N-acyl Chain Research Articles

Elucidation of Gut Microbiota-Associated Lipids Using LC-MS/MS and 16S rRNA Sequence Analyses.

SummaryHost-microbiota interactions create a unique metabolic milieu that modulates intestinal environments. Integration of 16S ribosomal RNA (rRNA) sequences and mass spectrometry (MS)-based lipidomics has a great potential to reveal the relationship between bacterial composition and the complex metabolic network in the gut. In this study, we conducted untargeted lipidomics followed by a feature-based molecular MS/MS spectral networking to characterize gut bacteria-dependent lipid subclasses in mice. An estimated 24.8% of lipid molecules in feces were microbiota-dependent, as judged by > 10-fold decrease in antibiotic-treated mice. Among these, there was a series of unique and microbiota-related lipid structures, including acyl alpha-hydroxyl fatty acid (AAHFA) that was newly identified in this study. Based on the integrated analysis of 985 lipid profiles and 16S rRNA sequence data providing 2,494 operational taxonomic units, we could successfully predict the bacterial species responsible for the biosynthesis of these unique lipids, including AAHFA.

Resolving Modifications on Sphingoid Base and N-Acyl Chain of Sphingomyelin Lipids in Complex Lipid Extracts.

Sphingomyelins (SMs) are an essential class of lipids widely existing in different organisms. The sphingoid base and N-acyl chain are two building blocks which can undergo different types of modifications during lipogenesis, including desaturation, hydroxylation, and methyl branching. Current lipidomic analysis methods cannot provide detailed information on these structural motifs. Herein, we developed a tandem mass spectrometric method by harnessing radical-directed dissociation (RDD) from collision-induced dissociation (CID) of the bicarbonate anion adduct of SM ([M + HCO3]-). A major RDD channel produced a high-abundance fragment carrying the intact N-acyl chain, termed as "N-acyl fragment", allowing the assignment of the sphingoid base/N-acyl composition and relative quantitation of compositional isomers of SM at high sensitivity. RDD also produced intrachain fragments in lower abundances, which helped localization of methyl branching and hydroxylation in SM. The acetone Paternò-Büchi (PB) reaction was found to be capable of derivatizing the Δ4 carbon-carbon double bond (C═C) in sphingosine (SPH) base and producing C═C diagnostic ions upon CID, albeit at much lower efficiencies than those of the isolated C═C in alkyl chains. A liquid chromatography-mass spectrometry workflow was developed by incorporating MS2 CID of SM via [M + HCO3]- and PB-MS2 CID. The capability of profiling SM with detailed structural information was demonstrated by analyzing complex lipid extracts from porcine brain and Caenorhabditis elegans. These results provided visualization of the sphingoid base/N-acyl compositional isomers of SM lipids and revealed large structural diversity from each sample. These included identification of the sphingadiene base [d18:1(Δ4,14)], C═C location isomers in N-acyls, C-2 hydroxylation of N-acyls, and iso-methyl branched SPH base.

Different rates of flux through the biosynthetic pathway for long-chain versus very-long-chain sphingolipids

The backbone of all sphingolipids (SLs) is a sphingoid long-chain base (LCB) to which a fatty acid is N-acylated. Considerable variability exists in the chain length and degree of saturation of both of these hydrophobic chains, and recent work has implicated ceramides with different LCBs and N-acyl chains in distinct biological processes; moreover, they may play different roles in disease states and possibly even act as prognostic markers. We now demonstrate that the half-life, or turnover rate, of ceramides containing diverse N-acyl chains is different. By means of a pulse-labeling protocol using stable-isotope, deuterated free fatty acids, and following their incorporation into ceramide and downstream SLs, we show that very-long-chain (VLC) ceramides containing C24:0 or C24:1 fatty acids turn over much more rapidly than long-chain (LC) ceramides containing C16:0 or C18:0 fatty acids due to the more rapid metabolism of the former into VLC sphingomyelin and VLC hexosylceramide. In contrast, d16:1 and d18:1 ceramides show similar rates of turnover, indicating that the length of the sphingoid LCB does not influence the flux of ceramides through the biosynthetic pathway. Together, these data demonstrate that the N-acyl chain length of SLs may not only affect membrane biophysical properties but also influence the rate of metabolism of SLs so as to regulate their levels and perhaps their biological functions.

