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Sphingolipid Biosynthesis Martina Leipelt and Alfred H. Merrill, Jr.Georgia Institute of Technology, Atlanta, Georgia, USA

Sphingolipids are a complex family of compounds thatperform

diverse structural and regulatory functions for eukaryotes and

someprokaryotes and viruses. They share a common structural

feature, a sphingoid base backbone that is synthesized de novo

from serine and a fatty acyl-coenzyme A, then converted into

ceramides, phosphosphingolipids, glycosphingolipids, and

other species, including protein adducts. Several diseases result

from disruption of de novo sphingolipid biosynthesis by

environmental factors or hereditary defects, but modulation

of sphingolipid biosynthesis is also being exploredas a means tocontrol other diseases, including sphingolipid storage diseases

and cancer.

Structures and Nomenclature

Sphingolipids can be divided into several major cat-egories: the sphingoid bases and their simple derivatives,ceramides, and more complex sphingolipids (Figure 1).The International Union of Pure and Applied Chemists(IUPAC) has recommended a systematic nomenclaturefor sphingolipids. The root name “sphingosin,” inreference to the sphinx, was given by J. L. W.

Thudichum in 1884 “in commemoration of the manyenigmas which it presented to the inquirer.”

SPHINGOID B ASES

The structure of sphingosine, the major sphingoid baseof mammals, is (2S, 3R, 4E)-2-aminooctadec-4-ene-1,3-diol (it is also called D-erythro-sphingosine andE-sphing-4-enine) (Figure 1). This is only one of manysphingoid bases found in nature, which vary in alkylchain length and branching, the number and positions of double bonds, the presence of additional hydroxylgroups, and other features. The structural variationhas functional significance; for example, sphingoid basesin skin have additional hydroxyls at position 4 and/or 6that can interact with neighboring molecules, therebystrengthening the permeability barrier of skin.

Sphingoid bases function as intra- and extracellularsignals and second messengers in the form of freesphingoid bases, sphingoid base 1-phosphates (Figure 1),and possibly other species. Nonetheless, sphingoid basesare present in cells primarily as the backbones of morecomplex sphingolipids.

CERAMIDES

Ceramides are fatty acid derivatives of sphingoid bases(Figure 1). The fatty acids are typically saturated ormono-unsaturated with chain lengths from 14 to 26carbon atoms (or even longer in the special case of skin),and sometimes have a hydroxyl group on the a- or v -carbon atom. These structural features favor thesegregation of ceramides and some complex sphingoli-pids into specialized regions of the membrane (called

“rafts” and “caveolae”) that participate in cell signaling,nutrient transport, and other functions.

Ceramides also serve as second messengers thatregulate cell growth, senescence, and programmed celldeath (apoptosis). Their biologic activity depends on thetype of sphingoid base and fatty acid; for example,dihydroceramides (i.e., without the 4,5-double bond of the sphingosine backbone) (Figure 1) are less potent thanceramides as inducers of apoptosis, whereas phytocer-amides (i.e., with 4-hydroxysphinganine or “phyto-sphingosine” as the backbone) are more potent.

MORE COMPLEX PHOSPHO- ANDGLYCO-SPHINGOLIPIDS

The major phosphosphingolipids of mammals aresphingomyelins (ceramide phosphocholines) (Figure 1),whereas insects contain mainly ceramide phosphoetha-nolamines and fungi have phytoceramidephosphoinosi-tols and inositol phosphates. Some aquatic organismsalso contain sphingolipids in which the phosphate hasbeen replaced by a phosphono- or arsenate group.

