Glycoconjugate Journal 20, 39–47, 2004
C© 2004 Kluwer Academic Publishers. Manufactured in The Netherlands.
Glycosphingolipids and cell death
Meryem Bektas and Sarah Spiegel
Department of Biochemistry, Virginia Commonwealth University School of Medicine, Richmond, VA 23298, USA
Sphingolipids have been implicated in various cellular processes including growth, cell-cell or ligand-receptor interac-
tions, and differentiation. In addition to their importance as reservoirs of metabolites with important signaling properties,
sphingolipids also help provide structural order to plasma membrane lipids and proteins within the bilayer. Glycosylated
sphingolipids, and sphingomyelin in particular, are involved in the formation of lipid rafts. Although it is well accepted that
ceramide, the backbone of all sphingolipids, plays a critical role in apoptosis, less is known about the biological functions
of glycosphingolipids. This review summarizes current knowledge of the involvement of glycosphingolipids in cell death
and in other pathological processes and diseases.
Published in 2004.
Keywords: glycosphingolipids, apoptosis, GD3, glucosylceramide
Introduction
Glycosphingolipids (GSL) are lipid components of membranes
that are important for the proper development of vertebrates.
They are involved in multiple processes, including cell type
specific adhesion, cell-cell interaction, embryogenesis, and de-
velopment and differentiation of neuronal cells and leukocytes
[1]. GSL can also serve as binding sites for several viruses,
bacteria, and bacterial toxins [2]. Different tissues display dif-
ferent GSL patterns on the cell surface which can be dramat-
ically altered during development [1]. A further modulation
can be seen during pathological processes such as tumor de-
velopment. GM3/GD3, for example, is a melanoma-associated
antigen involved in metastasis [3–5]. On the other hand, gluco-
sylceramide (GlcCer) expression is associated with multidrug
resistance in many cancer cells [6–8].
GSL are predominantly located at the plasma membrane and
the early endosomes of the Golgi complex. In the plasma mem-
brane, it has recently been shown that sphingolipid-derived
molecules aggregate and form a less fluid and more ordered
phase, referred to as membrane rafts, which are formed in the
Golgi compartment and targeted to the plasma membrane. Rafts
are considered to be small, mobile lateral assemblies of sphin-
golipids, particularly enriched in sphingomyelin and choles-
terol, but also containing ceramide and GPI-anchored proteins.
To whom correspondence should be addressed: Dr. Sarah Spiegel,
Department of Biochemistry, Virginia Commonwealth University School
of Medicine, Richmond, VA 23298-0614, USA. Tel: (804) 828-9330; Fax:
(804) 828-8999; E-mail: sspiegel@vcu.edu
They have important roles in concentrating and modulating
specific signaling molecules, such as Src-tyrosine kinase fam-
ily members, growth receptors, and death receptors [9–12].
The role of rafts will not be discussed here as it has been
the subject of recent excellent reviews [9,13]. Less complex
sphingolipid-derived molecules, including ceramide, ceramide-
1-phosphate, sphingosine, and sphingosine-1-phosphate (S1P),
are known signaling molecules in diverse receptor and non-
receptor-mediated signaling pathways. These bioactive lipid
mediators are formed as a result of stimuli-induced metabolism
of complex sphingolipids. Ceramide has mainly been impli-
cated in signaling pathways leading to suppression of growth,
cellular senescence, differentiation, and apoptosis, whereas
ceramide-1-phosphate mediates cell survival and is involved
in synaptic vesicular fusion in neuronal cells, as well as neu-
trophil phagolysosome formation [14]. S1P has many biological
actions and, importantly, acts counter to ceramide to mediate
cell growth and survival, as well as influencing directed cell
movement [15,16]. The biological effects of sphingosine may
vary among cell types but it has been associated with negative
effects on cell growth and survival and has been implicated as an
inhibitor of protein kinase C and other protein kinases [17,18].
Biosynthesis and structure of glycosphingolipids
The de novo biosynthesis of GSL is initiated at the cytosolic sur-
face of the endoplasmic reticulum (ER) by the condensation of
L-serine and palmitoyl coenzyme A to form 3-ketosphinganine
catalyzed by serine palmitoyltransferase (SPT), a pyridoxal
phosphate-dependent enzyme [19,20]. SPT has lower activity

40 Bektas and Spiegel
than the other enzymes involved in biosynthesis and is rate-
limiting and seems to be a key enzyme controlling cellular
sphingolipid content. In the ensuing NADPH-dependent reac-
tion, 3-ketosphinganine is reduced to D-erythro-sphinganine
by 3-ketosphinganine reductase. The enzyme sphinganine-N -
acyltransferase (ceramide synthase) transfers a long-chain fatty
acid to the amino group of 3-ketosphinganine, resulting in the
formation of D-erythro-dihydroceramide. The latter enzyme
shows selectivity for stearic acid and is also able to acylate sph-
ingosine derived from the “salvage pathway” of sphingolipid
catabolism [21]. A double bond is then introduced between car-
bon atoms 4 and 5 by a desaturase to form ceramide [22]. All
four enzymes of ceramide biosynthesis are located at the cy-
tosolic surface of the ER membrane [23,24]. Recently, major
progress has been made in cloning the enzymes of the de novo
pathway.
