Glycosphingolipids and cell death

Sphingolipids have been implicated in various cellular processes including growth, cell-cell or ligand-receptor interactions, and differentiation. In addition to their importance as reservoirs of metabolites with important signaling properties, sphingolipids also help provide structural order to plasma membrane lipids and proteins within the bilayer. Glycosylated sphingolipids, and sphingomyelin in particular, are involved in the formation of lipid rafts. Although it is well accepted that ceramide, the backbone of all sphingolipids, plays a critical role in apoptosis, less is known about the biological functions of glycosphingolipids. This review summarizes current knowledge of the involvement of glycosphingolipids in cell death and in other pathological processes and diseases. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Sphingolipids and gangliosides of the nervous system in membrane function and dysfunction

Simple sphingolipids such as ceramide and sphingomyelin (SM) as well as more complex glycosphingolipids play very important roles in cell function under physiological conditions and during disease development and progression. Sphingolipids are particularly abundant in the nervous system. Due to their amphiphilic nature they localize to cellular membranes and many of their roles in health and disease result from membrane reorganization and from lipid interaction with proteins within cellular membranes. In this review we discuss some of the functions of sphingolipids in processes that entail cellular membranes and their role in neurodegenerative diseases, with an emphasis on SM, ceramide and gangliosides.     

Sphingolipid transport in eukaryotic cells

Sphingolipids constitute a sizeable fraction of the membrane lipids in all eukaryotes and are indispensable for eukaryotic life. First of all, the involvement of sphingolipids in organizing the lateral domain structure of membranes appears essential for processes like protein sorting and membrane signaling. In addition, recognition events between complex glycosphingolipids and glycoproteins are thought to be required for tissue differentiation in higher eukaryotes and for other specific cell interactions. Finally, upon certain stimuli like stress or receptor activation, sphingolipids give rise to a variety of second messengers with effects on cellular homeostasis. All sphingolipid actions are governed by their local concentration. The intricate control of their intracellular topology by the proteins responsible for their synthesis, hydrolysis and intracellular transport is the topic of this review.     

The cell biology of glycosphingolipids

Glycosphingolipids, a family of heterogeneous lipids with biophysical properties conserved from fungi to mammals, are key components of cellular membranes. Because of their tightly packed backbone, they have the ability to associate with other sphingolipids and cholesterol to form microdomains called lipid rafts, with which a variety of proteins associate. These microdomains are thought to originate in the Golgi apparatus, where most sphingolipids are synthesized, and are enriched at the plasma membrane. They are involved in an increasing number of processes, including sorting of proteins by allowing selectivity in intracellular membrane transport. Apart from being involved in recognition and signaling on the cell surface, glycosphingolipids may fulfill unexpected roles on the cytosolic surface of cellular membranes.     

A simple and novel method for tritium labeling of gangliosides and other sphingolipids

A very simple method for introducing tritium specifically into the ceramide portion of gangliosides, neutral glycosphingolipids and sphingomyelin has been developed using potassium boro [3H]hydride and palladium as catalyst. In this way six different gangliosides, five different neutral glycosphingolipids and sphinomyelin with high specific radioactivity were prepared for the first time. This simple procedure which does not require sophisticated apparatuses is suitable for large scale preparation of tritium-labeled sphingolipids as well as for nanogram quantities of individual sphingolipids so as to serve for analytical purposes. During the course of the labeling procedure no degradation of even the most labile trisialosyl-gangliotetraosylceramide has been observed. The yield of tritiated compounds is almost quantitative. The specific radioactivity depends on the unsaturation of the ceramide moiety and the specific activity of the boro[3H]hydride employed.     

Sphingolipids in tumor metastases and angiogenesis

This review article summarizes data on the involvement of sphingolipids (sphingosine-1-phosphate, sphingosine-1-phosphocholine, neutral glycosphingolipids, and gangliosides) in tumor metastases and angiogenesis.     

Very long chain sphingolipids: Tissue expression, function and synthesis

Forecort membranes of all mammalian cells contain, in addition to phosphoglycerolipids and cholesterol, substantial amounts of sphingolipids. In most cells the acyl moieties of these sphingolipids are of long chain type (C16-24) and often saturated. However, epidermal keratinocytes and male germ cells largely express sphingolipids with very long chain-acyl moieties (C26-C36) during their differentiation and maturation. This expression seems to depend on CerS3, one of six ceramide synthases. However, the complex biosynthetic sequence for both epidermal and testicular sphingolipids has not been elucidated completely. Whereas it is established that omega-hydroxylated very long chain-sphingolipids are essential for proper skin barrier function, the role of polyunsaturated very long chain-sphingolipids (ceramides, sphingomyelins and glycosphingolipids) in differentiating spermatocytes/spermatids is just beginning to be revealed.     

