Crystal structure and glycan-binding ability of galectin domain of OsGALT1.

N- linked glycan structures vary across different organisms, and Lewis A glycan represents a unique N-glycoform in plants. The GT31 enzyme GALT1 catalyzes Lewis A glycan synthesis by transferring galactose from UDP-galactose to the terminal GlcNAc of N-glycan branches. Structurally, the plant GALT1 protein is characterized by the presence of both a galactosyltransferase catalytic domain and a galectin-like domain. However, the specific ligands recognized by its plant-specific galectin domain, as well as the functional role of this domain in catalysis, remain poorly understood. In this study, we identified two putative Oryza sativa GALT1 proteins and determined the crystal structures of their galectin domains at resolutions of 2.35 Å and 2.20 Å, respectively. Both structures adopt a fold consisting of two layers of β-sheets, closely resembling the fold of human galectin-4. Isothermal titration calorimetry revealed that the OsGALT1-1 galectin domain binds weakly to galactose and Gal-β-1,3-GlcNAc, whereas the OsGALT1-2 galectin domain shows no detectable binding ability. Molecular dynamics simulations further suggested that an Asn-to-Asp substitution between OsGALT1-1 and OsGALT1-2 galectin domains may weaken hydrogen-bond interactions, thereby compromising the binding stability. Collectively, these findings indicate that the OsGALT1-1 galectin domain functions as a galactoside-binding lectin and facilitates the modification of bi-antennary N-glycans to form Lewis A structures.