Sphingolipids: Chemistry and Biochemistry
An Introduction

1. Sphingolipid Basics

The sphingolipids comprise a wide range of complex lipids in which the defining component is a long-chain or sphingoid base, which in living tissues is usually linked to a fatty acid via an amide bond. J.L.W. Thudichum, a German chemist working in London, first coined the root term “sphingo-” in 1884 following his discovery of the first glycosphingolipids, because the enigmatic nature of the molecules reminded him of the riddle of the sphinx. Regretfully, the importance of his work was not recognized until 25 years after his death, and it was 1947 before the term “sphingolipide” was introduced by Herbert Carter and colleagues. While they are much less enigmatic than they once were, sphingolipids are extremely versatile molecules that continue to fascinate as new knowledge is gained of their functions in healthy (and diseased) animal and plant tissues. They are found in only a few bacterial genera, but they are present in Sphingomonas, Sphingobacterium and a few other species, and many pathogenic species utilize host sphingolipids to promote infections. Novel sphingolipid structures continue to be reported, and as an example at the last count, more than 200 of the complex sphingolipids classified as gangliosides, with variations in the complex carbohydrate component alone, had been characterized in vertebrates.

Sphingolipid building blocks

Long-chain or sphingoid bases, of which sphingosine (illustrated) is typical, are the basic elements and are the simplest possible functional sphingolipids. They can differ in chain-length and in the presence of various functional groups including double bonds of both the cis- and trans-configuration at different locations in the aliphatic chain, and likewise ceramides, which contain sphingoid bases linked to fatty acids by amide bonds, vary appreciably in the compositions of both aliphatic components depending on their biological origins. Long-chain bases and ceramides have important biological properties of their own, in relation to intra- and inter-cellular molecular signalling, especially in animal cells, while another relatively simple sphingolipid, sphingosine-1-phosphate, is now recognized as a key factor in countless aspects of animal metabolism. The concentrations of these bioactive lipids respond rapidly to the action of specific stimuli and then regulate downstream effectors and targets.

Ceramides are the precursors of a multitude of sphingo-phospho- and sphingo-glycolipids with an immense range of functions in tissues, and the properties and functions of these complex sphingolipids are quite distinct from those of the comparable glycerophospho- and glyceroglycolipids. In animals, sphingomyelin has structural similarities to phosphatidylcholine, but has very different physical and biological properties, while the complex oligoglycosylceramides and gangliosides (glycosphingolipids), of which glucosylceramide is the precursor, have no true parallels among the glyceroglycolipids.

Complex sphingolipids

Complex sphingolipids are synthesised in the endoplasmic reticulum and Golgi, but they are located mainly in the plasma membrane of most mammalian cells where they have a structural function and also serve as adhesion sites for proteins from the extracellular tissue. The glycosphingolipids are especially important for myelin formation in the brain, but sphingolipids have intracellular functions in all cellular compartments, including the nucleus. The first five carbon atoms of the sphingoid base in sphingolipids have a highly specific stereochemistry and constitute a key feature that has been termed the ‘sphingoid motif’, which in comparison to other lipid species facilitates a relatively large number of molecular interactions with other membrane lipids via hydrogen-bonding, charge-pairing, hydrophobic and van der Waals forces. In membranes, a distinctive property of sphingolipids is that they spontaneously form transient nanodomains termed 'rafts', usually in conjunction with cholesterol, where such proteins as enzymes and receptors congregate to carry out their signalling and other functions. Thus, in addition to their direct effects on metabolism, sphingolipids influence innumerable aspects of biochemistry indirectly via their physical properties.

While it may be obvious that a well-balanced sphingolipid metabolism is important for health in animals, increasing evidence has been acquired to demonstrate that impaired sphingolipid metabolism and function are involved in the pathophysiology of many of the more common human diseases, which include diabetes, various cancers, microbial infections, Alzheimer's disease and other neurological syndromes, and diseases of the cardiovascular and respiratory systems. In humans, several important genetic defects in sphingolipid metabolism or sphingolipidoses have been detected, especially storage diseases associated with the lysosomal compartment where sphingolipids are catabolized. Sphingolipids and their metabolism are therefore likely to prove of ever-increasing interest to scientists.

