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Synthesis of Sphingosine and the Ceramides

The sphingolipids, like the phospholipids, are composed of a polar head group and two nonpolar tails. The core of sphingolipids is the long-chain amino alcohol, sphingosine. The sphingolipids include the sphingomyelins and glycosphingolipids (the cerebrosides, sulfatides, globosides and gangliosides). Sphingomyelins are the only sphingolipid class that are also phospholipids. Sphingolipids are components of all membranes but are particularly abundant in the myelin sheath.

 

 

 

 

 

 

 

 

 

 

 

Structures of sphingosine and a ceramide

"n" indicates any fatty acid may be N-acetylated at this position.

Top: Sphingosine
Bottom: Basic composition of a ceramide

The initiation of the synthesis of the sphingoid bases (sphingosine, dihydrosphingosine, and the various ceramides) takes place via the condensation of palmitoyl-CoA and serine as shown in the Figure below. This reaction occurs on the cytoplasmic face of the endoplasmic reticulum (ER) and is catalyzed by the pyridoxal phosphate-dependent enzyme, serine palmitoyltransferase (SPT). The product of this reaction is 3-ketosphinganine (3-ketodihydrosphingosine). SPT is the rate-limiting enzyme of the sphingolipid biosynthesis pathway. Active SPT is a heterodimeric enzyme composed of two main catalytic subunits. The two main catalytic subunits of SPT are SPTLC1 and SPTLC2, or the SPTLC2L isoform which is also called SPTLC3. The SPTLC1 subunit is present in all catalytically active SPT enzymes. Depending on the tissue in which the SPT complex is expressed there will be either the SPTLC2 or the SPTLC3 subunit in the complex. The LC in the enzyme designations refers to Long-Chain base subunit of SPT. The SPTLC1 gene is located on chromosome 9q22.2 and is composed of 17 exons that generate three alternatively spliced mRNAs. The SPTLC2 gene is located on chromosome 14q24.3 and is composed of 13 exons that encode a 562 amino acid protein. The SPTLC3 gene is located on chromosome 20p12.1 and is composed of 15 exons that encode a 552 amino acid protein.

An additional protein associates with the catalytic subunits to greatly enhance the activity of the enzyme complex as well as to confer acyl-CoA preference to the complex. There are two distinct activity enhancing subunits of SPT referred to as SPT small subunit A (SPTSSA) and SPT small subunit B (SPTSSB. The SSSPTA gene is located on chromosome 14q13.1 and is composed of 2 exons that encode a protein of 71 amino acids. The SPTSSB gene is located on chromosome 3q26.1 and is composed of 4 exons that encode a protein of 76 amino acids.

Following formation of 3-ketosphinganine this compound is reduced to sphinganine (dihydrosphingosine) via the action of 3-ketosphinganine reductase (3-ketodihydrosphingosine reductase). The 3-ketosphinganine reductase enzyme is encoded by the KDSR gene which is located on chromosome 18q21.3 and is composed of 11 exons that encode a protein of 332 amino acids. Sphinganine is then fatty acylated generating dihydroceramide. The acylation of sphinganine (also called dihydrosphingosine) occurs through the activities of six different ceramide synthases (CerS) in humans. These CerS enzymes introduce fatty acids of varying lengths [designated by the –(CH2)n– in the structure] and degrees of unsaturation. In other organisms the ceramide synthases are referred to as sphinganine N-acyl transferases. The actions of CerS and the role of ceramide in biological responses is covered below. Dihydroceramide is then unsaturated in the original palmitic acid portion of the molecule by the enzyme dihydroceramide desaturase 1 (DES1). The official designation for DES1 is delta(4)-desaturase, sphingolipid 1 which is encoded by the DEGS1 gene located on chromosome 1q42.11 that is composed of 5 exons encoding a protein of 323 amino acids.

Following conversion to ceramide, sphingosine is released via the action of ceramidase. Sphingosine can be re-converted to a ceramide by condensation with a fatty-acyl-CoA catalyzed by the various CerS. There are at least two ceramidase genes in humans both of which are defined by their pH range of activity: acid and neutral. Acid ceramidase (N-acylsphingosine amidohydrolase) is encoded by the ASAH1 gene. The ASAH1 gene is located on chromosome 8p22 and is composed of 16 exons that generate three alternatively spliced mRNAs. When studied in mice it has been shown that the ASAH1 gene is critical for early embryo survival and for removing ceramide from the embryo to prevent the default apoptosis pathway. Defects in the human ASAH1 gene result in the lysosomal storage disease Farber lipogranulomatosis. Neutral ceramidase (non-lysosomal ceramidase) is encoded by the ASAH2 gene and the enzyme is expressed in the apical membranes of the proximal and distal tubules of the kidney, endosome-like organelles in heptocytes, and in the epithelial cells of the gut. The ASAH2 gene is located on chromosome 10q11.21 and is composed of 22 exons that generate two alternatively spliced mRNAs. By studying the effects of ASAH2 knock-out mice it has been determined that neutral ceramidase is involved in the catabolism of dietary sphingolipids and the regulation of bioactive sphingolipid metabolites in the intestinal tract.

