Cell density-dependent inhibition of
growth and neural differentiation in the human neuroblastoma cell line
SK-N-MC are associated with a ganglioside sialidase-mediated increase
of GM1 and lactosylceramide at the cell surface.
Because these glycolipids expose galactose residues, we have initiated
the study of the potential role of galectins in such cellular events.
Using specific antibodies, galectin-1 but not galectin-3 was found to
be present at the cell surface. Assessment of
carbohydrate-dependent binding revealed a saturable amount
of ligand sites approaching 2.6 × 106 galectin-1
molecules bound/cell. Presence during cell culture of the sialidase
inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid or of
the GM1-binding cholera toxin B subunit effected a decrease
of the presentation of galectin-1 ligands by 30-50%. The assumption
that GM1 is a major ligand for galectin-1 was reinforced by
the correlation between the number of
carbohydrate-dependent 125I-iodinated
GM1-neoganglioprotein binding sites and the amount of
immunoreactive surface galectin-1, the marked sensitivity of probe
binding to the presence of anti-galectin-1 antibody, and the inhibition
of cell adhesion to surface-immobilized GM1 by the
antibody. The results open the possibility that the
carbohydrate-dependent interaction between ganglioside
GM1 and galectin-1 may relay sialidase-dependent alterations in this cell system.
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INTRODUCTION |
Gangliosides can exert a variety of cellular functions that
include the triggering and modulation of transmembrane signaling and
the mediation of recognition of receptor molecules in homotypic and
heterotypic associations (1-4). Owing to this versatility in
regulatory processes, it is fitting that the presentation of gangliosides at the cell surface is subject to control mechanisms that
involve biosynthesis, endocytic uptake, recirculation, and lysosomal
degradation (5, 6). Moreover, the structure of the oligosaccharide
chains of gangliosides can be remodeled in situ by the
action of a cell surface sialidase in the course of transformation,
differentiation and cell contact formation (7-12). In human
neuroblastoma cells (SK-N-MC), the activity of this sialidase was
directed toward gangliosides with terminal sialic acids, yielding a
shift from higher sialylated species to GM1 and a
conversion of GM3 to lactosylceramide (13). Such
alterations of the ganglioside profile are apparently of profound
impact on the behavior of the neuroblastoma cells, because the
selective inhibition of the ganglioside sialidase activity by
2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en)1 led to marked
changes in cellular morphology, a complete release from contact
inhibition of growth, and a loss of differentiation markers (14, 15).
Because the underlying molecular events are unknown, we have now
addressed questions on the presence and nature of potential receptors
for GM1 and/or lactosylceramide that are generated by the
action of the ganglioside sialidase on the surface of neuroblastoma
cells.
The current literature prompts investigation of the role of galectins
in this respect. Galectins are a widely expressed group of animal
lectins that are known to be involved in the regulation of cell
adhesion and immune functions as well as proliferation and apoptosis
(for reviews see Refs. 16-22). Especially galectin-1, a homodimeric
"proto-type" lectin with one carbohydrate recognition domain per
subunit of 14 kDa, and galectin-3, a monomeric "chimera-type" lectin (29-35 kDa) with one carbohydrate recognition domain, have been
shown to bind to a distinct constituent of the GM1 sugar chain: the
2,3-sialylated lactosyl sequence. A
galactosyl-
1,3-N-acetylgalactosaminyl sequence had
notable inhibitory potency in asialofetuin-dependent binding
assays only for galectin-3, and the limited spatial accessibility in
solid phase assays appears to preclude binding of both galectins to
lactosylceramide (23-25). Notably galectin-1 from bovine heart and
from rat and human brain has been demonstrated to bind to glycolipids
with lactosamine structures and to a ganglioside mixture in solid phase
assays and to associate with the carbohydrate portion of
lysoganglioside GM1 immobilized on Spherosil-DEAE-dextran
beads (26-28). Galectin-1 is also readily applicable in the detection of accessible ligand sites in tissue sections and on cells (18, 29-31).
