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PubMed2003

Sphingolipids are essential for differentiation but not growth in Leishmania.

Sphingolipids (SLs) play critical roles in eukaryotic cells in the formation of lipid rafts, membrane trafficking, and signal transduction. Here we created a SL null mutant in the protozoan parasite Leishmania major through targeted deletion of the key de novo biosynthetic enzyme serine palmitoyltransferase subunit 2 (SPT2). Although SLs are typically essential, spt2- Leishmania were viable, yet were completely deficient in de novo sphingolipid synthesis, and lacked inositol phosphorylceramides and other SLs. Remarkably, spt2- parasites maintained 'lipid rafts' as defined by Triton X-100 detergent resistant membrane formation. Upon entry to stationary phase spt2- failed to differentiate to infective metacyclic parasites and died instead. Death occurred not by apoptosis or changes in metacyclic gene expression, but from catastrophic problems leading to accumulation of small vesicles characteristic of the multivesicular body/multivesicular tubule network. Stage specificity may reflect changes in membrane structure as well as elevated demands in vesicular trafficking required for parasite remodeling during differentiation. We suggest that SL-deficient Leishmania provide a useful biological setting for tests of essential SL enzymes in other organisms where SL perturbation is lethal.

Acyltransferases↗
PubMed2001

Sphingolipid activator proteins: proteins with complex functions in lipid degradation and skin biogenesis.

Sphingolipid activator proteins (SAPs or saposins) are essential cofactors for the lysosomal degradation of membrane-anchored sphingolipids. Four of the five known proteins of this class, SAPs A--D, derive from a single precursor protein and show high homology, whereas the fifth protein, GM2AP, is larger and displays a different secondary structure. Although the main function of all five proteins is assumed to lie in the activation of lipid degradation, their specificities and modes of action seem to differ considerably. It has recently been demonstrated that the action of the proteins is highly enhanced by the presence of acidic lipids in the target membranes. These results have some interesting implications for the topology of lysosomal degradation of lipids and may provide new insights into the function of these interesting proteins, which are ubiquitously expressed in the different tissues of the body. Recent studies indicated that the SAPs play an important role in the biogenesis of the epidermal water barrier, which has been demonstrated by the analysis of the skin phenotype displayed by SAP-knockout mice. The results obtained so far have led to some new insights into the formation of the epidermal water permeability barrier and may lead to a better understanding of this complex process.

Epidermis↗
PubMed2004

Mutation in saposin D domain of sphingolipid activator protein gene causes urinary system defects and cerebellar Purkinje cell degeneration with accumulation of hydroxy fatty acid-containing ceramide in mouse.

The sphingolipid activator proteins (saposins A, B, C and D) are small homologous glycoproteins that are encoded by a single gene in tandem within a large precursor protein (prosaposin) and are required for in vivo degradation of some sphingolipids with relatively short carbohydrate chains. Human patients with prosaposin or specific saposin B or C deficiency are known, and prosaposin- and saposin A-deficient mouse lines have been generated. Experimental evidence suggests that saposin D may be a lysosomal acid ceramidase activator. However, no specific saposin D deficiency state is known in any mammalian species. We have generated a specific saposin D(-/-) mouse by introducing a mutation (C509S) into the saposin D domain of the mouse prosaposin gene. Saposin D(-/-) mice developed progressive polyuria at around 2 months and ataxia at around 4 months. Pathologically, the kidney of saposin D(-/-) mice showed renal tubular degeneration and eventual hydronephrosis. In the nervous system, progressive and selective loss of the cerebellar Purkinje cells in a striped pattern was conspicuous, and almost all Purkinje cells disappeared by 12 months. Biochemically, ceramides, particularly those containing hydroxy fatty acids accumulated in the kidney and the brain, most prominently in the cerebellum. These results not only indicate the role of saposin D in in vivo ceramide metabolism, but also suggest possible cytotoxicity of ceramide underlying the cerebellar Purkinje cell and renal tubular cell degeneration.

Animals↗
PubMed1976

Chemical and hemolytic properties of sphingolipids modified by ozonolysis and reduction.

