Evidence for subsites in the galectins involved in sugar binding at the nonreducing end of the central galactose of oligosaccharide ligands: sequence analysis, homology modeling and mutagenesis studies of hamster galectin-3

A model of the carbohydrate recognition domain CRD, residues 111-245, ofhamster galectin-3 has been made using homology modeling and dynamicsminimization methods. The model is based on the known x-ray structures ofbovine galectin-1 and human galectin-2. The oligosaccharidesNeuNAc-alpha2,3-Gal-beta1,4-Glc and GalNAc-alpha1, 3-[Fuc-alpha1,2]-Gal-beta1,4-Glc, known to be specific high-affinity ligandsfor galectin-3, as well as lactose recognized by all galectins were dockedin the galectin-3 CRD model structure and a minimized binding conformationfound in each case. These studies indicate a putative extendedcarbohydrate-binding subsite in the hamster galectin- 3 involving Arg139,Glu230, and Ser232 for NeuNAc-alpha2,3-; Arg139 and Glu160 forfucose-alpha1,2-; and Arg139 and Ile141 for GalNAc-alpha1,3- substituentson the primary galactose. Each of these positions is variable within thewhole galectin family. Two of these residues, Arg139 and Ser232, wereselected for mutagenesis to probe their importance in this newly identifiedputative subsite. Residue 139 adopts main-chain dihedral anglescharacteristic of an isolated bridge structural feature, while residue 232is the C-terminal residue of beta- strand-11, and is followed immediatelyby an inverse gamma-turn. A systematic series of mutant proteins have beenprepared to represent the residue variation present in the alignedsequences of galectins-1, - 2, and -3. Minimized docked models weregenerated for each mutant in complex with NeuNAc-alpha2,3-Gal-beta1,4-Glc,GalNAc-alpha1, 3-[Fuc- alpha1,2]-Gal-beta1,4- Glc, and Gal-beta1,4-Glc.Correlation of the computed protein-carbohydrate interaction energies foreach lectin- oligosaccharide pair with the experimentally determinedbinding affinities for fetuin and asialofetuin or the relative potencies oflactose and sialyllactose in inhibiting binding to asiolofetuin isconsistent with the postulated key importance of Arg139 in recognition ofthe extended sialylated ligand.