Sphingolipids in lower animals.
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Animals↗
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Sphingolipid metabolism and signal transduction: inhibition of in vitro phospholipase activity by sphingosine.
Sphingosine inhibits protein kinase C activity in vitro and has been used to implicate this enzyme in signal transduction and cell function. We report that sphingosine directly inhibits phospholipases A2 and D. Sphingosine inhibits Ca(2+)-dependent phospholipases A2 from Naja naja, porcine pancreas, Crotalus adamanteus, human disc and neutrophil in a dose-dependent manner with IC50 values ranging from 5-40 microM using [1-14C]oleate-labelled autoclaved E. coli (20 microM) as substrate. Inhibition is comparable using the same concentrations (20 microM) of [1-14C]oleate-labelled C. albicans or E. coli, or aqueous dispersions of 1-acyl-2-[1-14C]linoleoylglycerophosphoethanolamine or -choline. Sphinganine and stearylamine are as inhibitory as sphingosine; monoolein is less inhibitory (IC50 = 70 microM), while octylamine, N-acetylsphingosine, sphingomyelin and ceramide have no effect. Inhibition is relieved by increasing concentrations of substrate phospholipid. The molar ratio of sphingosine to phospholipid required for 50% inhibition ranges from 0.5 to 1.0 with 2-100 microM E. coli phospholipid. In contrast, sphingosine has a biphasic effect on the hydrolysis of E. coli by S. chromofuscus phospholipase D; concentrations less than or equal to 25 microM stimulate activity while concentrations greater than 25 microM are inhibitory. Addition of Triton X-100 eliminates both the stimulatory and inhibitory effects of sphingosine on phospholipase D activity.
Animals↗
Sphingolipids and cell function.
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Animals↗
Diagnostic and therapeutic applications of sphingolipid hydrolyzing enzymes.
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Antibodies↗
Sphingolipid-to-glycerophospholipid conversion in SPL-null cells implies the existence of an alternative isozyme.
Sphingosine-1-phosphate (S1P) lyase catalyzes the cleavage of the bioactive lipid molecule S1P to phosphoethanolamine and hexadecenal, both of which are utilized as glycerophospholipid precursors. Until now, only one gene, SPL, has been identified as encoding a S1P lyase. In the present study, SPL-null F9 cells were able to convert radiolabeled dihydrosphingosine to glycerophospholipids, albeit at much lower efficiency than parent cells. Lysates prepared from the SPL-null cells exhibited weak but significant dihydrosphingosine-1-phosphate lyase activity in vitro. These results provide evidence of the existence of an alternative S1P lyase.
Aldehyde-Lyases↗
Sphingolipids in human lens membranes: an update on their composition and possible biological implications.
The unique nature of the most abundant phospholipids in human lens membranes remained overlooked until the 1990s when it was possible to discern dihydrosphingomyelins (DHSMs) from the more common sphingomyelins (SMs). Unlike in other mammalian membranes, DHSMs comprise nearly half of the phospholipids in adult human lenses. Compared to SMs with a trans double bond between carbons 4 and 5 of the sphingoid backbone, the absence of this unsaturation site in DHSMs allows the participation of the OH group on C3 in intermolecular H-bonds and leads to stronger interlipid interactions with both neighboring DHSMs and cholesterol. Phospholipid compositional changes with age and lens region observed in mammals with various life spans and lens growth rates, suggest that the highest levels of DHSMs along with the lowest amounts of phosphatidylcholines and SMs are found in lenses with the lowest growth rate, namely human lenses. The participation of phospholipid metabolites in the control of mitosis and elongation of lens cells is plausible and deserves investigation.
Adult↗
Sphingolipid C4 hydroxylation influences properties of yeast detergent-insoluble glycolipid-enriched membranes.
Sphingoid base C4 hydroxylation is required for syringomycin E action on the yeast plasma membrane. Detergent-insoluble glycolipid-enriched membranes (DIGs) from a yeast strain lacking C4 hydroxylated sphingoid bases (sur2delta) are composed of linear membrane fragments instead of vesicular structures observed for wild-type DIGs, though they have similar lipid compositions and amounts of DIG marker proteins. Light-scattering bands collected from sur2delta after centrifugation of Triton X-100-treated cell lysates in continuous density gradients have lower buoyant densities than that of the wild-type. The results show that C4 hydroxylation influences the physical and structural properties of DIGs and suggest that syringomycin E interacts with lipid rafts.