Bacterial lyso-form lipoproteins are synthesized via an intramolecular acyl chain migration

All bacterial lipoproteins share a variably acylated N-terminal cysteine residue. Gram-negative bacterial lipoproteins are triacylated with a thioether-linked diacylglycerol moiety and an N-acyl chain. The latter is transferred from a membrane phospholipid donor to the α-amino terminus by the enzyme lipoprotein N-acyltransferase (Lnt), using an active-site cysteine thioester covalent intermediate. Many Gram-positive Firmicutes also have N-acylated lipoproteins, but the enzymes catalyzing N-acylation remain uncharacterized. The integral membrane protein Lit (lipoprotein intramolecular transacylase) from the opportunistic nosocomial pathogen Enterococcus faecalis synthesizes a specific lysoform lipoprotein (N-acyl S-monoacylglycerol) chemotype by an unknown mechanism that helps this bacterium evade immune recognition by the Toll-like receptor 2 family complex. Here, we used a deuterium-labeled lipoprotein substrate with reconstituted Lit to investigate intramolecular acyl chain transfer. We observed that Lit transfers the sn-2 ester-linked lipid from the diacylglycerol moiety to the α-amino terminus without forming a covalent thioester intermediate. Utilizing Mut-Seq to analyze an alanine scan library of Lit alleles, we identified two stretches of functionally important amino acid residues containing two conserved histidines. Topology maps based on reporter fusion assays and cysteine accessibility placed both histidines in the extracellular half of the cytoplasmic membrane. We propose a general acid base-promoted catalytic mechanism, invoking direct nucleophilic attack by the substrate α-amino group on the sn-2 ester to form a cyclic tetrahedral intermediate that then collapses to produce lyso-lipoprotein. Lit is a unique example of an intramolecular transacylase differentiated from that catalyzed by Lnt, and provides insight into the heterogeneity of bacterial lipoprotein biosynthetic systems.

Synthesis and evaluation of sulfonyl piperazine LpxH inhibitors

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is essential in lipid A biosynthesis and has emerged as a promising target for the development of novel antibiotics against multidrug-resistant Gram-negative pathogens. Recently, we reported the crystal structure of Klebsiella pneumoniae LpxH in complex with 1 (AZ1), a sulfonyl piperazine LpxH inhibitor. The analysis of the LpxH-AZ1 co-crystal structure and ligand dynamics led to the design of 2 (JH-LPH-28) and 3 (JH-LPH-33) with enhanced LpxH inhibition. In order to harness our recent findings, we prepared and evaluated a series of sulfonyl piperazine analogs with modifications in the phenyl and N-acetyl groups of 3. Herein, we describe the synthesis and structure–activity relationship of sulfonyl piperazine LpxH inhibitors. We also report the structural analysis of an extended N-acyl chain analog 27b (JH-LPH-41) in complex with K. pneumoniae LpxH, revealing that 27b reaches an untapped polar pocket near the di-manganese cluster in the active site of K. pneumoniae LpxH. We expect that our findings will provide designing principles for new LpxH inhibitors and establish important frameworks for the future development of antibiotics against multidrug-resistant Gram-negative pathogens.

Targeted analysis of ceramides and cerebrosides in yellow lupin seeds by reversed-phase liquid chromatography coupled to electrospray ionization and multistage mass spectrometry

Ceramides (Cer) and cerebrosides are important sphingolipids (SL) involved in many biological processes. Herein, the SL content of yellow lupin seeds (Lupinus luteus) was determined by liquid–liquid extraction, mild alkaline hydrolysis (1 h at 37 °C) and reversed-phase liquid chromatography with negative electrospray ionization coupled to either an orbital-trap (Fourier-transformed, FT) or linear ion-trap (LIT) mass spectrometry (RPLC-ESI/MS). The chemical identity of SL including the sugar residues and sphingoid backbone (SB) was inferred by collision-induced dissociation multiple-stage mass spectrometry (MSn, n = 2,3). Up to 52 Cer and 47 cerebrosides were successfully recognized and quantified in sample extracts of L. luteus seeds also counting isobaric species. As reported for other vegetables, a hydroxylated SB was observed whereby the N-acyl chains showed a high occurrence of very-long-chain moieties; phytosphingosine and 4-hydroxy-8-sphingenine were the predominant SB paired mainly with oleic acid (C18:1), hydroxylated behenic acid (C22:0;1) and hydroxylated lignoceric acid (C24:0;1).

