A convenient oxidation of natural glycosphingolipids to their "ceramide acids" for neoglycoconjugation. Bovine serum albumin-glycosylceramide acid conjugates as investigative probes for HIV gp120 coat protein-glycosphingolipid interactions.

A new method to cleave the double bond of sphingolipids has been developed. Using limited concentrations of KMnO4 and an excess of NaIO4, in a neutral aqueous tert-butanol solvent system gave nearly quantitative yields of the oxidized product. A variety of natural glycosphingolipids (GSLs): GlcC, GalC, SGC, LC, Gb3C, Gb4C, Gg4C, Gb5C, and GM1C, gave the corresponding acids: 2-hydroxy-3-(N-acyl)-4-(O-glycosyl)-oxybutyric acids, i.e. "glycosyl ceramide acids" (GSL.CCOOH) in excellent yields (80-90%). Deacyl GSLs (dGSLs) were oxidized to acids containing the oligosaccharides devoid of hydrocarbon chains, i.e. "ceramide oligosaccharides" (dGSL. NRR1CCOOH, where R = R1 = H; R = H, R1 = CH3CO; or R = R1 = Me). The efficacy of this method was demonstrated by transforming natural GSLs: GlcC, GalC, GalS, SGC, LC, Gb3C, and Gb4C into neoglycoproteins via coupling glycosyl ceramide acids (except GalS, which was coupled directly) to bovine serum albumin (BSA). Mass spectroscopic analysis of GalC-BSA conjugates, (GalC.CONH)nBSA and (GalS.NHCO)nBSA gave a value of 9 +/- 1 and 16 +/- 2 for n. Neoglycoconjugates derived from GlcC, GalC (type I and II and the behenic analog), SGC, LC, and Gb3C were recognized by the recombinant human immunodeficiency virus coat protein gp120 (rgp120). The GalS conjugate showed significantly reduced binding, and the Gb4C conjugate showed no binding. Thus, rgp120/GSL-BSA interaction requires a terminal galactose and/or glucose residue. Terminal N-acetylgalactosamine containing GSLs are not bound. The ceramide acid conjugates provide a more effective scaffold for presentation of glycone for rgp120 binding than those derived from dGSLs. The retention of receptor specificity of the glycoconjugates was validated by retention of the expected binding specificity of VT1 and VT2e for Gb3C and Gb4C conjugates, respectively. These studies open a new vista in the generation of glycoconjugates from GSLs and further emphasize the role of aglycone in glycolipid recognition.     

Reduction of plasma glycosphingolipid levels has no impact on atherosclerosis in apolipoprotein E-null mice

Glycosphingolipids (GSLs) have been implicated as potential atherogenic lipids. Studies in apolipoprotein E-null (apoE(-/-)) mice indicate that exacerbated tissue GSL accumulation resulting from alpha-galactosidase deficiency promotes atherosclerosis, whereas the serine palmitoyl transferase inhibitor myriocin (which reduces plasma and tissue levels of several sphingolipids, including sphingomyelin, ceramide, sphingosine-1-phosphate, and GSLs) inhibits atherosclerosis. It is not clear whether GSL synthesis inhibition per se has an impact on atherosclerosis. To address this issue, apoE(-/-) mice maintained on a high-fat diet were treated with a potent glucosylceramide synthesis inhibitor, d-threo-1-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidino-propanol (EtDO-P4), 10 mg/kg/day for 94 days, and lesion development was compared in mice that were treated with vehicle only. EtDO-P4 reduced plasma GSL concentration by approximately 50% but did not affect cholesterol or triglyceride levels. Assessment of atherosclerotic lesions at four different sites indicated that EtDO-P4 had no significant impact on lesion area. Thus, despite the previously observed positive correlations between plasma and aortic GSL concentrations and the development of atherosclerosis, and the in vitro evidence implying that GSLs may be pro-atherogenic, our current data indicate that inhibition of GSL synthesis does not inhibit atherosclerosis in vivo.     

