Neurochemical Research, Vol. 27, Nos. 7/8, August 2002 (© 2002), pp.

629–636

Understanding the Stepwise Synthesis of Glycolipids*

Hugo J. F. Maccioni,1,2 Claudio G. Giraudo,1 and José Luis Daniotti1

(Accepted March 18, 2002)

Glycolipid expression is highly regulated during development and differeniation. The control
relies mainly on transcriptional modulation of key glycosyltransferases acting at the branching
points of the pathway of biosynthesis. Transferases are Golgi residents that depend on N-gly-
cosylation and oligosaccharide processing for proper folding in the endoplasmic reticulum. The
N-terminal domain bears information for their transport to the Golgi, retention in the organelle
and differential concentration in sub-Golgi compartments. In the Golgi, some transferases as-
sociate forming functional multienzyme complexes. It is envisaged that the machinery for syn-
thesis in the Golgi complex, and its dynamics, constitute a potential target for fine tuning of the
control of glycolipid expression according to cell demands.

KEY WORDS: Glycolipid synthesis; glycosyltransferases; Golgi complex organization.

INTRODUCTION Ganglioside Expression in Developing Neural Cells

Glycosphingolipids constitute a large and hetero-
geneous family of amphypathic lipids anchored to the
extracytosolic leaflet of cell membranes through the
ceramide moiety. At the plasma membrane, they ex-
pose the sugar-containing hydrophilic portion to the
extracellular space, contributing to the complexity of
the glycocalix. Gangliosides are a particular glycolipid
class characterized by containing a variable number of
sialic acid residues. Nearly all animal cells contain at
least some ganglioside class in their membranes, but
the plasma membrane of cells from the central nervous
system are particularly rich in these compounds.
* Special issue dedicated to Dr. Guido Tettamanti.
1
Centro de Investigaciones en Química Biológica de Córdoba,
CIQUIBIC (UNC-CONICET), Departamento de Química Biológ-
ica, Facultad de Ciencias Químicas, Universidad Nacional de Cór-
doba, 5000 Córdoba, Argentina.
2
Tel: 54-351-4334168/4334171; Fax: 54-351-4334074; E-mail:
maccioni@dqbfcq.uncor.edu
The composition of central nervous system gan-
gliosides is regulated during development in a cell
type– and region-dependent manner (1– 4). An illustra-
tive example is the developing vertebrate retina, which
from an early outfold of the forebrain epithelium gives
rise to the optic nerve and the optic vesicle. Later in-
vagination of the optic vesicle neuroepithelium results in
the formation of an outer layer, from which the neural
retina is generated, and an inner layer from which the
retinal pigment epithelium is generated (5). At the limit-
ing zone between the inner and outer epithelial layers,
the ciliary margin, the cells behave as multipotential pre-
cursors. Descendants displaced toward the outer epithe-
lial layer give rise to most of the different cell types of
the neural retina while those displaced toward the inner
epithelial layer give rise to cells of the pigment epithe-
lium. In chickens, GM3 and GD3, in decreasing order of
concentration, are the major gangliosides in both epithe-
lial layers around 3 days of development (6). As devel-

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630
opment progresses GD3 becomes the major ganglioside
in the whole neural retina and it is then progressively
substituted by GD1a, which is the major ganglioside by
embryonic day 15 and in the adult (2). Cells that follow
the route of differentiation towards the retinal pigmented
epithelium, on the other hand, present a pattern of gan-
glioside composition totally different: they maintain
GM3 expression and practically do not express any other
ganglioside even in the adult stage (6).
Developmental Control of Ganglioside Expression
Ganglioside compositional changes as those de-
scribed above constitute a general phenomenon in the
developing central nervous system (1). They rely on
the balance between the activities of glycosyltrans-
ferases acting at the branching points of the pathway
of biosynthesis in which common precursors are used
for synthesis of different products (2,3,7,8). As shown
in Figure 1, LacCer, GM3 and GD3 are common sub-
strates for GalNAc-T and for, respectively, Sial-T1
and Sial-T2 (it is not clear at the present if Sial-T2 or
a different enzyme, Sial-T3, acts on GD3 to form
GT3). The relative activities of the two enzymes act-
ing at these branching points influence whether the
common substrates will be mainly used as precursors
Fig. 1. Schematic representation of the biosynthesis of gangliosides
(for details, see reference 21).
Maccioni, Giraudo, and Daniotti
of either the “o,” “a,” “b,” or “c” series gangliosides.
In the case of the developing chick embryo retina
mentioned above, the pattern dominated by GM3 in
pigmented epithelium cells corresponds with very low
activity values of both GalNAc-T and Sial-T2 (6). In
neural retina cells at early developmental stages, the
high GD3 expression corresponds with a reduced ac-
tivity of GalNAc-T and a high activity of Sial-T2. As
development proceeds, a drop in Sial-T2 activity and
a concomitant increase of GalNAc-T activity results
in a high GalNAc-T/Sial-T2 activity ratio. As a result,
the common precursor GM3 is conveyed towards the
synthesis of GM2 and complex gangliosides of the
“a” pathway (2,7). Similar relationships between
transferase activities and glycolipid expression were
found in non neural tissues (9) and during oncogenic
transformation (10–13). Activities, in turn, depend on
the transcriptional regulation of the respective genes
and also on posttranscriptional events. Among post-
transcriptional events, for some of these enzymes the
relative saturation with the cognate sugar nucleotide
donors (14–16) or their phosphorylation state (17–20)
have been shown to modulate activities (for review,
see references 21 and 22).
