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

Abstract

Cell density-dependent inhibition of growth and neural differentiation in the human neuroblastoma cell line SK-N-MC are associated with a ganglioside sialidasemediated increase of GM 1 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 carbohydratedependent binding revealed a saturable amount of ligand sites approaching 2.6 ؋ 10 6 galectin-1 molecules bound/cell. Presence during cell culture of the sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid or of the GM 1 -binding cholera toxin B subunit effected a decrease of the presentation of galectin-1 ligands by 30 -50%. The assumption that GM 1 is a major ligand for galectin-1 was reinforced by the correlation between the number of carbohydrate-dependent 125 I-iodinated GM 1 -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 surfaceimmobilized GM 1 by the antibody. The results open the possibility that the carbohydrate-dependent interaction between ganglioside GM 1 and galectin-1 may relay sialidase-dependent alterations in this cell system.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 GM 1 and a conversion of GM 3 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 GM 1 and/or lactosylceramide that are generated by the action of the ganglioside sialidase on the surface of neuroblastoma cells.The current literature prompts inves...

Galectin-1 binds oncogenic H-Ras to mediate Ras membrane anchorage and cell transformation

“…Concomitantly, phosphorylated (active) ERK increased as well (155%, Figure 7a). Activation of Ras or of ERK by galectin-1 expression was not blocked by 100 mM lactose or by neutralizing galectin-1 Ab (Kopitz et al, 1998) (Figure 7b) implying that neither the lectin function of galectin-1 nor extracellular galectin-1 were involved in Ras and ERK activation in this set-up. Thus, expression of galectin-1 apparently increased membrane association of Ras and triggered the Ras-ERK pathway, which is essential for Ras transformation (Shields et al, 2000).…”

“…H-Ras and galectin-1 are members of two families of proteins known to be associated with human malignancies (Andre et al, 1999;Barondes et al, 1994;Bresalier et al, 1997;Hadari et al, 2000;Kopitz et al, 1998;Ohannesian et al, 1995;Polyak et al, 1997;van den Brule et al, 1995;Wells and Mallucci, 1991;Yamaoka et al, 1991Yamaoka et al, , 2000. Perhaps it is not less important to note that both Ras proteins (Bar-Sagi and Hall, 2000;Downward, 1998;Shields et al, 2000) and galectins (Hughes, 1999;Perillo et al, 1998), share in common a number of regulatory activities including regulation of cell growth, apoptosis, cell adhesion cell migration and metastasis.…”

“…By sequencing p14 we identi®ed it as rat galectin-1 (Figure 1c), a b-galactoside-binding protein previously implicated in cell transformation (Andre et al, 1999;Barondes et al, 1994;Kopitz et al, 1998;Ohannesian et al, 1995;van den Brule et al, 1995;Wells and Mallucci, 1991;Yamaoka et al, 1991Yamaoka et al, , 2000. Speci®c anti galectin-1 antibody (Ab) (Kopitz et al, 1998;Yamaoka et al, 2000) con®rmed that p14 released by DTT from the 34 ± 43 kDa Ras-immunoreactive band is galectin-1 (Figure 1d). Moreover, we repeated the cross-linking/ two-step gel procedure using an enriched plasma membrane preparation of EJ cells and found that both H-Ras(12V) and galectin-1 were released from the Ras immunoreactive 34 ± 43 kDa complex of the plasma membrane (Figure 1e).…”

“…The Coomassie-stained p14 was analysed by microsequencing at the Technion Protein Center (Haifa, Israel). The second gels were also used for immunoblotting with pan-Ras Ab (Haklai et al, 1998) and/or with 2 mg/ml rabbit anti-galectin-1 Ab (Kopitz et al, 1998;Yamaoka et al, 2000). Ras-galectin-1 complexes were immunoprecipitated from the entire MonoQ pool of cross-linked preparations or from Triton X-100 extracts of EJ cell membranes without cross-linking using pan-Ras Ab (2 mg) and protein GSepharose or galectin-1 Ab (20 mg) and protein A-Sepharose (Haklai et al, 1998).…”

Galectin-1 Interacts with the α5β1 Fibronectin Receptor to Restrict Carcinoma Cell Growth via Induction of p21 and p27

“…So far, however, the functional relevance of integrin binding for Gal-1-dependent growth-regulatory effects has not been conclusively demonstrated. Of interest, not an integrin but the pentasaccharide of ganglioside G M1 serves as Gal-1 contact site responsible for growth inhibition of neuroblastoma cells (6,40).…”