Organelle biogenesis and intracellular lipid transport in eukaryotes.

The inter- and intramembrane transport of phospholipids, sphingolipids, and sterols involves the most fundamental processes of membrane biogenesis. Identification of the mechanisms involved in these lipid transport reactions has lagged significantly behind that for intermembrane protein traffic until recently. Application of methods that include fluorescently labeled and spin-labeled lipid analogs, new cellular fractionation techniques, topographically specific chemical modification techniques, the identification of organelle-specific metabolism, permeabilized cell methodology, and yeast molecular genetics has contributed to revealing a diverse biochemical array of transport processes for lipids. Compelling evidence now exists for ATP-dependent, ATP-independent, vesicle-dependent, and vesicle-independent transport processes that are lipid and membrane specific. ATP-dependent transport processes include the transbilayer movement of phosphatidylserine and phosphatidylethanolamine at the plasma membrane and the transport of phosphatidylserine from its site of synthesis to the mitochondria. ATP-independent processes include the transbilayer movement of virtually all lipids at the endoplasmic reticulum, the movement of phosphatidylserine between the inner and outer mitochondrial membranes, and the transfer of nascent phosphatidylcholine and phosphatidylethanolamine to the plasma membrane. The ATP-independent movement of lipids between organelles is believed to be due to the action of lipid transfer proteins, but this still remains to be proved. Vesicle-based transport mechanisms (which are also inherently ATP dependent) include the transport of nascent cholesterol, sphingomyelin, and glycosphingolipids from the Golgi apparatus to the plasma membrane and the recycling of sphingolipids and selected pools of phosphatidylcholine from the plasma membrane to the cell interior. The vesicles involved in cholesterol transport to the plasma membrane are different from those involved in bulk protein transport to the cell surface. The vesicles involved in recycling sphingomyelin to and from the cell surface are different from those involved in the assembly of newly synthesized sphingolipids into the plasma membrane. The preliminary characterization of these lipid translocation processes suggests divergent rather than unifying mechanisms for lipid transport in organelle assembly.     

Evidence for de novo sphingolipid biosynthesis in Toxoplasma gondii

Glycolipids are important components of cellular membranes involved in various biological functions. In this report, we describe the identification of the de novo synthesis of glycosphingolipids by Toxoplasma gondii tachyzoites. Parasite-specific glycolipids were identified by metabolic labelling of parasites with tritiated serine and galactose. These glycolipids were characterised as sphingolipids based on the labelling protocol and their insensitivity towards alkaline treatment. Synthesis of parasite glycosphingolipids were inhibited by threo-phenyl-2-palmitoylamino-3-morpholino-1-propanol and L-cycloserine, two well-established inhibitors of de novo sphingolipid biosynthesis. The identified glycolipids were insensitive towards treatment with endoglycoceramidase II indicating that they might belong to globo-type glycosphingolipids. Taken together, we provide evidence for the first time that T. gondii is capable of synthesising glycosphingolipids de novo.     

Synthesis of potentially caged sphingolipids, possible precursors of cellular modulators and second messengers

An increasing number of sphingolipids, glycosphingolipids and some of their degradation products have been recognized in recent years as second messengers involved in signal transduction and as modulators of numerous cellular functions. These can be converted into inert, caged compounds, introduced into cells and tissues and subsequently photolysed to active compounds thus enabling the study of fast biological processes. The novel, potentially caged compounds synthesized here are substituted 2-nitrobenzyl urethans and 2-nitrobenzyl amines derived from sphingosine, dihydrosphingosine, N-methylsphingosine, N-methyldihydrosphingosine, psychosine and glucosylsphingosine. Upon irradiation of the afore mentioned compounds they release, or are expected to release, the free biologically active amines.     