At a macromolecular level, it should be considered
that reactions indicated in Figure 1 occur in the Golgi
complex. In this organelle the transferases form part of
a complex, poorly understood machinery for synthesis.
In the following paragraphs we present a brief descrip-
tion of the information, some of which is just emerging,
about the events leading to the organization of such ma-
chinery. Knowledge of these events is fundamental as a
previous step to understand its dynamics and ability
to act as a unit capable of fine tuning of ganglioside
expression in response to regulatory clues.
The Synthesis in the Golgi Complex
Glycosyltransferases, sugar nucleotide transporters
and intermediates of the synthesis are integral com-
ponents of the Golgi membranes, but their precise
structural relationships are still under investigation.
Ceramide synthesized at the cytosolic face of the ER
is (23) transported to the Golgi and used there for syn-
thesis of glucosylceramide-derived glycolipids by Golgi
resident glycosyltransferases. Galactosylceramide (Gal-
Cer constitutes an exception, since it is synthesized in
the lumen of the ER from translocated ceramide) (24).
The first acting transferase on glucosylceramide-
derived glycolipids, ceramide glucosyltransferase (Glc-
T), is a type III membrane protein with its catalytic site
oriented toward the cytoplasm (23,25). Glucosylce-
Understanding the Stepwise Synthesis of Glycolipids
ramide (GlcCer) formed at the cytoplasmic surface of
cis Golgi membranes translocates somewhere along the
Golgi stack to the lumenal surface of the membranes.
There, it is used as substrate by the subsequent glyco-
syltransferases that build up the higher order neutral
glycolipid derivatives and gangliosides. These trans-
ferases share with glycoprotein glycosyltransferases the
typical type II membrane topology, consisting of a short
cytosolic tail, a transmembrane region, and a luminally
oriented catalytic domain (26). They also share the
property of a differential concentration along sub com-
partments of the Golgi complex.
Sub-Golgi Localization of Different Transfer Steps
Different methodologies have been applied to
map the sub-Golgi localization of the different steps
that, starting from GlcCer, end up with the synthesis of
complex gangliosides. The information coming from
the approaches mentioned in the following paragraphs
produced a reasonably clear picture of the sub-Golgi
localization of ganglioside glycosyltransferases.
Endogenous Acceptor Labeling Studies. Experi-
ments of labeling the endogenous acceptors of isolated
Golgi membranes from chicken neural retina cells,
showed that transferases acting beyond the synthesis of
GlcCer overlap and are functionally coupled in the
trans Golgi network (TGN). The synthesis of GlcCer,
however, was uncoupled from the synthesis of LacCer
(16). GlcCer synthesis in vitro in these membranes oc-
curs all along Golgi subfractions, but the synthesized
GlcCer molecules were essentially inaccessible to the
lumenal LacCer synthase (27). These experiments indi-
cated that a translocating activity exists in vivo, most
probably in cis Golgi subcompartments, that positions
GlcCer in the lumenal surface of the Golgi membranes;
this was an elusive activity in vitro, and direct evidence
for it is still lacking. Translocating activities, despite
being essential activities, have in general remained elu-
sive (28). The recent identification of a protein that
translocates polyprenol linked oligosaccharides from
the cytosolic side of ER membranes to the lumen of the
ER (29) will probably open the way to the discovery of
other translocating activities.
GlcCer moves from the cis Golgi to the distal
Golgi, presumably via transport vesicles (30) or as part
of a cisternal maturation process (31). In the distal Golgi,
GlcCer is topologically available for elongation in vitro
up to the stage of GD1a if the membranes are incubated
with appropriate sugar nucleotide donors (16). These ex-
periments clearly indicated that all reactions necessary
for the conversion of GlcCer into GD1a colocalize and
are functionally coupled in the isolated Golgi vesicles.
631
The origin of these vesicles was anticipated to be the
trans-Golgi network, since one of the enzymes operating
in the vesicles, GalNAc-T, was mapped beyond the
block imposed by Brefeldin A (BFA) on the metabolic
labeling of gangliosides (see later).
Subfractionation Studies. Subfractionation by dif-
ferential or/and isopycnic centrifugation followed by
determination of the codistribution of the enzyme activ-
ities with established markers of Golgi membranes has
been widely applied. Former studies in rat liver cells in-
dicated that glycolipid glycosyltransferases localize
along the Golgi stack following the order in which they
act in the pathway of synthesis (32,33). Later on, and in
agreement with the results obtained in chicken neural
retina cells discussed above, all the enzymes that cat-
alyze the steps of synthesis from LacCer to complex
gangliosides were localized in the late Golgi compart-
ment of liver cells (34). In both studies with liver cells,
considerable overlap among subfractions was evident
and it was not clear if this was due to limitations of the
separation technique or to true overlapping distribution
of the enzymes.