Novel class of glycosphingolipids involved in male fertility

Mice require testicular glycosphingolipids (GSLs) for proper spermatogenesis. Mutant mice strains deficient in specific genes encoding biosynthetic enzymes of the GSL pathway including Galgt1 (encoding GM2 synthase) and Siat9 (encoding GM3 synthase) have been established lacking various overlapping subsets of GSLs. Although male Galgt1-/- mice are infertile, male Siat9-/- mice are fertile. Interestingly, GSLs thought to be essential for male spermatogenesis are not synthesized in either of these mice strains. Hence, these GSLs cannot account for the different phenotypes. A novel class of GSLs was observed composed of eight fucosylated molecules present in fertile but not in infertile mutant mice. These GSLs contain polyunsaturated very long chain fatty acid residues in their ceramide moieties. GSLs of this class are expressed differentially in testicular germ cells. More importantly, the neutral subset of this new GSL class strictly correlates with male fertility. These data implicate polyunsaturated, fucosylated GSLs as essential for spermatogenesis and male mouse fertility.     

Systematic analyses of free ceramide species and ceramide species comprising neutral glycosphingolipids by MALDI-TOF MS with high-energy CID

Free ceramides and glycosphingolipids (GSLs) are important components of the membrane microdomain and play significant roles in cell survival. Recent studies have revealed that both fatty acids and long-chain bases (LCBs) are more diverse than expected, in terms of i) alkyl chain length, ii) hydroxylation and iii) the presence or absence of double bonds. Electrospray ionization mass spectrometry and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) have been well utilized to characterize sphingolipids with high throughput, but reports to date have not fully characterized various types of ceramide species such as hydroxyl fatty acids and/or trihydroxy-LCBs of both free ceramides and the constituent ceramides in neutral GSLs. We performed a systematic analysis of both ceramide species, including LCBs with nona-octadeca lengths using MALDI-TOF MS with high-energy collision-induced dissociation (CID) at 20 keV. Using both protonated and sodiated ions, this technique enabled us to propose general rules to discriminate between isomeric and isobaric ceramide species, unrelated to the presence or absence of sugar chains. In addition, this high-energy CID generated ^3,5A ions, indicating Hex1-4Hex linkage in the sugar chains. Using this method, we demonstrated distinct differences among ceramide species, including free ceramides, sphingomyelins, and neutral GSLs of glucosylceramides, galactosylceramides, lactosylceramides, globotriaosylceramides and Forssman glycolipids in the equine kidneys.     

Degradation of glycosphingolipids in oyster: ceramide glycanase and ceramidase in the hepatopancreas of oyster, <Emphasis Type="Italic">Crassostrea virginica</Emphasis>

The hepatopancreas of oyster, Crassostrea virginica, was found to contain two unique glycosphingolipid (GSL) cleaving enzymes, ceramide glycanase (CGase) and ceramidase. These two enzymes were found to be tightly associated together through the consecutive purification steps including gel filtration, hydrophobic interaction and cation-exchange chromatographies. They were separated only by preparatory SDS-PAGE. The purified CGase was found to have a molecular mass of 52 kDa and pH optimum of 3.2–3.3. This enzyme prefers to hydrolyze the acidic GSLs, II³SO₃LacCer and gangliosides over the neutral GSLs. Oyster ceramidase was found to have a molecular mass of 88 kDa and pH optimum of 4–4.5. Since oyster ceramidase greatly prefers ceramides with C₆ to C₈ fatty acids, C₆-ceramide (N-hexanoyl-D-sphingosine) was used as the substrate for its purification and characterization. The oyster acid ceramidase also catalyzed the synthesis of ceramide from a sphingosine and a fatty acid. For the synthesis, C₁₆ and C₁₈ fatty acids were the best precursors. The amino acid sequences of the two cyanogenbromide peptides derived from the purified ceramidase were found to have similarities to those of several neutral and alkaline ceramidases reported. The tight association of CGase and ceramidase may indicate that CGase in oyster hepatopancreas acts as a vehicle to release ceramide from GSLs for subsequent generation of sphingosines and fatty acids by ceramidase to serve as signaling factors and energy source.     