By using 125I-iodinated galectins and specific
anti-galectin antibodies, we have now assessed the possible occurrence
on neuroblastoma cells of galectins and galectin-binding sites and the
importance of cell surface galectin-1 for cell adhesion to
GM1-bearing polystyrene beads. In addition, the existence
and nature of potential cell surface GM1 docking sites was
probed in similar studies with a water-soluble GM1-derived
neoganglioprotein in the absence and presence of anti-galectin
antibodies. In order to evaluate the contribution of lactosylceramide
to galectin-1 binding, Viscum album L. agglutinin, the
galactoside-binding lectin from mistletoe that interacts with a variety
of galactose-terminating sugar chains (32-34) but fails to bind to
GM1 in microplate
assays,2 was used in
conjunction with the GM1-specific cholera toxin B subunit.
Our results clearly support the view that GM1 is a major ligand for galectin-1 that is present on the surface of human neuroblastoma cells and is accessible for
GM1-neoganglioprotein binding. Interestingly, galectin-1
and galectin-3 appear to share this property, a finding of potential
physiological relevance beyond this cell system.
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MATERIALS AND METHODS |
Purification and Sources of the Lectins--
The
galactoside-binding lectins from V. album L. and galectin-1
from human lung were purified by affinity chromatography on lactosylated Sepharose 4B and further chromatographic steps, as described (35, 36). Traces of contaminants such as galactoside-binding immunoglobulin G were removed from galectin-1 by ion exchange chromatography on DEAE-Sepharose CL-6B and by subsequent affinity chromatography on protein A-Sepharose CL-4B. Murine galectin-3 was
expressed from plasmid prCBP35s in Escherichia coli JA221 cells as outlined elsewhere (37) and similarly purified by affinity chromatography. Cholera toxin B subunit was purchased from Sigma.
Preparation of Neoganglioprotein and Lectin-specific
Antibodies--
The lysoganglioside of purified GM1 was
obtained by deacylation in 1 M KOH followed by selective
re-N-acetylation and covalently linked as sphingosine
N-alkyl(sulfosuccinimidyl)ester derivative to
carbohydrate-free bovine serum albumin with an incorporation yield of
2-3 units/carrier molecule as described (38, 39). Polyclonal
antibodies against the mammalian galectins-1 and -3 were raised in
rabbits, purified from serum by protein A-Sepharose 4B chromatography,
and checked for specificity and lack of cross-reactivity as described
elsewhere (40, 41).
Iodination of Lectins, Lectin-specific Immunoglobulin G, and
Neoganglioprotein--
100 µg of protein was incubated at room
temperature for 15 min in 50 mM sodium phosphate, pH 7.0, with 74 MBq carrier-free Na125I (Amersham Pharmacia
Biotech) in the presence of two IodobeadsTM (Pierce). In the cases of
the galectins and the V. album agglutinin, 100 mM lactose was included in the iodination mixture for
protection of their binding domains. Labeled proteins were separated
from free iodine and lactose by gel filtration on Sephadex G-25 (PD-10 column, Amersham Pharmacia Biotech). Specific radioactivities were 278 KBq/µg for galectin-1, 212 KBq/µg for galectin-3, 209 KBq/µg for
V. album L. agglutinin, 35 KBq/µg for cholera toxin B
subunit, 843 KBq/µg for GM1-neoganglioprotein, 243 KBq/µg for anti-galectin-1 IgG, and 226 KBq/µg for anti-galectin-3
IgG. Binding of radiolabeled galectins and V. album L. agglutinin to lactosylated Sepharose 4B always exceeded 90%, proving
the lack of significant damage by the iodination procedure.