Pharmacological Studies. Drugs that impair the
function of the Golgi complex or the vesicular flow
along the ER–Golgi complex–plasma membrane path-
way have also been used to map the different transfer
steps along Golgi subcompartments. Monensin, a car-
boxylic ionophore that catalyses cation-for-proton ex-
change across membranes causes swelling of trans
Golgi cisternae and TGN, where H1-ATPases concen-
trate (35). As a consequence, biosynthetic steps of cis
and medial Golgi are less affected by Monensin than
those of the trans Golgi and TGN. It was found that
the synthesis of complex gangliosides was more af-
fected by monensin than the synthesis of the simple
gangliosides GM3 and GD3 (36–38). BFA inhibits
loading of the ADP ribosylation factor (ARF) with
GTP, impairing the recruitment of coatomer proteins
on Golgi membranes. This causes a redistribution of
the proximal Golgi (cis, medial, and trans) into the ER
and blocks the transport from these compartments to
the distal Golgi (TGN), which is fused with early en-
dosomes (39). The metabolic labeling of glycolipids in
different cell types in the presence of BFA (40 – 42)
indicate that reactions for synthesis of simple ganglio-
sides, although they may act in the distal Golgi, act
also in proximal Golgi, while those acting beyond
GalNAc-T were absent from proximal Golgi. (For a re-
view, see reference 21).
Metabolic and Immunocytochemical Studies. De-
termination of the sub-Golgi localization of glycolipid
glycosyltransferases by classic immunocytochemistry
has been impaired by the lack of suitable antibodies
632
against the native forms. to this matter, The heterolo-
gous expression of epitope tagged glycosyltransferases
combined with immunolocalization studies at the light
and electron microscope was used as an alternative ap-
proach. GalNAc-T (Fig. 1) has been carefully exam-
ined through this approach in several CHO-K1 cell
clones stably expressing a c-myc tagged at different
levels of activity (43). It was found that BFA com-
pletely blocked the synthesis of GM2, GM1, and
GD1a in moderately expressing clones, leading to ac-
cumulation of GM3. This indicated that GalNAc-T
does not localize in proximal Golgi compartments and
in presence of BFA resulted uncoupled from the en-
zyme that glycosylate ceramide up to the stage of
GM3, now merged in the ER membranes. This is not
an indication that these enzymes reside exclusively in
the proximal Golgi; however, transport of gangliosides
toward the plasma membrane is also blocked by BFA
(42), implying a prolongation in the time of residence
of intermediates in membranes of the ER. Thus, with
just a fraction of the total of the enzymes Glc-T, Gal-T1,
and Sial-T1 in the ER all incoming ceramide would be
converted in GM3.
Direct visualization of GalNAc-T in these cells by
c-myc immunostaining confirmed the absence of this
enzyme from the proximal Golgi and its presence in
TGN membranes. Studies of co localization with estab-
lished markers of medial Golgi (Mannosidase II, ManII)
and TGN-endosomal (mannose-6-P-receptor, M6PR)
membranes in cells treated with BFA showed GalNAc-
T colocalized with the TGN marker M6PR but not with
the medial Golgi marker ManII in cells of moderately
expressing clones. However, in highly expressing
clones it colocalized also with ManII, suggesting that
the mechanism that concentrates it to the TGN involves
steps that, when saturated, lead to its mislocalization to
the cis, medial, or trans Golgi (43). GalNAc-T forms
disulfide bonded dimers (44). It is possible that upon
heavy expression large oligomers may be formed either
in the ER or in the Golgi that escape to the mechanisms
that control their fine sub Golgi localization (see later).
Similar studies, carried out with epitope tagged Sial-T2
showed the presence of this enzyme in proximal but
also in distal compartments of the Golgi complex (45).
Assembling the Machinery for Synthesis of
Glycolipids
An emerging question from the studies com-
mented above is to know how the particular distribu-
tion of the different glycosyltransferases along the
Maccioni, Giraudo, and Daniotti
Golgi subcompartments is achieved. Glycosyltrans-
ferases synthesized in the ER move toward the Golgi
complex. They are retained in the organelle while their
glycolipid products follow the exocytic membrane
flow. The molecular signals that participate in the fold-
ing of these proteins in the ER during or after synthe-
sis, in their transport from the ER to the Golgi, in their
retention in the organelle forming particular gradient
concentrations along cisternae and particular associa-
tions between them, are unsettled issues at present.
