Journal Article

N-Glycoengineering of insect cells for tri-antennary N-glycan biosynthesis

Misaki, Ryo, Sawada-Choi, Reimi Lai Sang, Sana, Yuto, Fujiyama, Kazuhito, Nishiguchi, Naokuni, Kajiura, Hiroyuki

2026

Overview

Insect cells are attractive hosts for biopharmaceutical production due to their high productivity and mammalian-like post-translational modifications. However, their insect-specific N -glycans differ from mammalian types, thereby reducing product desirability. Here, with an emphasis on engineering N -glycosylation in insect cells, we aimed to develop a more tractable Spodoptera frugiperda Sf9 cell platform as a practical alternative to insect-based systems for engineering the production of tri-antennary N -glycans. A database search revealed that silkworm possesses N -acetylglucosaminyltransferase IV (GNTIV), a putative glycosyltransferase essential for tri-antennary N -glycan synthesis; however, it was not functional in vitro. Then, human GNTIV was introduced into insect cells, resulting in the production of small amounts of tri-antennary N -glycans. This suggested the need for additional factors to efficiently biosynthesize tri-antennary N -glycans. Subsequently, additional insect-derived glycosyltransferases, such as active GNTI and/or GNTII, were co-expressed with GNTIV. Co-expression of three N -acetylglucosaminyltransferases effectively led to the increased biosynthesis of tri-antennary N -glycans. On the other hand, trimming of the N -glycan structure was also observed due to the action of one or more endogenous glycosylhydrolases, which hydrolyze the terminal N -acetylglucosamine residue in insect cells. These facts indicate that effective tri-antennary N -glycan biosynthesis in insect cells requires not only the introduction of exogenous glycosyltransferases but also the knockdown or knockout of endogenous glycosylhydrolase(s).

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 631/337/458/1524 / 631/45/221 / Animals / Biosynthesis / Bombyx - genetics / Enzymes / Eukaryotes / Genes / Genetic engineering / Genomes / GlcNAc extension / Glycoproteins / Glycosylation / Glycosyltransferase / Humanities and Social Sciences / Humans / Insect cells / Insects / Mammals / multidisciplinary / N-Acetylglucosamine / N-Acetylglucosaminyltransferase IV / N-Acetylglucosaminyltransferases - genetics / N-Acetylglucosaminyltransferases - metabolism / N-Acetylglucosinyldiphosphodolichol N-acetylglucosaminyltransferase / N-Acetylhexosaminidase / N-glycans / N-Glycosylation / Pharmaceuticals / Polysaccharides / Polysaccharides - biosynthesis / Post-translation / Proteins / Science / Science (multidisciplinary) / Sf9 Cells / Spodoptera - genetics