed on the GLTP motif has emerged. Human GLTP-motifs have evolved to function not only as glucosylceramide binding/transferring domains for phosphoinositol 4-phosphate adaptor protein-2 during glycosphingolipid biosynthesis but also as selective binding/transfer proteins for ceramide-1-phosphate. The latter, known as ceramide-1-phosphate transfer protein, recently has been shown to form GLTP-fold while critically regulating Group-IV cytoplasmic phospholipase A2 activity and pro-inflammatory eicosanoid production.

Nanoscale packing differences in sphingomyelin and phosphatidylcholine revealed by BODIPY fluorescence in monolayers: physiological implications.

Phosphatidycholines (PC) with two saturated acyl chains (e.g., dipalmitoyl) mimic natural sphingomyelin (SM) by promoting raft formation in model membranes. However, sphingoid-based lipids, such as SM, rather than saturated-chain PCs have been implicated as key components of lipid rafts in biomembranes. These observations raise questions about the physical packing properties of the phase states that can be formed by these two major plasma membrane lipids with identical phosphocholine headgroups. To investigate, we developed a monolayer platform capable of monitoring changes in surface fluorescence by acquiring multiple spectra during measurement of a lipid force-area isotherm. We relied on the concentration-dependent emission changes of 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY)-labeled PC to detect nanoscale alterations in lipid packing and phase state induced by monolayer lateral compression. The BODIPY-PC probe contained an indacene ring with four symmetrically located methyl (Me) substituents to enhance localization to the lipid hydrocarbon region. Surface fluorescence spectra indicated changes in miscibility even when force-area isotherms showed no deviation from ideal mixing behavior in the surface pressure versus cross-sectional molecular area response. We detected slightly better mixing of Me4-BODIPY-8-PC with the fluid-like, liquid expanded phase of 1-palmitoyl-2-oleoyl-PC compared to N-oleoyl-SM. Remarkably, in the gel-like, liquid condensed phase, Me4-BODIPY-8-PC mixed better with N-palmitoyl-SM than dipalmitoyl-PC, suggesting naturally abundant SMs with saturated acyl chains form gel-like lipid phase(s) with enhanced ability to accommodate deeply embedded components compared to dipalmitoyl-PC gel phase. The findings reveal a fundamental difference in the lateral packing properties of SM and PC that occurs even when their acyl chains match.

Arabidopsis accelerated cell death 11, ACD11, is a ceramide-1-phosphate transfer protein and intermediary regulator of phytoceramide levels.

The accelerated cell death 11 (acd11) mutant of Arabidopsis provides a genetic model for studying immune response activation and localized cellular suicide that halt pathogen spread during infection in plants. Here, we elucidate ACD11 structure and function and show that acd11 disruption dramatically alters the in vivo balance of sphingolipid mediators that regulate eukaryotic-programmed cell death. In acd11 mutants, normally low ceramide-1-phosphate (C1P) levels become elevated, but the relatively abundant cell death inducer phytoceramide rises acutely. ACD11 exhibits selective intermembrane transfer of C1P and phyto-C1P. Crystal structures establish C1P binding via a surface-localized, phosphate headgroup recognition center connected to an interior hydrophobic pocket that adaptively ensheaths lipid chains via a cleft-like gating mechanism. Point mutation mapping confirms functional involvement of binding site residues. A π helix (π bulge) near the lipid binding cleft distinguishes apo-ACD11 from other GLTP folds. The global two-layer, α-helically dominated, "sandwich" topology displaying C1P-selective binding identifies ACD11 as the plant prototype of a GLTP fold subfamily.

Non-vesicular trafficking by a ceramide-1-phosphate transfer protein regulates eicosanoids.