Glycosphingolipids are classified on the basis of carbohydrate composition: (1) neutral glycosphingoli-pids contain one or more uncharged sugars such asglucose (abbreviated Glc, hence, glucosylceramide isGlcCer), galactose (Gal), N-acetylglucosamine(GlcNAc), N-acetylgalactosamine (GalNAc), and fucose(Fuc); and (2) acidic glycosphingolipids contain ionizedfunctional groups (phosphate or sulfate) attached toneutral sugars, or charged sugar residues such as sialicacid (N-acetylneuraminic acid). The latter are calledgangliosides, and the number of sialic acid residues isusually denoted with a subscript letter (i.e., mono-, di- ortri-) plus a number reflecting the subspecies within that

Encyclopedia of Biological Chemistry, Volume 4. q 2004, Elsevier Inc. All Rights Reserved. 76



category (see examples in Figure 1). For a few glyco-sphingolipids, historically assigned names as antigensand blood group structures are still in common usage(e.g., Lexis x and sialyl Lewis x).

PROTEIN A DDUCTS

Some sphingolipids are covalently attached to protein,e.g., v -hydroxy-ceramides and -GlcCers are attached tosurface proteins of skin and inositolphosphoceramidesare used as membrane anchors for some fungal proteins,in a manner somewhat analogous to the glycosylpho-sphatidylinositol (GPI) anchors that are attached to

proteins in other eukaryotes.

De novo Synthesis of the

Ceramide Backbone

Sphingolipid biosynthesis is widespread among eukary-otic cells, and it appears that new synthesis (i.e., de novo)is relied upon more than reutilization of sphingolipidsfrom exogenous sources, such as food. The biosynthetic

pathway for such a diverse family of compounds(conservatively estimated to be in the tens of thousands)is obviously complex; however, its fundamental featurescan be summarized in Figures 2 and 3.

SERINE P AL MITOY LT RAN SFERASE

Serine palmitoyltransferase (SPT) catalyzes the initialstep of the pathway which, for many organisms, isthe condensation of serine and palmitoyl-CoA to form3-ketosphinganine (Figure 2). However, for organismsthat produce sphingoid bases with other alkyl chainlengths (such as the C14 species of insects), the firstenzyme of the pathway utilizes a different cosubstrate(dodecanoyl-CoA, in this example) and could berenamed “serine dodecanoyltransferase.”

SPT is a pyridoxal 50 phosphate-dependent enzymecomprised of two gene products (termed SPTLC1 andSPTLC2 for humans, and LCB1 and LCB2 for yeast); athird has also been identified in yeast, but does notapp ear to h ave a ho mo lo gu e in mammals. Inmost organisms, SPT is associated mainly with the

HO

O

O

OH

OHO

OO

OH

O

OH HO

OOH

HO

AcNH4

HO

OOH

HO

OHH

OH

O

HNAc

OH

HO2C

HO

HO H

H

Cer

GlucoseGalactose

N-acetyl galactosamine

Galactose

N-acetyl neuraminic acid

GM3

GM2

GM1

Lactosylceramide (LacCer)

Glucosylceramide (GlcCer)

31

4

3

1

2

1 1 1́

O---_---

Ceramide (N-acylsphingosine)

OH

NH

D-erythro -sphingosine

O

P(O2 –

)O-choline

Phosphosphingolipids:

Sphingomyelin

Glycosphingolipids(examples):

Gangliosides:

Examples of other sphingoid

base backbones:

OH

NH2

4-hydroxysphinganine(phytosphingosine)

OH

OH

NH2

D-erythro -sphinganine(dihydrosphingosine)

OH

HO

FIGURE 1 Structures of representative sphingolipids. Shown are several examples of sphingoid bases (sphingosine, sphinganine and

4-hydroxysphinganine, boxed in red), ceramide (in green), sphingomyelin (blue), and neutral (GlcCer and LacCer) and acidic (gangliosides GM1,GM2, and GM3) glycosphingolipids.

