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

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年3月16日

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

摘要

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).