Phosphorylated sphingolipids ceramide-1-phosphate (C1P) and sphingosine-1-phosphate (S1P) have emerged as key regulators of cell growth, survival, migration and inflammation. C1P produced by ceramide kinase is an activator of group IVA cytosolic phospholipase A2α (cPLA2α), the rate-limiting releaser of arachidonic acid used for pro-inflammatory eicosanoid production, which contributes to disease pathogenesis in asthma or airway hyper-responsiveness, cancer, atherosclerosis and thrombosis. To modulate eicosanoid action and avoid the damaging effects of chronic inflammation, cells require efficient targeting, trafficking and presentation of C1P to specific cellular sites. Vesicular trafficking is likely but non-vesicular mechanisms for C1P sensing, transfer and presentation remain unexplored. Moreover, the molecular basis for selective recognition and binding among signalling lipids with phosphate headgroups, namely C1P, phosphatidic acid or their lyso-derivatives, remains unclear. Here, a ubiquitously expressed lipid transfer protein, human GLTPD1, named here CPTP, is shown to specifically transfer C1P between membranes. Crystal structures establish C1P binding through a novel surface-localized, phosphate headgroup recognition centre connected to an interior hydrophobic pocket that adaptively expands to ensheath differing-length lipid chains using a cleft-like gating mechanism. The two-layer, α-helically-dominated 'sandwich' topology identifies CPTP as the prototype for a new glycolipid transfer protein fold subfamily. CPTP resides in the cell cytosol but associates with the trans-Golgi network, nucleus and plasma membrane. RNA interference-induced CPTP depletion elevates C1P steady-state levels and alters Golgi cisternae stack morphology. The resulting C1P decrease in plasma membranes and increase in the Golgi complex stimulates cPLA2α release of arachidonic acid, triggering pro-inflammatory eicosanoid generation.

GLTP-fold interaction with planar phosphatidylcholine surfaces is synergistically stimulated by phosphatidic acid and phosphatidylethanolamine.

Among amphitropic proteins, human glycolipid transfer protein (GLTP) forms a structurally-unique fold that translocates on/off membranes to specifically transfer glycolipids. Phosphatidylcholine (PC) bilayers with curvature-induced packing stress stimulate much faster glycolipid intervesicular transfer than nonstressed PC bilayers raising questions about planar cytosol-facing biomembranes being viable sites for GLTP interaction. Herein, GLTP-mediated desorption kinetics of fluorescent glycolipid (tetramethyl-boron dipyrromethene (BODIPY)-label) from lipid monolayers are assessed using a novel microfluidics-based surface balance that monitors lipid lateral packing while simultaneously acquiring surface fluorescence data. At biomembrane-like packing (30-35 mN/m), GLTP uptake of BODIPY-glycolipid from POPC monolayers was nearly nonexistent but could be induced by reducing surface pressure to mirror packing in curvature-stressed bilayers. In contrast, 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE) matrices supported robust BODIPY-glycolipid uptake by GLTP at both high and low surface pressures. Unexpectedly, negatively-charged cytosol-facing lipids, i.e., phosphatidic acid and phosphatidylserine, also supported BODIPY-glycolipid uptake by GLTP at high surface pressure. Remarkably, including both 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (5 mol%) and POPE (15 mol%) in POPC synergistically activated GLTP at high surface pressure. Our study shows that matrix lipid headgroup composition, rather than molecular packing per se, is a key regulator of GLTP-fold function while demonstrating the novel capabilities of the microfluidics-based film balance for investigating protein-membrane interfacial interactions.

Enhanced selectivity for sulfatide by engineered human glycolipid transfer protein.

Human glycolipid transfer protein (GLTP) fold represents a novel structural motif for lipid binding/transfer and reversible membrane translocation. GLTPs transfer glycosphingolipids (GSLs) that are key regulators of cell growth, division, surface adhesion, and neurodevelopment. Herein, we report structure-guided engineering of the lipid binding features of GLTP. New crystal structures of wild-type GLTP and two mutants (D48V and A47D‖D48V), each containing bound N-nervonoyl-sulfatide, reveal the molecular basis for selective anchoring of sulfatide (3-O-sulfo-galactosylceramide) by D48V-GLTP. Directed point mutations of "portal entrance" residues, A47 and D48, reversibly regulate sphingosine access to the hydrophobic pocket via a mechanism that could involve homodimerization. "Door-opening" conformational changes by phenylalanines within the hydrophobic pocket are revealed during lipid encapsulation by new crystal structures of bona fide apo-GLTP and GLTP complexed with N-oleoyl-glucosylceramide. The development of "engineered GLTPs" with enhanced specificity for select GSLs provides a potential new therapeutic approach for targeting GSL-mediated pathologies.