Dynamics of sphingolipids and the serine palmitoyltransferase complex in rat oligodendrocytes during myelination

Myelin is a unique lipid-rich membrane structure that accelerates neurotransmission and supports neuronal function. Sphingolipids are critical myelin components. Yet sphingolipid content and synthesis have not been well characterized in oligodendrocytes, the myelin-producing cells of the CNS. Here, using quantitative real-time PCR, LC-MS/MS-based lipid analysis, and biochemical assays, we examined sphingolipid synthesis during the peak period of myelination in the postnatal rat brain. Importantly, we characterized sphingolipid production in isolated oligodendrocytes. We analyzed sphingolipid distribution and levels of critical enzymes and regulators in the sphingolipid biosynthetic pathway, with focus on the serine palmitoyltransferase (SPT) complex, the rate-limiting step in this pathway. During myelination, levels of the major SPT subunits increased and oligodendrocyte maturation was accompanied by extensive alterations in the composition of the SPT complex. These included changes in the relative levels of two alternative catalytic subunits, SPTLC2 and -3, in the relative levels of isoforms of the small subunits, ssSPTa and -b, and in the isoform distribution of the SPT regulators, the ORMDLs. Myelination progression was accompanied by distinct changes in both the nature of the sphingoid backbone and the N-acyl chains incorporated into sphingolipids. We conclude that the distribution of these changes among sphingolipid family members is indicative of a selective channeling of the ceramide backbone toward specific downstream metabolic pathways during myelination. Our findings provide insights into myelin production in oligodendrocytes and suggest how dysregulation of the biosynthesis of this highly specialized membrane could contribute to demyelinating diseases.

Esfingolipidoak eta kolesterola, egitura lipidoak baino gehiago

Esfingolipidoek, beren buru polarrean dauzkaten talde polarren bidez, geruza bikoitzean existitzen diren beste lipido batzuekin sare konplexu bat eratzeko ahalmena daukate. Sare konplexu honen eraketa, -OH eta -NH- taldeekin hidrogeno-zubiak eratuz lortzen dute hain zuzen ere. Azken urteotan, lipido-urmaelaren hipotesiaren barruan, esfingomielinaren eta kolesterolaren arteko elkarrekintza espezifikoa asko ikertzen ari da, eta ikerketa honen emaitza da, estres-egoeren aurrean, zelulen mintzetan, esfingomielina-zeramida konplexuak sortzen direla proposatu izana. Gainera, kolesterol-zeramida elkarrekintzak ere gertatzen direla erakusten duten artikuluak gero eta ugariagoak dira. Existitzen diren esfingolipido moten inguruan jartzen ari den arretak gaiaren inguruko jakinduria handitzera garamatza. Izan ere, azkenaldian, deoxiesfingolipidoak bezalako esfingolipido aldakorren eta N-azil katearen luzerak edota lipidoen asegabetze-mailek daukaten efektuaren inguruko ikerketa gehiago egiten ari dira. Aldakortasun horiek garrantzitsuak izan daitezke zeramidarekin lotutako droga terapeutiko edota gaixotasun ezberdinen tratamenduak garatzeko; horien artetik garrantzitsuenak minbiziarekin lotutako terapiak dira.

New fluorogenic probes for neutral and alkaline ceramidases

New fluorogenic ceramidase substrates derived from the N-acyl modification of our previously reported probes (RBM14) are reported. While none of the new probes were superior to the known RBM14C12 as acid ceramidase substrates, the corresponding nervonic acid amide (RBM14C24:1) is an efficient and selective substrate for the recombinant human neutral ceramidase, both in cell lysates and in intact cells. A second generation of substrates, incorporating the natural 2-(N-acylamino)-1,3-diol-4-ene framework (compounds RBM15) is also reported. Among them, the corresponding fatty acyl amides with an unsaturated N-acyl chain can be used as substrates to determine alkaline ceramidase (ACER)1 and ACER2 activities. In particular, compound RBM15C18:1 has emerged as the best fluorogenic probe reported so far to measure ACER1 and ACER2 activities in a 96-well plate format.