Ceramide is a precursor of both GSL and sphingomyelin.
In the synthesis of sphingomyelin, a phosphocholine group
is transferred from phosphatidylcholine to ceramide. Sphin-
gomyelin synthesis occurs in several cellular compartments,
although most is synthesized on the lumenal side of the Golgi
complex [25–27]. In vertebrates, GSL synthesis is initiated
by coupling a glucose [28] or galactose [29] residue in a β-
glycosidic linkage to the C1-hydroxyl of ceramide. Specific
glycosyltransferases catalyze [30,31] the transfer of additional
single nucleotide activated sugars onto ceramide forming more
complex GSL (Figure 1) [30–32]. Most of the GSL of ver-
tebrates arise from glucosylation rather than galactosylation of
LacCer GM3 GD3 GT3
GlcCer
Cer
GA2
GA1
GM1b
GM2
GM1a
GD1a
GD2
GD1b
GT1b
GT2
GT1c
GQ1c
L-serine + palmitoyl-CoA
3-ketosphinganine
dihydrosphingosine
dihydroceramide
0-series a-series b-series c-series
GD1αGD1c GT1αGT1a GP1c GP1cαGQ1αGQ1b
GalCerSulfatide
SM
Figure 1. Simplified scheme of glycosphingolipid biosynthesis.
For detailed descriptions of the biosynthetic pathways, see [38]
and text.
ceramide. Glucosylation is rate-limiting for ganglioside biosyn-
thesis. This glucosyltransferase seems to be crucial during em-
bryogenesis as the knock out of the respective gene has been
shown to be lethal [33].
Regarding topology of GSL synthesis, ceramide must be
transported from the ER to the Golgi complex where it is glu-
cosylated on the cytosolic surface of the Golgi-compartment
[30,34–36]. Galactosylation of ceramide in the formation of
glycosphingolipids of the galacto-series has been localized to
the ER and Golgi. Transfer of a sulfate headgroup results in for-
mation of sulfatides. The galacto-series gangliosides are found
predominantly in the nervous system where they are important
in development and normal functioning of the CNS [37–39].
Transport of ceramide from the ER to Golgi can occur by means
of vesicular and non-vesicular mechanisms [40–42] and subse-
quent addition of sugar residues occurs on the lumenal face of
the Golgi catalyzed by distinct glycosyltransferases [43].
Almost all gangliosides are structurally and biosynthetically
derived from lactosylceramide which is formed by the transfer
of a galactosyl residue to glucosylceramide. Sequential addition
of one, two or three sialic acids to lactosylceramide results
in formation of GM3, GD3 and GT3, respectively, precursors
for more complex ganglio-series gangliosides. Sphingolipids
are targeted to their cellular sites by both vesicular and non-
vesicular trafficking [44].
Mechanisms of GD3-induced apoptosis
Ceramide is a well-known participant in the progression of pro-
apoptotic signals initiated by Fas and tumor necrosis factor-α
(TNF-α) through activation of their respective death receptors.
The mitochondria also has a central role in ceramide-mediated
cell death [45,46]. Generation of ceramide at the mitochon-
dria, but not at other organelles, was shown to be involved
in apoptosis of MCF-7 human breast cancer cells [47]. Fas
cross-linking, TNF-α, and cell-permeable ceramide analogs all
induce transient intracellular ceramide accumulation. An el-
egant study has shown that the intracellular ceramide accu-
mulated due to Fas cross-linking is rapidly converted to GD3
by enhanced GD3 synthase activity in lymphoid and myeloid
cell lines [48]. Antisense RNA against GD3 synthase prevents
apoptosis, implying the need for newly-synthesized GD3. On
the other hand, enforced expression of GD3 synthase was suf-
ficient to trigger apoptosis. In this study, other gangliosides,
such as GD1a, GT1b or GM1, failed to mimic GD3-induced
cell killing [48]. Use of broad-spectrum caspase inhibitors re-
vealed that caspases upstream of GD3 synthesis were crucial
for Fas-induced apoptosis, suggesting modulatory interaction
between GD3 and caspases. With respect to the mechanism
by which GD3 induces apoptosis, changes in the mitochon-
drial membrane potential (m) and increased reactive oxy-
gen species (ROS) production have been demonstrated [48–51].
As a consequence of decreased m , permeability of the in-
ner mitochondrial membrane increases, causing the collapse of
the ion gradient along the membrane and depolarization of the