JBC Invitrogen Ultrasensitive Cytokine Assays

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J Biol Chem, Vol. 273, Issue 18, 11205-11211, May 1, 1998


Galectin-1 Is a Major Receptor for Ganglioside GM1, a Product of the Growth-controlling Activity of a Cell Surface Ganglioside Sialidase, on Human Neuroblastoma Cells in Culture*

Jürgen KopitzDagger , Carolina von ReitzensteinDagger , Maria Burchert§, Michael CantzDagger , and Hans-Joachim Gabius§

From the Dagger  Institut für Pathochemie und Neurochemie, Klinikum der Ruprecht-Karls-Universität, Im Neuenheimer Feld 220, D-69120 Heidelberg, Germany and the § Institut für Physiologische Chemie, Tierärztliche Fakultät, Ludwig-Maximilians-Universität, Veterinärstrasse 13, D-80539 München, Germany

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha 2,3-sialylated lactosyl sequence. A galactosyl-beta 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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

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. open circle , total binding; triangle , 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); bullet , 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 (bullet ) or presence (triangle ) 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%.

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 (open circle , square ) or presence of 250 µM NeuAc2en (bullet , black-square) and galectin-1 binding was assayed in the absence (open circle , bullet ) or presence (square , black-square) of an excess of unlabeled cholera toxin B subunit as described under "Materials and Methods."

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 (bullet ) or presence (triangle ) of galectin-1-specific antibody, as described under "Materials and Methods." Results are the means of three determinations ± S.D.

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.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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, alpha 7beta 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.

    ACKNOWLEDGEMENT

We gratefully acknowledge Dr. J. L. Wang for kindly providing the expression vector for murine galectin-3.

    FOOTNOTES

* This work was supported by Grants Ca 76/7-3 and Ga 349/7-1 of the Deutsche Forschungsgemeinschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Inst. für Pathochemie und Neurochemie, Im Neuenheimer Feld 220, D-69120 Heidelberg. Fax: 49-6221-564228.

1 The abbreviation used is: NeuAc2en, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid.

2 H.-J. Gabius and H. Wiegandt, unpublished observation.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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