Various sphingolipids were chemically modified in their sphingosine base by ozonolysis and reduction. The derivatives obtained from Forssman globoside, globoside I, galactosyl ceramide, and sphingomyelin were purified and all were found to be hemolytic. The presence of cholesterol could inhibit this activity. The simultaneous cleavage at a double bond in the fatty acid as well as in the sphingosine of Forssman globoside resulted in the formation of a more polar compound with no detectable hemolytic activity. The haptenic reactivity was retained after ozonolysis and reduction of Forssman globoside, as shown by precipitin line formation in agar gel with appropriate antibodies. The results indicate that this modification procedure may be useful in studies of the physiological and immunological properties of sphingolipids.

Chemical Phenomena↗
PubMed2001

Characterization of an Arabidopsis cDNA encoding a subunit of serine palmitoyltransferase, the initial enzyme in sphingolipid biosynthesis.

Serine palmitoyltransferase (SPT; EC 2.3.1.50) catalyzes the condensation of serine with palmitoyl-CoA to form 3-ketosphinganine in the first step of de novo sphingolipid biosynthesis. In this study, we describe the cloning and functional characterization of a cDNA from Arabidopsis thaliana encoding the LCB2 subunit of SPT. The Arabidopsis LCB2 (AtLCB2) cDNA contains an open reading frame of 1,467 nucleotides, encoding 489 amino acids. The predicted polypeptide contains three transmembrane helices and a highly conserved motif involved in pyridoxal phosphate binding. Expression of this open reading frame in the Saccharomyces cerevisiae mutant strains defective in SPT activity resulted in the expression of a significant level of sphinganine, suggesting that AtLCB2 cDNA encodes SPT. Southern blot analysis and inspection of the complete Arabidopsis genome sequence database suggest that there is a second LCB2-like gene in Arabidopsis. Expression of a green fluorescent protein (GFP) fusion product in suspension-cultured tobacco BY-2 cells showed that AtLCB2 is localized to the endoplasmic reticulum. AtLCB2 cDNA may be used to study how sphingolipid synthesis is regulated in higher plants.

Acyltransferases↗
PubMed1997

Lipid biochemistry: functions of glycerolipids and sphingolipids in cellular signaling.

Products of glycerolipid and sphingolipid metabolism are now known to fulfill second messenger functions in a variety of cellular signaling pathways. Evidence for glycerolipid-derived second messengers was first obtained from the "phosphatidylinositol cycle," which involves a signal-dependent hydrolysis of phosphatidylinositol bisphosphate yielding diacylglycerol and inositol trisphosphate. The role of diacylglycerol in the regulation of protein kinase C activity and its site of interaction with PKC are now well known. Recently, another glycerolipid second messenger, phosphatidic acid, was found to interact with the protooncogenic Raf-1 kinase. In cultured cells, a signal-induced generation of phosphatidic acid was critical for Raf-1 translocation to the cell membrane. Thus, different glycerolipid second messengers appear to regulate distinct targets with exquisite specificity. Analogous to the PI cycle, a "sphingomyelin cycle" was also found to exist, generating sphingolipid second messengers. Ceramide, derived from the agonist-induced hydrolysis of sphingomyelin, is a potent biomolecule with effects in multiple cell signaling pathways. The steroid hormone progesterone stimulated sphingomyelin hydrolysis in Xenopus oocytes. Ceramide, derived from the "sphingomyelin cycle," was sufficient for meiotic cell cycle progression in the oocytes. These results demonstrate the various effects of lipid-derived second messengers and promise exciting discoveries into the roles of lipids in cell signaling.

Animals↗
PubMed2005

Loss of sphingosine kinase-1 activates the intrinsic pathway of programmed cell death: modulation of sphingolipid levels and the induction of apoptosis.

