Crystal structure and sugar-binding ability of the C-terminal domain of N-acetylglucosaminyltransferase IV establish a new carbohydrate-binding module family.

N-glycans are modified by glycosyltransferases in the endoplasmic reticulum and Golgi apparatus. N-acetylglucosaminyltransferase IV (GnT-IV) is a Golgi-localized glycosyltransferase that synthesizes complex-type N-glycans in vertebrates. This enzyme attaches N-acetylglucosamine (GlcNAc) to the α-1,3-linked mannose branch of the N-glycan core structure via a β-1,4 linkage. Deficiency of this enzyme is known to cause abnormal cellular functions, making it a vital enzyme for living organisms. However, there has been no report on its 3-dimensional structure to date. Here, we demonstrated that the C-terminal regions (named CBML) of human GnT-IVa and Bombyx mori ortholog have the ability to bind β-N-acetylglucosamine. In addition, we determined the crystal structures of human CBML, B. mori CBML, and its complex with β-GlcNAc at 1.97, 1.47, and 1.15 Å resolutions, respectively, and showed that they adopt a β-sandwich fold, similar to carbohydrate-binding module family 32 (CBM32) proteins. The regions homologous to CBML (≥24% identity) were found in GnT-IV isozymes, GnT-IVb, and GnT-IVc (known as GnT-VI), and the structure of B. mori CBML in complex with β-GlcNAc indicated that the GlcNAc-binding residues were highly conserved among these isozymes. These residues are also conserved with the GlcNAc-binding CBM32 domain of β-N-acetylhexosaminidase NagH from Clostridium perfringens despite the low sequence identity (<20%). Taken together with the phylogenetic analysis, these findings indicate that these CBMLs may be novel CBM family proteins with GlcNAc-binding ability.