Cell Fractionation↗
Development and function of CD1d-restricted NKT cells: influence of sphingolipids, SAP and sex.
Natural killer T (NKT) cells are innate-like lymphocytes with immunoregulatory properties. They express an invariant and conserved T-cell receptor that recognizes glycolipids presented by CD1d. Manipulation, activation or loss of NKT cells has profound effects on diverse immune functions and can influence desirable responses to infectious organisms and tumors, as well as undesirable responses to autoantigens. Here, we discuss recent progress in the identification of natural endogenous and exogenous CD1d-presented antigens, the understanding of factors that regulate development of NKT cells in the thymus and of factors that influence their function in the periphery. Clinical implications of these findings are briefly touched on.
Animals↗
Shift in sphingolipid metabolism leads to an accumulation of ceramide in senescence.
Ceramide mediates the effects of several agonists leading to differentiation, apoptosis or senescence. We previously showed that ceramide becomes elevated in senescent fibroblasts. In the present study, senescent cultures of Wi-38 fibroblasts and human umbilical-vein endothelial cells were compared to low-passage cultures in order to identify which of the several pathways is predominantly responsible for the increased ceramide. We found that senescent cells take up the ceramide precursor [(3)H]palmitic acid and convert it to ceramide at essentially equivalent rates to their low-passage counterparts, suggesting that, as a whole, the inherent steps are unaltered. Analysis of subsequent steps, however, revealed changes in ceramide metabolism. The rate of ceramide conversion to sphingomyelin was reduced while glucosylceramide synthesis differed between the cell lines, while the rate of the reverse reactions tended to be increased in senescent cells. We also found a decrease in acidic but not alkaline ceramidase. The data show an overall change in favor increased ceramide levels. Of all of the pathways, neutral sphingomyelinase appears to be the most likely source of the senescence-associated ceramide. The relevance to mitosis and apoptosis are discussed.
Aging↗
Preventing the binding of pathogens to the host by controlling sphingolipid metabolism.
The binding of many pathogens and toxins to human cells can be inhibited by (1) depleting host cells of their surface glycosphingolipids; (2) coating the binding sites on pathogens (adhesins) with glycosphingolipid-like substances (decoys); (3) coating the host's glycosphingolipids with substances that compete with the pathogen for binding. Details of using these methods are described.
Bacterial Adhesion↗
Interactions between nitric oxide and sphingolipids and the potential consequences in physiology and pathology.
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Animals↗
Involvement of sphingolipids in apoptin-induced cell killing.
The potential anti-tumor agent Apoptin activates apoptosis in many human cancers and transformed cell lines, but is believed to be less potent in primary cells. Although caspase 3 is activated during apoptin-induced apoptosis, the mechanism of tumor cell killing remains elusive. We now show that apoptin-mediated cell death involves modulation of the sphingomyelin-ceramide pathway. Treating cells with Ad-GFPApoptin resulted in increased ceramide accumulation and enhanced expression of acid sphingomyelinase (ASMase) with a concomitant increase in ASMase activity and decreased sphingomyelin. Using confocal microscopy, ASMase, normally present in the endosomal/lysosomal compartment, was observed to translocate to the cell's periphery. Cotreatment of Ad-GFPApoptin-infected cells with the ASMase inhibitor desipramine (2.5 muM) attenuated (30%; P<0.01) apoptin-induced cell death. Apoptin was also able to induce a significant decline in sphingosine content by inhibition of ceramide deacylation through down-regulation of acid ceramidase at the protein level. Supporting the role of ceramide in apoptin action, treatment of cells with the combination of an exogenous cell-permeable ceramide analog (C6-ceramide) and Ad-GFPApoptin infection yielded a significant increase (P<0.01) in apoptosis over either treatment modality alone. Together, these data suggest that apoptin modulates ceramide/sphingolipid metabolism as part of its mechanism of action.
Adenoviridae↗
Sphingolipid-derived signalling modulators: interaction with phosphatidylserine.