Synthesis of sphingosine

Pathway for ceramides and sphingosine synthesis. The synthesis of ceramides can occur via the de novo pathway or the hydrolysis pathway. The de novo pathway begins with the transamination of palmitoyl-CoA via a condensation reaction with serine. This reaction, catalyzed by serine palmitoyltransferase, represents the rate-limiting step in ceramide synthesis. The hydrolysis pathway (not shown) to ceramides utilizes sphingomyelins as the substrates through the action of sphingomyelinase. Ceramides can serve as the substrates for sphingosine synthesis and sphingosine can serve as a substrate for ceramide synthesis, as depicted, through the actions of ceramidases and ceramide synthases.

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Sphingomyelin Synthesis and Metabolism

Sphingomyelins are sphingolipids that are also phospholipids. Sphingomyelins are important structural lipid components of nerve cell membranes. The predominant sphingomyelins contain palmitic or stearic acid N-acylated at carbon 2 of sphingosine.

Structure of a sphingomyelin

A sphingomyelin

The sphingomyelins are synthesized by the transfer of phosphorylcholine from phosphatidylcholine to a ceramide in a reaction catalyzed by sphingomyelin synthases (SMS). There are two SMS genes in humans identified as SMS1 and SMS2. SMS1 is found in the trans-Golgi apparatus while SMS2 is predominantly associated with the plasma membrane. The SMS1 enzyme is encoded by the SGMS1 gene located on chromosome 10q11.2 which is composed of 15 exons that encode a 413 amino acid protein with five transmembrane-spanning domains. The SMS2 enzyme is encoded by the SGMS2 gene located on chromosome 4q25 which is composed of 12 exons that generate three alternatively spliced mRNAs, all of which encode the same 365 amino acid protein.

Metabolism of sphingomyelins

Metabolism of the sphingomyelins. The interconversion of ceramides and sphoingomyelins occurs as a result of the actions of sphingomyelinases and sphingomyelin synthases. The fatty acid incorporated into a ceramide by sphingomyelin synthases is most commonly derived from a phosphatidylcholine, PC.

As shown in the Figure above, sphingomyelins are degraded via the action of sphingomyelinases resulting in release of ceramides and phosphocholine. The sphingomyelinase in humans functions at acidic pH and is, therefore, referred to as acid sphingomyelinase (ASMase or aSMase). The human ASMase is encoded for by the sphingomyelin phosphodiesterase-1 gene (gene symbol: SMPD1) wich is located on chromosome 11p15.4–11p15.1 and is composed of 6 exons that generate two alternatively spliced mRNAs. These mRNAs encode ASMase isoform 1 which is a 631 amino acid glycoprotein and ASMase isoform 2 which is a 620 amino acid glycoprotein. Defects in the SMPD1 gene result in the lysosomal storage disease known as Niemann-Pick disease. There are in fact two major forms of Niemann-Pick (NP) disease. NP disease caused by acid sphingomyelinase deficiencies comprises types A and B, referred to as NPA and NPB. The other form of NP disease comprises types C1 and C2, the former due to defects in the NPC1 gene and the latter due to defects in a gene identified as NPC2.

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Metabolism of the Ceramides

The overall level of ceramides in a cell is a balance between the need for sphingosine and sphingosine derivatives, such as sphingosine-1-phosphate (discussed in detail below), and the sphingomyelins. With respect to the sphingomyelins they serve a dual purpose of being important membrane phospholipids and as a reservoir for ceramides. The conversion of both dihydrosphingosine (sphinganine) and sphingosine to ceramide is catalyzed by the ceramide synthases (CerS). As indicated above there are six CerS in humans identified as CerS1 through CerS6 encoded for by six genes. CerS were originally referred to as Lass genes (for Longevity Assurance) based on their homology to the yeast gene, longevity assurance gene-1 (LAG1). LAG1 was so-called because deletion of the gene in yeast prolonged their life-span. An additional related gene in yeast is referred to as LAC1 and when both genes are deleted yeast exhibit poor growth or die. A human gene, originally identified as UOG-1 (upstream of growth and differentiation factor-1), was shown to complement a LAG1 deletion in yeast and when overexpressed in mammalian cells resulted in increased ceramide synthesis. The ceramides synthesized by the enzyme contained exclusively stearic acid (a C18 fatty acid). Subsequent studies demonstrated that other human LAG homologs, originally identified as translocating chain-associating membrane proteins (TRH) synthesized ceramides with varying fatty acyl chain length. Each of these genes are now identified as CerS.