Activation of sphingosine kinase-1 (SK1) by overexpression or agonist stimulation promotes cell proliferation, survival, and anti-apoptosis. Studies on the function of endogenous SK1 are lacking. Endogenous SK1 has been shown to be down-regulated under stress, and knockdown of the enzyme reduces the percentage of viable MCF-7 breast cancer cells (Taha, T. A. et al. 2004. J. Biol. Chem. 279, 20546-20554). In this study, we examined the mechanisms by which SK1 loss affects the growth of cells. Knockdown of the enzyme by small interfering RNA caused cell cycle arrest and induced apoptosis. Cell death involved effector caspase activation, cytochrome c release and Bax oligomerization in the mitochondrial membrane, thus placing SK1 knockdown upstream of the mitochondrial pathway of apoptosis. SK1 knockdown also induced significant increases in ceramide levels in whole cells and in mitochondria enriched fractions of cells. Inhibition of de novo sphingolipid biosynthesis with myriocin significantly attenuated Bax oligomerization and downstream caspase activation after SK1 loss. These studies for the first time implicate endogenous SK1 as an important survival enzyme in MCF-7 cells and link the biological consequences of knocking down the enzyme to its biochemical role as a regulator of sphingolipid metabolism.

Adenocarcinoma↗
PubMed1996

L-cycloserine, an inhibitor of sphingolipid biosynthesis, inhibits HIV-1 cytopathic effects, replication, and infectivity.

Drugs that reduce viral production or prevent viral spread by interference with the host's cellular components are unlikely to induce resistance, in contrast to treatment modalities that interact with the HIV-1 life cycle. Two features make L-cycloserine (L-CS) a candidate drug of this kind: (a) L-CS is a potent inhibitor of the sphingolipid pathway (b) sphingolipids, galactocerebrosides, and sulfatides have been shown, by others, to bind gp120. In a feasibility and efficacy study, we have found that L-CS inhibits HIV-1 replication in a CD4+ lymphoid cell line (CEM) as documented by the reduction of syncytium formation, the number of HIV-1 infected cells, and p24 protein production. This observation may lead to a new strategy for the treatment of HIV-1 infection.

Antimetabolites↗
PubMed2005

A sphingolipid elicitor-inducible mitogen-activated protein kinase is regulated by the small GTPase OsRac1 and heterotrimeric G-protein in rice 1[w].

Mitogen-activated protein kinase (MAPK) cascades are activated in plants during responses to pathogens or to pathogen-derived elicitors and mediate intracellular stress responses. Here, we show that a rice (Oryza sativa) MAPK, OsMAPK6, was posttranslationally activated in a cell culture by a sphingolipid elicitor. Suppression of OsMAPK6 expression by RNA interference resulted in a strong reduction of pathogen-induced Phe ammonia-lyase mRNA, whereas the mRNA level of another rice MAPK, OsMAPK5a, was highly increased. Silencing of a small GTPase, OsRac1, by RNA interference or loss-of-function mutation (d1) of the heterotrimeric G-protein alpha-subunit gene resulted in a strong reduction of the OsMAPK6 protein levels and of kinase activation by a sphingolipid elicitor. Furthermore, coimmunoprecipitation experiments with OsRac1 and OsMAPK6 proteins showed that OsMAPK6 is closely associated with the active form of OsRac1, but not with inactive forms of OsRac1. These results indicate that these two G-proteins regulate an elicitor-inducible MAPK in rice at the protein level.

Amino Acid Sequence↗
PubMed1996

Inhibitors of sphingolipid synthesis modulate interferon (IFN)-gamma-induced intercellular adhesion molecule (ICAM)-1 and human leukocyte antigen (HLA)-DR expression on cultured normal human keratinocytes: possible involvement of ceramide in biologic action of IFN-gamma.