Characterization of the lateral distribution of fluorescent lipid in binary-constituent lipid monolayers by principal component analysis.

Lipid lateral organization in binary-constituent monolayers consisting of fluorescent and nonfluorescent lipids has been investigated by acquiring multiple emission spectra during measurement of each force-area isotherm. The emission spectra reflect BODIPY-labeled lipid surface concentration and lateral mixing with different nonfluorescent lipid species. Using principal component analysis (PCA) each spectrum could be approximated as the linear combination of only two principal vectors. One point on a plane could be associated with each spectrum, where the coordinates of the point are the coefficients of the linear combination. Points belonging to the same lipid constituents and experimental conditions form a curve on the plane, where each point belongs to a different mole fraction. The location and shape of the curve reflects the lateral organization of the fluorescent lipid mixed with a specific nonfluorescent lipid. The method provides massive data compression that preserves and emphasizes key information pertaining to lipid distribution in different lipid monolayer phases. Collectively, the capacity of PCA for handling large spectral data sets, the nanoscale resolution afforded by the fluorescence signal, and the inherent versatility of monolayers for characterization of lipid lateral interactions enable significantly enhanced resolution of lipid lateral organizational changes induced by different lipid compositions.

Glycolipid acquisition by human glycolipid transfer protein dramatically alters intrinsic tryptophan fluorescence: insights into glycolipid binding affinity.

Glycolipid transfer proteins (GLTPs) are small, soluble proteins that selectively accelerate the intermembrane transfer of glycolipids. The GLTP fold is conformationally unique among lipid binding/transfer proteins and serves as the prototype and founding member of the new GLTP superfamily. In the present study, changes in human GLTP tryptophan fluorescence, induced by membrane vesicles containing glycolipid, are shown to reflect glycolipid binding when vesicle concentrations are low. Characterization of the glycolipid-induced "signature response," i.e. approximately 40% decrease in Trp intensity and approximately 12-nm blue shift in emission wavelength maximum, involved various modes of glycolipid presentation, i.e. microinjection/dilution of lipid-ethanol solutions or phosphatidylcholine vesicles, prepared by sonication or extrusion and containing embedded glycolipids. High resolution x-ray structures of apo- and holo-GLTP indicate that major conformational alterations are not responsible for the glycolipid-induced GLTP signature response. Instead, glycolipid binding alters the local environment of Trp-96, which accounts for approximately 70% of total emission intensity of three Trp residues in GLTP and provides a stacking platform that aids formation of a hydrogen bond network with the ceramide-linked sugar of the glycolipid headgroup. The changes in Trp signal were used to quantitatively assess human GLTP binding affinity for various lipids including glycolipids containing different sugar headgroups and homogenous acyl chains. The presence of the glycolipid acyl chain and at least one sugar were essential for achieving a low-to-submicromolar dissociation constant that was only slightly altered by increased sugar headgroup complexity.

Side chain oxygenated cholesterol regulates cellular cholesterol homeostasis through direct sterol-membrane interactions.

Side chain oxysterols exert cholesterol homeostatic effects by suppression of sterol regulatory element-binding protein maturation and promoting degradation of hydroxymethylglutaryl-CoA reductase. To examine whether oxysterol-membrane interactions contribute to the regulation of cellular cholesterol homeostasis, we synthesized the enantiomer of 25-hydroxycholesterol. Using this unique oxysterol probe, we provide evidence that oxysterol regulation of cholesterol homeostatic responses is not mediated by enantiospecific oxysterol-protein interactions. We show that side chain oxysterols, but not steroid ring-modified oxysterols, exhibit membrane expansion behavior in phospholipid monolayers and bilayers in vitro. This behavior is non-enantiospecific and is abrogated by increasing the saturation of phospholipid acyl chain constituents. Moreover, we extend these findings into cultured cells by showing that exposure to saturated fatty acids at concentrations that lead to endoplasmic reticulum membrane phospholipid remodeling inhibits oxysterol activity. These studies implicate oxysterol-membrane interactions in acute regulation of sterol homeostatic responses and provide new insights into the mechanism through which oxysterols regulate cellular cholesterol balance.

