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, of hamster galectin-3 has been made using homology modeling and dynami cs minimization methods, The model is based on the known x-ray structu res of bovine galectin-1 and human galectin-2. The oligosaccharides Ne uNAc-alpha 2,3-Gal-beta 1,4-Glc and GalNAc-alpha 1,3-[Fuc-alpha 1,2]-G al-beta 1,4-Glc, known to be specific high-affinity ligands for galect in-3, as web as lactose recognized by all galectins were docked in the galectin-3 CRD model structure and a minimized binding conformation f ound in each case, These studies indicate a putative extended carbohyd rate-binding subsite in the hamster galectin-3 involving Arg139, Glu23 0, and Ser232 for NeuNAc-alpha 2,3-; Arg139 and Glu160 for fucose-alph a 1,2-; and Arg139 and Ile141 for GalNAc-alpha 1,3- substituents on th e primary galactose, Each of these positions is variable within the wh ole galectin family. Two of these residues, Arg139 and Ser232, were se lected for mutagenesis to probe their importance in this newly identif ied putative subsite, Residue 139 adopts main-chain dihedral angles ch aracteristic of an isolated bridge structural feature, while residue 2 32 is the C-terminal residue of beta-strand-11, and is followed immedi ately by an inverse gamma-turn, A systematic series of mutant proteins have been prepared to represent the residue variation present in the aligned sequences of galectins-1, -2, and 3. Minimized docked models w ere generated for each mutant in complex with Neu-NAc-alpha 2,3-Gal-be ta 1,4-Glc, GalNAc-alpha 1,3-[Fuc-alpha 1,2]-Gal-beta 1,4-Glc, and Gal -beta 1,4-Glc. Correlation of the computed protein-carbohydrate intera ction energies for each lectin-oligosaccharide pair with the experimen tally determined binding affinities for fetuin and asialofetuin or the relative potencies of lactose and sialyllactose in inhibiting binding to asiolofetuin is consistent with the postulated key importance of A rg139 in recognition of the extended sialylated ligand.