We previously described the synthesis of two deuterium-labelled sphingoid bases, which made it possible to perform NMR spectroscopy on this family of signalling modulators in membranes (Rigby, A.C, Barber, K.R and Grant, C.W.M. (1995) Biochim. Biophys. Acta 1240, 75-82). In the present work we sought to test the concept that such mediators may display altered physical behaviour through association with anionic lipids - as a possible mechanism of involvement in signal transduction. Lyso-dihydrogalactosylceramide with deuterium nuclei at C4 and C5 of the sphingosine backbone and at C'3 and C'4 of the galactose ring ([2H4]lyso-GalCer), and N,N-dimethylsphingosine with deuterated amino-methyl groups ([2H6]dimethylsphingosine), were assembled as minor components into unsonicated fluid bilayer membranes of 1-palmitoyl-2-oleoylphosphatidylcholine/cholesterol. The effect of (anionic) phosphatidylserine was considered in this zwitterionic host matrix. The results present a picture of rapidly reversible interaction. The (-) charged phosphatidylserine exerted readily-measurable control over the orientation of the carbohydrate residue of [2H4]lyso-GalCer. In contrast, surrounding (-) charges exerted little spectral influence at the level of C4 and C5 of the lyso-GalCer, membrane-inserted, backbone; or at the level of the amino group of dimethylsphingosine. It would appear that packing alterations induced by the phosphatidylserine/sphingoid base association can translate into sizeable spatial constraints in the neighbouring aqueous domain.
Cholesterol↗
Molecular cloning of the sphingolipid activator protein-1 (SAP-1), the sulfatide sulfatase activator.
A cDNA coding for SAP-1 was isolated from a lambda gt11 human hepatoma expression library using polyclonal antibodies raised against human SAP-1. Three positive clones were isolated with inserts of approximately 0.3 Kb (S1.1), 2 Kb (S1.2) and 2.2 Kb (S-1.3). The latter 2 contained an internal EcoRI site. All three clones cross-hybridized with one another, indicating sequence homology. The nucleotide sequence of S-1.1 was determined. Colinearity was established between 19 amino acids obtained by sequencing the amino terminus of pure SAP-1 and 57 bp from the 5' end of S-1.1. The open reading frame of S-1.1 coded for 67 amino acids. One glycosylation site was found 21 residues from the amino terminus, and no stop codons were found. S-1.1 codes for a mature polypeptide chain with a calculated molecular weight of 8955 daltons, corresponding to approximately 99% of mature SAP-1.
Amino Acid Sequence↗
Partitioning of pyrene-labeled phospho- and sphingolipids between ordered and disordered bilayer domains.
Here we have studied how the length of the pyrene-labeled acyl chain (n) of a phosphatidylcholine, sphingomyelin, or galactosylceramide affects the partitioning of these lipids between 1), gel and fluid domains coexisting in bovine brain sphingomyelin (BB-SM) or BB-SM/spin-labeled phosphatidylcholine (PC) bilayers or 2), between liquid-disordered and liquid-ordered domains in BB-SM/spin-labeled PC/cholesterol bilayers. The partitioning behavior was deduced either from modeling of pyrene excimer/monomer ratio versus temperature plots, or from quenching of the pyrene monomer fluorescence by spin-labeled PC. New methods were developed to model excimer formation and pyrene lipid quenching in segregated bilayers. The main result is that partition to either gel or liquid-ordered domains increased significantly with increasing length of the labeled acyl chain, probably because the pyrene moiety attached to a long chain perturbs these ordered domains less. Differences in partitioning were also observed between phosphatidylcholine, sphingomyelin, and galactosylceramide, thus indicating that the lipid backbone and headgroup-specific properties are not severely masked by the pyrene moiety. We conclude that pyrene-labeled lipids could be valuable tools when monitoring domain formation in model and biological membranes as well as when assessing the role of membrane domains in lipid trafficking and sorting.
Fluorometry↗
Modulation of nanotube formation by structural modifications of sphingolipids.