Fatty Acid Compositions of Ceramides and Sphingomyelins in Mammalian Tissues and Cultured Cells

Sphingolipids consist of a sphingoid base N-linked to a fatty acyl chain. Among them, sphingomyelins (SM) are major components of mammalian cells, while ceramide (Cer) plays an important role as a lipid second messenger. We have performed a quantitative lipidomic study of Cer and SM species in different mammalian tissues (adipose tissue, liver, brain and blood serum of human, mice, rat and dog), as well as in cell cultures of mammalian origin (primary hepatocytes, immortalized MDCK cells, mice melanoma b16 cells, and mice primary CD4+ T lymphocytes) using an ultra-high performance liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC-ToF-MS)-based platform. The data have been compared with published, in general semi-quantitative, results from 20 other samples, with good agreement. The sphingoid base was predominantly d18-1 sphingosine (2-amino-4-octadecene-1,3-diol) in all cases. The fatty acid composition of SM was clearly different from that of Cer. In virtually all samples the most abundant Cer species were those containing C24:0 and C24:1 in their N-acyl chains, while the main species contained in SM was C16:0. Brain was the most divergent tissue, in which Cer and SM C18:0 were very abundant.

The fatty acids of sphingomyelins and ceramides in mammalian tissues and cultured cells: Biophysical and physiological implications.

Sphingolipids consist of a sphingoid base N-linked to a fatty acyl chain. Among them, sphingomyelins (SM) are major components of mammalian cells, while ceramide (Cer) plays an important role as a lipid second messenger. We have performed a quantitative lipidomic study of Cer and SM species in different mammalian tissues (adipose tissue, liver, brain and blood serum of human, mice, rat and dog), as well as in cell cultures of mammalian origin (primary hepatocytes, immortalized MDCK cells, mice melanoma b16 cells, and mice primary CD4 + T lymphocytes) using an ultra-high performance liquid chromatography coupled to time-of-flight mass spectrometry (UHPLC-ToF-MS)-based platform. The data have been compared with published, in general semi-quantitative, results from 20 other samples, with good agreement. The sphingoid base was predominantly d18-1 sphingosine (2-amino-4-octadecene-1,3-diol) in all cases. The fatty acid composition of SM was clearly different from that of Cer. In virtually all samples the most abundant Cer species were those containing C24:0 and C24:1 in their N-acyl chains, while the main species contained in SM was C16:0. Brain was the most divergent tissue, in which Cer and SM C18:0 were very abundant.

Novel Polyphenols That Inhibit Colon Cancer Cell Growth Affecting Cancer Cell Metabolism

New series of polyphenols with a hydrophilic galloyl-based head and a hydrophobic N-acyl tail, linked through a serinol moiety, have been synthesized and tested against colon cancer cell growth. Our structure activity relationship studies revealed that galloyl moieties are essential for growth inhibition. Moreover, the length of the N-acyl chain is crucial for the activity. Introduction of a (Z) double bond in the acyl chain increased the anticancer properties. Our findings demonstrate that 16, the most potent compound within this series, has inhibitory effects on colon cancer cell growth and metabolism (glycolysis and mitochondrial respiration) at the same time that it activates 5'AMP-activated kinase (AMPK) and induces apoptotic cell death. Based on these results, we propose that 16 might reprogram colon cancer cell metabolism through AMPK activation. This might lead to alterations on cancer cell bioenergy compromising cancer cell viability. Importantly, these antiproliferative and proapoptotic effects are selective for cancer cells. Accordingly, these results indicate that 16, with an unsaturated C18 chain, might be a useful prototype for the development of novel colon cancer cell growth inhibitors affecting cell metabolism.