To elucidate the participation of sphingosine and ceramide in the biologic action of cytokines on epidermal keratinocytes, we studied whether inhibitors of sphingolipid synthesis modulate interferon (IFN)-gamma-induced intercellular adhesion molecule (ICAM)-1 and human leukocyte antigen (HLA)-DR expression on cultured normal human keratinocytes. Pretreatment of keratinocytes with L-cycloserine or fumonisin B1, but not 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), significantly suppressed both ICAM-1 and HLA-DR expression induced by IFN-gamma. Because the synthesis of all kinds of sphingolipids is blocked by L-cycloserine and all except that of sphinganine by fumonisin B1, whereas PDMP inhibits the synthesis of glucosylceramide and glycosphingolipids, the result suggests the participation of ceramide and/or sphingosine in IFN-gamma-induced ICAM-1 and HLA-DR expression. Exogenous C2-ceramide reversed the effects of L-cycloserine and fumonisin B1. On the other hand, sphingosine reversed the effect of L-cycloserine, but not of fumonisin B1. These results indicate that ceramide participates in this pathway, as fumonisin B1, but not L-cycloserine, inhibits the synthesis of ceramide from sphingosine. In addition, reverse transcriptase polymerase chain reaction showed that L-cycloserine reduced the mRNA for ICAM-1, HLA-DR alpha, and HLA-DR beta induced by IFN-gamma, and C2-ceramide and sphingosine antagonized the effect of L-cycloserine. Furthermore, the degradation rate of fluorescent sphingomyelin into ceramide in keratinocytes was increased by IFN-gamma, suggesting that IFN-gamma activates sphingomyelin hydrolysis in keratinocytes. These observations suggest the possible role of ceramide in IFN-gamma-induced ICAM-1 and HLA-DR expression on keratinocytes. Ceramide may function as an endogenous modulator mediating the cytokine signals in keratinocytes.

Base Sequence↗
PubMed1998

Lipid second messengers derived from glycerolipids and sphingolipids, and their role in smooth muscle function.

The processes that link activation of an external receptor to the internal mechanisms that elicit a physiological response have been the subject of extensive investigation. It has been established that rather than just being an inert barrier to protect the cell from environmental damage, there are populations of phospholipids located within the plasma membrane that act as a reservoir for signalling molecules and when a receptor binds its appropriate activating ligand a chain of events is initiated which leads to the breakdown of these lipids and the release of second messengers. Such processes are rapid enough for physiological responses to be effected. The purpose of this review is to examine the profile of lipid second messengers derived from glycerophospholipids and sphingolipids. In the former class are included phosphoinositide and phosphatidylcholine and the latter includes sphingomyelin. Hydrolysis of such parent compounds is mediated by phospholipases and the profile of metabolites appears to be agonist specific and modulated by a number of mechanisms including heterotrimeric G-protein subunits, small G-proteins, alterations in intracellular calcium concentration, protein kinase C and tyrosine kinases. The recent interest in sphingolipids, particularly in vascular smooth muscle cells, has been provoked by the observation that ceramide and sphingoid base formation is observed in response to vasoconstrictor hormones.

Animals↗
PubMed1995

Topical sphingolipids in antisepsis and antifungal therapy.

Sphingosine and sphinganine, free sphingolipids of the stratum corneum, are, in vitro, strongly inhibitory for both bacteria and fungi. Whether or not they are suitable, indeed active, in vivo was examined: (i) on human volunteers, first as a preventative antiseptic against subsequently applied Staphylococcus aureus and Candida albicans, and second as a restorative antiseptic against the previously expanded normal skin flora; and (ii) on guinea-pigs as therapy for experimental C. albicans and Trichophyton mentagrophytes infections. In the antiseptic studies, which involved 200 micrograms/cm2 of sphinganine in ethanol (50 microliters of a 1.6% solution), up to three-log reductions in the population of target micro-organisms were obtained, compared with vehicle and untreated controls (P < 0.001). The daily application of sphingosine as 1.5% ethanol-petrolatum ointment was able to diminish inflammation slightly in dermatophyte-infected guinea-pigs (P = 0.02-0.05), although the animals remained culture positive over the 3-week sampling period. The candida infections, treated daily with 1.5% sphinganine in ethanol, showed no improvement in inflammation compared with controls, except for 2 days of the 2-week observation period (P = 0.01-0.03); however, by the fourth day of therapy the yeast was eliminated in 75% of animals. No gross toxicity was observed among animals or human volunteers. These experiments further support simple sphingolipids as important antimicrobial agents of the cutaneous barrier and point toward a new biochemical approach in treating infectious disease.

Animals↗
PubMed2004

Rafts and sphingolipid biosynthesis in the kinetoplastid parasitic protozoa.