Resveratrol directly targets COX-2 to inhibit carcinogenesis.

Targeted molecular cancer therapies can potentially deliver treatment directly to a specific protein or gene to optimize efficacy and reduce adverse side effects often associated with traditional chemotherapy. Key oncoprotein and oncogene targets are rapidly being identified based on their expression, pathogenesis and clinical outcome. One such protein target is cyclooxygenase-2 (COX-2), which is highly expressed in various cancers. Research findings suggest that resveratrol (RSVL; 3,5,4'-trihydroxy-trans-stilbene) demonstrates nonselective COX-2 inhibition. We report herein that RSVL directly binds with COX-2 and this binding is absolutely required for RSVL's inhibition of the ability of human colon adenocarcinoma HT-29 cells to form colonies in soft agar. Binding of COX-2 with RSVL was compared with two RSVL analogues, 3,3',4',5',5-pentahydroxy-trans-stilbene (RSVL-2) or 3,4',5-trimethoxy-trans-stilbene (RSVL-3). The results indicated that COX-2 binds with RSVL-2 more strongly than with RSVL, but does not bind with RSVL-3. RSVL or RSVL-2, but not RSVL-3, inhibited COX-2-mediated PGE(2) production in vitro and ex vivo. HT-29 human colon adenocarcinoma cells express high levels of COX-2 and either RSVL or RSVL-2, but not RSVL-3, suppressed anchorage independent growth of these cells in soft agar. RSVL or RSVL-2 (not RSVL-3) suppressed growth of COX-2(+/+) cells by 60% or 80%, respectively. Notably, cells deficient in COX-2 were unresponsive to RSVL or RSVL-2. These data suggest that the anticancer effects of RSVL or RSLV-2 might be mediated directly through COX-2.

New BODIPY lipid probes for fluorescence studies of membranes.

Many fluorescent lipid probes tend to loop back to the membrane interface when attached to a lipid acyl chain rather than embedding deeply into the bilayer. To achieve maximum embedding of BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) fluorophore into the bilayer apolar region, a series of sn-2 acyl-labeled phosphatidylcholines was synthesized bearing 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me(4)-BODIPY-8) at the end of C(3)-, C(5)-, C(7)-, or C(9)-acyl. A strategy was used of symmetrically dispersing the methyl groups at BODIPY ring positions 1, 3, 5, and 7 to decrease fluorophore polarity. Iodide quenching of the phosphatidylcholine probes in bilayer vesicles confirmed that the Me(4)-BODIPY-8 fluorophore was embedded in the bilayer. Parallax analysis of Me(4)-BODIPY-8 fluorescence quenching by phosphatidylcholines containing iodide at different positions along the sn-2 acyl chain indicated that the penetration depth of Me(4)-BODIPY-8 into the bilayer was determined by the length of the linking acyl chain. Evaluation using monolayers showed minimal perturbation of <10 mol% probe in fluid-phase and cholesterol-enriched phosphatidylcholine. Spectral characterization in monolayers and bilayers confirmed the retention of many features of other BODIPY derivatives (i.e., absorption and emission wavelength maxima near 498 nm and approximately 506-515 nm) but also showed the absence of the 620-630 nm peak associated with BODIPY dimer fluorescence and the presence of a 570 nm emission shoulder at high Me(4)-BODIPY-8 surface concentrations. We conclude that the new probes should have versatile utility in membrane studies, especially when precise location of the reporter group is needed.

Lactosylceramide: lateral interactions with cholesterol.