Each CerS exhibits fatty acyl chain length specificity as well as differential tissue distribution. CerS1 is specific for stearic acid (C18) and is expressed in brain, skeletal muscle, and testis. CerS2 is specific for C20–C26 fatty acids and is expressed in the liver and kidney. CerS3 is specific for C22–C26 fatty acids and is expressed in the skin and testis. CerS4 is specific for C18–C20 fatty acids and is ubiquitously expressed but with highest levels in liver, heart, skin, and leukocytes. CerS5 is specific for palmitic acid (C16) and is ubiquitously expressed at low levels. CerS6 is specific for myristic (C14) and palmitic acid and is expressed at low levels in all tissues. CerS1 is structurally and functionally distinct from the other five CerS all of which contain a homeobox-like domain.

The biological significance of ceramide synthesis and the activity of the CerS is demonstrated by studies in several different types of human cancers. In this regard CerS1 appears to most significant. Head and neck squamous cell carcinomas (HNSCC) exhibit a downregulation of C18-ceramide levels when compared to adjacent normal tissue. In addition, a balance between the levels of C16- and C18-ceramides is associated with the state of clinical progression of HNSCC. In the chemotherapy of certain cancers, CerS1 activity may also play a role. Enhanced expression of CerS1 has been shown to sensitize cells to a variety of chemotherapeutic drugs such as cisplatin, vincristine, and doxorubicin. In HNSCC CerS1 is involved in regulating apoptotic cell death via doxorubicin-induced caspase activation. When myeloid leukemia cells are treated with Gleevec® (imatinib) there is an increased production of C18-ceramide which is involved in the induction of apoptosis. The proposed mechanism for ceramide involvement in apoptotic processes involves the activation of the aspartate protease cathepsin D. Cathepsin D is associated with membranes and when activated by ceramides is released to the cytosol where it triggers the mitochondrial apoptosis pathway. Further evidence for the role of ceramides in negative growth responses is seen in cell cultures to which ceramide analogues are added. These types of assays demonstrate that ceramides induce oxidative stress, growth arrest, and apoptosis and/or necrosis.

When derived from the sphingomyelins, ceramides are the products of the action of acid sphingomyelinase (ASMase). The importance of sphingomyelin as a source of ceramide can be evidenced by the fact that the activation of the ASMase pathway is a shared response to the effects of cytokines, stress, radiation, chemotherapeutic drugs, and pathogenic and cytotoxic agents. Several receptor- and non-receptor-mediated pathways, activated in response to stress, such as those involving death ligands like TNF-α and TNF-related apoptosis inducing ligands (TRAIL), are coupled to the activation of ASMase activity. The induction of ASMase, in response to apoptotic triggers, results in increased production of ceramides which then can initiate aspects of the apoptosis pathways as described above.

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Ceramides and Insulin Resistance

Numerous lines of evidence over the past 10 years have shown that various inducers of cellular stress such as inflammatory activation, excess saturated fatty acid intake, and chemotherapeutics, result in increased rates of ceramide synthesis. In addition, there is ample evidence demonstrating that the accumulation of cellular ceramides is associated with the pathogenesis of diseases such as obesity, diabetes, atherosclerosis, and cardiomyopathy. For example, studies in mice have correlated endogenous ceramides and glucosylceramides with the antagonism of insulin-stimulated glucose uptake and synthesis. In animal models of obesity, evidence shows that genetic or pharmacological inhibition of ceramide or glucosylceramide biosynthesis leads to increased peripheral insulin sensitivity while at the same time reducing the severity of pathologies associated with insulin resistance including diabetes, atherosclerosis, hepatic steatosis, and/or cardiomyopathy. With respect to overall lipid homeostasis and the role of adipose tissue in disease pathology, studies have revealed roles for the adipokines leptin, adiponectin, and TNF-α in the modulation of cellular ceramide levels.

An enhanced systemic inflammatory status as well as cellular stress have both been associated with insulin resistance. With respect to biological lipids, excess lipid intake, especially saturated fatty acids, leads to mitochondrial and endoplasmic reticulum (ER) stress. Increased fat oxidation in mitochondria leads to the production of reactive oxygen species (ROS) which are known to result in insulin resistance. Both mitochondrial and ER stress can result in apoptosis. Excess fatty acid intake also interferes with normal insulin receptor-mediated signal transduction resulting in insulin resistance. Excess saturated fatty acid intake, particularly palmitic acid, results in increased ceramide synthesis which has been shown to be both a cause and effector of pancreatic β-cell stress resulting in impaired insulin secretion. For more information on the role of fats and mitochondrial stress in insulin resistance visit the Insulin Functions page. Obesity, which results in insulin resistance and development of type 2 diabetes, has long been associated with low-grade systemic inflammation. The correlation between obesity, ceramide synthesis, and insulin resistance is discussed below.