Neuroblastoma Cell Culture and Immunocytochemical Galectin
Localization--
Neuroblastoma cells (strain SK-N-MC) were cultured
in Eagle's minimal essential medium supplemented with 10% fetal calf
serum (PAA Laboratories, Cölbe, Germany) as described (15). Cell surface localization of galectins in SK-N-MC cells was tested according
to Avellana-Adalid et al. (42). Briefly, cells were grown on
glass coverslips, treated with galectin-specific IgG (10 µg/ml,
2 h, 37 °C) before fixation, and stained with 20 µg/ml fluorescein isothiocyanate-labeled second antibody (fluoresceinylated goat F(ab') anti-rabbit IgG, Santa Cruz Biotechnology, Santa Cruz, CA)
for 1 h at 20 °C. Photographs were taken using an Ortholux microscope (Leitz, Stuttgart, Germany).
Cell Binding Studies with 125I-labeled Probes and
Ganglioside-coated Magnetic Beads--
Cells grown to confluency in
96-well tissue culture plates (Falcon Primaria, Heidelberg, Germany)
for 5 days (approximately 105 cells/well) were incubated
for 16 h in medium without fetal calf serum. For the binding
assays, the medium consisted of serum-free Eagle's minimal essential
medium (100 µl/well) fortified with 25 mM HEPES, pH 7.4, 0.01% bovine serum albumin to block protein-binding sites, and the
labeled proteins at nonsaturating concentrations. Cultures were
incubated for 2 h at 37 °C and washed three times with 200 µl
of 20 mM phosphate-buffered saline, pH 7.4, and the cells
were solubilized in 100 µl of 0.2 M NaOH, and their
radioactivity was determined in a liquid scintillation counter
(TRI-CARB 1600TR, Canberra-Packard, Dreieich, Germany). For
quantitation of carbohydrate-independent adsorption of labeled probes
their binding was also assayed in presence of a mixture of 150 mM lactose/0.5 mg/ml asialofetuin for galectins and
V. album L. agglutinin or an at least 25-fold excess of
unlabeled ligand for cholera toxin B subunit or
GM1-neoganglioprotein, respectively. Presaturation of the
antibody preparation by addition of an excess of galectin-1 and
preadsorption of cells with preimmune serum excluded
antigen-independent IgG binding. The difference between total binding
and unspecific adsorption was considered as
carbohydrate-dependent binding. When the inhibition of ligand binding by antibodies or cholera toxin B subunit was assayed, 25 µg
of the respective inhibitor protein was added per well 1 h prior
to a radioactive ligand to allow presaturation of the binding
sites.
For assessment of cell adhesion to surface-immobilized glycolipids,
neuroblastoma cells (6 days after subculture) were metabolically labeled for 24 h with 100 µCi of
[33P]orthophosphate (Amersham Pharmacia Biotech). Cells
were collected by treatment with 0.2 mM EDTA in
phosphate-buffered saline for 10 min, centrifuged (200 × g for 5 min), and resuspended in HEPES-buffered Dulbecco's
modified Eagle's medium containing 5 mg/ml bovine serum albumin
(105 cells/250 µl; specific radioactivity 877 dpm/104 cells). Adhesion of 105 labeled cells
to 5 × 107 magnetic beads (carboxylated magnetic
polystyrene monodisperse microspheres, 3.2-µm diameter
average, Spherotech, Libertyville, IL) adsorbed with a mixture
of phospatidylcholine and cholesterol (carrier), and the
respective glycolipids was measured as described by Yang et
al. (43). Aliquots of the cell suspensions were treated for 30 min with anti-galectin-1-specific IgG (10 µg/ml
medium) prior to the binding assay.
