Probing hydrogen‐bonding interactions of bovine heart galectin‐1 and methyl β‐lactoside by use of engineered ligands

Abstract

The binding of different synthetic monodeoxy, 0-methyl and fluorodeoxy derivatives of methyl P-lactoside to galectin-1 from bovine heart has been studied to probe the role of hydrogen bonding in the recognition and binding. The energetic contributions of the hydroxyl groups of methyl ,8-lactoside directly involved in the interaction have been estimated and the nature of the protein residues involved has been predicted on the basis of the free energy data. Interpretations of the results have been sustained by molecular modeling of the three-dimensional structure of the sugars in solution. One side of the disaccharide molecule is not involved (HO-6 and HO-2') or only marginally involved (HO-3') in hydrogen bonding. Moreover, 0-methylation at these positions causes an enhancement of the binding, suggesting favourable interactions of the methyl groups which may come into contact with hydrophobic residues at the periphery of the combining site. Hydrogen-bonding interactions are almost exclusively restricted to the other side of the molecule : the C-4' and C-6' hydroxyl groups act as donors of the strongest hydrogen bonds to charged groups of the lectin, while the C-3 hydroxyl group participates in a strong hydrogen bond with a neutral group. The results also suggest that the N-acetyl NH group in N-acetyllactosamine, as well as the hydroxyl group at position C-2 in methyl /3-lactoside, are involved in a polar interaction with neutral groups of the combining site. This hydrogen-bonding pattern contrasts markedly with that previously reported for the two galactose-specific Ricinus communis lectins. The recognition of different epitopes of the same basic structure underlies the differences in the oligosaccharide-binding specificities of galectin-1 and the R. communis lectins.Bovine heart muscle contains a soluble 14-kDa lectin, recently named galectin-1 [l], which is a member of a family of metal-independent P-galactoside-binding lectins (galectins), widely distributed in animal tissues. Although a specific function of these lectins has not been unequivocally demonstrated, they have been suggested to be involved in cellular-recognition events such as differentiation, development and metastasis of malignant cells [2-41, via recognition of specific cellular glycoconjugates. The recent finding of homologous lectins in some of the most primitive multicellular animals such as sponge [5] and nematode [6], suggests that the galectin family existed at a very early stage in evolution [7] and, consequently, that these lectins might be involved in 'essential minimum' functions of multicellular animals. Their highly conserved carbohydrate-binding specificity reflects the biological importance of the recognition of P-galactoside-containing structures.In general, the specificity and affinity in the molecular recognition of carbohydrates by proteins is achieved mainly through hydrogen bonds, with added contributions from van der Waal's contacts and stacking interactions of aromatic residues with the hydrophobic patches of the sugar [8-...

Domain versatility in plant AB‐toxins: Evidence for a local, pH‐dependent rearrangement in the 2γ lectin site of the mistletoe lectin by applying ligand derivatives and modelling

“…Plastic microwells were coated with 50 ll of asialofetuin solution for 16 h at 4°C and binding of 125 I-VAA to the wells was assayed essentially as described [12], except that the buffer used was 5 mM sodium phosphate buffer, pH 7.2, 0.2 M NaCl. For binding assays with biotinylated VAA, precoated wells were incubated with 50 ll of lectin solution for 2 h at 20°C, and the extent of bound lectin was monitored by measuring the amount of streptavidin associated to the wells after incubation for 1 h at 20°C with 50 ll (15000 cpm) of 125 I-streptavidin solution in the same buffer.…”

Different Architecture of the Combining Site of the Two Chicken Galectins Revealed by Chemical Mapping Studies with Synthetic Ligand Derivatives

“…Materials-Sources for saccharides were as indicated in detail previously (18). The chicken ␤-galactoside-specific lectins, CL-14 from adult intestine and CL-16 from adult liver, were isolated by successive steps of affinity and ion exchange chromatography (8).…”

“…Quantitative Binding Studies-Binding of the 125 I-labeled chicken lectins was assayed using asialofibrin films on the surface of plastic microwells essentially as described (18) except that the buffer used was 5 mM sodium phosphate buffer, pH 7.2, containing 0.2 M NaCl, 2 mM 2-mercaptoethanol, and 1% bovine serum albumin. For binding assays with biotinylated lectins, precoated wells were incubated with 50 l of 3 g of lectin/ml solution for 2 h at 25°C, and the extent of bound lectin was monitored by measuring the amount of streptavidin associated to the wells after incubation for 1 h at 25°C with 50 l (15000 cpm) of 125 I-streptavidin solution in the same buffer and thorough washing steps to remove unbound radioactive material.…”

“…The chicken ␤-galactoside-specific lectins, CL-14 from adult intestine and CL-16 from adult liver, were isolated by successive steps of affinity and ion exchange chromatography (8). Radioiodination of the lectins using IODO-GEN (Pierce Eurochemie) was carried out under activity-preserving conditions in the presence of 0.1 M lactose as described for bovine galectin-1 (18). CL-14 was also radioiodinated in the presence of lactose using Bolton-Hunter reagent (Amersham Int.)…”

“…X-ray crystallography has revealed a similar but not identical carbohydrate-binding geometry for galectin-1 (16) and galectin-2 (17) that involves key interactions of the hydroxyl groups at positions 4 and 6 of galactose and at position 3 of glucose in N-acetyllactosamine. It is essential to emphasize that a similar mode of binding has been observed in solution by chemical mapping studies in which the interaction of different monodeoxy, O-methyl and fluorodeoxy derivatives of methyl ␤-lactoside to galectin-1 was investigated (18). Therefore, it is reasonable to suggest that such a deliberate chemical mapping study will prove very informative for the analysis of protein-carbohydrate interactions in solution (19,20).…”