Eleven residues determine the acyl chain specificity of ceramide synthases

Lipids display large structural complexity, with ∼40,000 different lipids identified to date, ∼4000 of which are sphingolipids. A critical factor determining the biological activities of the sphingolipid, ceramide, and of more complex sphingolipids is their N-acyl chain length, which in mammals is determined by a family of six ceramide synthases (CerS). Little information is available about the CerS regions that determine specificity toward different acyl-CoA substrates. We previously demonstrated that substrate specificity resides in a region of ∼150 residues in the Tram-Lag-CLN8 domain. Using site-directed mutagenesis and biochemical analyses, we now narrow specificity down to an 11-residue sequence in a loop located between the last two putative transmembrane domains (TMDs) of the CerS. The specificity of a chimeric protein, CerS5(299-309→CerS2), based on the backbone of CerS5 (which generates C16-ceramide), but containing 11 residues from CerS2 (which generates C22-C24-ceramides), was altered such that it generated C22-C24 and other ceramides. Moreover, a chimeric protein, CerS4(291-301→CerS2), based on CerS4 (which normally generates C18-C22 ceramides) displayed significant activity toward C24:1-CoA. Additional data supported the notion that substitutions of these 11 residues alter the specificities of the CerS toward their cognate acyl-CoAs. Our findings may suggest that this short loop may restrict adjacent TMDs, leading to a more open conformation in the membrane, and that the CerS acting on shorter acyl-CoAs may have a longer, more flexible loop, permitting TMD flexibility. In summary, we have identified an 11-residue region that determines the acyl-CoA specificity of CerS.

Glycosphingolipidomics of donkey milk by hydrophilic interaction liquid chromatography coupled to ESI and multistage MS.

Hydrophilic interaction liquid chromatography coupled to negative-ion electrospray linear ion-trap multiple-stage MS (HILIC-ESI-MSn , n=2,3) was used to characterize polar lipids occurring in donkey milk. Besides the detection of abundant phospholipids, the structural characterization and content evaluation of minor glycosphingolipids (GSLs) were assessed. We report an unprecedented characterization of 11 hexosyl-ceramides (HexCer), 10 Hex2 Cer, and 4 Hex3 Cer. CID-MS/MS spectra in negative ion mode mainly afford information on the monosaccharide number and ceramide constitution (i.e., N-acyl residue and long-chain base), whereas MS/MS/MS spectra on the ceramide anions allow to recognize for each GSL the sphingoid base. The occurrence of sphingosine (S), sphinganine (DS), and phytosphingosine (P) was inferred from the fragmentation patterns. The milk samples exhibit a relatively high number of phytosphingosine substitutes, perhaps because of the feeding of donkeys, mainly based on pasture grass. However, the incidence of hydroxylated species on the α-carbon of the acyl chain was also revealed. The fatty acid composition of N-acyl chains showed high values of long-chain saturated fatty acids such as 20:0, 22:0, 23:0, and 24:0. An average content of GSL is also provided and three representative mono-, di-, and tri-HexCer in donkey milk are the following: HexCer 18:0/24:1 phytosphingosine nonhydroxylated [PN] at m/z 862.6 as chloride adduct [M+Cl]- , and content 225.9±2.8μg 100mL-1 ; Hex2 Cer 18:0/16:0 sphinganine nonhydroxylated [DSN] at m/z 862.7 as deprotonated adduct [M-H]- , and content 70.8±1.4μg 100mL-1 ; and Hex3 Cer 18:1/24:1 [SN] at m/z 1132.8 as [M-H]- , and content 38.5±0.7μg 100mL-1 .

Lipotoxic very-long-chain ceramides cause mitochondrial dysfunction, oxidative stress, and cell death in cardiomyocytes.