Summary relatively large rafts are a feature of activated mammalian cells. These studies allow us to consider the functional role of lipid rafts in kinetoplastid parasites, which are particularly rich in lipid-anchored surface molecules. Morphological, biochemical and genetic studies indicate that lipid rafts (and sphingolipid biosythesis) are important in the differentiation of extracellular Leishmania to mammalian-infective metacyclic promastigotes, perhaps orchestrating the clearly observable reorganization of the plasma membrane during this process that leads to an activated metacyclic primed for invasion. However, the first step in the sphingolipid biosynthetic pathway (mediated by serine palmitoyltransferase), and at least regulated, de novo sphingoid base and ceramide synthesis, are not essential for the pathogenesis of intramacrophage Leishmania amastigotes.

Animals↗
PubMed1992

Metabolic effects of short-chain ceramide and glucosylceramide on sphingolipids and protein kinase C.

Recent studies have identified a potential role for glucosylceramide (GlcCer) in growth promotion and hormonal signalling. In an effort to demonstrate a growth-promoting activity of GlcCer, we prepared a GlcCer having a short-chain acid (octanoyl), in the belief that this glycolipid could be absorbed more readily and more uniformly by cultured cells. By using a mixture of two specific lecithins, dioleoylglycerophosphocholine and 1-stearoyl-2-palmitoylglycerophosphocholine, we were able to prepare dispersions containing a high molar proportion of the GlcCer and the related ceramide, octanoyl sphingosine. Unexpectedly, both sphingolipids inhibited protein and DNA synthesis in Madin-Darby canine kidney cells and produced large increases in the levels of the natural lipids, GlcCer, ceramide, free sphingosine, and an amine that may be glucosylsphingosine (GlcSph). Decreases were seen in the level of sphingomyelin and the proportion of protein kinase C in the cell membranes. The level of lactosylceramide was diminished by octanoyl GlcCer but elevated considerably by octanoyl sphingosine. Diacylglycerols were increased by the lecithins in the liposomes, but the exogenous sphingolipids had no effect. Octanoyl sphingosine labeled in the sphingoid base yielded labeled GlcCer and sphingomyelin labeled in both long-chain and very-long-chain fatty acid families, as well as the octanoyl version. The two families of ceramides, however, had relatively little radioactivity. Some of these changes are attributed to rapid hydrolysis of the added lipids with the formation, particularly from the ceramide, of sphingosine and its anabolic metabolite, GlcSph. Several observations support the idea that the octanoyl sphingosine inhibited the phosphocholinetransferase that synthesizes sphingomyelin while the octanoyl GlcCer inhibited GlcCer beta-glucosidase and GlcCer galactosyltransferase. The use of unnatural short-chain lipids in the study of cell growth and other phenomena may result in unexpected changes in related metabolites and the findings from such experiments should therefore be interpreted cautiously.

Animals↗
PubMed1991

Incorporation of very-long-chain fatty acids into sphingolipids of cultured neural cells.

We examined effects of exogenous very-long-chain fatty acids on lipids of cultured chick neurons and astrocytes. When chick neurons were incubated in chemically defined medium containing 10 microM nervonic acid (C24:1) for 7 days, it was found that a major fatty acid moiety of gangliosides and sphingomyelin was nervonic acid itself, which was not normally detected in the sphingolipid fraction. This alteration in the fatty acid composition apparently occurred in each ganglioside species. Under these experimental conditions, nervonic acid was not found in the glycerophospholipid fraction, and the amounts of triacylglycerol and free nervonic acid increased. Addition of behenic acid (C22:0) or erucic acid (C22:1) also induced changes in the fatty acid composition of gangliosides. When chick astrocytes were incubated in the presence of 10 microM nervonic acid for 7 days, no significant change was observed in the fatty acid composition of gangliosides. These studies indicate that the manipulation of the fatty acid moiety of sphingolipids in cultured neurons is possible.

Animals↗
PubMed1998

Regulatory roles for sphingolipids in the growth of polarized neurons.

