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Long-Chain (Sphingoid) Bases



1.   Structures and Occurrence

Long-chain bases (sphingoids or sphingoid bases) are the characteristic or defining structural unit of the sphingolipids. The bases are long-chain aliphatic amines, containing two or three hydroxyl groups, and often a distinctive trans-double bond in position 4. To be more precise, they are 2-amino-1,3-dihydroxy-alkanes or alkenes with (2S,3R)-erythro stereochemistry, with various further structural modifications.

Structures of sphingoid bases

In animal tissues, the most commonest or abundant of these is sphingosine ((2S,3R,4E)-2-amino-4-octadecen-1,3-diol) or 4E-sphingenine, i.e. with a C18 aliphatic chain, hydroxyl groups in positions 1 and 3 and an amine group in position 2; the double bond in position 4 has the trans (or E) configuration. This was first characterized in 1947 by Professor Herbert Carter, who was also the first to propose the term “sphingolipides” for those lipids containing sphingosine. It is usually accompanied by the saturated analogue, dihydrosphingosine (or sphinganine).

For shorthand purposes, a nomenclature similar to that for fatty acids can be used; the chain length and number of double bonds are denoted in the same manner with the prefix 'd' or 't' to designate di- and trihydroxy bases, respectively. Thus, sphingosine is denoted as d18:1 and phytosphingosine is t18:0. The position of the double bond may be indicated by a superscript, i.e. 4-sphingenine is d18:1Δ4t or 4E-d18:1. While alternative nomenclatures are occasionally seen in publications, they are not recommended.

The long-chain base composition of individual lipids can vary markedly between species, tissues, organelles and even different membranes within a single organelle. For example, the data in Table 1 is perhaps from an extreme example, but it illustrates that remarkable differences that can exist between lipids in rat liver mitochondria. Only part of the data from the paper cited is listed, but it illustrates that 3-keto-sphinganine, produced in the first step of sphingosine biosynthesis (see below) and normally a minor component of sphingolipids - often not detectable, can vary from 28 to 100% of the sphingoid bases depending on the lipid class and membrane within the organelle.

Table 1. Long chain base composition of some lipid components of mitochondria from rat liver.
  Base (%)
d18:1 d18:0-3keto t21:1 (phyto) Unidentified
 
  Ceramidesa 18 28 53 -
  Glucosylceramidesa 3 95 - 3
  Lactosylceramidesb 100
a whole mitochondria; b mitochondrial inner membrane
Data from Ardail, D. et al. FEBS Letts, 488, 160-164 (2001).

The compositions of long-chain bases of sphingomyelins of some animal tissues are listed in Table 2 of our web page on sphingomyelins, where the main C18 components are accompanied by small amounts of C16 to C19 dihydroxy bases, though the latter attain higher proportions in tissues of ruminant animals. In gangliosides from human brain and intestinal tissues, eicosasphingosine (2S,3R,4E-d20:1) occurs in appreciable concentrations with variable amounts in different regions and membranes. Shorter-chain bases are found in many insect species, and in the fruit fly, Drosophila melanogaster, which is widely used in genetic experiments, the main components are C14 bases, while nematodes produce C17 iso-branched bases.

Phytosphingosine or 4D-hydroxy-sphinganine ((2S,3R,4R)-2-amino-octadecanetriol) is a common long-chain base of mainly plant origin; it is a saturated C18-trihydroxy compound, although unsaturated analogues, for example with a trans (or occasionally a cis (Z)) double bond in position 8, i.e. dehydrophytosphingosine or 4D‑hydroxy-8-sphingenine, tend to be much more abundant (see Table 2 of our web page on ceramide monohexosides for tabulated data on two plant species). In many plant species, there are lipid class preferences also, and dihydroxy long-chain bases are more enriched in glucosylceramides than in glycosylinositolphosphoceramides, for example. This is true in the model plant Arabidopsis thaliana, where the data listed for whole tissue is probably representative largely of the latter lipid (Table 2 below)

Table 2. Sphingolipid long-chain base composition of whole tissue and glycosylceramides from Arabidopsis thaliana.
  Base (%)
  t18:1 (8Z) t18:1 (8E) t18:0 d18:1 (8Z) d18:1 (8E) d18:0
 
  Whole tissue 12 70 13 4 1
  Glycosylceramides 44 22 5 28 2
Data from Sperling, P. et al. Plant Physiol. Biochem., 43, 1032-1038 (2005)

Other plant long-chain bases have double bonds in position 4, which can be of either the cis or trans configuration, although trans-isomers are by far the more common, while the base d18:2Δ4E,8Z/E is found in many species. In A. thaliana, Δ4 long-chain bases are found mainly in the flowers and pollen and then exclusively as a component of the glucosylceramides. In general, the composition is dependent on species, but typically it is composed of up to eight different C18-sphingoid bases, with variable geometry of the double bond in position 8, i.e. (E/Z)-sphing-8-enine (d18:1Δ8), (4E,8E/Z)-sphinga-4,8-dienine (d18:2Δ4,8) and (8E/Z)-4-hydroxy-8-sphingenine (t18:1Δ8); d18:1Δ4, d18:0 and t18:0 are only present in small amounts.