The ability of ceramides to interfere with insulin receptor signaling is the result of blocking the receptors' ability to activate the downstream effector kinase, PKB/AKT. There are three members of the PKB/AKT family of serine/threonine kinases identified as AKT1 (PKB, also PKBα), AKT2 (PKBβ), and AKT3 (PKBγ). Experiments in cell culture, involving both adipocytes and skeletal muscle cells, have shown that ceramides inhibit insulin-stimulated glucose uptake by blocking translocation of GLUT4 to the plasma membrane as well as by interfering with glycogen synthesis. The blockade of PKB/AKT activation is central to the effects of ceramides and can be demonstrated by constitutive overexpression of the kinase which negates the effects of ceramides. This PKB/AKT-blocking action of ceramides has been shown in all cell types tested.

Several lines of evidence have solidified the model of ceramides leading to insulin resistence as a consequence of blockade of PKB/AKT activation. Administration of ceramide to cells in culture blocks the translocation of PKB/AKT to the plasma membrane. This inhibition of translocation is the result of the phosphorylation of a regulatory site in the plekstrin homology (PH domain) of PKB/AKT. The phosphorylation leads to reduced affinity of the kinase for phosphoinositides. The kinase responsible for the ceramide-induced phosphorylation of PKB/AKT is likely to be the atypical PKC isoform PKCζ since this kinase is activated by ceramides in vitro. Additional evidence pointing to a link between ceramides and activation of PKCζ is that mutation of a target serine (S34) in the kinase, to alanine, confers resistance to ceramide action. Also, ceramide addition has been shown to stabilize interactions between PKB/AKT and PKCζ via their recruitment to plasma membrane rafts or caveolae. Another mechanism by which ceramides impact the activity of PKB/AKT is by activating protein phosphatase 2A (PP2A) to dephosphorylate the kinase. Experiments that were designed to specifically inhibit PP2A were shown to prevent the effects of ceramide on PKB/AKT in a number of different cell types. In some cell types, both mechanisms are functional, while in other cell culture systems either PKCζ or PP2A is the central mediator of ceramide effects.

Palmitic acid (C16:0) is the most abundant saturated fatty aid in the circulation. The role of saturated fatty acids in increased levels of ceramides has been demonstrated by adding palmitate to cultured muscle cells. In this system the addition of palmitate results in increased ceramide accumulation while simultaneously inhibiting PKB/AKT. Ceramide synthesis was indeed required for the effect of palmitate addition on the activity of PKB/AKT since pharmacological inhibition of ceramide synthesis or siRNA-mediated knockdown of several enzymes required for ceramide biosynthesis (serine palmitoyltransferase, ceramide synthases, or dihydroceramide desaturase) completely blocks the effects of palmitate on insulin signaling.

An alternative means to examine the effects of ceramides on insulin sensitivity is to block the pathways of ceramide metabolism. Treatment of cells with acid ceramidase inhibitors results in increased endogenous ceramide levels while simultaneously blocking insulin-mediated activation of PKB/AKT. Under conditions of ceramidase inhibition there is an exagerated effect of palmitic acid addition on insulin resistance. Conversely, if one overexpresses acid ceramidase, the inhibition of insulin signaling induced by palmitate addition is completely blocked.

The cellular effects of glucosylceramide, although similar to ceramides themselves, does exhibit cell-type specificity. Glucosylceramide is the precursor for a complex family of gangliosides, for example the GM3 ganglioside. Adipocytes are highly sensitive to the inhibitory effects of glucosylated sphingolipids on insulin actions, whereas muscle cells are unaffected. Addition of GM3 ganglioside to adipocytes inhibits insulin activation of IRS-1. In addition, TNF-α treatment induces GM3 accumulation in membrane lipid rafts allowing for association with the insulin receptor through caveolin-1 present in the rafts. The antagonistic effects of TNF-α can be prevented by first depleting cells of glucosylated ceramides. Obesity is associated with adipose tissue enrichment in the complex gangliosides, GM2, GM1, and GD1a. The significance of the accumulation of these gangliosides has been demonstrated in mice lacking GM3 synthase which generates the major ganglioside precursor. These mice are protected from insulin resistance and glucose intolerance when fed a high-fat diet. Treatment of genetically obese or diet-induced obese mice with highly specific glucosylceramide synthase (UDP-glucose ceramide glucosyltransferase, UGCG) inhibitors results in improved glucose tolerance and increased insulin sensitivity in muscle and liver. Collectively, these studies strongly implicate a role for glucosylated ceramides in increased adipose tissue inflammation, peripheral insulin resistance, and hepatic steatosis.

The most potent reagent used to study the effects of the manipulation of enzymes involved in sphingolipid biosynthesis is the compound myriocin [2-Amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxoicos-6-enoic acid]. Myriocin is a highly specific inhibitor of serine palmitoyltransferase (SPT), which is the first and rate-limiting enzyme in the de novo pathway of ceramide synthesis. See the Figure above showing sphingosine and ceramide synthesis. Myriocin (also known as antibiotic ISP-1 and thermozymocidin) was isolated from themophilic fungi such as Mycelia sterilia and Isaria sinclairii. Extracts from these fungi have been used in traditional Chinese medicine as a treatment for numerous conditions including diabetes. Myriocin can be administered chronically to rodents and it appears to be well tolerated. Addition of myriocin to animals that are models of obesity prevents insulin resistance and the development of diabetes, atherosclerosis, and cardiomyopathy. In addition, myriocin improvesd glucose tolerance, insulin sensitivity and ameliorates hypertension when administered to rodents.