Accumulating data support a role for bioactive lipids as mediators of lipotixicity in cardiomyocytes. One class of these, the ceramides, constitutes a family of molecules that differ in structure and are synthesized by distinct enzymes, ceramide synthase (CerS)1-CerS6. Data support that specific ceramides and the enzymes that catalyze their formation play distinct roles in cell function. In a mouse model of diabetic cardiomyopathy, sphingolipid profiling revealed increases in not only the CerS5-derived ceramides but also in very long chain (VLC) ceramides derived from CerS2. Overexpression of CerS2 elevated VLC ceramides caused insulin resistance, oxidative stress, mitochondrial dysfunction, and mitophagy. Palmitate induced CerS2 and oxidative stress, mitophagy, and apoptosis, which were prevented by depletion of CerS2. Neither overexpression nor knockdown of CerS5 had any function in these processes, suggesting a chain-length dependent impact of ceramides on mitochondrial function. This concept was also supported by the observation that synthetic mitochondria-targeted ceramides led to mitophagy in a manner proportional to N-acyl chain length. Finally, blocking mitophagy exacerbated cell death. Taken together, our results support a model by which CerS2 and VLC ceramides have a distinct role in lipotoxicity, leading to mitochondrial damage, which results in subsequent adaptive mitophagy. Our data reveal a novel lipotoxic pathway through CerS2.-Law, B. A., Liao, X., Moore, K. S., Southard, A., Roddy, P., Ji, R., Szulc, Z., Bielawska, A., Schulze, P. C., Cowart, L. A. Lipotoxic very-long-chain ceramides cause mitochondrial dysfunction, oxidative stress, and cell death in cardiomyocytes.

Molecular structure, supramolecular organization and thermotropic phase behavior of N-acylglycine alkyl esters with matched acyl and alkyl chains

N-Acylglycines (NAGs), the endogenous single-tailed lipids present in rat brain and other mammalian tissues, play significant roles in cell physiology and exhibit interesting pharmacological properties. In the present study, a homologous series of N-acylglycine alkyl esters (NAGEs) with matched chains were synthesized and characterized. Results of differential scanning calorimetric studies revealed that all NAGEs exhibit a single sharp phase transition and that the transition enthalpy and entropy show a linear dependence on the N-acyl and ester alkyl chain length. The structure of N-myristoylglycine myristyl ester (NMGME), solved by single-crystal X-ray diffraction, showed that the molecule adopts a linear geometry and revealed that the structure of N-myristoyl glycyl moiety in NMGME is identical to that in N-myristoylglycine. The molecules are packed in layers with the polar functional groups of the ester and amide functionalities located at the center of the layer. The crystal packing is stabilized by NH⋯O hydrogen bonds between the amide CO and NH groups of adjacent molecules as well as by CH⋯O hydrogen bonds between the amide carbonyl and the methylene CH adjacent to the ester carbonyl of neighboring molecules as well as between ester carbonyl and methylene group of the glycine moiety of adjacent molecules. Powder X-ray diffraction studies showed a linear dependence of the d-spacings on the acyl chain length, suggesting that all NAGEs adopt a structure similar to the packing exhibited in the crystal lattice of NMGME.

The many faces (and phases) of ceramide and sphingomyelin II - binary mixtures.

A rather widespread idea on the functional importance of sphingolipids in cell membranes refers to the occurrence of ordered domains enriched in sphingomyelin and ceramide that are largely assumed to exist irrespective of the type of N-acyl chain in the sphingolipid. Ceramides and sphingomyelins are the simplest kind of two-chained sphingolipids and show a variety of species, depending on the fatty acyl chain length, hydroxylation, and unsaturation. Abundant evidences have shown that variations of the N-acyl chain length in ceramides and sphingomyelins markedly affect their phase state, interfacial elasticity, surface topography, electrostatics, and miscibility, and that even the usually conceived "condensed" sphingolipids and many of their mixtures may exhibit liquid-like expanded states. Their lateral miscibility properties are subtlety regulated by those chemical differences. Even between ceramides with different acyl chain length, their partial miscibility is responsible for a rich two-dimensional structural variety that impacts on the membrane properties at the mesoscale level. In this review, we will discuss the miscibility properties of ceramide, sphingomyelin, and glycosphingolipids that differ in their N-acyl or oligosaccharide chains. This work is a second part that accompanies a previous overview of the properties of membranes formed by pure ceramides or sphingomyelins, which is also included in this Special Issue.

The many faces (and phases) of ceramide and sphingomyelin I - single lipids.