Phytosphingosine is found in significant amounts in intestinal cells of animals also, with much smaller relative proportions in kidney and skin. Although non-mammalian sphingoid bases in general tend to be poorly absorbed from the intestines, a proportion of the phytosphingosine and related sphingoid bases found in animal tissues may enter via the food chain.

The number of different long-chain bases that has been found in animals, plants and microorganisms now amounts to over one hundred, and many of these may occur in a single tissue or organism, but almost always as part of a complex lipid as opposed to in the free form. The aliphatic chains can contain from 14 to as many as 27 carbon atoms, and they can be saturated, monounsaturated and diunsaturated, with double bonds of either the cis or trans configuration. Sphingoid bases with three double bonds, such as sphinga-4E,8E,10E-trienine, have been found in a dinoflagellate and some marine invertebrates. In addition, long-chain bases can have branched chains with methyl substituents (omega-1 (iso), omega-2 (anteiso) or other positions), hydroxyl groups in position 5 or 6, ethoxy groups in position 3, and even a cyclopropane ring. Similarly, saturated and monoenoic, and straight- and branched-chain trihydroxy bases are found. N-Methyl, N,N-dimethyl and N,N,N-trimethyl derivatives of sphingoid bases have been detected in mouse brain. Of these, N,N-dimethylsphingosine is of particular interest in that it inhibits protein kinase C, sphingosine kinase and many other enzyme systems.

Yeasts and fungi tend to have distinctive and characteristic long-chain base compositions. For example, fungi have 9-methyl-4E,8E-sphingadienine as the main sphingoid base in the glucosylceramides but not in the ceramide phosphoinositol glycosides, while yeasts contain mainly the saturated C18 bases sphinganine and phytosphingosine. Only a few bacteria contain sphingolipids, and saturated long-chain bases are often reported, although some similar to those in fungi have been found in the Gram-negative Myxobacterium Sorangium cellulosum.

Structure of 9-methylsphinga-4,8-dienine

Sphingoid bases are surface-active amphiphiles, with critical micellar concentrations of about 20 μM. In that they bear a small positive charge at neutral pH, they are unusual amongst lipids, although their pKa (9.1) is lower than in simple amines as a consequence of intra-molecular hydrogen bonding. Together with their relatively high solubility (> 1μM), this enables them to cross membranes or move between membranes with relative ease. In so doing, they increase the permeability of membranes to small solutes.

The complex sphingolipids are discussed elsewhere in these web pages, but in most the sphingoid base is linked via the amine group to a fatty acid, including very-long-chain saturated and 2-hydroxy components, i.e. to form a ceramide, while a polar head group is attached to the primary hydroxyl moiety to produce more complex sphingolipids. An important exception is sphingosine-1-phosphate, which is not acylated and has signalling functions in cells akin to those of lysophospholipids.


2.   Biosynthesis and Metabolism

Sphinganine: The basic mechanism for the biosynthesis of sphinganine involves condensation of palmitoyl-coenzyme A with serine, catalysed by a membrane-bound enzyme serine palmitoyltransferase, requiring pyridoxal 5’-phosphate, on the cytosolic side of the endoplasmic reticulum in animal cells to form 3-keto-sphinganine as illustrated. This is believed to be the key regulatory or rate-limiting step in sphingolipid biosynthesis and is conserved in all organisms studied to date. Elimination of this enzyme is embryonically fatal in mammals and fruit flies. In mammals, serine palmitoyltransferase is a heterodimer composed of two main subunits, designated SPTLC1 with either SPTLC2 or SPTLC3, together with two small subunits SSSPTA and SSSPTB; the active site is at the interface between the two subunits. The SPTLC1-SPTLC2 complex has a strong preference for C16-CoA as substrate, while the SPTLC1-SPTLC3 complex uses both C14-CoA and C16-CoA with a slight preference for C14-CoA. Addition of either of the two small subunits to the complexes changes the substrate preferences substantially. The activity of the enzyme is governed partly by the availability of serine and partly by ORM-like (ORMDL) proteins, which are ubiquitously expressed trans-membrane proteins located in the endoplasmic reticulum.