SPHINGOLIPID BIOSYNTHESIS 77



endoplasmic reticulum, as are the other enzymes of ceramide biosynthesis. SPT activity is affected by a widerange of factors: sphingosine 1-phosphate, endotoxinand cytokines, heat shock, UVB irradiation, cytotoxicdrugs (including many cancer chemotherapeutic drugs),retinoic acid, and a number of small molecule inhibitorsproduced by microorganisms (one of which, ISP1 ormyriocin, is often used to block de novo sphingolipidsynthesis by cells in culture). The mechanisms of SPTregulation are not fully understood, but include (forexample) both acute modulation by heat shock andincreased expression of SPT mRNA by cytokines.Mutations in human SPTLC1 cause hereditary sensoryneuropathy type I (HSN1), which is the most commonhereditary disorder of peripheral sensory neurons.

CERAMIDE S YNT HASE

3-Ketosphinganine is rapidly converted to sphinganineby an NADPH-dependent reductase, then ceramidesyntase(s) acylate sphinganine to dihydroceramidesusing fatty acyl-CoA’s varying in length from C16 to

C30 (and usually saturated or mono-unsaturated)(Figure 2). Ceramide synthase is actually a family of enzymes, each of which appears to arise from a differentgene and to utilize a particular subset of fatty acyl-CoA’s(e.g., TRH4 utilizes palmitoyl-CoA whereas UOG1 usesstearoyl-CoA).

Ceramide synthase is activated by a number of stimuli,including cancer chemotherapeutic drugs and irra-diation, and the increased production of ceramide isthought to mediate the toxicity of these treatments.Ceramide synthase is also the target of a number of mycotoxins (fumonisins), which are produced by fungithat grow on corn and, when consumed, result inspectrum of diseases that are important to agriculture(equine leukoencephalomalacia and porcine pulmonaryedema) as well as human cancerand possibly birthdefects.

DIHYDROCERAMIDE DESATURASE

Insertion of double bond(s) into the sphingoid basebackbones occurs mainly after formation of dihydrocer-amide(s) (Figure 2). For mammals, introduction of the

Sphinganine

Dihydro-

ceramide

Palmitoyl-CoA

3-keto-sphinganine

3-ketosphinganine

reductase

NH

HCH2OH

O

HO

H(-)OOC CH2OH

NH3(+)

O

SCoA

O HCH2OH

NH3(+)

HCH2OH

HO

NH3(+)

NH

HCH2OH

O

HOCH3(CH2)10CH2Ceramide

(Dihydro)ceramide synthase

Dihydroceramide

desaturase

Serine+

Serine palmitoyltransferase

CH3(CH2)10CH2

CH3(CH2)10CH2

CH3(CH2)9-19*CH2

CH3(CH2)9-19*CH2

CH3(CH2)10CH2

CH3(CH2)10CH2

NADPH

Fatty acyl-CoA

NAD[P]H

Sphingomyelin

Glucosylceramide

(Galactosylceramide)

Sphingomyelin synthase

Glc(Gal)ceramide synthase(s)

Dihydrosphingomyelin

Dihydroglucosylceramide

(DHGalactosylceramide)

Sphingomyelin synthase

Glc(Gal)ceramide synthase(s)

Sphingosine

HCH2OH

HO

NH3(+)

CH3(CH2)10CH2

Ceramidase

Sphingosine kinase

Sphingosine

1-phosphate

HCH2OPO3

2 –

NH3(+)

CH3(CH2)10CH2

HO

Sphingosine kinase

Sphinganine

1-phosphate

HCH2OPO3

2 –

NH3(+)

CH3(CH2)10CH2

HO

FIGURE 2 The de novo biosynthetic pathway for sphingoid bases, ceramide, (dihydro)sphingomyelins and (dihydro)glucosylceramides. The

color coding distinguishes the enzyme names (in red) and the metabolites (in blue).

78 SPHINGOLIPID BIOSYNTHESIS



4,5-double bond is catalyzed by two pyridine nucleo-tide-dependent desaturases (DES1 and DES2), one of which may also be responsible for addition of the 4-hydroxyl-group of phytoceramides.