There are appreciable differences in sphingolipid compositions and metabolism between animal and plant cells, both with respect to the aliphatic components and especially the polar head groups, although there are some important similarities. While sphingomyelin is the most abundant sphingolipid in animals, it does not occur in plants and fungi. Although less is known of the role they play in plants, it has become apparent that complex sphingolipids are much more abundant in plant membranes than was once believed, and it is now recognized that they are key components of the plasma membrane and endomembrane system.

2. Some General Comments on Sphingolipid Metabolism

The biosynthesis and catabolism of sphingolipids involves many intermediate metabolites, all of which have distinctive biological activities of their own. In animals, the relationships between these metabolites have been rationalized in terms of a ‘sphingomyelin, sphingolipid or ceramide cycle’.

The sphingomyelin cycle

Many different enzymes (and their isoforms) are involved, and their activities depend on numerous factors, including intracellular locations and mechanisms of activation. Each of the various compounds in these pathways has characteristic metabolic properties, and these are discussed in more detail on the web pages describing the individual sphingolipids. Thus, free sphingosine and other long-chain bases, which are the primary precursors of ceramides and thence of all the complex sphingolipids, function as mediators of many cellular events, for example by inhibiting the important enzyme protein kinase C. Ceramides are involved in cellular signalling, especially in the regulation of apoptosis, cell differentiation, transformation and proliferation, and most stress conditions. In contrast, sphingosine-1-phosphate and ceramide-1-phosphate promote cellular division (mitosis) as opposed to apoptosis, so that the balance between these lipids and ceramide and/or sphingosine levels in cells is critical and necessitates exquisite control in each cellular compartment.

The ‘structural’ sphingolipids, such as sphingomyelin, monoglycosylceramides, oligoglycosylceramides, gangliosides and sulfatides, all have unique and characteristic biological functions, some of which are due to their physical properties and location within raft nanodomains of membranes. Most of the reactions in the sphingomyelin cycle are reversible and the relevant enzymes are located in the endoplasmic reticulum, Golgi, plasma membrane and mitochondria, but the more complex sphingolipids are catabolized in the lysosomal compartment. Sphingolipids are especially important in providing the permeability barrier in skin, where they are characterized by the presence of ultra-long fatty acyl components as well as fatty acyl groups linked to a hydroxyl group at the terminal end of the N‑linked fatty acids (thereby generating a three‑acyl-chain rather than a two‑chain molecule).

Metabolic pathways that are comparable to those of the sphingomyelin cycle are believed to occur in plants, although they have not been studied as extensively as those in animals, and sphingolipid metabolites such as sphingosine-1-phosphate (or analogues) have been linked to programmed cell death, signal transduction, membrane stability, host-pathogen interactions and stress responses.

Sphingolipid biosynthetic pathways in plants

Plants have a unique range of complex sphingolipids in their membranes, such as ceramide phosphorylinositol and the phytoglycosphingolipids, and these are now known to constitute a higher proportion of the total lipids than has hitherto been supposed, although their functions have hardly been explored. While sphingolipids are produced by relatively few bacterial species, sulfono-analogues of long-chain bases and ceramides (capnoids) are produced by some species, but for convenience, these are discussed with the sulfonolipids

3. Fatty acid Components of Sphingolipids

The fatty acids of sphingolipids are very different from those of glycerolipids, and they are linked mainly via amide bonds, which are chemically much more stable than O-ester bonds. In general, these consist of very-long-chain (up to C₂₆) odd- and even-numbered saturated or monoenoic acyl groups and related 2(R)-hydroxy components, while even longer fatty acids (C₂₈ to C₃₆) occur in spermatozoa and the epidermis. The dienoic acid 15,18‑tetracosadienoate (24:2(n‑6)) derived from elongation of linoleic acid is found in the ceramides and other sphingolipids of several tissues, but at relatively low levels. Polyunsaturated fatty acids are only rarely present, although sphingomyelins of testes and spermatozoa are exceptions in that they contain such fatty acids, which are even longer in chain-length (up to 34 carbon atoms) and include 28:4(n‑6) and 30:5(n‑6). Skin ceramides also contain unusual very-long-chain fatty acids, while yeast sphingolipids are distinctive in containing mainly C₂₆ fatty acids.