Biosynthesis of sphinganine

The keto group is then reduced to a hydroxyl by a specific reductase, also on the cytosolic side of the endoplasmic reticulum, a step that must occur rapidly as these intermediates are rarely encountered in tissues. The enzymes are presumed to be in a similar location in plant cells.

Sphingosine and ceramides: The free sphinganine is rapidly N-acylated by acyl-coA to form dihydroceramides by dihydroceramide synthases, which in animals are located on the endoplasmic reticulum, presumably on the cytoplasmic surface. Animals and plants have multiple isoforms of this enzyme, for which the abbreviated term ‘ceramide synthase’ is now widely applied as they utilize most other sphingoid bases, such as those produced by hydrolysis of sphingolipids, as substrates. They are unique gene products located on different chromosomes with considerable variation in the expression of the enzymes in different cell types within each tissue. Each isoenzyme has distinct specificities for the chain-length of the fatty acyl-CoA moieties but to a limited extent only for the base, suggesting that ceramides containing different fatty acids have differing roles in cellular physiology.

For example, humans and mice have six ceramide synthases, of which ceramide synthase 2 is most abundant and is specific for coA esters of very-long-chain fatty acids (C20 to C26); it is most active in lung, liver and kidney. Ceramide synthase 1 is specific for 18:0 and is located mainly in brain with lower levels in skeletal muscle and testes. Ceramide synthase 3 is responsible for the unusual ceramides of skin and testes and uses C26-CoA and higher including polyunsaturated-CoAs with the latter tissue, while ceramide synthase 4 (skin, liver, heart and leukocytes) uses C18 to C22-CoAs. Ceramide synthases 5 (lung epithelia and brain gray and white matter) and 6 (intestine, kidney and lymph nodes) generate C16 and C18 ceramides. However, hydroxylation and the presence or otherwise of double bonds in the acyl-coAs do not appear to influence the specificity of the ceramide synthases. Also, the expression of mRNA expression for ceramide synthases does not always correlate with the fatty acid composition of sphingolipids in a particular tissue, suggesting that other factors are involved in determining which molecular species are formed. One such is acyl-coenzyme A-binding protein (ACBP), which facilitates the synthesis of ceramides containing very-long fatty acids and stimulates ceramide synthases 2 and 3 especially. There is much more discussion of ceramides and ceramide biosynthesis in a specific web page on this lipid.

Biosynthesis of long-chain bases via ceramide

Insertion of the trans-double bond in position 4 to produce sphingosine occurs only after the sphinganine has been esterified in this way to form a ceramide as illustrated above. The desaturases were first characterized in plants, and this subsequently simplified the isolation of the appropriate enzymes in humans and other organisms. Two dihydroceramide desaturases have now been identified in animals and designated 'DEGS1 and DEGS2'. Both enzymes insert trans double bonds in position 4, but DEGS2 is a dual function enzyme that also acts as a hydroxylase to generate phytoceramides. Distribution of the enzymes in tissues is very different, with DEGS1 expressed ubiquitously but highest in liver, Harderian gland, kidney and lung. DEGS2 expression is largely restricted to skin, intestine and kidney, where phytoceramides are more important. Ceramide synthesis and desaturation occur at the cytosolic surface of the endoplasmic reticulum.

A considerable family of Δ4-sphingolipid desaturases has now been identified, and an early study by Stoffel and colleagues demonstrated that Δ4-desaturation involves first syn-removal of the C(4)- HR and then the C(5)-HS hydrogens. This appears to have been the first evidence that desaturases in general operate in this stepwise fashion. In plants, two distinct types of sphingoid Δ8 desaturase have been characterized that catalyse the introduction of a double bond at position 8,9 of phytosphinganine to form both trans and cis isomers in the ratio of 7:1. It appears that the trans isomer is formed when the hydrogen on carbon 8 is removed first, and the cis when carbon 9 is the point of attack. While the main group of Δ8-desaturases requires a 4-hydroxysphinganine moiety as substrate, the second does not.

Scottish thistlePhytosphingosine is formed from sphinganine, produced as above, by hydroxylation in position 4, possibly via the free base in plants although it can be formed both from sphinganine and a ceramide substrate in yeasts. Sphinganine linked to ceramide is the substrate for 4-hydroxylation in intestinal cells. Much remains to be learned of the processes involved, but it is known that the enzyme responsible is closely related to a Δ4 desaturase. Indeed, it has been shown that there are bifunctional Δ4-desaturase/4-hydroxylases in Candida albicans and mammals (DEGS2 discussed above) with which both 4-hydroxylation and Δ4-desaturation are initiated by removal of the proR C-4 hydrogen.