Structural studies on the neutral glycosphingolipids of Manduca sexta

Glycosphingolipids (GSLs) have been implicated as playing major roles in cellular interactions and control of cell proliferation in muticellular organisms. Moreover GSLs and other sphingolipids such as sphingomyelins, ceramides and sphingosines serve a variety of roles in signal transduction. Hence, identification of structures of GSLs in different biota will shed light in understanding their physiological role. During this study, the major glycosphingolipid component present in the extracts of stage-12 and stage-17/18 metamorphosing adults of Manduca sexta was identified as mactosyl ceramide. We report the isolation of several ceramide disaccharides, a ceramide trisaccharide and a ceramide tetrasaccharide. The GSL structures were confirmed by high-resolution mass spectrometry and tandem mass spectrometry. The identity of the monosaccharides was proved using exoglycosidases. The predominant sphingosine chain-length varied from C-14 (tetradecasphing-4-enine) to C-16 (hexadecasphing-4-enine) in these GSLs. Sphingosines of both chain lengths were accompanied by their doubly unsaturated counterparts tetradecasphinga-4,6-diene and hexadecasphinga-4,6-diene. It is also interesting to note the presence of tetradecasphinganine and hexadecasphinganine in minute amounts in the form of a GSL in the extracts of M. sexta. The varying degrees of unsaturation in the sphingosine moiety of GSLs in M. sexta may be biologically significant in insect metamorphosis. The ceramide trisaccharides and ceramide tetrasaccharide belong to the arthro-series, The observation of fucose in the M. sexta GSLs is the first report of the presence of fucose in an arthroseries GSL.     

Glucosinolate structures in evolution

By 2000, around 106 natural glucosinolates (GSLs) were probably documented. In the past decade, 26 additional natural GSL structures have been elucidated and documented. Hence, the total number of documented GSLs from nature by 2011 can be estimated to around 132. A considerable number of additional suggested structures are concluded not to be sufficiently documented. In many cases, NMR spectroscopy would have provided the missing structural information. Of the GSLs documented in the past decade, several are of previously unexpected structures and occur at considerable levels. Most originate from just four species: Barbarea vulgaris, Arabidopsis thaliana, Eruca sativa and Isatis tinctoria. Acyl derivatives of known GSLs comprised 15 of the 26 newly documented structures, while the remaining exhibited new substitution patterns or chain length, or contained a mercapto group or related thio-functionality.GSL identification methods are reviewed, and the importance of using authentic references and structure-sensitive detection methods such as MS and NMR is stressed, especially when species with relatively unknown chemistry are analyzed. An example of qualitative GSL analysis is presented with experimental details (group separation and HPLC of both intact and desulfated GSLs, detection and structure determination by UV, MS, NMR and susceptibility to myrosinase) with emphasis on the use of NMR for structure elucidation of even minor GSLs and GSL hydrolysis products. The example includes identification of a novel GSL, (R)-2-hydroxy-2-(3-hydroxyphenyl)ethylglucosinolate.Recent investigations of GSL evolution, based on investigations of species with well established phylogeny, are reviewed. From the relatively few such investigations, it is already clear that GSL profiles are regularly subject to evolution. This result is compatible with natural selection for specific GSL side chains. The probable existence of structure-specific GSL catabolism in intact plants suggests that biochemical evolution of GSLs has more complex implications than the mere liberation of a different hydrolysis product upon tissue disruption.     

Non-natural glycosphingolipids and structurally simpler analogues bind HIV-1 recombinant Gp120

Interactions of recombinant gp120 (rgp120) with non-natural glycosphingolipids (GSLs) and structurally simpler analogues have been studied using a competitive adhesion assay. Conjugates of cellobiosyl ceramide and melibiosyl ceramide were synthetically prepared as water-soluble GSL analogues. These ligands were screened against a panel of biologically relevant analogues, and the results show that their interactions with rgp120 are comparable to natural cellular receptors. Glycolipid interactions with rgp120 were probed further by the synthesis and testing of structurally simpler analogues that were obtained by reductive amination of lactose, cellobiose, and melibiose with a biotinylated amino ethylene glycol moiety. RGp120 did not recognize conjugates lacking a lipid component. However, palmitoylation of the secondary amino alditols yielded compounds with comparable rgp120 affinity to the natural cellular receptor, galactosyl ceramide (GalCer). Taken together, the SAR showed that both a hydrophobic and a hydrophilic component are required for rgp120 recognition. Moreover, structural variability in the carbohydrate headgroup did not significantly alter rgp120 recognition indicating that this interaction is not highly specific.     

