SPHINGOLIPIDS


AN INTRODUCTION TO SPHINGOLIPIDS AND MEMBRANE RAFTS


1.  Sphingolipids

The sphingolipids comprise a complex range of lipids in which fatty acids are linked via amide bonds to a long-chain base or sphingoid. The root term “sphingo-” was first coined by J.L.W. Thudichum in 1884 because the enigmatic nature of the molecules reminded him of the riddle of the sphinx. The term “sphingolipide” was introduced by Herbert Carter and colleagues in 1947. While they are perhaps less enigmatic than they once were, sphingolipids are extremely versatile molecules and surprises are certainly expected as new knowledge is gained of their functions in healthy and diseased animal and plant tissues. They are also found in a few bacterial genera (but especially Sphingomonas and Sphingobacterium).

Figure 1 - formulae

A long-chain base, such as sphingosine, is the simplest possible functional sphingolipid, but ceramides, which contain a fatty acid linked by an amide bond, are not only important molecules in their own right, but are the precursors of phospholipids and glycolipids with an immense range of functions in tissues. These are quite distinct from the properties of the complex glycerolipids. For example, sphingomyelin has structural similarities to phosphatidylcholine, but has very different physical and biological properties, while the complex oligoglycosylceramides and gangliosides have no true parallels among the glycerolipids.

Complex sphingolipids are located mainly in the plasma membrane of mammalian cells where they have a structural function, although they also serve as adhesion sites for proteins from the extracellular tissue. However, they have analogous intracellular functions in all cellular compartments, including the nucleus. It is now evident that sphingolipids and their metabolites have important roles in signal transduction.

In recent years, it has become apparent that sphingolipids are involved in the patho-physiology of many of the more common human diseases including diabetes, many different cancers, microbial infections, Alzheimer's disease and other neurological syndromes, and diseases of the cardiovascular and respiratory systems. Sphingolipids and their metabolism are therefore likely to prove of ever increasing interest to scientists.


2.  General Comments on Sphingolipid Metabolism

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

Sphingomyelin cycle

Many different enzymes (and their isoforms) are involved, and their activities depend on a number of factors, including intracellular location 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 on the individual compounds. 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, and especially in the regulation of apoptosis, and 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.

Similarly, the ‘structural’ sphingolipids, such as sphingomyelin, monoglycosylceramides, oligoglycosylceramides, gangliosides and sulfatides, all have unique and characteristic biological functions, most of which are due to their physical properties and location within membranes (see below).

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 (sphingomyelin itself does not occur in plants). However, sphingolipid metabolites such as sphingosine-1-phosphate have been linked to programmed cell death, signal transduction, membrane stability, host-pathogen interactions and stress responses, for example.

sphingolipid pathway in plants

Plants also have a unique range of complex lipids 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 had hitherto been supposed. The functions of these have hardly been explored.


3.  Fatty acid Components of Sphingolipids

The fatty acids of sphingolipids are very different from those of glycerolipids, consisting of very-long-chain (up to C26) odd- and even-numbered saturated or monoenoic and related 2-R-hydroxy components, while even longer fatty acids (C28 to C36) occur in spermatozoa and the epidermis. In plants, 2-hydroxy acids predominate sometimes accompanied by small amounts of 2,3-dihydroxy acids. Polyunsaturated fatty acids are only rarely present, although sphingomyelins of testes and spermatozoa are exceptions in that they contain polyunsaturated fatty acids, which are even longer in chain-length (up to 34 carbon atoms), including 28:4(n-6) and 30:5(n-6). Skin ceramides also contain unusual fatty acids, while yeast sphingolipids are distinctive in containing mainly C26 fatty acids.

Very-long-chain saturated and monoenoic fatty acids are produced by specific elongases (ELOVL), 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 (see our webpage on long-chain bases). Hydroxylation is effected by a fatty acid 2-hydroxylase in mammals, i.e. an NAD(P)H-dependent monooxygenase, which is an integral membrane protein of the endoplasmic reticulum. It converts free long-chain fatty acids to free 2-hydroxy acids in vitro and probably also in vivo. For example, 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. Similarly, in skin, 2-hydroxy and non-hydroxy fatty acids as their CoA esters are used with equal facility for ceramide biosynthesis by ceramide synthases. It is possible that a proportion of the odd-chain fatty acids in brain at least are synthesised by peroxisomal α-oxidation of the 2-hydroxy acids. In plants, it is possible 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 have been found in Arabidopsis, with one specific for very-long-chain fatty acids and one for palmitic acid.

