Long-Chain or 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

The commonest or most abundant of these in animal tissues 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. Alternative nomenclatures are occasionally seen in publications.

The compositions of long-chain bases of sphingomyelins of some animal tissues are listed in Table 2 of our web page on sphingomyelins. 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. Eicosasphingosine (2S,3R,4E-d20:1) is found in appreciable concentrations in gangliosides from human brain and intestinal tissues, for example, with variable amounts in different regions and membranes in these tissues 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. 3-Keto-sphingoid bases, produced in the first step of sphingosine biosynthesis (see below), are only rarely detected in tissues.

A common long-chain base of mainly plant origin is a saturated C18 trihydroxy compound phytosphingosine or 4D-hydroxy-sphinganine ((2S,3R,4R)-2-amino-octadecanetriol), 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). There are also lipid class preferences. In many plant species, dihydroxy long-chain bases are more enriched in glucosylceramides than in glycosylinositolphosphoceramides, but in the model plant Arabidopsis thaliana trihydroxy bases predominate in both classes and comprise nearly 90% of the total in the leaves.

Table 1. Sphingolipid long-chain base composition of leaves from A. thaliana
Base%Base%
t18:1 (8Z) 20 d18:1 (8Z) 1
t18:1 (8E) 64 d18:1 (8E) 8
t18:0 6 d18:0 1
 
Data from Chen et al. Plant Cell, 18, 3576-3593 (2006)

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 may plant species. In Arabidopsis, Δ4 long-chain bases are found mainly in the flowers and pollen and then exclusively as a component of the glucosylceramides.

Phytosphingosine is found in significant amounts in intestinal cells of animals also, with much smaller relative proportions in kidney and skin.

The number of different long-chain bases that has been found in animals, plants and microorganisms must now number 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. A proportion of of the phytosphingosine and related sphingoid bases found in animal tissues may enter via the food chain, although nonmammalian sphingoid bases in general tend to be poorly absorbed from the intestines.

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 base sphinganine. In plants, 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 (d18:18), (4E,8E/Z)-sphinga-4,8-dienine (d18:24,8) and (8E/Z)-4-hydroxy-8-sphingenine (t18:18); d18:14, d18:0 and t18:0 are only present in small amounts.

structure of 9-methylsphinga-4,8-dienine

In addition, many organisms produce sphingosine-like compounds that can interfere with sphingolipid metabolism, such as the mycotoxin fumonisins discussed below. N-Methyl, N,N-dimethyl and N,N,N-trimethyl derivatives of sphingoid bases have been detected in mouse brain. N,N-Dimethylsphingosine is of particular interest in that it inhibits protein kinase C, sphingosine kinase and many other enzyme systems.

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

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 (see below), 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.

myriocin

Sphingoid bases are surface-active amphiphiles, with critical micellar concentrations of about 20 μM. They are unusual amongst lipids in that they bear a small positive charge at neutral pH, though their pKa (9.1) is lower than in simple amines as a consequence of intramolecular hydrogen bonding. This together with their relatively high solubility (>1 μM) 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 has signalling functions in cells akin to those of lysophospholipids.

 

2.   Biosynthesis and Metabolism

The basic mechanism for the biosynthesis of sphinganine involves condensation of palmitoyl-coenzyme A with serine, catalysed by a membrane-bound enzyme requiring pyridoxal 5’-phosphate, serine palmitoyltransferase, on the cytosolic side of the endoplasmic reticulum in animal cells as illustrated to form 3-keto-sphinganine. This is believed to be the key regulatory or rate-limiting step in ceramide biosynthesis, and elimination of this enzyme is embryonically fatal in mammals and fruit flies. The specificity of the enzyme controls the chain length of the base, as other fatty acyl-coA groups can be utilized but with lower efficiency. 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.

Biosynthesis of sphinganine

The free sphinganine is rapidly N-acylated by acyl-coA to form dihydroceramides by dihydroceramide synthases, which in animals are located on the cytosolic face of the endoplasmic reticulum. Animals and plants have multiple isoforms of this enzyme, for which the abbreviated term ‘ceramide synthase’ is now widely applied as they can also utilize other sphingoid bases produced by hydrolysis of sphingolipids. Each isoenzyme has characteristic tissue distributions and distinct specificities for the chain length of the fatty acyl-CoA moieties and to a limited extent 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) and is most active in the central nervous system, while ceramide synthase 1 is specific for 18:0 and is located exclusively in brain and skeletal muscle. Ceramide synthase 3 is responsible for the unusual ceramides of skin and testes and uses C26-CoA and higher, while ceramide synthase 4 uses C18 to C22-CoAs and ceramide synthases 5 and 6 generate C16 ceramide.

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 (see also our web pages on ceramides per se), 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.

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. Two distinct types of sphingoid Δ8 desaturase have been characterized in plants 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. The main group of Δ8 desaturases requires a 4-hydroxysphinganine moiety as substrate, but the second does not. Fungi produce trans Δ8 isomers only. Δ4 and Δ8 desaturases do not occur in the yeast S. cerevisiae.

