Kajiura H.Nishiguchi N.Sawada-Choi R.L.S.Sana Y.Misaki R.Fujiyama K.Mahidol University2026-05-252026-05-252026-12-01Scientific Reports Vol.16 No.1 (2026)https://repository.li.mahidol.ac.th/handle/123456789/116851Insect 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).MultidisciplinaryArticleSCOPUS10.1038/s41598-026-41152-82-s2.0-1050388870312045232241839924