Folding. Conceptual translation of glycosyltrans-
ferase cDNAs disclose a variable number of N-glyco-
sylation sequons (Asp-X-Ser/Thr) (21). Studies on the
occupancy of these sequons and on the eventual role of
these oligosaccharides in GalNAc-T, Gal-T2, and Sial-
T2 (45–48) revealed that N-linked oligosaccharides
are fundamental for the enzyme activity and the sub-
cellular localization of these transferases. The case of
the mouse b1,3 galactosyltransferase (Gal-T2), which
contains just one N-glycosylation site (Asn 143), clearly
illustrates this observation. Inhibition of N-glycosyla-
tion by either an inhibitor of en bloc glycosylation
(Tunicamycin) in the ER or by elimination of the
N-glycosylation site by site-directed mutagenesis re-
sulted in the synthesis of a polypeptide of 40 kDa that
lacks enzyme activity and is unable to exit the ER
(Fig. 2). Inhibition of N-glycan trimming by Cas-
tanospermine (an inhibitor of ER glucosidase I and II)
partially impaired the exiting from the ER of an en-
zyme with reduced enzyme activity showing reduction
of the Km and Vmax values for the substrates (48).
It is clear that N-glycosylation and N-glycan
trimming, probably through known oligosaccharide-
mediated quality control processes (49), is relevant for
the control of Gal-T2 folding in the ER and necessary
for a conformation competent for its enzyme activity
and exiting from the ER. Similar results with inhibitors
of N-glycosylation were obtained with Sial-T2; in this
case, release of the N-glycans from the solubilized en-
zyme by treatment with N-glycanase resulted also in
decreased thermal stability (47).
Transport from the ER to the Golgi Complex.
Membrane bound proteins, and secreted proteins
move through the exocytic pathway as components of
the vesicular flow. From the site of synthesis in the
ER they are conveyed to the Golgi complex and are
either retained as residents of the organelle or con-
tinue moving in traffic towards post Golgi compart-
ments and the plasma membrane. Concentration of
cargo in the exiting sites of the ER, and formation of
COPII coated transport vesicles is a complex process
that begins with the recruitment of several cytosolic
Understanding the Stepwise Synthesis of Glycolipids
Fig. 2. Subcellular localization of Gal-T2-HA and Gal-T2-
HAAsn143Gln. Cells expressing Gal-T2-HA (A, B), or Gal-T2-
HAAsn143Gln (C, D) were double immunostained for Gal-T2-HA
(A, C) and for ManII (B, D) and examined by conventional fluo-
rescence microscopy. Arrows point to cells transiently expressing
Gal-T2-HA. Note that while in control cells Gal-T2 showed typical
Golgi localization (A); in cells transfected with the mutated cDNA
(C) the immunoreactivity was mainly located in the endoplasmic
reticulum.
proteins. The process is initiated by conversion to the
GTP form of a small GTP binding protein, Sar1,
which translocates from the cytosol to the ER mem-
brane. Binding of Sar-1 is followed by recruitment of
other cytosolic proteins necessary for formation of the
cargo-containing COPII vesicles that initiate the jour-
ney to the Golgi complex (50).
Studies from this laboratory have shown that the
N-terminal domain of glycolipid glycosyltransferases
is sufficient for conveying reporter proteins (i.e., GFP)
out from the ER toward the Golgi complex, and to con-
centrate them in the organelle (48,51). Whether the cy-
tosolic domain, or the membrane spanning domain or
the stem region, or appropriate combinations of them
are relevant for these glycosyltransferases to become a
cargo of the transporting vesicles remains to be estab-
lished. Preliminary experiments from this laboratory
indicate that the cytosolic domains are important for
exiting the ER in the way to the Golgi, since deletion
mutants fail to concentrate in the Golgi, and remain in
the ER (52).
Distribution along Sub-Golgi Compartments.
Several lines of evidence point to the transmembrane
633
domains (TMDs) of glycoprotein glycosyltransferases
as preventing leakage from the Golgi complex. This is
also true for glycolipid glycosyltransferases (48,51).
The lack of homology among TMDs led to the pro-
posal of two models, which are not mutually exclu-
sive, to explain the TMD mediated retention of these
proteins in the Golgi. One model proposes that associ-
ations between glycosyltransferases through their
amino terminal domains (Ntd) may contribute to the
formation of aggregates that by virtue of their greater
size are excluded from transport vesicles leaving the
Golgi, and are thus retained in the organelle (53). The
other model proposes that the intrinsic properties of
the TMD of Golgi enzymes drives their exclusion from
transport vesicles and their differential partition in
Golgi lipid domains, from which proteins destined for
transport are excluded (54).
As mentioned earlier, a concentration gradient of
the glycosyltransferases acting on the elongation of
GlcCer does exists, with GalNAc-T concentrating in
the TGN (43), and Sial-T2 more evenly distributed
along proximal and distal compartments (45). The Ntds
of these enzymes seem to carry on information for that
particular distribution. In cells expressing chimeras be-
tween these Ntds and the GFP showed that the GFP
fused to the Ntd of Sial-T2 behaved as a proximal
Golgi protein, redistributing to the ER in the presence
of BFA. Fused to the Ntd of GalNAc-T, on the other
hand, GFP fluorescence appeared condensed with late
endosomes, as TGN located proteins did. It also re-
mains to be elucidated which elements within the Ntds
are relevant for establishing the particular concentra-
tion gradients of these two enzymes.