Lactosylceramide (LacCer) is a key intermediate in glycosphingolipid metabolism and is highly enriched in detergent-resistant biomembrane fractions associated with microdomains, i.e., rafts and caveolae. Here, the lateral interactions of cholesterol with LacCers containing various homogeneous saturated (8:0, 16:0, 18:0, 24:0) or monounsaturated acyl chains (18:1, 24:1) have been characterized using a Langmuir-type film balance. Cholesterol-induced changes in lateral packing were assessed by measuring changes in average molecular area, i.e., area condensations, and in lateral elasticity, i.e., surface compressional moduli (C S(-1)) with emphasis on high surface pressures (> or = 30 mN/m) that mimic biomembrane conditions. Cholesterol most dramatically affected the lateral packing elasticity of LacCers with long saturated acyl chains at sterol mole fractions > or = 0.3, consistent with liquid-ordered (LO) phase formation. The lateral elasticity within the LacCer-cholesterol LO-phase was much lower than that observed within pure LacCer condensed, i.e., gel, phase. The magnitude of the cholesterol-induced reduction in lateral elasticity was strongly mitigated by cis monounsaturation in the LacCer acyl chain. At identical high sterol mole fractions, higher lateral elasticity was observed within LacCer-cholesterol mixtures compared with galactosylceramide-cholesterol and sphingomyelin-cholesterol mixtures. The results show how changes to sphingolipid headgroup and acyl chain structure contribute to the modulation of lateral packing elasticity in sphingolipid-cholesterol LO-phases.

Small angle X-ray scattering (SAXS) and differential scanning calorimetry (DSC) studies of amide phospholipids.

Varying chemically the structure of phospholipids in the region between hydrophobic and hydrophilic segments is expected to have a strong influence on the interaction with water and the phase behavior. This is studied in this work with the motivation to investigate these lipids as potential inhibitors of phospholipase A2. Thus the amide phospholipids L-ether-amide-PC (1-O-hexadecyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine), L-ester-amide-PC (1-palmitoyl-2-N-palmitoyl-2-amino-2-deoxy-sn-glycero-3-phosphocholine) and L-ether-amide-PE (1-O-hexadecyl-2-N-palmitoyl-2-deoxy-sn-glycero-3-phosphoethanolamine) have been synthesized and characterized. The phase behavior and thermal transitions in buffer dispersions are examined by a combination of high-sensitivity differential scanning calorimetry (DSC) and small angle X-ray scattering (SAXS) experiments between 10 and 80 degrees C at pH 8.9. The onset temperatures determined from DSC measurements agree well with the starting temperatures of changes in the repeat distance obtained by SAXS measurements. The phases observed are lamellar both below and above the main phase transition. The phase transition temperatures and enthalpies depend strongly on the substitutions in sn-1 position and head group structure. The lamellar repeat distance in gel and liquid-crystalline phases increases with increasing temperature for L-ester-amide-PC and L-ether-amide-PC, whereas the temperature dependence is opposite for the L-ether-amide-PE. The observed behavior is discussed and compared with that of DPPC and DPPE, indicating the strong dependence of hydration and phase behavior on head group structure.

Direct observations of the cleavage reaction of an L-DPPC monolayer catalyzed by phospholipase A2 and inhibited by an indole inhibitor at the air/water interface.

The enzymatic hydrolysis of an L-dipalmitoylphosphatidylcholine (L-DPPC) monolayer at the air/water interface, catalyzed by phospholipase A(2) (PLA(2)), serves as a model for biospecific interfacial reactions. The cleavage of L-DPPC was investigated by Brewster angle microscopy. Different types of domain defects were observed to form in the coexisting liquid expanded and liquid condensed phases during the hydrolysis reaction. The adsorption of the enzyme was quantitatively recorded as the increase of the surface pressure over a fixed molecular area with time. In the case of L-DPPC, the surface pressure first increases and then starts to decrease, which indicates that a soluble product (lysolipid) is produced during the catalytic cleavage reaction. The increase and decrease of the surface pressure, which corresponds to the change of shape and number density of domains, indicated the occurrence of the following processes: adsorption of PLA(2), cleavage reaction, and rearrangement of substrate and product molecules at the interface. Addition of a PLA(2) inhibitor to the lipid monolayer leads to a fast surface pressure increase after enzyme injection. The surface pressure reaches a maximum value and then does not change for a long time. During this period, no change in the domain shape and number density was observed, which indicates that the enzyme is inhibited for a certain period of time. The experimental results provide the possibility of a direct way to prove inhibitor activity.