Discovery of a lectin domain that regulates enzyme activity in mouse N-acetylglucosaminyltransferase-IVa (MGAT4A)

N -Glycosylation is a common post-translational modification, and the number of GlcNAc branches in N -glycans impacts glycoprotein functions. N -Acetylglucosaminyltransferase-IVa (GnT-IVa, also designated as MGAT4A) forms a β1-4 GlcNAc branch on the α1-3 mannose arm in N -glycans. Downregulation or loss of GnT-IVa causes diabetic phenotypes by dysregulating glucose transporter-2 in pancreatic β-cells. Despite the physiological importance of GnT-IVa, its structure and catalytic mechanism are poorly understood. Here, we identify the lectin domain in mouse GnT-IVa’s C-terminal region. The crystal structure of the lectin domain shows structural similarity to a bacterial GlcNAc-binding lectin. Comprehensive glycan binding assay using 157 glycans and solution NMR reveal that the GnT-IVa lectin domain selectively interacts with the product N -glycans having a β1-4 GlcNAc branch. Point mutation of the residue critical to sugar recognition impairs the enzymatic activity, suggesting that the lectin domain is a regulatory subunit for efficient catalytic reaction. Our findings provide insights into how branching structures of N -glycans are biosynthesized. X-ray crystallography together with NMR and computer modelling shed light on the structure and catalytic mechanism of GnTIVa, a key enzyme involved in GlcNAc branch synthesis, that bears an unusual C-terminal lectin domain that regulates its catalytic activity.