Intracellular transport,organelle biogenesis and establishment of golgi identity: Impact of brefeldin a on the activity of lipid synthesizing enzymes

• 1.1. The effect of brefeldin A (BFA) on generation of transport vesicles, synthesis of phospho-glycerides, sphingosine and ceramides, and utilization of the sphingolipid precursors in the formation of sphingomyelin and glycosphingolipids in Golgi was investigated.
• 2.2. In the presence of 5–10 gmg/ml BFA, the incorporation of [³H]palmitate into glycerides, phosphoglycerides and sphingolipids decreased 45–60%, and the production of endoplasmic reticulum transport vesicles was reduced 30–50%.
• 3.3. In Golgi membranes, the presence of 5–10 gmg/ml BFA in the mixture, assembled to generate Golgi vesicles, evoked inhibitory effect on the synthesis of sphingomyelin, glycosphingolipids and phosphatidylcholine. On average, the synthesis of the sphingolipids and phosphatidylcholine and production of Golgi transport vesicles declined to 30–40%.
• 4.4. Addition of 5–10 gmg/ml BFA to the assay mixture prepared to measure the activity of cytidylyltransferase, phosphocholine diacylglyceroltransferase, and serine palmitoyltransferase, caused up to 50% inhibition of the enzymes involved in the synthesis of phosphatidylcholine and up to 70% inhibition of the enzyme generating 3-ketosphinganine.
• 5.5. The results suggest that BFA inhibits the synthesis of phosphoglycerides and sphingolipids. This, at first, is displayed in reduced production of endoplasmic reticulum and Golgi transport vesicles, while the depletion of sphingolipids abrogates the identity of Golgi membranes.

     

Thermotropic behavior and lateral distribution of very long chain sphingolipids

Sphingolipids containing very long acyl chains are abundant in certain specialized tissues and minor components of plasma membranes in most mammalian cells. There are cellular processes in which these sphingolipids are required, and the function seems to be mediated through sphingolipid-rich membrane domains. This study was conducted to explore how very long acyl chains of sphingolipids influence their lateral distribution in membranes. Differential scanning calorimetry showed that 24:0- and 24:1-sphingomyelins, galactosylceramides and glucosylceramides exhibited complex thermotropic behavior and partial miscibility with palmitoyl sphingomyelin. The T_m was decreased by about 20 °C for all 24:1-sphingolipids compared to the corresponding 24:0-sphingolipids. The ability to pack tightly with ordered and extended acyl chains is a necessity for membrane lipids to partition into ordered domains in membranes and thus the 24:1-sphingolipids appeared less likely to do so. Fluorescence quenching measurements showed that the 24:0-sphingolipids formed ordered domains in multicomponent membranes, both as the only sphingolipid and mixed with palmitoyl sphingomyelin. These domains had a high packing density which appeared to hinder the partitioning of sterols into them, as reported by the fluorescent cholesterol analog cholestatrienol. 24:0-SM was, however, better able to accommodate sterol than the glycosphingolipids. The 24:1-sphingolipids could, depending on head group structure, either stabilize or disrupt ordered sphingolipid/cholesterol domains. We conclude that very long chain sphingolipids, when present in biological membranes, may affect the physical properties of or the distribution of sterols between lateral domains. It was also evident that not only the very long acyl chain but also the specific molecular structure of the sphingolipids was of importance for their membrane properties.     

Differential labelling of sphingolipids by [3H]serine and ([3H]methyl)-methionine in fish leukocytes

Long chain bases are constituents of all sphingolipids and their biosynthesis is presumed to occur via the initial condensation of serine with palmitoyl-CoA. The biosynthesis of phytosphingosine, a long chain base containing three hydroxyl groups, has been less studied than sphingosine but is assumed to occur by hydroxylation of sphinganine. We report in this paper that the label from ([3H]methyl)-methionine is preferentially incorporated into phytosphingosine bases of neutral glycosphingolipids, whereas the label from [3H]serine is mainly incorporated into the sphingoid base of sphingomyelin. These results show that in fish leukocytes the biosynthesis of individual sphingoid bases and their downstream sphingolipid products follow different pathways of metabolism. Our observations suggest that in fish leukocytes the synthesis of the constitutive long chain bases of sphingomyelin and complex glycosphingolipids is coordinately regulated and may be localized in separate compartments.     