Induction of Ganglioside Biosynthesis and Neurite Outgrowth of Primary Cultured Neurons by l-threo-1-Phenyl-2-Decanoylamino-3-Morpholino-1-Propanol

Abstract: We reported previously that stereoisomers of 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), the d - threo and l - threo forms, exerted inhibitory and stimulatory effects on glycosphingolipid (GSL) biosynthesis in B16 melanoma cells, respectively. In the present study, the primary cultured rat neocortical explants were treated with l - or d - threo -PDMP. These isomers exhibited opposite effects on neurite outgrowth: d -PDMP was inhibitory at concentrations ranging from 5 to 20 µ M , whereas l -PDMP was stimulatory over the same concentration range, and the maximal effect was observed at 10–15 µ M . Rat neocortical explants were doubly labeled with [¹⁴C]serine and [³H]galactose at 15 µ M l - or d -PDMP. l -PDMP increased the incorporations of both labels into sphinganine, sphingosine, ceramide, sphingomyelin, neutral GSLs, and gangliosides, whereas d -PDMP inhibited the glucosylation of ceramide resulting in a reduction of ganglioside biosynthesis and accumulation of precursors of glucosylceramide, ceramide, and sphingomyelin. To clarify the stimulatory effect of l -PDMP on GSL biosynthesis, serine palmitoyltransferase, sphingosine N -acyltransferase, glucosylceramide synthase, lactosylceramide synthase, GM3 synthase, and GD3 synthase were quantified in cell lysates of explants pretreated with this agent. Serine palmitoyltransferase was fully activated up to 150% of the control. Furthermore, marked increases in the activities of lactosylceramide synthase (200%), GM3 synthase (240%), and GD3 synthase (300%) were observed. These results suggest that the neurotrophic action of l -PDMP may be ascribable to its stimulatory effect on the biosynthesis of GSLs, especially that of gangliosides.     

Increased glycosphingolipid levels in serum and aortae of apolipoprotein E gene knockout mice

The apolipoprotein E gene knockout (apoE-/-) mouse develops atherosclerosis that shares many features of human atherosclerosis. Increased levels of glycosphingolipid (GSL) have been reported in human atherosclerotic lesions; however, GSL levels have not been studied in the apoE-/- mouse. Here we used HPLC methods to analyze serum and aortic GSL levels in apoE-/- and C57BL/6J control mice. The concentrations of glucosyl ceramide (GlcCer), lactosyl ceramide (LacCer), GalNAcbeta1-4Galbeta1-4Glc-Cer (GA2), and ceramide trihexoside (CTH) were increased by approximately 7-fold in the apoE-/- mouse serum compared with controls. The major serum ganglioside, N-glycolyl GalNAcbeta1-4[NeuNAcalpha2-3]Galbeta1-4Glc-Cer (N-glycolyl GM2), was increased in concentration by approximately 3-fold. A redistribution of GSLs from HDL to VLDL populations was also observed in the apoE-/- mice. These changes were accompanied by an increase in the levels of GSLs in the aortic sinus and arch of the apoE-/- mice. The spectrum of gangliosides present in the aortic tissues was more complex than that found in the lipoproteins, with the latter represented almost entirely by N-glycolyl GM2 and the former comprised of NeuNAcalpha2-3Galbeta1-4Glc-Cer (GM3), GM2, N-glycolyl GM2, GM1, GD3, and GD1a. In conclusion, neutral GSL and ganglioside levels were increased in the serum and aortae of apoE-/- mice compared with controls, and this was associated with a preferential redistribution of GSL to the proatherogenic lipoprotein populations. The apoE-/- mouse therefore represents a useful model to study the potential role of GSL metabolism in atherogenesis.     