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 (see the comments on rafts below). For example, synthetic glycerolipids containing very-long-chain fatty acids (C26) specifically allow growth in yeast mutants lacking sphingolipids, probably by stabilizing the proton-pumping enzyme H+-ATPase. Similarly, ceramides containing different fatty acids can be used in highly specific ways. For example, 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 stress.


4.  Membrane Rafts and Caveolae

Raft structure and organization

It is impossible to understand the functions of sphingolipids without some understanding of their distinctive physical location within membranes. Sphingolipids are located only in the outer (exoplasmic) leaflet of the plasma membrane bilayer, while glycerophospholipids such as phosphatidylinositol, phosphatidylserine and phosphatidylethanolamine occur only in the inner (cytoplasmic) leaflet under normal circumstances. Cholesterol is believed to occur in roughly equal proportions in both leaflets. Further, sphingomyelin and other sphingolipids together with cholesterol are located in an intimate association in specific sub-domains or 'rafts' (or related invaginated structures termed 'caveolae') of membranes. These are laterally segregated regions that form as a result of selective affinities between sphingolipids and membrane proteins, which act to compartmentalize the latter and thereby separate different biochemical functions. It has become evident that such rafts do not represent a single monolithic structure, but a heterogeneous collection of domains differing in their protein and lipid compositions as well as in their stability with respect to time. Many aspects of raft structure especially are uncertain and controversial, because of the technical difficulties involved in their study, although new insights are being obtained by super-resolution fluorescence microscopy.

The packing of cholesterol with the saturated acyl chains of sphingolipids is thermodynamically favoured over that with unsaturated acyl chains, and cholesterol is essential to the process of raft formation. If one of either the sphingolipid or cholesterol is depleted by any means, the other follows and vice versa. Indeed, there is evidence that in animal tissues sphingomyelin regulates the capacity of membranes to absorb cholesterol and thereby controls its flux between the plasma membrane and regulatory pathways in the endoplasmic reticulum.

A formal definition of rafts has been proposed, i.e.

"Membrane rafts are small (10-200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein-lipid interactions."

Earlier definitions have tended to depend on the methods used experimentally to isolate raft preparations and especially their resistance to non-ionic detergents, i.e. their insolubility in cold 1% Triton X-100, for example. They have then been described as detergent-resistant membranes or 'DRM'. This has resulted in much confusion and controversy in the literature. DRM certainly contain raft material, but it is now considered that they should not be treated as rafts per se, as more robust methods of studying raft formation are available.

As much as 50% of the plasma membrane may consist of such rafts, and the apical membrane of epithelial cells especially may behave like a large raft. As sphingolipids containing long, largely saturated acyl chains, they pack more tightly together, thus giving sphingolipids much higher melting temperatures than membrane glycerophospholipids. This tight acyl chain packing is essential for raft lipid organization, since the differential packing facility of sphingolipids and cholesterol in comparison with glycerophospholipids is believed to lead to phase separation in the membrane, giving rise to sphingolipid-rich regions ('liquid-ordered' phase) surrounded by glycerophospholipid-rich domains ('liquid-disordered' phase). This ordering is responsible for the resistance to attack by detergents. As these rafts are relatively small (approximately 50 nm diameter and containing roughly 3000 sphingomyelin molecules) and mobile, they are not easy to study by microscopic methods. They are thicker than normal membranes (46 versus 40 angstroms). Sphingolipids tend to have more free hydroxyl groups, both in the long-chain bases and fatty acid components than glycerolipids, and these enter into hydrogen bonding and contribute to the stability of rafts. The presence of very-long-chain fatty acid components (e.g. C26) is believed to be essential. Cholesterol interacts particularly strongly with sphingomyelin, and much less with glycosphingolipids.