Fatty acids of sphingolipids

In plants and yeasts, a similar range of chain-lengths occur as in animals, but 2-hydroxy acids predominate sometimes accompanied by small amounts of 2,3‑dihydroxy acids; saturated fatty acids are most abundant, and the sphingolipids of rice (Oryza sativa) contain only saturated components, but monoenes are present in higher proportions in the Brassica family (including the model plant Arabidopsis) and a few other species. Some fungal species contain monoenoic fatty acids with a trans-3 double bond and/or a hydroxyl group.

Very-long-chain saturated and monoenoic fatty acids for sphingolipid biosynthesis are produced from medium-chain precursors by elongases (ELOVL) in the endoplasmic reticulum of cells in mammals, and there is increasing evidence that specific isoforms are involved in the biosynthesis of certain ceramides. For example, ELOVL1 has been linked to the production of ceramides with C₂₄ fatty acids (saturated and unsaturated), while ELOVL4 is responsible for the ultra-long-chain fatty acids in skin. Yeasts possess three elongation enzymes: Elo1 (for medium to long-chain fatty acids), Elo2 (up to C₂₂) and Elo3 (up to C₂₆).

The hydroxyl group is believed to add to the hydrogen-bonding capacity of the sphingolipids, and it helps to stabilize membrane structures and strengthen the interactions with membrane proteins. In mammals, hydroxylation is accomplished by a fatty acid 2-hydroxylase (FA2H), i.e., an NAD(P)H-dependent monooxygenase, which is an integral membrane protein of the endoplasmic reticulum and converts unesterified long-chain fatty acids to 2‑hydroxy acids, although further uncharacterized enzymes are believed to exist. The catalytic region consists of four conserved histidines that form a di-metal ion centre and adds the hydroxyl group stereospecifically to produce only the (R)-enantiomer. Experimental evidence has been obtained that is consistent with 2‑hydroxylation occurring at the fatty acid level prior to incorporation into ceramides in the brain of mice and in the urinary bladder of mice and humans where the enzyme is expressed at high levels. In skin, 2‑hydroxy and non-hydroxy fatty acids as their CoA esters are used with equal facility for ceramide biosynthesis by ceramide synthases. As mutations in the fatty acid 2‑hydroxylase in humans and mice give rise to demyelination disorders, such as leukodystrophy, it is evident that sphingolipids containing 2‑hydroxy acids have unique functions in membranes that cannot be substituted by non-hydroxy analogues. Some of the odd-chain fatty acids in brain may be synthesised by peroxisomal α-oxidation of the 2‑hydroxy acids.

Scottish thistle In plants, it appears that 2‑hydroxyl groups are inserted into fatty acyl chains while they are linked to ceramide, as ceramide synthase does not accept hydroxy fatty acids in vitro at least. Two fatty acid 2‑hydroxylases (di-iron-oxo enzymes) have been found in Arabidopsis, with one specific for very-long-chain fatty acids (FAH1) and one for long chain fatty acids (FAH2). In fungi, a hydroxyl group is inserted at C2 of the fatty acid in a dihydroceramide intermediate. The double bond is introduced by a sphingolipid fatty acid desaturase in Arabidopsis, and this is structurally distinct from that in the moss Physcomitrium patens.

Although the fatty acids are only occasionally considered in terms of the biological functions of sphingolipids, their influence is considerable, especially but not only in relation to their physical properties and function in membranes. Very-long-chain fatty acids may play a role in stabilizing highly curved membrane domains as is required during cell division, and the hydrophobic nature of the fatty acyl groups (together with the long-chain bases) enables the hydrogen bonding that is essential for the formation of raft nanodomains in membranes. As a rule, lipid bilayers containing sphingolipids with 2-hydroxy-fatty acyl or 4-hydroxy-sphingoid base moieties tend to generate condensed and more stable gel phases with higher melting temperatures than their non-hydroxylated equivalents, because they have a more extended and strengthened intermolecular hydrogen bonding network. Changes in fatty acid composition are seen in some disease states, and increased concentrations of fatty acids >C₂₄ are a feature of adrenoleukodystrophy, an X-linked genetic disorder.