Ceramides, the simplest kind of two-chained sphingolipids, contain a single hydroxyl group in position 1 of the sphingoid base. Sphingomyelins further contain a phosphocholine group at the OH of position 1 of ceramide. Ceramides and sphingomyelins show a variety of species depending on the fatty acyl chain length, hydroxylation, and unsaturation. Because of the relatively high transition temperature of sphingomyelin compared to lecithin and, particularly, of ceramides with 16:0-18:0 saturated chains, a widespread idea on their functional importance refers to formation of rather solid domains enriched in sphingomyelin and ceramide. Frequently, and especially in the cell biology field, these are generally (and erroneously) assumed to occur irrespective on the type of N-acyl chain in these lipids. This is because most studies indicating such condensed ordered domains employed sphingolipids with acyl chains with 16 carbons while scarce attention has been focused on the influence of the N-acyl chain on their surface properties. However, abundant evidence has shown that variations of the N-acyl chain length in ceramides and sphingomyelins markedly affect their phase state, interfacial elasticity, surface topography, electrostatics and miscibility and that, even the usually conceived "condensed" sphingolipids and many of their mixtures, may exhibit liquid-like expanded states. This review is a summarized overview of our work and of related others on some facts regarding membranes composed of single molecular species of ceramide and sphingomyelin. A second part is dedicated to discuss the miscibility properties between species of sphingolipids that differ in N-acyl and oligosaccharide chains.

Sphingolipids in marine microalgae: Development and application of a mass spectrometric method for global structural characterization of ceramides and glycosphingolipids in three major phyla

Sphingolipid compositions are crucial for the structural and physiological properties of microalgae membranes. In the present study, we developed a quadrupole time-of-flight (Q-TOF) mass spectrometric method based on MSE data collection for the identification of sphingolipids with high efficiency, selectivity, sensitivity and mass accuracy and applied this method for precise structural identification and quantitative profiling of ceramides and glycosphingolipids in total lipid extracts from 17 strains of microalgae, including 11 strains of diatom, 3 strains of dinoflagellate and 3 strains of haptophyta. Using this method, four species of sphingolipids including 27 ceramides, 13 monosaccharide ceramides, 18 disaccharide ceramides and 18 trisaccharide ceramides were identified. The compositions of sphingolipid-included glycosyl moieties, long chain bases and N-acyl chains showed a significant difference among different microalgae categories. Some long chain bases including d19:2, d19:3 and d19:4, glycosyl moieties including disaccharide and trisaccharide, and N-acyl chains such as 14:0, 14:1, 24:0, 24:1, h18:1, h19:1 and h22:0–2 can be chosen as the molecular signature for microalgae from three major phyla. This methodology will be useful for a wide range of physiological and pathological studies of sphingolipids. Furthermore, the diversity of sphingolipid structure could provide a new criterion for microalgae chemotaxonomy.

Complex Effects of 24:1 Sphingolipids in Membranes Containing Dioleoylphosphatidylcholine and Cholesterol.

The effects of C24:1 sphingolipids have been tested in phospholipid bilayers containing cholesterol. Confocal microscopy, differential scanning calorimetry, and atomic force microscopy imaging and force curves have been used. More precisely, the effects of C24:1 ceramide (nervonoyl ceramide, nCer) were evaluated and compared to those of C16:0 ceramide (palmitoyl ceramide, pCer) in bilayers composed basically of dioleoylphosphatidylcholine, sphingomyelin (either C24:1, nSM or C16:0, pSM) and cholesterol. Combination of equimolecular amounts of C24:1 and C16:0 sphingolipids were also studied under the same conditions. Results show that both pCer and nCer are capable of forming segregated gel domains. Force spectroscopy data point to nCer having a lower stiffening effect than pCer, while the presence of nSM reduces the stiffness. DSC reveals Tm reduction by nSM in every case. Furthermore, pSM seems to better accommodate both ceramides in a single phase of intermediate properties, while nSM partial accommodation of ceramides generates different gel phases with higher stiffnesses caused by interceramide cooperation. If both pSM and nSM are present, a clear preference of both ceramides toward pSM is observed. These findings show the sharp increase in complexity when membranes exhibit different sphingolipids of varying N-acyl chains, which should be a common issue in an actual cell membrane environment.