 |
RESULTS |
Immunological Detection of Galectins at the Cell
Surface--
Specific polyclonal antibodies to galectin-1 and
galectin-3 that had rigorously been checked for lack of
cross-reactivity to the other galectin were used for immunodetection
studies. Immunofluorescent monitoring of unfixed cultured SK-N-MC
neuroblastoma cells clearly revealed the presence of galectin-1 on the
cells (Fig. 1). Galectin-3, however, was
not detected, and a preimmune IgG preparation likewise failed to bind
(results not shown). To estimate the quantity of cell
surface-associated galectin-1, binding studies with iodinated galectin-1-specific antibody were performed and showed saturation kinetics. Scatchard analysis of the data yielded a straight line indicative of a single class of noninteracting sites, and a value of
5 × 104 bivalent immunoglobulin G molecules bound per
cell at saturation (Fig. 2). Owing to the
large size of the probe relative to that of the 28-kDa lectin, the
number of accessible galectin-1 molecules should rather be considered
as an underestimation. Because blotting analysis of samples of the
fetal calf serum (up to 100 µg of protein) used in the culture medium
failed to detect any galectin-1 down to a detection limit of 100 pg, it
can be excluded that the immunoreactive galectin-1 on the cell surface
originated from the serum supplement (results not shown).

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Fig. 1.
Immunofluorescence detection of galectin-1 on
the surface of human SK-N-MC neuroblastoma cells. Cells were
cultured on glass coverslips for 5 days before the cells were treated
with polyclonal anti-galectin-1 IgG (A), fixed, and stained
with fluorescein isothiocyanate-labeled second antibody. For
counterstaining of DNA propidium iodide was used. For controls
(B), untreated cells were exposed to second antibody.
Fluorescence was observed with an Ortholux microscope (Leitz).
Magnification, 1200×.
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Fig. 2.
Evaluation of binding of
125I-iodinated galectin-1-specific antibody to
the surface of neuroblastoma cells by Scatchard analysis. Cells
were cultured for 5 days in 96-well plates before specific binding of
iodinated anti-galectin-1 IgG was assayed at a cell density of
105 cells/well as described under "Materials and
Methods." Results are the means of three determinations ± S.D.
Inset, binding curve.
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Quantitation of Cell Surface Ligands for Lectins--
To confer
any regulatory activity, it would be essential that ligand sites for
the galectin are present on the cell surface. Thus, we iodinated
galectin-1 under activity-preserving conditions and determined the
extent of carbohydrate-inhibitable binding. For comparison, we studied
galectin-3, V. album L. agglutinin (a tetrameric
galactoside-binding lectin from mistletoe that fails to bind to
GM1 immobilized on thin-layer chromatography plates), and
the GM1-specific cholera toxin B subunit in similar assays with aliquots from the same batch of cells under identical experimental conditions.
The specific binding of the iodinated lectins to the surface of
cultured cells was saturable in each case (Fig.
3, A-D). As a further control
of specificity of galectin-1 association, cells were preincubated with
an excess of unlabeled galectin-3 prior to being exposed to iodinated
galectin-1. Based on the similarities of the carbohydrate recognition
domains of the two galectins, mutual blocking of carbohydrate ligands
could be expected. Indeed, galectin-3 completely prevented access of
the labeled galectin-1 to the surface determinants (data not shown).
Because the same was true in the reversed setting, both galectins thus
appeared to recognize an at least very similar set of carbohydrate
ligands. To obtain KD values and the number of bound
lectin molecules/cell at saturation, Scatchard analyses were performed
that resulted in straight lines (Fig. 4,
A-D). As expected from the competition experiment,
galectins-1 and -3 exhibited similar binding capacities for the
neuroblastoma cell surface (Fig. 4, A and B, and
Table I). Compared with the ganglioside
GM1-specific B subunit of cholera toxin, the affinity of
the galectins to their surface ligands was clearly lower (Table I),
probably due to multivalency of the bacterial lectin (44). The size
difference between the choleratoxin B subunit and the mammalian
galectins should be kept in mind as a possible reason for the
Bmax value dissimilarities (44). The pronounced
differences between the mistletoe lectin and the galectins indicated
the presence of different ligands, although the considerably larger
spatial extension of the plant protein (molecular mass, 120 kDa) should
not be neglected as a possible source for the noted difference.