Genetic manipulation of several enzymes in ceramide metabolism has also been shown to insulin sensitizing. In mice heterozygous for the SPT subunit SPTLC2 there is a reduction in peripheral ceramide levels and improved insulin sensitivity when these animals are fed a high-fat diet. Similar results are seen in mice heterozygous for dihydroceramide desaturase 1 (DES1). Both SPT and DES1 are required for ceramide biosynthesis. As described above, a large family of ceramide synthases (CerS) have been identified in mammals. CerS1 is the most abundant isoform expressed in skeletal muscle and is involved primarily in the synthesis of C18:0 ceramides. The level of expression of CerS1 was shown to be significantly elevated in mice fed a high-fat diet. This increase in CerS1 expression was associated with alterations in ceramide levels and reduced glucose tolerance.

Collectively these data demonstrate a complex interrelationship between sphingosine and ceramide metabolism and insulin resistance. As pointed out ceramides can be deacetylated by ceramidases to form sphingosine. As discussed below, sphingosine can be phosphorylated to S1P which is an important biologically active lipid. Ceramides can also be glucosylated by the enzyme, UDP-glucose ceramide glucosyltransferase (also known as glucosylceramide synthase, GCS), forming glucosylceramides which then serve as the building blocks of complex glycosphingolipids. Ceramides can also act as substrates for the sphingomyelin synthases yielding sphingomyelins or they can be phosphorylated (by ceramide kinase) to yield ceramide-1-phosphate. Thus, it is clear that multiple products of the actions of SPT, CerS, and DES1 could all potentially contribute to the development of insulin resistance and diabetes.

As pointed out earleir, obesity is associated with a low-grade systemic inflammatory state. One of the mechanisms involved in this inflammatory status is the activation of toll-like receptors (TLRs). TLR activation leads to enhanced transcription of pro-inflammatory cytokines such as TNF-α and interleukin-6 (IL6). Saturated fatty acids are known to activate TLR4 and this activation is requisite for lipid induction of TNF-α and other cytokines. When TLRs are knocked-out in mice the animals are protected from lipid-induced insulin resistance. The signal transduction cascade initiated by TLR activation involves the downstream effectors IKKβ and NFκB. TLR4 activation has been shown to selectively and strongly increase the levels of sphingolipids within cells. Several studies have shown that ceramide is indeed an obligate intermediate linking TLR4 activation to the induction of insulin resistance.

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The Glycosphingolipids

Glycosphingolipids, or glycolipids, are composed of a ceramide backbone with a wide variety of carbohydrate groups (mono- or oligosaccharides) attached to carbon 1 of sphingosine. The four principal classes of glycosphingolipids are the cerebrosides, sulfatides, globosides and gangliosides.

Cerebrosides have a single sugar group linked to ceramide. The most common of these is galactose (galactocerebrosides), with a minor level of glucose (glucocerebrosides). Galactocerebrosides are found predominantly in neuronal cell membranes. By contrast glucocerebrosides are not normally found in membranes, especially neuronal membranes; instead, they represent intermediates in the synthesis or degradation of more complex glycosphingolipids.

Galactocerebrosides are synthesized from ceramide and UDP-galactose. Excess lysosomal accumulation of glucocerebrosides is observed in Gaucher disease. Glucocerebrosides are only intermediates in the synthesis of complex gangliosides or are found at elevated levels only in disease states such as Gaucher disease, where there is a defect in the catabolism of the complex gangliosides. Thus, the presence of high concentrations of glucocerebrosides in cells such as monocytes and macrophages is indicative of a metabolic defect.

Structure of a glucosylcerebroside

A glucocerebroside

Sulfatides: The sulfuric acid esters of galactocerebrosides are the sulfatides. Sulfatides are synthesized from galactocerebrosides and activated sulfate, 3'-phosphoadenosine 5'-phosphosulfate (PAPS) via the action of enzymes of the sulfotransferase family. Humans express 50 different sulfotransferase genes, 37 of which encode membrane-bound enzymes and 13 of whcich encode cytosolic enzymes. These enzymes are not only involved in the synthesis of sulfatides but in the generation of sulfate esters and sulfo-amines such as those found in the glycosaminoglycans. Excess accumulation of sulfatides is observed in metachromatic leukodystrophy (sulfatide lipodosis).

Structure of 3'-phosphoadenosine 5'-phosphosulfate (PAPS)

Globosides: Globosides represent cerebrosides that contain additional carbohydrates, predominantly galactose, glucose or GalNAc. Lactosyl ceramide is a globoside found in erythrocyte plasma membranes. Globotriaosylceramide (also called ceramide trihexoside) contains glucose and two moles of galactose and accumulates, primarily in the kidneys, of patients suffering from Fabry disease.