Inhibition of very long acyl chain sphingolipid synthesis modifies membrane dynamics during plant cytokinesis

Plant cytokinesis requires intense membrane trafficking and remodeling to form a specific membrane structure, the cell plate that will ultimately separate the daughter cells. The nature and the role of lipids involved in the formation of the cell plate remain unclear. Plant membranes are particularly rich in sphingolipids such as glucosyl-ceramides with long (16 carbons) or very long (24 carbons) acyl chains. We reveal here that inhibition of the synthesis of sphingolipids with very long acyl chains induces defective cell plates with persistent vesicular structures and large gaps. Golgi-derived vesicles carrying material toward the cell plate display longer vesicle–vesicle contact time and their cargos accumulate at the cell plate, suggesting membrane fusion and/or recycling defects. In vitro fusion experiments between artificial vesicles show that glycosphingolipids with very long acyl chains stimulate lipid bilayer fusion. Therefore we propose that the very long acyl chains of sphingolipids are essential structural determinants for vesicle dynamics and membrane fusion during cytokinesis.     

Sphingolipid metabolism in neural cells

Sphingolipids were discovered more than a century ago in the brain. Cerebrosides and sphingomyelins were named so because they were first isolated from neural tissue. Although glycosphingolipids and especially those containing sialic acid in their oligosaccharide moiety are particularly abundant in the brain, sphingolipids are ubiquitous cellular membrane components. They form cell- and species-specific profiles at the cell surfaces that characteristically change in development, differentiation, and oncogenic transformation, indicating the significance of these lipid molecules for cell-cell and cell-matrix interactions as well as for cell adhesion, modulation of membrane receptors and signal transduction. This review summarizes sphingolipid metabolism with emphasis on aspects particularly relevant in neural cell types, including neurons, oligodendrocytes and neuroblastoma cells. In addition, the reader is briefly introduced into the methodology of lipid evaluation techniques and also into the putative physiological functions of glycosphingolipids and their metabolites in neural tissue.     

Sphingolipid metabolism in neural cells

Sphingolipids were discovered more than a century ago in the brain. Cerebrosides and sphingomyelins were named so because they were first isolated from neural tissue. Although glycosphingolipids and especially those containing sialic acid in their oligosaccharide moiety are particularly abundant in the brain, sphingolipids are ubiquitous cellular membrane components. They form cell- and species-specific profiles at the cell surfaces that characteristically change in development, differentiation, and oncogenic transformation, indicating the significance of these lipid molecules for cell-cell and cell-matrix interactions as well as for cell adhesion, modulation of membrane receptors and signal transduction. This review summarizes sphingolipid metabolism with emphasis on aspects particularly relevant in neural cell types, including neurons, oligodendrocytes and neuroblastoma cells. In addition, the reader is briefly introduced into the methodology of lipid evaluation techniques and also into the putative physiological functions of glycosphingolipids and their metabolites in neural tissue.     

Membrane dynamics and cell polarity: the role of sphingolipids

In recent years, glycosphingolipids (GSLs) have attracted widespread attention due to the appreciation that this class of lipids has a major impact on biological life. Inhibition of the synthesis of glucosylceramide, which serves as a precursor for the generation of complex glycosphinglipids, is embryonic lethal. GSLs play a major role in growth and development. Metabolites of sphingolipids, such as ceramide, sphinganine, and sphingosine, may function as second messengers or regulators of signal transduction that affect events ranging from apoptosis to the (co)regulation of the cell cycle. In addition, GSLs can provide a molecular platform for clustering of signal transducers. The ability of sphingolipids, with or without cholesterol, to form microdomains or rafts is critical in sorting and membrane transport that underlies the biogenesis of polarized membrane domains. Here, a brief summary is presented of some recent developments in this field, with a particular emphasis on raft assembly and membrane transport in the establishment of membrane polarity.     

Differential labelling of sphingolipids by [H]serine and ([H]methyl)-methionine in fish leukocytes

Long chain bases are constituents of all sphingolipids and their biosynthesis is presumed to occur via the initial condensation of serine with palmitoyl-CoA. The biosynthesis of phytosphingosine, a long chain base containing three hydroxyl groups, has been less studied than sphingosine but is assumed to occur by hydroxylation of sphinganine. We report in this paper that the label from ([³H]methyl)-methionine is preferentially incorporated into phytosphingosine bases of neutral glycosphingolipids, whereas the label from [³H]serine is mainly incorporated into the sphingoid base of sphingomyelin. These results show that in fish leukocytes the biosynthesis of individual sphingoid bases and their downstream sphingolipid products follow different pathways of metabolism. Our observations suggest that in fish leukocytes the synthesis of the constitutive long chain bases of sphingomyelin and complex glycosphingolipids is coordinately regulated and may be localized in separate compartments.     

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.