Physical and Functional Relationships in Golgi
Membranes. The experiments of labeling the endoge-
nous glycolipids of intact Golgi membranes in vitro
described above clearly showed that some transfer
steps of the pathway (Fig. 1) colocalize and are func-
tionally coupled. Since these experiments were carried
out in the absence of cytosolic proteins necessary for
sustaining vesicular coupling among compartments, it
was concluded that these transfer steps colocalize
functionally in the same Golgi vesicle. Many years ago
it was postulated that ganglioside glycosyltransferases
form multienzyme complexes on which glycolipid
oligosaccharides grow by the successive addition of
sugar moieties (55,56). For many years it was difficult
to demonstrate the existence of such complexes. It was
not possible to discard that acceptors and transferases
for each step could be spatially segregated along mem-
brane cisternae but still be able to collide with each
other due to their rapid lateral diffusion (57). More-
634
over, tubular connections between different cisternae
may allow diffusion of intermediates and coupling of
products of one step with enzymes working in the next
step located in a different cisternae.
The presence of complexes between GalNAc-T
and Gal-T2 in Golgi membranes of CHO-K1 cells
that constitutively express tagged forms of these en-
zymes were examined in our laboratory (51). Ex-
periments of coimmunoprecipitation showed that a
fraction of GalNAc-T specifically coimmunoprecipi-
tates Gal-T2 and vice versa, that a fraction of Gal-T2
coimmunoprecipitates GalNAc-T. The complexes ef-
ficiently transformed GM3 into GM1 on incubation
with UDP-GalNAc and UDP-[3 H]-Gal in vitro, indi-
cating that GalNAc-T and Gal-T2 are constituents of
an integral membrane enzyme complex that may en-
hance glycolipid glycosylation efficiency (Fig. 3).
Interaction between these two enzymes occurs with
at least participation of the N-terminal domains since
when the full-length form of one of them was co-
transfected with the N-terminal domain of the other,
it was still possible to coimmunoprecipitate one with
another. In addition, a dominant negative effect was
exerted by the truncated form of Gal-T2 over the full
length Gal-T2, as evidenced by inhibition of the
amount of GM1 formed in immunoprecipitates of
triple transfectants.
The existence of these complexes in vivo was con-
firmed by experiments of fluorescence resonance en-
ergy transfer (FRET) between chimeric constructs of
the N-terminal domain of the two enzymes and mutants
of the GFP with spectral overlap. The chimeras local-
ized to the Golgi complex in an association close
enough to allow FRET between the fluorofores (Fig. 4).
Another interesting possibility emerging from these ex-
periments is that these macromolecular associations
may, by virtue of their size, be excluded from the small
transport vesicles budding from the Golgi towards the
plasma membrane, thus contributing to the mechanism
of retention of the transferases in the Golgi complex.
Further work will be necessary to know how other
transferases behave in terms of associations. Among
many possibilities, it will be interesting to know if all
of them participate of a unique multienzyme complex
or if complexes of different transferases exist. In the
second possibility, they may have different sub-Golgi
location and coupling between them may present in-
teresting alternatives. Furthermore, it will be interest-
ing to know whether these complexes concentrate in
particular lipid domains of the Golgi membranes, as do
other proteins (29).
Maccioni, Giraudo, and Daniotti
Fig. 3. Immunocomplexes from double transfectants efficiently
convert GM3 to GM1. Truncated Gal-T2 affects the efficiency of
conversion. Immunocomplexes from double (GalNAc-T/Gal-T2)
(lane 1) or triple (GalNAc-T/Gal-T2/Gal-T21–52) (lane 2) trans-
fectants were incubated with 100 mM UDP-GalNAc, 10 mM UDP-
[3H]Gal (1 3 106 cpm), 20 mM MnCl2, 100 mM sodium cacodylate
pH 7.2, 3 mM CDP-choline, and 20 mg Triton CF-54–Tween 80
(2:1, w/w) for 2 h at 37°C in the presence of 400 mM GM3.
Reactions products were isolated, run on HPTLC, and subjected to
PhosphorImaging. Cochromatographed radioactive glycolipid stan-
dards are shown at right.
SUMMARY
Ganglioside expression is highly regulated dur-
ing development and differentiation. The control re-
lies mainly on transcriptional and posttranscriptional
modulation of key glycosyltransferases acting at the
branching points of the pathway of biosynthesis.
These transferases are Golgi resident proteins that de-
pend on proper N-glycosylation and oligosaccharide
processing for proper folding in the ER and on deter-
minants on the N-terminal domain for their transport
to the Golgi, retention in the organelle, and differen-
tial concentration in the sub-Golgi compartments.
Coimmunoprecipitation and fluorescence resonance
energy transfer experiments indicate that within the
Golgi, some transferases associate forming functional
Fig. 4. Gal-T21–52-ECFP and GalNAc-T1–27-EYFP undergo FRET in
living cells. Cells expressing Gal-T21–52 ECFP (Donor) and GalNAc-
T1–27-EYFP (Acceptor) fusion proteins were observed with the
filters for ECFP (Donor) or EYFP (Acceptor) or FRET. Arrows
indicate colocalization of the truncated forms of Gal-T2 and GalNAc-
T in the Golgi region. Arrowheads indicate regions of the Golgi in
which truncated Gal-T2 and GalNAc-T forms were close enough as
to undergo FRET.