In Arabidopsis, three different isoforms of ceramide synthase have been identified and denoted LOH1, LOH2 and LOH3. Phytosphingosine is used efficiently by LOH1 and LOH3, but only LOH2 uses sphinganine efficiently; LOH2 and 3 prefer unsaturated long-chain bases. Marked fatty acid specificity is also observed, with LOH2 almost completely specific for palmitoyl-CoA, while LOH1 shows greatest activity for 24:0- and 26:0-CoAs; none utilize unsaturated acyl-CoAs efficiently. In plants, fatty acid desaturases and hydroxylases are also closely related, and sphingolipid fatty acid α-hydroxylation is believed to occur on the ceramide, as opposed to the free acyl chain. However, the substrates for desaturation of long-chain bases in plants (free bases or ceramides) are still uncertain.

Unesterified sphingosine: Although sphingosine per se is absorbed by enterocytes during digestion of dietary sphingolipids in animals and some of this is converted to complex sphingolipids, synthesis of sphingoid bases de novo is essential in most organisms and inhibition of the biosynthetic pathways affects growth and viability. However, this can be tissue specific, as deletion of the liver-specific SPTLC2 in mice, had no effect on liver function, while a comparable deletion adipocyte-specific SPTLC1 caused major tissue defects. Presumably, the latter tissue is unable to take up enough sphingolipid from the circulation to remedy the problem. The essentiality of sphingosine base synthesis in plants has also been demonstrated in studies with mutants in which specific enzymes have been deleted.

A cycle of reactions occurs in tissues by which sphingoid bases are incorporated via ceramide intermediates into sphingolipids (see the web pages on individual sphingolipids), which are utilized for innumerable functions, before being broken down again to their component parts. It is worth noting that all the free sphingosine in tissues must arise by this route, in particular by the action of ceramidases on ceramides. The levels of free sphingoids and their capacities to function as lipid mediators are controlled mainly by enzymic re-acylation to form ceramides, although some is acted upon by sphingosine kinases to produce sphingosine-1-phosphate.

Free sphingosine production from ceramide

Fungal sphingoid bases: Fungi produce trans Δ8 isomers only, but Δ4 and Δ8 desaturases do not occur in the widely studied yeast S. cerevisiae. In the biosynthesis of sphingoid bases in fungi, the double bonds in positions 4 and 8 and the methyl group in position 9 are inserted sequentially into the sphinganine portion of a ceramide, the last by means of an S-adenosylmethionine-dependent methyltransferase similar to plant and bacterial cyclopropane fatty acid synthases. For example, in the yeast Pichia pastoris, there is a distinct ceramide synthase, which utilizes dihydroxy sphingoid bases and C16/C18 acyl-coenzyme A as substrates to produce ceramides. The long-chain-base components of the ceramide are then desaturated in situ by a Δ4 desaturase and the fatty acid components are hydroxylated in position 2. Further desaturation of the long-chain base component by a Δ8 desaturase occurs before the methyl group in position 9 is introduced by an S-adenosylmethionine-dependent sphingolipid C-9 methyltransferase. As a final step a trans-double bond may be introduced into position 3 of the fatty acid component. These ceramides are used exclusively for the production of glucosylceramides, and it is believed that a separate ceramide synthase with a different specificity and/or membrane location produces the ceramide precursors for ceramide phosphorylinositol.

It has been established that long-chain bases with 4-hydroxyl groups are necessary for the viability of the filamentous fungus Aspergillus nidulans and for growth in plants such as Arabidopsis thaliana. The presence of an 8E double bond confers aluminium tolerance to yeasts and plants. However, a trans-4 double bond in the sphingoid base does not appear to be essential for growth and development in Arabidopsis.

Catabolism of sphingosine and other long-chain bases occurs after conversion to sphingosine-1-phosphate and analogues as discussed in our web page on this metabolite. In yeasts, an alternative means of detoxification has been reported in which an excess of phytosphingosine is first acetylated and then converted to a vinyl ether prior to export from the cells.


3.   1-Deoxy-Sphingoid Bases

Some sphingoid base analogues are distinctive in that they lack the hydroxyl group on carbon 1. For example, fungal toxins that have structural similarities to sphingoid bases (e.g. fumonisin B1 illustrated) are found in maize and other crop plants where they are a cause of disease. When ingested by humans and other animals, they can exacerbate or cause a number of disease states (including oesophageal cancer, non-alcoholic steatohepatitis and diabetes) by inhibiting the dihydroceramide synthase. The result is an accumulation of sphinganine and sphinganine-1-phosphate together with a reduction in the amounts of ceramides and complex sphingolipids.