Structure and function of glycosphingolipids and sphingolipids: recollections and future trends

Based on development of various methodologies for isolation and characterization of glycosphingolipids (GSLs), we have identified a number of GSLs with globo-series or lacto-series structure. Many of them are tumor-associated or developmentally regulated antigens. The major question arose, what are their functions in cells and tissues? Various approaches to answer this question were undertaken. While the method is different for each approach, we have continuously studied GSL or glycosyl epitope interaction with functional membrane components, which include tetraspanins, growth factor receptors, integrins, and signal transducer molecules. Often, GSLs were found to interact with other carbohydrates within a specific membrane microdomain termed "glycosynapse", which mediates cell adhesion with concurrent signal transduction. Future trends in GSL and glycosyl epitope research are considered, including stem cell biology and epithelial-mesenchymal transition (EMT) process.     

Glycosphingolipids of purified human lymphocytes.

Biochemical analysis of the glycosphingolipids (GSLs) of human lymphocytes revealed qualitative and quantitative variations among purified lymphocytes from different tissues. The major neutral GSLs of tonsil lymphocytes are glucosyl ceramide (CMH), lactosyl ceramide (CDH), trihexosyl ceramide (CTH), and globoside. Thymocytes and peripheral blood lymphocytes (PBL) contain only traces of CTH and globoside, and PBL contain more CMH and CDH per cell than tonsil lymphocytes. Thymocytes and PBL contain relatively large amounts of more complex neutral GSLs that are present in only trace amounts in tonsil lymphocytes. Peripheral blood lymphocytes contained three and five times more lipid-bound sialic acid than thymocytes and toncil lymphocytes, respectively. Thymocytes and PBL contained mostly hematoside, whereas tonsil lymphocytes contained more complex gangliosides in addition to hematoside. The observed differences in GSL content among these cells may be related to their content of B cells, which comprise approximately 50% of tonsil lymphocytes, 10% of PBL and 0-2% of thymus cells, and/or the known differences in functional capacities of cells in different lymphoid organs. These findings suggest that cell surface GSLs may serve as markers for identification of functional subpopulations of human lymphocytes.     

Characterization of neutral glycosphingolipids from porcine erythrocyte membranes

1. Six neutral GSL fractions were purified from porcine erythrocyte membranes. 2. They were identified to be LacCer (14% of total neutral GSLs), 2-hydroxy acid-rich and -poor Gb3Cer (3 and 7%, respectively) and Gb4Cer (71%) by means of NMR spectrometry. 3. Monohexosylceramides (5%) were composed of GlcCer and GalCer with near amount. 4. All these GSL classes contained a high concentration (more than 20% of total acids in each class) of 2-hydroxy fatty acids. 5. GalCer and GlcCer contained considerable amounts of C16- and C18-acids, and of C18-phytosphingosine, whereas C24-acids and C18-sphingosine were predominant in the other GSLs. 6. A minor GSL fraction (less than 1% of total neutral GSLs) which migrated more slowly than Gb5Cer on a thin layer plate and composed of several GSL components contained L-fucose.     

A facile method for controlling the reaction equilibrium of sphingolipid ceramide N-deacylase for lyso-glycosphingolipid production

Lyso-glycosphingolipids (lyso-GSLs), the N-deacylated forms of glycosphingolipids (GSLs), are important synthetic intermediates for the preparation of GSL analogs. Although lyso-GSLs can be produced by hydrolyzing natural GSLs using sphingolipid ceramide N-deacylase (SCDase), the yield for this reaction is usually low because SCDase also catalyzes the reverse reaction, ultimately establishing an equilibrium between hydrolysis and synthesis. In the present study, we developed an efficient method for controlling the reaction equilibrium by introducing divalent metal cation and detergent in the enzymatic reaction system. In the presence of both Ca²⁺ and taurodeoxycholate hydrate, the generated fatty acids were precipitated by the formation of insoluble stearate salts and pushing the reaction equilibrium toward hydrolysis. The yield of GM1 hydrolysis can be achieved as high as 96%, with an improvement up to 45% compared with the nonoptimized condition. In preparative scale, 75 mg of lyso-GM1 was obtained from 100 mg of GM1 with a 90% yield, which is the highest reported yield to date. The method can also be used for the efficient hydrolysis of a variety of GSLs and sphingomyelin. Thus, this method should serve as a facile, easily scalable, and general tool for lyso-GSL production to facilitate further GSL research.     