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Fig. 3.
Binding of
125I-iodinated lectins to the surface of
neuroblastoma cells. Cells were cultured in 96-well plates for 5 days (final cell density, 105 cells/well) before binding of
iodinated ligands was measured as described under "Materials and
Methods." A, galectin-1; B, galectin-3;
C, V. album L. agglutinin; D, cholera
toxin B subunit. , total binding; , binding in the presence of
150 mM lactose/0.5 mg/ml asialofetuin (A,
B, and C) or 0.25 mg/ml unlabeled cholera toxin B
subunit (D); , specific binding. Results are the means of
three independent determinations ± S.D.
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Fig. 4.
Specific binding of
125I-iodinated lectins to the surface of
neuroblastoma cells cultured in the absence ( ) or presence ( ) of
250 µM NeuAc2en prior to performing the binding assays.
A, cholera toxin B subunit; B, galectin-1;
C, galectin-3; D, V. album L. agglutinin. Each point represents the mean of three measurements ± S.D.
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Table I
Determination of the apparent affinity constant (KD) and the
apparent number of bound lectin molecules per cell at saturation
(Bmax) for human neuroblastoma cells cultured in the absence or
in the presence of the surface ganglioside sialidase inhibitor
2-deoxy-2,3-dehydro-N-acetylneuraminic acid
Data are derived from the experiments shown in Fig. 4 where each point
of the binding curve represents the mean of three independent
measurements and ± S.D. was always <10%.
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Modulation of Lectin Binding by Ganglioside Sialidase
Inhibition--
Because the addition of the ganglioside sialidase
inhibitor NeuAc2en to neuroblastoma cell cultures had been shown to
cause drastic changes in the relative abundance of membrane
gangliosides and to trigger pronounced morphological alterations and a
release of the cells from growth arrest (14, 15), it appeared likely that it also would affect the presence of lectin-binding sites at the
cell surface. To test this reasoning, cells were exposed to the
inhibitor under conditions where no generation of ganglioside GM1 from higher sialylated species and no ganglioside
GM3 desialylation was detectable. Indeed, binding of
cholera toxin B subunit was 55% reduced (Fig. 4A and Table
I). Remarkably, a significant decrease of binding was also seen with
the galectins-1 and -3. As shown in Fig. 4 (B and
C) and in Table I, the number of galectin-binding sites but
not their apparent affinities decreased by approximately 30% in the
presence of NeuAc2en, whereas the GM1-unreactive probe V. album L. agglutinin showed no marked change of binding
parameters (Fig. 4D and Table I). The experiments in the
presence of the sialidase inhibitor thus indicate that cholera toxin B
subunit and the galectins-1 and -3 share a ligand structure whose level of expression is sensitive to sialidase activity changes. This conclusion would be strengthened if the bacterial lectin were capable
of interfering with cell surface binding of galectin-1. Indeed, a
reduction of binding by more than 50% was seen when an excess of
cholera toxin B subunit was employed to block access of the galectin-1
to ganglioside GM1 (Fig. 5),
a decrease that was stronger than that produced by the sialidase
inhibitor. The combination of both effectors did not further decrease
the binding of galectin-1 (Fig. 5). The results indicate that
galectin-1 binds to other cholera toxin-unreactive glycoligands with an
affinity similar to that measured for the effector-sensitive sites.
Apparently, other products of the ganglioside sialidase action, in
particular lactosylceramide, which is extensively produced by
ganglioside GM3 desialylation, do not contribute to
galectin-1 binding, because no significant additional effect of
NeuAc2en was observed after blocking of ganglioside GM1 by
the cholera toxin B subunit. To judge if the
sialidase-dependent elevation of GM1 might be
of any functional significance for galectin-1-binding, it would be supportive to show that ganglioside GM1 can interact with
the cell surface. Moreover, this binding capacity, if mediated by galectin-1, should be abolished by galectin-1-specific antibody.