Gangliosides: Gangliosides are very similar to globosides except that they also contain NANA (sialic acid) in varying amounts. The specific names for gangliosides are a key to their structure. The letter G refers to ganglioside, and the subscripts M, D, T and Q indicate that the molecule contains mono-, di-, tri and quatra(tetra)-sialic acid. The numerical subscripts 1, 2 and 3 refer to the carbohydrate sequence that is attached to ceramide; 1 stands for GalGalNAcGalGlc-ceramide, 2 for GalNAcGalGlc-ceramide and 3 for GalGlc-ceramide.

Structure of a GM2-ganglioside

Structure of a GM2 ganglioside. The most common GM2 ganglioside is composed of the ceramide backbone, the typical GalNAc-Gal-Glc trisaccharide and the NANA (sialic acid) attached via an α(2,3) glycosidic linkage to the galactose residue. In the GM2 ganglioside trisaccharide core, glucose is attached to the ceramide backbone via a β1 glycosidic linkage, the galactose is attached to the glucose via a β(1,4) glycosidic linkage and the GalNAc is attached to the galactose via a β(1,4) glycosidic linkage.

Deficiencies in lysosomal enzymes, which normally are responsible for the degradation of the carbohydrate portions of various gangliosides, underlie the symptoms observed in rare autosomally inherited diseases termed lysosomal storage diseases, (also called the sphingolipidoses or lipid storage diseases) many of which are listed below.

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Sphingosine-1-phosphate (S1P) Signal Transduction

Lysophospholipids (LPs) are minor lipid components compared to the major membrane phospholipids such as phosphatidylcholline (PC), phosphatidylethanolamine (PE), and sphingomyelin. The LPs were originally presumed to be simple metabolic intermediates in the de novo biosynthesis of phospholipids. However, subsequent studies demonstrated that the LPs exhibited biological properties resembling those of extracellular growth factors or signaling molecules. The most biologically significant LPs are sphingosine 1-phosphate (S1P), lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), and sphingosylphosphorylcholine (SPC). More information on the biological activities of the lysophospholipids can be found in the Signal Transduction page.

Synthesis of S1P occurs exclusively from sphingosine via the action of sphingosine kinases. Sphingosine is phosphorylated in humans through the action of two related sphingosine kinases encode by the SPHK1 and SPHK2 genes. The intermediate in sphingosine synthesis, dihydrosphingosine, is also a substrate for sphinogosine kinases. The importance of the action of the sphingosine kinases can be shown by the fact that when both are knocked out in transgenic mice embryos are not viable due to the incomplete development of the brain and the vascular system. In vertebrates, S1P is secreted into the extracellular space by specific transporters, one of which is called spinster 2 homolog-2 encoded by the SPNS2 gene. Plasma levels of S1P are high, whereas interstitial fluids contain very low levels. This results in an S1P gradient in different compartments. Hematopoietic cells and vascular endothelial cells are the major sources of the high plasma S1P concentrations. Lymphatic endothelial cells are also thought to secrete S1P into the lymphatic circulation. The majority of plasma S1P is bound to HDL (65%) with another 30% bound by albumin. Recent work has demonstrated that the ability of HDL to induce vasodilation and migration of endothelial cells, as well as to serve a cardioprotective role in the vasculature is dependent on S1P. These studies suggest that the beneficial property of HDL to reduce the risk of cardiovascular disease may be due, in part, on its role as an S1P chaperone.

Degradation of S1P occurs through the action of S1P lyase or the S1P phosphatases (S1P phosphatase-1 and -2) as well as lysophospholipid phosphatase 3 (LPP3). The different S1P phosphatases remove the phosphate, thus, regenerating sphingosine which can re-enter the sphingolipid metabolic pathway. When used as a substrate for phospholipid synthesis, S1P is degraded by S1P lyase to yield hexadecenal and phosphoethanolamine. Phosphoethanolamine is the direct precursor for the synthesis of the phospholipid phosphatidylethanolamine (PE). The hexadecenal is converted into hexadecenoic acid by hexadecenal dehydrogenase and then into palmitoyl-CoA. The degradation of S1P by the S1P lyase pathway serves as an important pathway for the conversion of sphingolipids into glycerolipids.

Synthesis of sphingosine-1-phosphate

Metabolism of sphingosine-1-phosphate. Sphingosine-1-phosphate (S1P) can either be turned over and recycled or catabolized to phosphoethanolamine for use in phosphatidylethanolamine synthesis. The S1P recycling reaction begins with removal of the phosphate via the action of S1P phosphatases. The resulting sphingosine can then be rephosphorylated to S1P via the action of sphingosine kinase. The degradation of S1P to phosphoethanolamine involves the reaction catalyzed by S1P lyase.