Early developmental expression of the gene encoding glucosylceramide synthase,the enzyme controlling the first committed step of glycosphingolipid synthesis

Glycosphingolipids (GSLs) are ubiquitous plasma membrane components composed of a ceramide lipid anchor attached to one of a diverse complement of oligosaccharide structures. Fundamentally important activities have been attributed to GSLs including formation of plasma membrane structures involved in membrane trafficking, signal transduction and cell-cell interactions. Glucosylceramide synthase converts ceramide to glucosylceramide, a core structure of the vast majority of GSLs. Disruption of the gene encoding glucosylceramide synthase (Ugcg) caused embryonic lethality in mice during gastrulation. To further investigate the role of GSL synthesis during embryogenesis, we produced mice with a Lacz reporter gene inserted into the glucosylceramide synthase locus. These mice allowed the visualization of glucosylceramide synthase expression during early embryonic development.     

Glycosphingolipid synthesis is essential for MDCK cell differentiation

Glycosphingolipids (GSLs), which are highly concentrated at the apical membrane of polarized epithelial cells, are key components of cell membranes and are involved in a large number of processes. Here, we investigated the ability of hypertonicity (high salt medium) to induce Madin-Darby Canine Kidney (MDCK) cell differentiation and found an increase in GSL synthesis under hypertonic conditions. Then, we investigated the role of GSLs in MDCK cell differentiation induced by hypertonicity by using two approaches. First, cultured cells were depleted of GSLs by exposure to D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). Second, cells were transfected with an siRNA specific to glucosylceramide synthase, the key enzyme in GSL synthesis. Exposure of cells to both treatments resulted in the impairment of the development of the apical membrane domain and the formation of the primary cilium. Enzymatic inhibitions of the de novo and the salvage pathway of GSL synthesis were used to determine the source of ceramide responsible of the GSL increase involved in the development of the apical membrane domain induced by hypertonicity. The results from this study show that extracellular hypertonicity induces the development of a differentiated apical membrane in MDCK cells by performing a sphingolipid metabolic program that includes the formation of a specific pool of GSLs. The results suggest as precursor a specific pool of ceramides formed by activation of a Fumonisin B1-resistant ceramide synthase as a component of the salvage pathway.     

Self-recognition of N-linked glycans with multivalent GlcNAc, determined as ceramide mimetic conjugate

Aminoceramide mimetic was synthesized and conjugated to N-linked oligosaccharides having multivalent GlcNAc by reductive amination. Ceramide mimetic conjugates with "complex-type" glycan having five or six GlcNAc termini (termed Os Fr. B-Cer) were purified, analyzed by thin-layer chromatography (TLC), and finally characterized by MS/MS analysis through liquid chromatography/mass spectrometry. Binding of Os Fr. B-Cer placed on solid phase polystyrene surface with [3H]cholesterol-labeled liposomes containing Os Fr. B-Cer, or containing various glycosphingolipids (GSLs) was determined. The binding of Os Fr. B-Cer liposomes to Os Fr. B-Cer coated plate was significantly higher than binding of GM3 liposomes. Other GSL liposomes showed no binding. Thus, self-recognition of Os Fr. B-Cer was clearly demonstrated using ceramide mimetic conjugates.     

Determination of hydroxyoctadecadienoic acids

Oxidation of low-density lipoproteins (LDL) plays a crucial role in inflammatorydiseases and aging. The main oxidation products of LDL are stereoisomeric 9-hydroxy-10,12-octadecadienoic acids (9-HODEs) and 13-hydroxy-9,11-octadecadienoic acids (13-HODEs). Nevertheless the content of HODEs in natural oxidized LDL is low compared to other components, thus determination of HODEs requires a sample enrichment in most cases. Big losses are encountered during the necessary processing due to the instability of HODEs against acidic conditions. Therefore the use of labeled standards is required. Standards with an 18O label in the carboxylic group used previously may partly suffer a loss of the label by exchange with water. In this paper we describe an improved work-up procedure and the preparation of standards labeled with 18O in the hydroxylic group which is not exchangeable.