Removal of very-long-chain fatty acids from sphingolipids in mutants of Arabidopsis inhibits completely the development of seedlings. Among more specific interactions, it has been demonstrated that synthetic glycerolipids must contain very-long-chain fatty acids (C₂₆) to allow growth in yeast mutants lacking sphingolipids, probably by stabilizing the proton-pumping enzyme H⁺-ATPase. Ceramides containing different fatty acids can be used in highly specific ways, and in fungi, C₁₆ or C₁₈ hydroxy acids are used exclusively for synthesis of glucosylceramide, while those containing very-long-chain C₂₄ and C₂₆ hydroxy acids are used only for synthesis of glycosyl inositol phosphorylceramide anchors for proteins. In plants, sphingolipids containing 2-hydroxy acids are protective against oxidative and other biotic stresses.

4. Links between Glycerolipid and Sphingolipid Metabolism

Sphingolipid metabolism and glycerolipid metabolism have been widely treated as separate sciences until relatively recently, partly for historical reasons and partly because the analysis of the two lipid groups required different approaches and skills. However, there are many areas where the two overlap, not least because phosphatidylcholine is the biosynthetic precursor of sphingomyelin in animal cells, while in plants and fungi, phosphatidylinositol is the biosynthetic precursor of ceramide phosphorylinositol. In contrast, ethanolamine phosphate derived from the catabolism of sphingolipids via sphingosine 1-phosphate is recycled for the biosynthesis of phosphatidylethanolamine, and this is essential for survival in the protozoan parasite Trypanosoma brucei. In studies in vitro, sphingosine 1-phosphate has been shown to be an activator of the phospholipase C involved in the hydrolysis of the lipid mediator phosphatidylinositol 4,5-bisphosphate with formation of diacylglycerols and inositol triphosphate. In membranes, the location and functions of glycerophospholipids is influenced both positively and negatively by sphingolipid-rich domains or rafts.

There are several examples of phosphoinositides and other complex glycerolipids binding to enzymes of sphingolipid metabolism, either as part of a regulatory function that controls their activity or to facilitate their location to various membranes. Thus, sphingosine kinase 2, one of the enzymes responsible for the biosynthesis of sphingosine 1-phosphate, binds to phosphatidylinositol monophosphates, while the ceramide kinase responsible for the biosynthesis of ceramide 1-phosphate requires phosphatidylinositol 4,5-bisphosphate to function. The CERT protein involved in ceramide transport has a binding site for phosphatidylinositol 4‑phosphate. Sphingomyelin production at the trans-Golgi network triggers a signalling pathway leading to dephosphorylation of phosphatidylinositol 4-phosphate, interrupting transport of cholesterol and sphingomyelin. Again, the interactions are not solely in one direction as ceramide 1‑phosphate (with phosphatidylinositol 4,5-bisphosphate) binds to the specific phospholipase A₂ (cPLA₂α) responsible for the hydrolysis of phosphatidylinositol and thence the release arachidonic acid for eicosanoid production. Apart from the phosphoinositides, phosphatidylserine activates the neutral sphingomyelinase in brain.

The glyceroglycolipid seminolipid from male reproductive tissues and the sphingolipid sulfates have structural elements in common, and they use the same enzymes to introduce the carbohydrate and then the sulfate group to diacylglycerol and ceramide intermediates, respectively. In some types of glycosylphosphatidylinositols (protein anchors), the first mannose unit is decorated by the addition of a short oligosaccharide sequence starting with N‑acetylgalactosamine, before galactose is added by the action of a GM1 ganglioside synthase requiring the presence of lactosylceramide. Diacylglycerol acyltransferase 2 (DGAT2), a key enzyme in triacylglycerol biosynthesis, generates 1-O-acylceramides in skin and lipid droplets. Further, the biosynthesis of cholesteryl glycosides in animals involves a transfer of glucose from glucosylceramide to cholesterol by means of cellular β‑glucocerebrosidases.

	© Author: William W. Christie
	Contact/credits/disclaimer	Updated: May 3^rd, 2023

Sphingolipids: Chemistry and BiochemistryAn Introduction

1. Sphingolipid Basics

2. Some General Comments on Sphingolipid Metabolism

3. Fatty acid Components of Sphingolipids

4. Links between Glycerolipid and Sphingolipid Metabolism

Suggested Reading

Sphingolipids: Chemistry and Biochemistry
An Introduction