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...

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The mysteries of the functions of complex glycoconjugates have enthralled scientists over decades. Theoretical considerations have ascribed an enormous capacity to store information to oligosaccharides. In the interplay with lectins sugar-code words of complex carbohydrate structures can be deciphered. To capitalize on knowledge about this type of molecular recognition for rational marker/drug design, the intimate details of the recognition process must be delineated. To this aim the required approach is garnered from several fields, profiting from advances primarily in X-ray crystallography, nuclear magnetic resonance spectroscopy and computational calculations encompassing molecular mechanics, molecular dynamics and homology modeling. Collectively considered, the results force us to jettison the preconception of a rigid ligand structure. On the contrary, a carbohydrate ligand may move rather freely between two or even more low-energy positions, affording the basis for conformer selection by a lectin. By an exemplary illustration of the interdisciplinary approach including up-to-date refinements in carbohydrate modeling it is underscored why this combination is considered to show promise of fostering innovative strategies in rational marker/drug design.

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For proteins in solution the validity of certain crystallographic parameters can be ascertained by a combination of molecular-dynamics (MD) simulations and NMR spectroscopy. Using the laser photo-CIDNP (chemically induced dynamic nuclear polarization) technique as a measure for surface accessibility of histidine, tyrosine and tryptophan, the spectra of bovine galectin-I and Erythrina corallodendron lectin (EcorL) are readily reconcilable with the crystallographic data for these two proteins. The results emphasise the role of Trp68/Trp69 for carbohydrate binding in bovine galectin-l/chicken galectins and of Trp194 in murine galectin-3. This feature derived from the crystal structure of' bovine galectin-I is maintained in solution for the prototype human homologue, two avian galectins and the chimera-type murine galectin-3, as the spectra corroborate the CIDNP-inferable spatial parameters of the four calculated models for binding-site architecture. In EcorL, Tyr106/Tyr108 are constituents of the extended combining pocket, which can be shielded in solution by ligand presence. Discrepancies between results from modelling and CIDNP measurements concern primarily the lack of reactivity of histidine residues for human and avian prototype galectins and of Tyr82lTyr229 of the plant lectin. Site-directed mutagenesis of EcorL is assumed to provide information on the role of a certain residue for functional aspects. When singlesite mutants of EcorL ([Alal06]EcorL, [Alal08]EcorL, [Ala229]EcorL) were subjected to moleculardynamics (MD) simulations, the apparent surface accessibilities even of spatially separated amino acid side chains could non-uniformly be affected. This conclusion is supported by the assessment of the spectra for the mutant proteins. On the basis of these CIDNP-results modelling of the binding-site architecture of the lectin indicates the occurrence of notable alterations in the orientation of Tyr106/Tyr108 phenyl rings. The implied potential effect of single-site mutations on conformational features of a protein will deserve attention for the interpretation of studies comparing wild-type and mutant proteins.Keywords: galectin ; lectin ; Erythrina agglutinin ; NMR; molecular modeling.X-ray crystallography of proteins provides detailed information on three-dimensional structure. The precision of the derived spatial parameters of the protein in the crystal, however, should not be considered as proof for the solution structure. Among other factors, the often applied non-physiological conditions to favour crystal growth and the intermolecular interactions during packaging may lead to deviations from the solution structure(s) especially for surface group features (Wagner et al., 1992;MacArthur et al., 1994). Consequently, it is desirable to employ a sensitive spectroscopic method which in combination with mo- lecular-dynamics (MD) simulations can verify the applicability of distinct properties determined by crystallographic analysis for the solution structure. With a focus on surface epitopes, whose mobility can...