Synthesis of More

Complex Sphingolipids

Ceramides in their various forms (i.e., ceramides,dihydroceramides, phytoceramides, etc.) are at a keybranch point of complex sphingolipid biosynthesiswhere these intermediates are partitioned into eitherphosphosphingolipids or glycosphingolipids. For cellsthat produce more than one category of glycolipid (forexample, mammalian epithelial cells, which have bothGlcCer and GalCer), the glycolipid arm can havemultiple branches. The fate of a given intermediate isgoverned by the relative activities and selectivity of the

enzymes at this branch point as well as by the subcellularlocalization of the participants.

SPHINGOMYELIN AND OTHER

PHOSPHOSPHINGOLIPIDS

Sphingomyelins are synthesized by transfer of phosphor-ylcholine from phosphatidylcholine to ceramides(Figure 2). This reversible reaction links glycerolipidand sphingolipid metabolism and signaling, because

ceramides and diacylglycerols both function as metabolicintermediates and as intracellular second messengers.This may explain why cells produce dihydroceramides asthe initial products of de novo sphingolipid biosynthesissince that allows a relatively innocuous intermediate toaccumulate if later steps in the pathway slow.

Relatively little is known about the biochemistry of

sphingomyelin synthase, including whether the activitiesin the Golgi apparatus and plasma membranes representa single enzyme, or several different enzymes (twomammalian sphingomyelin synthase genes have beenidentified, SMS1 and SMS2). The regulation of sphingo-myelin biosynthesis is also intriguing – with changes indevelopment, neoplasia, and other normal and abnormalcell states.

Ceramide phosphorylethanolamines are synthesizedfrom phosphatidylethanolamine and ceramides in areaction analogous to sphingomyelin synthase (i.e.,transesterification with phosphatidylethanolamine),and once formed can be methylated to sphingomyelins insome species. Inositolphosphoceramides are also formedby transesterification (from phosphatidylinositols).

GLYCOSPHINGOLIPIDS

A pathway that is responsible for the biosynthesisof hundreds (to thousands) of different glycosphingoli-pids is obviously complex, but these compoundsare nonetheless produced using surprisingly few

LacCer GM3 GD3 GT3

GD2

GD1b

GT1b

GlcCerCer

GA2

GA1

GM1b GD1a

GM1a

GM2 GT2

GT1c

GQ1c

GalNAcT

GalT II

SAT IV

SAT V,

SAT X

SAT IIISAT IISAT I

GalT I

GlcT

GD1c GD1a GT1a GT1a GQ1b GQ1ba

GP1c GP1ca

0-series a-series b-series c-series

FIGURE 3 A representation of the combinatorial nature of glycosphingolipid biosynthesis. Shown are the reactions leading to the major

ganglioside series and the enzymes involved. The abbreviations refer to ceramide (Cer), glucosylceramide (GlcCer), lactosylceramide (LacCer) andthe different categories of gangliosides designated by “G” and subscripts for the number of sialic acids (M,D,T and Q representing 1,2,3 and 4,respectively) and other structural features. Abbreviations: GalNAcT, N-acetylgalactosaminetransferase; GalT, galactosyltransferase; GlcT,glucosyltransferase; and, SAT (sialyltransferase) with the Roman numerals reflecting the subtypes. (Modified from Kolter, T., Proia, R. L., and

Sandhoff, K. (2002). Combinatorial ganglioside biosynthesis. J. Biol. Chem. 277, 25859–25862.)




glycosyltransferases. Efficiency is achieved by a “combi-natorial” biosynthetic pathway that directs precursorsand intermediates toward the desired products bymodulating the activities of key combinations of enzymes (see Figure 3 for an illustration).