Understanding the Stepwise Synthesis of Glycolipids
multienzyme complexes, which may increase the effi-
ciency of the synthesis and also favor their retention in
the organelle. Whether or not all transferases acting on
the pathway of synthesis form part of a common mul-
tienzyme complex remains to be established. It is en-
visaged that the machinery for synthesis in the Golgi
complex and its dynamics constitute a potential target
for fine tuning of the control of ganglioside expression
according to cell demands.
ACKNOWLEDGMENTS
This work was supported in part by National Grants PMT-
PICT-0181 from CONICET, 89/96 from SECyT-UNC, 01-5185
from ANPCYT, Ramon Carrillo-Arturo Oñativia from MSPN
(Argentina), and Grants 75197 554001 from the Howard Hughes
Medical Institute and 10087 from Mizutani Foundation for Glyco-
science. H. J. F. M. and J. L. D. are Career Investigators, and C.G.G.
Fellow, of CONICET.
REFERENCES
1. Rösner, H. 1994. Ganglioside and brain development. Pages
19–36, In Glycobiology and the Brain, Pergamon Press, Oxford-
New York.
2. Panzetta, P., Maccioni, H. J. F., and Caputto, R. 1980. Synthe-
sis of retinal gangliosides during chick embryonic development.
J. Neurochem. 35:100–108.
3. Yu, R. K., Macala, L. J., Taki, T., Weinfield, H. M., and Yu,
F. S. 1988. Developmental changes in ganglioside composition
and synthesis in embryonic rat brain. J. Neurochem. 50:
1825 –1829.
4. Daniotti, J. L., Rosales Fritz, V. M., Kunda, P., Nishi, T., and
Maccioni, H. J. 1997. Cloning, characterization and develop-
mental expression of alpha2,8 sialyltransferase (GD3 synthase,
ST8Sia I) gene in chick brain and retina. Int. J. Dev. Neurosci.
15:767–776.
5. Coulombre, A. J. 1955. Correlation of structural and biochemi-
cal changes in the developing retina of the chick. Am. J. Anat.
26:153–190.
6. Daniotti, J. L., Landa, C. A. and Maccioni, H. J. F. 1994. Regu-
lation of ganglioside composition and synthesis is different in
developing chick retinal pigment epithelium and neural retina.
J. Neurochem. 62:1131–1136.
7. Daniotti, J. L., Landa, C. A., Rosner, H., and Maccioni, H. J. F.
1991 Dec. GD3 prevalence in adult rat retina correlates with the
maintenance of a high GD3-/GM2-synthase activity ratio
throughout development. J. Neurochem. 57:2054 –2058.
8. Bieberich, E. and Yu, R. K. 1999. Multi-enzyme kinetic analy-
sis of glycolipid biosynthesis. Biochim. Biophys. Acta, 1432:
113–124.
9. Lloyd, K. O. and Furukawa, K. 1998. Biosynthesis and functions
of gangliosides: recent advances. Glycoconj. J. 15:627–636.
10. Cumar, F. A., Brady, R. O., Kolodny, E. H., McFarland, V. W.,
and Mora, P. T. 1970. Enzymatic block in the synthesis of gan-
gliosides in DNA virus-transformed tumorigenic mouse cell
lines. Proc. Natl. Acad. Sci. U.S.A. 67:757–764.
11. Hakomori, S. 1981. Glycosphingolipids in cellular interaction,
differentiation, and oncogenesis. Annu. Rev. Biochem. 50:
733 –764.
635
12. Nagai, Y., Nakaishi, H., and Sanai, Y. 1986. Gene transfer as a
novel approach to the gene-controlled mechanism of the cellu-
lar expression of glycosphingolipids. Chem. Phys. Lipids 42:
91–103.
13. Sung, C. C., Pearl, D. K., Coons, S. W., Scheithauer, B. W.,
Johnson, P. C., and Yates, A. J. 1994. Gangliosides as diagnos-
tic markers of human astrocytomas and primitive neuroectoder-
mal tumors. Cancer 74:3010 –3022.
14. Martina, J. A. and Maccioni, H. J. 1996. UDP-sugar pyrophos-
phatase of rat retina: Subcellular localization and topography.
J. Neurosci. Res. 46:485– 491.
15. Martina, J. A., Daniotti, J. L., and Maccioni, H. J. F. 1995. A
UDP-GalNAc pyrophosphatase activity is developmentally reg-
ulated in the rat retina. J. Neurochem. 64:1274 –1280.
16. Maxzúd, M. K., Daniotti, J. L., and Maccioni, H. J. F. 1995.
Functional coupling of glycosyltransfer steps for synthesis of
gangliosides in Golgi membranes from neural retina cells.
J. Biol. Chem. 270:20207–20214.
17. Scheideler, M. A. and Dawson, G. 1986. Direct demonstration
of the activation of UDP-N-acetylgalactosamine: [GM3]N-
acetylgalactosaminyltransferase by cyclic AMP. J. Neurochem.
