Galectin-1 binding to LacNAc and 3'-O-substituted LacNAc ligands
Ford, Michael Garrett
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Galectins are a class of carbohydrate binding proteins, initially characterized by their affinity for galactose terminating ligands, that play important roles in cellular adhesion and development. There are considerable data on the types of saccharides which bind to the galectin-1 dimer, with higher affinity ligands containing the core sequence Gal-ß-(1,4)-GlcNAc or LacNAc. While initially described as binding specifically to galactose-terminating ligands, a number of important 3’-O-substituted structures are found which are physiologically significant. The capping of galactose with 3’-O-SO3 increases affinity, as does polymerization of LacNAc units as seen in polylactosamine structures found in basement membrane glycoproteins. The addition of sialic acid is another especially significant carbohydrate modification regulating the ability of glycans to bind to galectin-1. While glycans containing a Neu5Ac-a-(2,6)- LacNAc capping structure are unable to bind galectin-1, Neu5Ac-a-(2,3)-LacNAc terminated glycans can bind to galectin-1, with an affinity very similar to the LacNAc disaccharide. The competing actions of sialyltransferases can therefore act as a regulator of galectin-1 binding. The modification of lactosamine in N- and O-linked glycans is thought to be the determining switch in the induction of apoptosis by galectin- 1 in developing thymocytes. The clonal selection of immature thymocytes is dependent on this apoptotic process for the production of T-cells capable of discriminating self/nonself antigens. In order to provide a structural framework for the observations on galectin-1 ligand binding, we have utilized an X-ray structure of the galectin-1 LacNAc complex. Structural information on the complexes of the 3’-O-substituted ligands bound to the carbohydrate recognition domain of galectin-1 is obtained from molecular dynamics studies. The resulting molecular trajectories of galectin-1 – ligand complexes are not only able to reproduce geometric features of the X-ray structure, but also predict novel contacts between 3’-O- substituents and protein residues. Predicted binding energies of conformations taken from MD simulations are derived from the contributions to the binding free energy. The effect of 3’-O- modification of LacNAc on binding can also be studied directly, by observing the properties of the protein – ligand complexes in solution NMR spectroscopy. Monitoring chemical shift changes of backbone resonances during ligand titration is an established measure of changes in the local environment of protein residues due to ligand binding, and such experiments require the assignment of protein backbone resonances. In addition to the traditional suite of 3D experiments normally performed to assign a protein, we have undertaken an assignment strategy which incorporates additional angular information, in the form of the residual dipolar coupling. This approach not only assigned many residues in the galectin-1 binding site, but should also serve as a general tool for the assignment of larger proteins. Titration experiments on the assigned galectin-1 protein confirm a common binding site for LacNAc and Neu5Ac-LacNAc. The results provide a structural interpretation for the large body of in vitro binding data by providing a model in which galectin-1 mediated crosslinking of heterogeneous cell surface oligosaccharides is responsible for a variety of adhesion phenomena ascribed to this lectin.