Each of the LPs functions via interaction with specific G-protein coupled receptors (GPCRs) leading to autocrine or paracrine effects. The first GPCR shown to bind S1P was called S1P1. Currently there are five characterized S1P receptors. Because several of the LP receptors were independently identified in unrelated assays, there are several different names for some members of this receptor family. In particular, there is a group of genes that were originally identified as GPCRs and called endothelial differentiation genes (EDGs) that were later found to be the same as several of the LP receptors. Thus S1P1 is also known as EDG1, S1P2 as EDG5, S1P3 as EDG3, S1P4 as EDG6, and S1P5 as EDG8.

The biological activities attributed to S1P interaction with any of the five identified receptors are broad. These activities include involvement in vascular system and central nervous system development, viability and reproduction, immune cell trafficking, cell adhesion, cell survival and mitogenesis, stress responses, tissue homeostasis, angiogenesis, and metabolic regulation.

Sphingosine-1-phosphate (S1P) Receptors

S1P Receptor Alternative Name Gene Symbol G-Proteins Comments
S1P1 EDG1 S1PR1 Gi/o Expressed in brain, heart, spleen, liver, kidney, skeletal muscle, thymus, pancreatic β-cells, and numerous white blood cells. Within the immune system, activation of S1P1 has been shown to block B cell and T cell chemotaxis and infiltration into tissues. In addition S1P1 activation results in inhibition of late-stage maturation processes associated with T cells. Within the central nervous system S1P1 is involved in astrocyte migration and increased migration of neural stem cells. Within the vasculature S1P1 is involved in early vascular system development and endothelial cell functions such as adherens junction assembly and vascular smooth muscle cell development. Within the pancrease S1P1 functions in islet cell survival and insulin secretion.
S1P2 EDG5 S1PR2 Gi/o, Gq, G12/13 Expressed in the brain, heart, spleen, liver, lung, kidney, skeletal muscle, and thymus. S1P2 is involved in the development of epithelial cells, enhancing the survival of cardiac myocytes to ischemic-reperfusion injury, and hepatocyte proliferation and matrix remodeling. Within the vasculature S1P2 promotes mast cell degranulation and decreases vascular smooth muscle cell responses to PDGF-induced migration. In the eye S1P2 activation can result in pathologic angiogenesis and disruption in adherens junction formation.
S1P3 EDG3 S1PR3 Gi/o, Gq, G12/13 Expressed in the brain, heart, spleen, liver, lung, kidney, skeletal muscle, testis, and thymus. S1P3 activation is associated with a worsening of sepsis, increased inflammation and coagulation. However, with respect to cardiac tissues S1P3 promotes survival in response to ischemic-reperfusion injury.t
S1P4 EDG6 S1PR4 Gi/o, G12/13 Expressed in lymphoid tissues (leukocytes) and within a restricted subset of cells in the lung that includes airway smooth muscle cells. The primary responses to S1P4 activation are increased T cell migration and secretion of cytokines.
S1P5 EDG8 S1PR5 Gq, G12/13 Expressed in the brain, spleen, and the skin. Within the brain S1P5 activation is associated with inhibition of migration of oligodendrocyte progenitors while increasing the survival of oligodendrocytes. S1P5 also stimulates natural killer (NK) cell trafficking.

Recent studies have shown that SPHK2 (which contains a nuclear localization signal) and its product S1P are found in the nucleus associated with transcriptional co-repressor complexes that contain histone deacetylase 1 (HDAC1) and HDAC2. The S1P-containing HDAC complexes are prevented from deacetylating lysine residues in the histone tails, thereby altering gene expression patterns. One gene whose expression is upregulated in cells with S1P-HDAC complexes is the cell cycle regulating protein p21 which is an inhibitor of cyclin-dependent kinases (CDK) and is involved in p53-mediated apoptosis. This observation suggests that enhancing SPHK2-S1P-associated complexes in the nucleus could be of clinical benefit in p53-deficient types of cancer. Given that mutations in p53 are some of the most commonly occurring genetic abnormalities in cancer, this could prove highly significant.

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Clinical Significances of Sphingolipids

Some of the most devastating inborn errors in metabolism are those associated with defects in the enzymes responsible for the lysosomal degradation of membrane glycosphingolipids which are particularly abundant in the membranes of neural cells. Many of these disorders lead to severe psycho-motor retardation and early lethality. Because the disorders are caused by defective lysosomal enzymes, with the result being lysosomal accumulation of pathway intermediates, these are often referred to as lysosomal storage diseases. The mucopolysaccharidoses are another class of disorders that are members of the lysosomal storage diseases.