46:1639–1643.
18. Takamiya, K., Yamamoto, A., Yamashiro, S., Furukawa, K.,
Haraguchi, M., Okada, M., Ikeda, T., Shiku, H., and Furukawa,
K. 1995. T cell receptor mediated stimulation of mouse thymo-
cytes induces up-regulation of the GM2/GD2 synthase gene.
FEBS Lett. 358:79–83.
19. Gu, X., Preuss, U., Gu, T., and Yu, R. K. 1995. Regulation of
sialyltransferase activities by phosphorylation and dephospho-
rylation. J. Neurochem. 64:2295 – 2302.
20. Bieberich, E., Freischutz, B., Liour, S. S., and Yu, R. K. 1998.
Regulation of ganglioside metabolism by phosphorylation and
dephosphorylation. J. Neurochem. 71:972–979.
21. Maccioni, H. J. F., Daniotti, J. L., and Martina, J. A. 1999. Or-
ganisation of ganglioside synthesis in the Golgi apparatus.
Biochim. Biophys. Acta 1437:101–118.
22. Yu, R. K. and Bieberich, E. 2001. Regulation of glycosyltrans-
ferases in ganglioside biosynthesis by phosphorylation and de-
phosphorylation. Mol. Cell. Endocrinol. 177:19–24.
23. Futerman, A. H. and Pagano, R. E. 1991. Determination of the
intracellular sites and topology of glucosylceramide synthesis in
rat liver. Biochem. J. 280:295–302.
24. Sprong, H., Kruithof, B., Leijendekker, R., Slot, J. W., van
Meer, G., and van der Sluijs, P. 1998. UDP-galactose:ce-
ramide galactosyltransferase is a class I integral membrane
protein of the endoplasmic reticulum. J. Biol. Chem. 273:
25880 –25888.
25. Watanabe, R., Wu, K., Paul, P., Marks, D. L., Kobayashi, T.,
Pittelkow, M. R., and Pagano, R. E. 1998. Up-regulation of glu-
cosylceramide synthase expression and activity during human
keratinocyte differentiation. J. Biol. Chem. 273: 9651–9655.
26. Colley, K. J. 1997. Golgi localization of glycosyltransferases:
More questions than answers. Glycobiology 7:1–13.
27. Maxzud, M. K. and Maccioni, H. J. 2000. Glucosylceramide
synthesized in vitro from endogenous ceramide is uncoupled
from synthesis of lactosylceramide in Golgi membranes from
chicken embryo neural retina cells. Neurochem. Res. 25:
145–152.
28. Sprong, H., van der Sluijs, P., and van Meer, G. 2001. How pro-
teins move lipids and lipids move proteins. Nat. Rev. Mol. Cell
Biol. 2:504 –513.
29. Helenius, J., Ng, D. T., Marolda, C. L., Walter, P., Valvano,
M. A., and Aebi, M. 2002. Translocation of lipid-linked oligo-
saccharides across the ER membrane requires Rft1 protein. Nature
415:447– 450.
30. Rothman J. E. 1994. Mechanisms of intracellular protein trans-
port. Nature 372:55–63.
31. Pelham, H. R. and Rothman, J. E. 2000. The debate about trans-
636
port in the Golgi—two sides of the same coin? Cell 102:
713 –719.
32. Trinchera, M., Pirovano, B., and Ghidoni, R. 1990. Sub-Golgi
distribution in rat liver of CMP-NeuAc GM3 and CMP-NeuAc:
GT1b 28 sialyltransferases and comparison with the distribution
of the other glycosyltransferases activities involved in ganglio-
side biosynthesis. J. Biol. Chem. 265:18242–18247.
33. Trinchera M. and Ghidoni R. 1989. Two glycosphingolipid sia-
lyltransferases are localized in different sub Golgi compart-
ments in rat liver. J. Biol. Chem. 264:15766 –15769.
34. Lannert, H., Gorgas, K., Meissner, I., Wieland, F. T., and
Jeckel, D. 1998. Functional organization of the Golgi apparatus
in glycosphingolipid biosynthesis. Lactosylceramide and sub-
sequent glycosphingolipids are formed in the lumen of the late
Golgi. J. Biol. Chem. 273:2939–2946.
35. Mollenhauer, H. H., Morré, D. J., and Rowe, L. D. 1990. Alter-
ation of intracellular traffic by monensin: Mechanism, speci-
ficity and relationship to toxicity. Biochim. Biophys. Acta
1031:225 –246.
36. Miller-Prodraza, H. and Fishman, P. H. 1984. Effect of drugs
and temperature on biosynthesis and transport of glycosphyn-
golipids in cultured neurotumor cells. Biochim. Biophys. Acta
804:44 –51.
37. Nores, G. A. and Caputto, R. 1986. Regulation of ganglioside
and sialoglycoprotein biosynthesis. Effect of drugs affecting
membrane flow. Neurochem. Int. 8.