Phytosphingosine 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 with which both 4-hydroxylation and Δ4-desaturation are initiated by removal of the proR C-4 hydrogen. In plants, fatty acid desaturases and hydroxylases are also closely related. However, the substrates for desaturation in plants (free bases or ceramides) are still uncertain. 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.

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.

In the yeast Pichia pastoris, it has been established that 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 different specificities produces the ceramide precursors for ceramide phosphorylinositol. In plants, sphingolipid fatty acid α-hydroxylation is also believed to occur on the ceramide, as opposed to the free acyl chain.

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. This has been amply demonstrated in plants in studies with mutants in which specific enzymes have been deleted. Certain fungal toxins that have structural similarities to sphingoid bases (e.g. fumonisin B1 illustrated) are found in maize and other crop plants and can cause a number of disease states in humans (including oesophageal cancer) and other animals, as well as in plants, by inhibiting the dihydroceramide synthase, leading to an accumulation of sphinganine and sphinganine-1-phosphate together with a reduction in the amounts of complex sphingolipids.

fumonisin B1

1-Deoxysphingosine (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. Similar lipids are formed by condensation with alanine and glycine in a rare genetic disorder and are neurotoxic. Such compounds 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.

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. All the free sphingosine per se in tissues must arise by this route, in particular by the action of ceramidases (see the web page on ceramides). The levels of free sphingoids and their capacities to function as lipid mediators are controlled mainly by enzymic reacylation to form ceramides.

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.

 

3.   Biological Functions

Intracellular levels of free sphingoid bases are determined by the activities of ceramidase and sphingosine kinases. Although they are rarely found at greater than trace levels in tissues (typically 1-10 nmol/g wet tissue), they may have important functions as mediators of many cellular events, independently of their role as precursors of other lipids. 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.

They 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.

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.

 

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 modern mass spectrometry methods now appears to be a valuable alternative.

 

Suggested Reading

  • Christie, W.W. and Han, X. Lipid Analysis - Isolation, Separation, Identification and Lipidomic Analysis (4th edition), 446 pages (Oily Press, Bridgwater, U.K. and Woodhead Publishing Ltd, Cambridge, U.K.) (2010) - Woodhead Publishing Ltd.
  • Markham, J.E., Lynch, D.V., Napier, J.A., Dunn, T.M. and Cahoon, E.B. Plant sphingolipids: function follows form. Curr. Opin. Plant Biol., 16, 350-357 (2013) (DOI: 10.1016/j.pbi.2013.02.009).
  • Merrill, A.H. De novo sphingolipid biosynthesis: a necessary, but dangerous, pathway. J. Biol. Chem., 277, 25843-25846 (2002) (DOI: 10.1074/jbc.R200011200 ).
  • Merrill, A.H. Sphingolipids. In: Biochemistry of Lipids, Lipoproteins and Membranes (5th Edition). pp. 363-397 (Vance, D.E. and Vance, J.E. (editors), Elsevier, Amsterdam) (2008).
  • Merrill, A.H. Sphingolipid and glycosphingolipid metabolic pathways in the era of sphingolipidomics. Chem. Rev., 111, 6387–6422 (2011) (DOI: 10.1021/cr2002917).
  • Pruett, S.T., Bushnev, A., Hagedorn, K., Adiga, M., Haynes, C.A., Sullards, M.C., Liotta, D.C. and Merrill, A.H. Biodiversity of sphingoid bases (‘sphingosines’) and related amino alcohols. J. Lipid Res., 49, 1621-1639 (2008) (DOI: 10.1194/jlr.R800012-JLR200).
  • Sandhoff, R. Very long chain sphingolipids: Tissue expression, function and synthesis. FEBS Letts., 584, 1907-1913 (2010) (DOI: 10.1016/j.febslet.2009.12.032).
  • Sperling, P., Warnecke, D. and Heinz, E. Plant sphingolipids. In: Lipid Metabolism and Membrane Biogenesis. pp. 337-381 (ed. G. Daum, Springer-Verlag, Heidelberg) (2004) (DOI: 10.1016/S1388-1981(03)00033-7).
  • Ternes, P., Wobbe, T., Schwarz, M., Albrecht, S., Feussner, K., Riezman, I., Cregg, J.M., Heinz, E., Riezman, H., Feussner, I. and Warnecke, D. Two pathways of sphingolipid biosynthesis are separated in the yeast Pichia pastoris. J. Biol. Chem., 286, 11401-11414 (2011) (DOI: 10.1074/jbc.M110.193094).
  • Tidhar, R. and Futerman, A.H. The complexity of sphingolipid biosynthesis in the endoplasmic reticulum. Biochim. Biophys. Acta, 1833, 2511-2518 (2013) (DOI: 10.1016/j.bbamcr.2013.04.010).
  • Vaena de Avalos, S., Jones, J.A. and Hannun, Y.A. Ceramides. In: Bioactive Lipids. pp. 135-167 (edited by A. Nicolaou and G. Kokotos, The Oily Press, Bridgwater) (2004).

 

Updated January 27, 2014

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