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

Abstract

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.

Examination of differential glycoprotein preferences of N-acetylglucosaminyltransferase-IV isozymes a and b

“…Because the tissue distribution patterns of GnT-IVa and GnT-IVb are different ( 20 ), we first examined whether the expression of GnT-IVa and GnT-IVb is exclusive to each other or both enzymes simultaneously function in the same cells. We generated human embryonic kidney 293 (HEK293) GnT-IVa single KO (IVaKO) and GnT-IVa/GnT-IVb-double KO (DKO) cells ( 22 ) to discriminate these two possibilities and compared the levels of GnT-IV products by flow cytometry. For glycan detection, we used Datura stramonium agglutinin (DSA), which recognizes β1,4-linked oligomers of GlcNAc ( 23 ).…”

“…The amplified fragment was ligated to pcDNA6 myc-His A/mouse GnT-IVbLec, which had been digested with XhoI, using the NEBuilder HiFi DNA Assembly Master Mix, according to the manufacturer's protocol. The plasmid for the 6×His-J o u r n a l P r e -p r o o f tagged soluble GnT-IVa (pcDNA-IH/GnT-IVa) and pcDNA-IH/GnT-IVa∆lec were constructed, as described previously (23). For the construction of pcDNA-IH/GnT-IVb, the DNA fragment encoding the catalytic region (Ser61 to C-terminus) was amplified by PCR using pcDNA6 myc-His A/mouse GnT-IVb as a template and ligated to the EcoRV-XhoI sites of pcDNA-IH.…”

“…COS7, HEK293, and HEK293 GnT-IVa/GnT-IVb-double KO (DKO) cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 50 g/mL kanamycin under 5% CO 2 conditions at 37°C. The HEK293-DKO cell clone was established by a CRISPR system, as described previously (23).…”

“…We recently revealed that GnT-IVa, unlike other glycosyltransferases acting on N -glycans, uniquely has a lectin domain at its C terminus. We solved the crystal structure of the GnT-IVa lectin domain and found its structural similarity to a bacterial GlcNAc-binding lectin ( 22 ). Furthermore, this lectin domain specifically recognizes the product glycans having β1–4 GlcNAc branch synthesized by the catalytic domain, and the recognition of product glycans by this lectin domain is critical to the full enzymatic activity toward an oligosaccharide ( 22 ).…”

“…We solved the crystal structure of the GnT-IVa lectin domain and found its structural similarity to a bacterial GlcNAc-binding lectin ( 22 ). Furthermore, this lectin domain specifically recognizes the product glycans having β1–4 GlcNAc branch synthesized by the catalytic domain, and the recognition of product glycans by this lectin domain is critical to the full enzymatic activity toward an oligosaccharide ( 22 ). However, the importance of the lectin domain in GnT-IVb and the functional roles of lectin domains from GnT-IVa and GnT-IVb toward glycoprotein substrates remain unknown.…”