cartoonMembranes should be regarded as lipid-protein composites rather than a solution of protein in a lipid solvent. Indeed, membrane proteins are essential for raft formation, and the reggies/flottilins are especially important in that they promote the assembly of glycerophosphoinositol(GPI)-anchored proteins into rafts. However, many other proteins are present including tyrosine kinases, phosphatases and other signalling proteins. Palmitoylation or myristoylation can target proteins to lipid rafts (see our webpage on proteolipids), and for example flotillin is anchored in the lipid bilayer via N-terminal myristoyl and palmitoyl groups; it also binds to actin and the cytoskeleton and it is now evident that these are involved in raft formation. Other proteins with trans-membrane segments are targeted to rafts by specific amino acid sequences. These provide much of the important biological properties of rafts, and are also required to maintain their stability. The interplay of lipid-based raft units together with protein-mediated assembly of specific protein complexes generates functional domains with high biological activity in cell membranes. From comparisons of membrane solubility in different detergents, it appears that there may exist subsets of membrane raft domains, which differ in their molecular compositions. In particular, they may contain distinct ganglioside species as well as the glycosyl phosphatidylinositol anchors.

Rafts also contain some glycerophospholipids and these often consist of relatively simple molecular species with one saturated and one monounsaturated acyl chain. In addition, both ethanolamine plasmalogens, especially those containing arachidonic acid, and phosphatidylserine are enriched in rafts as compared to the plasma membrane as a whole.

Plants and yeasts: Specific micro-domains or rafts that are enriched in the characteristic plant sterols, sterol glucosides and sphingolipids, such as glucosylceramide and especially the glycosyl-inositol-phosphoceramides, have also been detected in the plasma membrane and Golgi apparatus of plant cells. These contain distinct sets of proteins, including those anchored by glycosyl phosphatidylinositol or glycosyl inositol phosphorylceramide. As in animal cells, such rafts may assist in positioning proteins in specific regions of the cell where they can function in development, membrane trafficking and signalling. In the plasma membrane of the yeast Saccharomyces cerevisiae raft-like structures have been identified, which do not contain ergosterol but are composed primarily of sphingolipids, possibly inositol phosphorylceramide, and contain glycosylphosphatidylinositol-anchored proteins. Micro-domains enriched in both ergosterol and sphingolipids have been found in the plasma membrane of other yeast species.

Caveolae

Those subdomains in the plasma membrane related to rafts and termed ‘caveolae’ lack glycosyl phosphatidylinositol-anchored proteins and are stabilized by particular membrane-spanning scaffolding proteins, the caveolins, a family of three palmitoylated hairpin-like proteins of which caveolin-1, a 24 kDa integral membrane protein with three palmitoylation sites at the C-terminal domain, is the main structural component. These aggregate into large complexes and organize flask-shaped invaginations 50 to 100 nm in diameter in membranes. In addition to the caveolins, caveolae are known to contain some specific signalling proteins, including Ras proteins, G-proteins and growth factor receptors, not present in other raft microdomains. The presence of caveolins rather than invaginations is now considered the defining property of caveolae.

Scottish thistleOther than the content of glycosyl phosphatidylinositol, the lipid composition of caveolae is similar to that of rafts in general in that they contain appreciable amounts of sphingolipids and cholesterol. However, the gangliosides GM1 and to some extent GM3 appear to be concentrated in caveolae. One hypothesis that cannot as yet be tested is that the neck region of caveolae with a strong positive curvature may be highly enriched in glycosphingolipids, which may be largely absent from their lumen. There is evidence that the caveolins interact directly with glycosphingolipids especially, and that the latter may have regulatory effects in the signalling functions of caveolae. It has been argued that caveolae best meet the accepted definition of a raft and should be used as a provisional standard against which other proposed raft structures should be judged, and it is believed that there may be different classes of caveolae with different compositions, physical forms and metabolic functions.

A general picture has emerged of caveolin-1 and sphingolipids forming the scaffold for an integrated network of interactions, which contribute to the regulation of cellular functions via their influence upon receptor molecules in the plasma membrane. Caveolae appear to have a role in controlling the level of free cholesterol in cells, and thence may affect signalling processes. They are especially abundant in endothelial cells, adipocytes, smooth muscle cells and fibroblasts, but have not been found in lymphocytes or mature neurons. In adipocytes, they may regulate the flux of fatty acids across the plasma membrane. Also in adipocytes, insulin is the main hormone that affects metabolism, and the receptor at the plasma membrane is located in caveolae with possible implications for diabetes, obesity and other metabolic disorders. In addition, caveolae function as an important route by which nutrients, such as folate and glucose, are able to cross the plasma membrane, although the mechanism is still obscure.