38. Rosales Fritz, V. M., Maxzud, M. K. and Maccioni, H. J. 1996.
GT3 synthesis in the proximal Golgi occurs in a compartment
different from those for GD3 and GM3 synthesis. J. Neurochem.
67:1393–1400.
39. Donaldson J. G. and Klausner R. D. 1994. ARF: A key regula-
tory switch in membrane traffic and organelle structure. Curr.
Opin. Cell Biol. 6:527–532.
40. Young, W. W., Jr., Lutz, M. S., Mills, S. E., and Lechler-
Osborn, S. 1990. Use of brefeldin A to define sites of gly-
cosphingolipid synthesis: GA2/GM2/GD2 synthase is trans to
the brefeldin A block. Proc. Natl. Acad. Sci. U.S.A. 87:
6838–6842.
41. van Echten, G., Iber, H., Stotz, H., Takatsuki, A., and Sandhoff,
K. 1990. Uncoupling of ganglioside biosynthesis by Brefeldin
A. Eur. J. Cell Biol. 51:135–139.
42. Rosales Fritz, V. M. and Maccioni, H. J. F. 1995. Effects of
brefeldin A on synthesis and intracellular transport of ganglio-
side GT3 by chick embryo retina cells. J. Neurochem. 65:
1859–1864.
43. Giraudo, C. G., Rosales Fritz, V. M., and Maccioni, H. J. 1999.
GA2/GM2/GD2 synthase localizes to the trans-golgi network of
CHO-K1 cells. Biochem. J. 342 (Pt 3):633 – 640.
Maccioni, Giraudo, and Daniotti
44. Li, J., Yen, T. Y., Allende, M. L., Joshi, R. K., Cai, J., Pierce,
W. M., Jaskiewicz, E., Darling, D. S., Macher, B. A., and Young,
W. W., Jr. 2000. Disulfide bonds of GM2 synthase homodimers.
Antiparallel orientation of the catalytic domains. J. Biol. Chem.
275:41476– 41486.
45. Daniotti, J. L., Martina, J. A., Giraudo, C. G., Zurita, A. R., and
Maccioni, H. J. 2000. GM3 alpha2,8-sialyltransferase (GD3
synthase): Protein characterization and sub-golgi location in
CHO-K1 cells. J. Neurochem. 74:1711–1720.
46. Haraguchi, M., Yamashiro, S., Furukawa, K., Takamiya, K.,
Shiku, H., and Furukawa, K. 1995. The effects of the site-directed
removal of N-glycosylation sites from beta-1,4-N-acetylgalacto-
saminyltransferase on its function. Biochem. J. 312: 273 –280.
47. Martina, J. A., Daniotti, J. L., and Maccioni, H. J. 1998. Influ-
ence of N-glycosylation and N-glycan trimming on the activity
and intracellular traffic of GD3 synthase. J. Biol. Chem. 273:
3725–3731.
48. Martina, J. A., Daniotti, J. L., and Maccioni, H. J. 2000. GM1
synthase depends on N-glycosylation for enzyme activity
and trafficking to the Golgi complex. Neurochem. Res. 25:
725–731.
49. Parodi, A. J. 1999. Reglucosylation of glycoproteins and qual-
ity control of glycoprotein folding in the endoplasmic reticulum
of yeast cells. Biochim. Biophys. Acta, 1426:287–295.
50. Aridor, M., Fish, K. N., Bannykh, S., Weissman, J., Roberts,
T. H., Lippincott-Schwartz, J., and Balch, W. E. 2001. The Sar1
GTPase coordinates biosynthetic cargo selection with endoplas-
mic reticulum export site assembly. J. Cell Biol. 152: 213–229.
51. Giraudo, C. G., Daniotti, J. L., and Maccioni, H. J. 2001. Phys-
ical and functional association of glycolipid N-acetyl-galac-
tosaminyl and galactosyl transferases in the Golgi apparatus.
Proc. Natl. Acad. Sci. U.S.A. 98:1625 –1630.
52. Giraudo, G. C. and Maccioni, H. J. F. Glycolipid glycosyltrans-
ferase N-terminus plays a role in sorting and sub-Golgi location.
XXXVII Reunión anual de la Sociedad Argentina de Investi-
gación Bioquímica y Biología Molecular, C67.
53. Fullekrug, J. and Nilsson, T. 1998. Protein sorting in the Golgi
complex. Biochim. Biophys. Acta 1404:77– 84.
54. Munro, S. 1998. Localization of proteins to the Golgi apparatus.
Trends Cell. Biol. 8:11–15.
55. Roseman, S. 1970. The synthesis of complex carbohydrates by
multiglycosyltransferase systems and their potential function in
intercellular adhesion. Chem. Phys. Lipids 5:270 –297.
56. Caputto, R., Maccioni, H. J. F., and Arce, A. 1974. Biosynthe-
sis of brain gangliosides. Mol. Cell Biochem. 4:97–106.
57. Cole, N. B., Smith, C. L., Sciaky, N., Terasaki, M., Edidin, M.,
and Lippincott-Schwartz, J. 1996. Diffusional mobility of Golgi
proteins in membranes of living cells. Science 273: 797–801.