Formula of fumonisin B1

1-Deoxysphinganine (2-amino,3-hydroxy-octadecane) and its N-acyl derivatives (ceramide analogues) accumulate in cells treated with fumonisin B1 as a result of condensation of palmitoyl-CoA with L-alanine catalysed by the serine palmitoyl transferase. In a rare genetic disorder (hereditary sensory and autonomic neuropathy type 1, HSAN1), similar lipids are formed in a reaction with alanine and glycine and are neurotoxic; they cause mitochondrial fragmentation and dysfunction. 1-Deoxy-sphingosine has a cis-double bond in position 14, and not a trans-double bond in position 4 as might have been expected.

Formulae of deoxy-sphingoid bases

Ceramides formed from deoxysphinganine are present in normal cells tissues at low levels, especially the liver, but they are not usually noticed because they are swamped by the much larger amounts of conventional ceramides. As it is cytotoxic for cancer cells in culture, 1-deoxysphinganine is under evaluation in phase I clinical trials. Lacking a terminal hydroxyl group, it cannot be catabolized by the normal pathway, but degradation can be initiated by the action of by cytochrome P450 4F enzymes.

In addition, some plants and animals, especially marine organisms, synthesise long-chain bases lacking the hydroxyl group in position 1 or 2, i.e. 1- or 2-deoxy-sphingoid bases. Among the more unusual of these are the C28 α,ω- or two-headed-sphingoid base-like compounds, such as calyxinin and oceanin (and their β-glycosides) found in sponges.

Formulae of calyxin and oceanin


4.   Biological Functions of Unesterified Sphingoid Bases

The primary function of sphingoid bases is to serve as a basic component of the sphingolipids. Independently of this, they are important mediators of many cellular events, although they are rarely found in unesterified form at greater than trace levels in tissues (typically 1-10 nmoles/g wet tissue). As discussed above, intracellular levels of free sphingoid bases are determined by the activities of ceramidase and sphingosine kinases. In animal cells, they inhibit protein kinase C indirectly, for example, by a mechanism involving inhibition of diacylglycerol synthesis. In addition, sphingoid bases are known to be potent inhibitors of cell growth, although they stimulate cell proliferation and DNA synthesis. They are involved in the process of apoptosis in a manner distinct from that of ceramides by binding to specific proteins and regulating their phosphorylation. Free sphingoid bases derived from ceramide in skin have antimicrobial properties.

Unesterified sphingoid bases may also have a protective role against cancer of the colon in humans. Thus, N,N-dimethylsphingosine and dihydrosphingosine are known to induce cell death in a variety of different types of malignant cells. In consequence, synthetic analogues of long-chain bases are being tested for their pharmaceutical properties, and a 1‑deoxy analogue termed ‘enigmol’, which cannot be degraded via the sphingosine-1-phosphate pathway, has shown promise against colon and prostate cancer. While sphingosine does not appear to participate in raft formation in membranes, it may rigidify pre-existing gel domains in mixed bilayers.

In the human adrenal cortex, sphingosine produced in situ by the acid ceramidase has a function in steroid production by serving as a ligand for steroidogenic factor 1 at the cell nucleus, which controls the transcription of genes involved in the conversion of cholesterol to steroid hormones.

Free sphingosine is believed to have a signalling role in plants by controlling pH gradients across membranes. In addition, free long chain bases (and the balance with the 1-phosphate derivatives) are essential for the regulation of apoptosis in plants.

Myriocin or 2-amino-3,4-dihydroxy-2-(hydroxymethyl)-14-oxoicos-6-enoic acid is a sphingoid metabolite of the thermophilic fungus Isaria sinclairii. It is a potent inhibitor of serine palmitoyltransferase, the first step in sphingosine biosynthesis, and it is also a powerful immunosuppressant. Via a programme of structural modification, a drug termed ‘fingolimod’ has been developed from this for the treatment of multiple sclerosis.

Formula of myriocin


4.   Analysis

The first step in the analysis of the sphingoid bases of sphingolipids is hydrolysis of any glycosidic linkage or phosphate bonds as well as the amide bond to the fatty acyl group. This should be accomplished by a procedure in which the minimum degradation or rearrangement of the bases occurs, such as O- or N-methylation. While many analysts claim that base-catalysed hydrolysis causes least disruption, rapid acid-catalysed methods are often preferred for convenience. Subsequently, the bases are best analysed by gas chromatography after derivatization to reduce their polarity. Analysis of long-chain bases in intact sphingolipids by liquid chromatography-mass spectrometry methods now appears to be a valuable alternative.


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