The following figure shows several of the pathways and intermediates in glycosphingolipid metabolism. Enzymes are indicated in green and the disease(s) associated with defects in the indicated enzyme are shown in blue. SAP-A, SAP-B, SAP-C, and SAP-D are the saposins which are a family of small glycoproteins. The saposins (A, B, C, and D) are all derived from a single precursor, prosaposin. The mature saposins, as well as prosaposin, activate several lysosomal hydrolases involved in the metabolism of various sphingolipids. Prosaposin is proteolytically processed to saposins A, B, C and D, within lysosomes but also exists as an integral membrane protein not destined for lysosomal entry. Uncleaved prosaposin can be found in many biological fluids such as seminal plasma, human milk, and cerebrospinal fluid, where it appears to have a different function. Each of the disease names in the image can be clicked to go to a descriptive page of that disease. The Table below the Figure lists some additional lysosomal storage diseases caused by defective sphingolipid metabolism. Click here for a large scale version of this image.

Pathways of sphingolipid degradation

Disorders Associated with Abnormal Sphingolipid Metabolism

Disorder Enzyme Deficiency Accumulating Substance Symptoms
Tay-Sachs disease HexA GM2 ganglioside infantile form: rapidly progressing mental retardation, blindness, early mortality
Sandhoff disease HexA and HexB globoside; GM2 ganglioside infantile form: same symptoms as Tay-Sachs, progresses more rapidly
Tay-Sachs AB variant
GM2 activator deficiency
GM2 activator (GM2A) GM2 ganglioside infantile form: same symptoms as Tay-Sachs
Gaucher disease acid β-glucosidase
(glucocerebrosidase)
glucocerebrosides hepatosplenomegaly, mental retardation in infantile form, long bone degeneration
Fabry disease α-galactosidase A globotriaosylceramide; also called ceramide trihexoside (CTH) kidney failure, skin rashes
Niemann-Pick diseases
Types A and B
Type C


sphingomyelinase
NPC1 protein


sphingomyelins
LDL-derived cholesterol
type A is severe disorder with heptosplenomegaly, severe neurological involvement leading to early death, type B only visceral involvement
Krabbe disease; globoid cell leukodystrophy (GLD) galactocerebrosidase galactocerebrosides mental retardation, myelin deficiency
GM1 gangliosidosis β-galactosidase-1 GM1 gangliosides mental retardation, skeletal abnormalities, hepatomegaly
Metachromatic leukodystrophy;
sulfatide lipodosis
arylsulfatase A sulfatides mental retardation, metachromasia of nerves
Fucosidosis α-fucosidase pentahexosylfucoglycolipid cerebral degeneration, thickened skin, muscle spasticity
Farber lipogranulomatosis acid ceramidase ceramides hepatosplenomegaly, painful swollen joints

One of the most clinically important classes of sphingolipids are those that confer antigenic determinants on the surfaces of cells, particularly the erythrocytes. The ABO blood group antigens are the carbohydrate moieties of glycolipids on the surface of cells as well as the carbohydrate portion of serum glycoproteins. When present on the surface of cells the ABO carbohydrates are linked to sphingolipid and are therefore of the glycosphingolipid class. When the ABO carbohydrates are associated with protein in the form of glycoproteins they are found in the serum and are referred to as the secreted forms. Some individuals produce the glycoprotein forms of the ABO antigens while others do not. This property distinguishes secretors from non-secretors, a property that has forensic importance such as in cases of rape.

Structures of the ABO blood group antigens

R represents the linkage to protein in the secreted forms, sphingolipid (ceramide) in the cell-surface bound form, open square: GlcNAc, open diamond: galactose, filled square: fucose, filled diamond: GalNAc. The linkage in the glycolipid form may include a glucose in a β-1,3 or β-1,4 to the initial galactose residue.

Structure of the ABO blood group carbohydrates

A significant cause of death in premature infants and, on occasion, in full term infants is respiratory distress syndrome (RDS) or hyaline membrane disease. This condition is caused by an insufficient amount of pulmonary surfactant. Under normal conditions the surfactant is synthesized by type II endothelial cells and is secreted into the alveolar spaces to prevent atelectasis following expiration during breathing. Surfactant is comprised primarily of dipalmitoyllecithin; additional lipid components include phosphatidylglycerol and phosphatidylinositol along with proteins of 18 and 36 kDa (termed surfactant proteins). During the third trimester the fetal lung synthesizes primarily sphingomyelin, and type II endothelial cells convert the majority of their stored glycogen to fatty acids and then to dipalmitoyllecithin. Fetal lung maturity can be determined by measuring the ratio of lecithin to sphingomyelin (L/S ratio) in the amniotic fluid. An L/S ratio less than 2.0 indicates a potential risk of RDS. The risk is nearly 75-80% when the L/S ratio is 1.5.

The carbohydrate portion of the ganglioside, GM1, present on the surface of intestinal epithelial cells, is the site of attachment of cholera toxin, the protein secreted by Vibrio cholerae.

These are just a few examples of how sphingolipids and glycosphingolipids are involved in various recognition functions at the surface of cells. As with the complex glycoproteins, an understanding of all of the functions of the glycolipids is far from complete.

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Last modified: May 26, 2017