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Fig. 5.
Effect of cholera toxin B subunit on binding
of 125I-labeled galectin-1 to neuroblastoma
cells. Cells were cultured in the absence ( , ) or presence
of 250 µM NeuAc2en ( , ) and galectin-1
binding was assayed in the absence ( , ) or presence ( , )
of an excess of unlabeled cholera toxin B subunit as described
under "Materials and Methods."
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Quantitation of Neoganglioprotein Binding to the Cell
Surface--
Because gangliosides in watery solution exist in the form
of micelles, they cannot be used as such in cell binding studies. Therefore, to test for the presence of ganglioside
GM1-binding sites on the cell surface, we synthesized a
water-soluble and iodinatable lysoganglioside GM1-albumin
conjugate (neoganglioprotein) by covalent bridging of carrier and
ligand parts with a homobifunctional cross-linker after base
treatment-induced generation of the lysoganglioside. As with the
lectins, binding of this type of probe was specific and saturable too
(Fig. 6), with about 1 × 105 neoganglioprotein molecules bound per cell and a
KD value of 3 × 10
8
M. Because the molecular weight of the galectin-1-detecting
immunoglobulin G is more than twice that of the neoganglioprotein with
ensuing consequences on assessment of neighboring determinants, the
number of 5 × 104 immunologically reactive galectin-1
molecules/cell can be considered not to differ largely from that of
GM1-neoganglioprotein binding sites. Treatment of the cells
with the sialidase inhibitor NeuAc2en did not affect these parameters
(data not shown). Consequently, the ligand property of the applied
probe is sufficient to allow interaction with receptor sites in the
presence of variable amounts of endogenous ligands. The rather low
KD value for the synthetic marker probably reflects
the well appreciated avidity effect of neoglycoproteins (45-47).

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Fig. 6.
Binding of
125I-iodinated lysoganglioside
GM1-bovine serum albumin conjugate to the
surface of neuroblastoma cells. Specific binding of the probe was
assessed in the absence ( ) or presence ( ) of galectin-1-specific
antibody, as described under "Materials and Methods." Results are
the means of three determinations ± S.D.
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When the polyclonal galectin-1-specific antibody was applied,
neoganglioprotein binding only reached about 25% of the original amount (Fig. 6). This drastic reduction is only explainable when galectin-1 and/or a spatially closely associated molecule are the sites
for the interaction with the carrier-bound lysoganglioside GM1. The nature of the remaining binding sites exhibiting a
higher affinity with a KD value of about 5 × 10
9 M for GM1-neoganglioprotein
is currently unclear.
Galectin-1-mediated Cell Binding to Surface-immobilized
Gangliosides--
To further support the notion that ganglioside
GM1 and galectin-1 form a recognition system, cell adhesion
to a panel of gangliosides presented on the surface of magnetic beads
was probed. Indeed, GM1 coated to the polystyrene surface
of the beads led to a marked elevation of the level of cell adhesion
relative to the other tested glycolipids and to glycolipid-free beads
(Table II). Notably, lactosylceramide
failed to act as a docking site. Preincubation of the cells with
galectin-1-specific antibody blocked the GM1-binding sites
on the cell surface (Table II), in line with the experiment described
in the previous paragraph.
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Table II
Cell adhesion to glycolipid-adsorbed magnetic beads
105 cells were incubated in presence of 5 × 107
coated beads ("Materials and Methods"). The results are the means
of four determinations ± S.D.