Over the past few years, our laboratory has focused on defining the regulatory roles of sphingolipids at various stages of neuronal development. These studies have been performed using primary cultures of hippocampal neurons, which are unique among neuronal cultures inasmuch as they develop by a well-characterized and stereotypic sequence of events that gives rise to fully differentiated axons and dendrites. The data demonstrate that sphingolipids play at least three distinct roles in regulating neuronal development, namely (1) that ceramide enhances the formation of minor neuronal processes from lamellipodia, (2) that glucosylceramide synthesis is required for both normal and accelerated axon growth, and (3) that at both of these stages of development, ceramide induces apoptotic cell death at high concentrations. These observations are consistent with the possibility that minor process formation and apoptosis are regulated by ceramide-dependent signaling pathways, whereas axonal growth may require GlcCer synthesis to support an intracellular transport pathway.

Animals↗
PubMed1999

Low-pH-dependent fusion of Sindbis virus with receptor-free cholesterol- and sphingolipid-containing liposomes.

There is controversy as to whether the cell entry mechanism of Sindbis virus (SIN) involves direct fusion of the viral envelope with the plasma membrane at neutral pH or uptake by receptor-mediated endocytosis and subsequent low-pH-induced fusion from within acidic endosomes. Here, we studied the membrane fusion activity of SIN in a liposomal model system. Fusion was followed fluorometrically by monitoring the dilution of pyrene-labeled lipids from biosynthetically labeled virus into unlabeled liposomes or from labeled liposomes into unlabeled virus. Fusion was also assessed on the basis of degradation of the viral core protein by trypsin encapsulated in the liposomes. SIN fused efficiently with receptor-free liposomes, consisting of phospholipids and cholesterol, indicating that receptor interaction is not a mechanistic requirement for fusion of the virus. Fusion was optimal at pH 5.0, with a threshold at pH 6.0, and undetectable at neutral pH, supporting a cell entry mechanism of SIN involving fusion from within acidic endosomes. Under optimal conditions, 60 to 85% of the virus fused, depending on the assay used, corresponding to all of the virus bound to the liposomes as assessed in a direct binding assay. Preincubation of the virus alone at pH 5.0 resulted in a rapid loss of fusion capacity. Fusion of SIN required the presence of both cholesterol and sphingolipid in the target liposomes, cholesterol being primarily involved in low-pH-induced virus-liposome binding and the sphingolipid catalyzing the fusion process itself. Under low-pH conditions, the E2/E1 heterodimeric envelope glycoprotein of the virus dissociated, with formation of a trypsin-resistant E1 homotrimer, which kinetically preceded the fusion reaction, thus suggesting that the E1 trimer represents the fusion-active conformation of the viral spike.

Animals↗
PubMed2002

Effects of membrane potential and sphingolipid structures on fusion of Semliki Forest virus.

Cells expressing the E1 and E2 envelope proteins of Semliki Forest virus (SFV) were fused to voltage-clamped planar lipid bilayer membranes at low pH. Formation and evolution of fusion pores were electrically monitored by capacitance measurements, and membrane continuity was tracked by video fluorescence microscopy by including rhodamine-phosphatidylethanolamine in the bilayer. Fusion occurred without leakage for a negative potential applied to the trans side of the planar membrane. When a positive potential was applied, leakage was severe, obscuring the observation of any fusion. E1-mediated cell-cell fusion occurred without leakage for negative intracellular potentials but with substantial leakage for zero membrane potential. Thus, negative membrane potentials are generally required for nonleaky fusion. With planar bilayers as the target, the first fusion pore that formed almost always enlarged; pore flickering was a rare event. Similar to other target membranes, fusion required cholesterol and sphingolipids in the planar membrane. Sphingosine did not support fusion, but both ceramide, with even a minimal acyl chain (C(2)-ceramide), and lysosphingomyelin (lyso-SM) promoted fusion with the same kinetics. Thus, unrelated modifications to different parts of sphingosine yielded sphingolipids that supported fusion to the same degree. Fusion studies of pyrene-labeled SFV with cholesterol-containing liposomes showed that C(2)-ceramide supported fusion while lyso-SM did not, apparently due to its positive curvature effects. A model is proposed in which the hydroxyls of C-1 and C-3 as well as N of C-2 of the sphingosine backbone must orient so as to form multiple hydrogen bonds to amino acids of SFV E1 for fusion to proceed.

Hydrogen Bonding↗