The addition of the carbohydrate headgroups iscatalyzed by glycosyltransferases that transfer a specificsugar from the appropriate sugar nucleotide (e.g., UDP-

Glc, UDP-Gal, etc.) to ceramide or the nonreducing endof the growing carbohydrate chain attached to ceramide.GlcCer and GalCer are synthesized by UDP-Glc(orGal):ceramide glucosyltransferases, hence, a majordeterminate of the types of glycosphingolipids made bya given cell type will be whether it expresses one or bothof these genes. Factors that regulate these enzymesinclude cell type, the nature of the ceramide substrate(ceramides witha-hydroxy fatty acids are mainly utilizedfor GalCer synthesis), and exposure of the cells toagonists such as endotoxin and acute phase responsemediators. A number of inhibitors of these glycosyl-transferases are being tested for efficacy in sphingolipid

storage diseases (caused by inherited defects in glyco-sphingolipid hydrolyases), based on the rationale thatslowing biosynthesis may counterbalance these defects.

Additional glycosyltransferases are responsible forsubsequent addition of sugars to make dihexosylcera-mides, trihexosylceramides, etc. as well as for addition of neutral sugars to gangliosides (Figure 3). Likewise,gangliosides are synthesized by the stepwise transfer of neutral sugars and sialic acids. In general, the enzymesresponsible for these reactions are located in the lumen of the Golgi apparatus, and the region corresponds to theorder in which the sugars are added. For example, thesialyltransferase catalyzing the synthesis of a simple

ganglioside (ganglioside GM3)isinthe cis-Golgi, whereasenzymes involved in terminal steps of more complexgangliosides are located in the more distal trans-Golginetwork.

Regulation of complex glycosphingolipid biosyn-thesis involves both transcriptional and posttranscrip-tional factors. For example, developmentally regulated,tissue selective variations in ganglioside amounts andtypes in mammalian tissues are under transcriptionalcontrol, but the activities of glycosyltransferases can befine tuned by posttranslational modification.

The biosynthesis of sulfatides (i.e., sulfated glyco-sphingolipids such as 30-sulfo-GalCer) is catalyzedby sulfotransferases (in this example: 30-phosphoadeny-lylsulfate:GalCer 30-sulfotransferase), which utilizethe activated sulfate donor 30-phosphoadenosine-50-phosphosulfate.

OTHER SPECIES

Although once thought to be only intermediates of sphin-golipid turnover, lysosphingolipids such as sphingosine

1-phosphate and sphingosylphosphocholine (lysosphin-gomyelin) are now known to be synthesized asimportant signaling molecules. Sphingosine 1-phosphateformation requires the release of sphingosine fromceramide (note that sphingosine is not a direct inter-mediate of de novo sphingolipid biosynthesis but firstappears in ceramide) by ceramidase(s) followed bytransfer of phosphate from ATP by sphingosine kinase(s)

(Figure 2). Less is known about the origin of sphingo-sylphosphocholine, although it is plausible that thiscould be made by a phospholipase A2-type cleavage of sphingomyelin, the transfer of phosphocholine tosphingosine, or both.

Sphingolipidomics

The large number and structural complexity of sphin-golipids has made quantitative analysis of all of themolecular species technically difficult, and heretofore

impossible with small samples such as cells in culture.However, it is now feasible to map the sphingolipid“metabolome” due to the relatively recent availability of tandem mass spectrometers of multiple configurations(e.g., tandem quadrupole, time-of-flight, and ion trapsas well as hybrids of these technologies) and modes of ionization (such as electrospray and matrix-assistedlaser-desorption ionization (MALDI)), especiallywhen combined with high-performance liquidchromatography. When complemented by the tools of genomics and proteomics, the new field of “sphingoli-pidomics” will finally be able to answer the manyriddles of how these molecules are made, and for

what functions.