Galactosylceramides (GalCers) containing nervonoyl (24:1(Delta15(cis))) acyl chains have the capacity to assemble into nanotubular microstructures in excess water (. Biophys. J. 69:1976-1986). To define the structural parameters that modulate nanotube formation, GalCer derivatives were synthesized that contained cis monounsaturated acyl chains with the formula X:1((X-9)). X indicates the total acyl carbon number (24, 22, 20, or 18), and 1 indicates a single cis double bond, the location of which is designated by the superscript (X-9). Deep etching of freeze-fractured 24:1(Delta15(cis)) GalCer dispersions followed by replica production and transmission electron microscopic analysis confirmed nanotube morphology (25-30-nm diameter). Control experiments revealed that tubule formation was promoted by cooling through the main enthalpic phase transition coupled with repetitive freeze-thaw cycling. Imparting a negative charge to the sugar headgroup of 24:1(Delta15)GalCer via sulfate dramatically altered mesomorpholgy and resulted in myelinic-like, multilamellar structures. Removal of the sugar headgroup (24:1(Delta15)Cer) resulted in flattened cylindrical structures with a cochleate appearance. Compared to these large-scale changes in morphology, more subtle changes were induced by structural changes in the acyl chain of 24:1(Delta15)GalCer. 22:1(Delta13)GalCer dispersions consisted of long, smooth tubules (35-40-nm diameters) with a strong tendency to self-align into bundle-like aggregates. In contrast, the microstructures formed by 20:1(Delta11)GalCer resembled helical ribbons with a right-handed twist. Ribbon widths averaged 30-35 nm, with helical pitches of 80-90 nm. 18:1(Delta9)GalCer displayed a variety of morphologies, including large-diameter multilamellar cylinders and liposome-like structures, as well as stacked, plate-like arrays. The results are discussed within the context of current theories of lipid tubule formation.
Freeze Etching↗
Proteomic characterisation of neuronal sphingolipid-cholesterol microdomains: role in plasminogen activation.
Sorting of certain membrane proteins requires a mechanism involving rafts, protein-lipid complexes enriched in glycosphingolipids and cholesterol. These microdomains remain at the plasma membrane of different cell types and play a role in signal transduction. Although recent reports have begun to describe molecules associated with rafts, their protein composition remains largely unknown, especially in neuronal cells. To address this question, we have purified detergent-insoluble raft fractions (DRMs) from primary cultures of hippocampal neurons. Bidimensional gel analysis and pharmacological raft lipid manipulation allowed the identification of neuronal raft proteins and their characterisation by MALDI-TOF analysis. Enolases were found among the proteins identified and functional studies demonstrate their participation in plasminogen binding. We also show the specific enrichment in rafts of several other plasminogen binding molecules and the exclusive activation of plasminogen to the protease plasmin in these microdomains. These observations suggest that neuronal rafts may play, in addition to intracellular signaling, a role in extracellular/membrane protein proteolysis.
Animals↗
Sphingolipids of the mycopathogen Sporothrix schenckii: identification of a glycosylinositol phosphorylceramide with novel core GlcNH(2)alpha1-->2Ins motif.
Acidic glycosphingolipid components were extracted from the yeast form of the dimorphic mycopathogen Sporothrix schenckii. Two minor and the major fraction from the yeast form (Ss-Y1, -Y2, and -Y6, respectively) have been isolated. By a combination of 1- and 2-D 1H-nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI-MS), and gas chromatography/mass spectrometry (GC/MS), Ss-Y6 was determined to be triglycosylinositol phosphorylceramide with a novel glycan structure, Manalpha1-->3Manalpha1-->6GlcNH(2)alpha1-->2Ins1-P-1Cer (where Ins=myo-inositol, P=phosphodiester). While the GlcNH(2)alpha1-->6Ins1-P- motif is found widely distributed in eukaryotic GPI anchors, the linkage GlcNH(2)alpha1-->2Ins1-P- has not been previously observed in any glycolipid. Ss-Y1 and Ss-Y2 were both found to have the known glycan structure Manalpha1-->3Manalpha1-->2Ins1-P-1Cer. Together with the results of a prior study [Toledo et al. (2001) Biochem. Biophys. Res. Commun. 280, 19-24] which showed that the mycelium form expresses GIPCs with the structures Manalpha1-->6Ins1-P-1Cer and Manalpha1-->3Manalpha1-->6Ins1-P-1Cer, these results demonstrate that S. schenckii can synthesize glycosylinositol phosphorylceramides with at least three different core linkages.
Amino Acid Motifs↗