Raft function

It should be recognized that lipid rafts in general are dynamic structures, which can be formed or undergo compositional changes during signalling events and are short-lived (milliseconds or less). There may also be some form of cross-talk between different raft populations, which can coalesce during activity. Thus in resting cells, sphingolipids may exist in small and highly dynamic domains, which on stimulation can stabilize and grow. In the process, they initiate biochemical reactions by promoting interactions between proteins. Raft domains may also aid membrane trafficking, for example by facilitating the transfer of specific proteins, such as the glycosyl phosphatidylinositol-anchored proteins, from the endoplasmic reticulum or Golgi to the plasma membrane.

Lipid rafts are believed to modulate signalling events in a number of different ways according to the composition of the specific subpopulations. Thus, the location of signalling molecules within one such micro-domain might in itself be a control mechanism, as a protein activated by phosphorylation within the raft and might be prevented from interacting with an inactivating phosphatase in another region of the membrane, for example. By concentrating all of the components of particular signalling pathways within one domain, lipid rafts could promote signalling in response to stimuli, while movement of signalling molecules in and out of the raft could control whether cells are able to respond to stimuli. Similarly, communication between different signalling pathways could be simplified if the relevant molecules were concentrated in the same lipid raft. In contrast, rafts might also regulate signals in a negative manner by sequestering signalling molecules in an inactive state. The physical properties of rafts may be key factors in these interactions. Thus, in response to receptor activation or other stimuli, sphingolipid compositions in rafts may be altered with effects on membrane architecture or morphology producing further downstream events.

cartoonRaft formation appears to be particularly important to the activity of T cells, i.e. lymphocytes derived from the thymus gland that are intimately involved in antibody production. Lipid rafts are a key element of membrane organization that appear to be crucial for the initiation of T cell signalling by enabling efficient interaction between antigens and receptors.

It is also apparent that the biological functions of neurotransmitters and their receptors, in particular acetylcholine and serotonin receptors, are highly dependent upon sphingolipids and cholesterol in raft domains. The lipids interact with receptors to alter their conformation ("chaperone-like" effect), thus regulating neurotransmitter binding and signal transducing functions amongst other events.

There is evidence that certain pathogens activate the acid sphingomyelinase that releases ceramide in membrane rafts transforming them into larger units. These can mediate the internalization of bacteria, viruses and parasites into host cells, to initiate programmed cell death (apoptosis) and release signalling molecules. They may also assist in the budding of viruses from infected cells. In effect, rafts and caveolae re-organize the receptor and intracellular signalling molecules in the cell membrane and enable the interaction of pathogens with cells. The lipids of viruses are derived from the host membranes, and for example, it has recently been demonstrated that the lipids of the HIV virus are enriched in sphingolipids that appear to be derived very specifically from rafts. Further, these lipids are essential for the infectivity of the virus. In contrast, rafts can assist cells to defeat infection by activation of transcription factors and the release of cytokines.

The problem of the actual size and lifetime of membrane rafts is not easily addressed. While a great deal of attention has been focused on rafts in the outer leaflet of the plasma membrane, little is known of the structural organization and properties of the corresponding inner leaflet or how the two layers interact.

Non-raft regions of membranes

A corollary of the existence of rafts, which are rich in cholesterol and with low levels of polyunsaturated fatty acids, is that micro-domains must also exist that are depleted in cholesterol and enriched in polyunsaturated fatty acids. Indeed, the rigid structure of cholesterol and the highly flexible chains of docosahexaenoic acid, for example, are incompatible and promote the lateral segregation of membranes into rafts. Micro-domains that are enriched in polyunsaturated lipids and cholesterol-poor are technically even less easy to study than rafts, but these may also contain distinctive proteins and have important biological functions. There are also suggestions that the interface between rafts and non-raft regions may attract a specific range of proteins.


Suggested Reading


W.W. Christie

James Hutton Institute (and Mylnefield Lipid Analysis), Invergowrie, Dundee (DD2 5DA), Scotland.

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