SEE A LSO THE FOLLOWING A RTICLES

Glycolipid-Dependent Adhesion Processes † LipidBilayer Structure † Lysophospholipid Receptors †

Protein Palmitoylation † Sphingolipid Catabolism

GLOSSARY

ceramide An N-acyl-derivative of sphingosine that is both ametabolic intermediate and a cell signaling molecule. In some

cases, the term is applied generically to any N-acyl-sphingoid base.glycosphingolipid A compound with a carbohydrate bound to a

sphingoid base (and most often, attached to position 1 of an

N-acyl-sphingoid base).

glycosyltransferase An enzyme that transfers a carbohydrate from adonor (usually a UDP-sugar) to an acceptor which, in the case of

sphingolipids, is either ceramide or a carbohydrate chain attachedto ceramide.

phosphosphingolipid A compound with a phosphate or phosphodie-

ster linked headgroup attached to a sphingoid base (or more often,to position 1 of an N-acyl-sphingoid base).

80 SPHINGOLIPID BIOSYNTHESIS



sphingoid base The backbone of more complex sphingolipids as well

as a cell signaling molecule. Structurally, a long-chain alkane (oralkene) with an amino at position 2, and (usually) hydroxyl groupsat positon 1 and 3 plus various alkyl chain lengths, degrees of

unsaturation, and additional hydroxyl groups.

sphingosine 1-phosphate A bioactive metabolite that serves as anintracellular and an extracellular signal as well as an intermediate

of sphingoid base catabolism.

FURTHER R EADING

Hannun, Y. A., and Obeid, L. M. (2002). The ceramide-centric

universe of lipid-mediated cell regulation: Stress encounters of thelipid kind. J. Biol. Chem. 277, 25847– 25850.

IUPAC-IUB Joint Commission on Biochemical Nomenclature (JCBN)

(1998). Nomenclature of glycolipids. Recommendations 1997. Eur. J. Biochem. 257, 293–298.

Kolter, T., Proia, R. L., and Sandhoff, K. (2002). Combinatorialganglioside biosynthesis. J. Biol. Chem. 277, 25859– 25862.

Merrill, A. H. Jr., (2002). De novo sphingolipid biosynthesis. A neces-sary, but dangerous, pathway. J. Biol. Chem. 277, 25843– 25846.

Merrill, A. H., Jr., and Sandhoff, K. (2002). Sphingolipids: Metabolism

and cell signaling. In New Comprehensive Biochemistry: Biochem-istry of Lipids, Lipoproteins, and Membranes (D. E. Vance and

J. E. Vance, eds.) Chapter 14, Elsevier, Amsterdam.

Spiegel, S., and Milstien, S. (2003). Sphingosine-1-phosphate: Anenigmatic signalling lipid. Nat. Rev. Mol. Cell Biol. 4, 397–407.

Sullards, M. C., Wang, E., Peng, Q., and Merrill, A. H., Jr. (2003).

Metabolomic profiling of sphingolipids in human glioma cell lines

by liquid chromatography tandem mass spectrometry. Cell Mol.

Biol. 49, 789–797.

Thudichum, J. L. W. (1884). A Treatise on the Chemical Constitution

of Brain. Bailliere, Tindall, and Cox, London.

BIOGRAPHY Martina Leipelt holds a Doctorate in Genetics from the University of

Hamburg, Germany. Her doctoral research with Professor E. Heinz

provided a systematic functional analysis of the glucosylceramide

synthase gene family with representatives of plants (Gossypium

arboreum), animals (Caenorhabditis elegans),and fungi (Magnaporthe

grisea, Candida albicans, and Pichia pastoris). She is currently a

postdoctoral fellow with Dr. Merrill.

Al Merrill is the Smithgall Institute Chair in Molecular Cell Biology

in the School of Biology and the Petit Institute for Bioengineering

and Biosciences at Georgia Institute of Technology. Dr. Merrill’s

laboratory, in collaboration with Dr. Ron Riley at the USDA,

discovered the inhibition of ceramide synthase by fumonisins and

identified the first diseases caused by disruption of de novo

sphingolipid biosynthesis His current research deals with sphingoli-

pidomics (http://www.lipidmaps.org).


http://www.lipidmaps.org/



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