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 |
DISCUSSION |
A relevant receptor molecule for sialidase-generated ganglioside
GM1 and lactosylceramide would have to fulfill several
requirements: presence at the cell surface, diminished binding after
treatment of the cells with the sialidase inhibitor NeuAc2en, a
reactivity toward a lysoganglioside GM1-exposing
neoganglioprotein, and mediation of cell binding to surfaces exposing
the respective glycolipids that should be sensitive to presence of a
receptor blocking antibody. Focusing on ganglioside GM1,
reduction of receptor binding must likewise occur in the presence of an
excess of the highly specific B-subunit of cholera toxin. Our results
clearly show that galectin-1 fulfills all of these prerequisites with
regard to ganglioside GM1. The immunofluorescence
experiments with specific polyclonal galectin-1 and -3 antibodies
extended the expression of cell surface galectin-1 from the murine
neuroblastoma cell line N1E115 as reported previously (42) to the
SK-N-MC human neuroblastoma cells studied here. Presence of galectin-3
in SK-N-MC cells, on the other hand, was not detected. The two
galectins were found to share a surface-exposed ligand structure with
cholera toxin B subunit that is highly specific for ganglioside
GM1. Because the presence of this blocking agent or of the
ganglioside sialidase inhibitor NeuAc2en reduced galectin-1 binding
considerably without further decrease by their combined use, it seems
unlikely that lactosylceramide plays a major role as a binding partner.
This conclusion is corroborated by a lack of effect of presence of the
sialidase inhibitor on the extent of association of mistletoe lectin to
cells, although as compared with galectin-1, its 4-fold higher
molecular weight and thus spatial extension might restrict access to
the sugar structures close to the lipid bilayer. A residual galectin-1
ligand capacity of the cells remained even after treatment with cholera
toxin B subunit and the sialidase inhibitor, pointing to additional
galectin-1-reactive glycostructures with KD values
similar to GM1. Ganglioside GM1 can thus be
considered as a major but apparently not the only galectin-1-reactive
structure on the surface of SK-N-MC neuroblastoma cells. Ganglioside
GM1 has so far not been described as a galectin ligand. Besides
lacto-N-neotetraosyl-containing neutral glycolipids, a
variety of glycoproteins such as laminin, tissue fibronectin, lysosome-associated membrane glycoproteins,
7
1-integrin, core 2 O-glycan
structure-bearing glycoproteins of T-cells, and highly glycosylated
mucins have been demonstrated to serve as ligands for galectin-1 and
often also for galectin-3 by virtue of suitable sugar sequences (22,
25, 27, 41, 48-57).
Because the proto-type galectin-1, like other dimeric or tetrameric
lectins (58, 59), can cross-link carbohydrate ligands to clusters with
defined stoichiometry, such spatial organization may also be relevant
for the responses triggered in this neuroblastoma cell system. No
galectin has so far been considered as a physiologically relevant
GM1 ganglioside receptor in any tested cell system (for review see Ref. 60). However, the enhancement of neuritogenesis and
cell adherence by the sugar portion of GM1 has been
tentatively ascribed to the presence of a 71-kDa membrane protein in
the case of murine cholinergic S20Y neuroblastoma cells (61-63). Based
on the application of the GM1-neoganglioprotein and the
binding of cells to surface-immobilized GM1 in the absence
and presence of galectin-1-specific antibody, as well as the total
number of immunologically assessed galectin-1 molecules and
glycocytochemically quantitated neoganglioprotein-binding sites, it
appears reasonable to conclude that the murine and the human lines
differ in their GM1 receptors. In this context it is
remarkable that the murine adrenergic N115 neuroblastoma line failed to
distinguish GM1 from other gangliosides in a cell adherence
assay, disclosing differences in the expression of ganglioside
receptors even between two murine cell lines (62). Overall, our present
results coalesce into the notion that galectin-1 is a major receptor
for the carbohydrate portion of ganglioside GM1 exposed on
the surface of cultured human SK-N-MC neuroblastoma cells. If and how
such interplay is involved in the ganglioside sialidase-dependent switch from proliferation to
differentiation will have to be the object of further study.
We gratefully acknowledge Dr. J. L. Wang
for kindly providing the expression vector for murine
galectin-3.