N-Glycosylation engineering of lepidopteran insect cells by the introduction of the β1,4-N-acetylglucosaminyltransferase III gene

Takahiro Okada, Hideyuki Ihara, Ritsu Ito, Miyako Nakano, Kana Matsumoto, Yoshiki Yamaguchi, Naoyuki Taniguchi, Yoshitaka Ikeda*

*Corresponding author for this work

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

The baculovirus-insect cell expression system is in widespread use for expressing post-translationally modified proteins. As a result, it is potentially applicable for the production of glycoproteins for therapeutic and diagnostic purposes. For practical use, however, remodeling of the biosynthetic pathway of host-cell N-glycosylation is required because insect cells produce paucimannosidic glycoforms, which are different from the typical mammalian glycoform, due to trimming of the non-reducing terminal β1,2-GlcNAc residue of the core structure by a specific β-N-acetylglucosaminidase. In order to establish a cell line which could be used as a host for the baculovirus-based production of glycoproteins with mammalian-type N-glycosylation, we prepared and characterized Spodoptera frugiperda Sf21 cells that had been transfected with the rat cDNA for β1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of a bisecting GlcNAc. As evidenced by structural analyses of N-glycans prepared from whole cells and the expressed recombinant glycoproteins, the introduction of GnT-III led to the production of bisected hybrid-type N-glycans in which the β1,2-GlcNAc residue at the α1,3-mannosyl branch is completely retained and which has the potential to be present in mammalian cells. These results and other related findings suggest that bisected oligosaccharides are highly resistant to β-N- acetylglucosaminidase activity of the S. frugiperda fused lobes gene product, or other related enzymes, which was confirmed in Sf21 cells. Our present study demonstrates that GnT-III transfection has the potential to be an effective approach in humanizing the N-glycosylation of lepidopteran insect cells, thereby providing a possible preliminary step for the generation of complex-type glycoforms if the presence of a bisecting GlcNAc can be tolerated.

Original languageEnglish
Pages (from-to)1147-1159
Number of pages13
JournalGlycobiology
Volume20
Issue number9
DOIs
Publication statusPublished - Sep 2010

Keywords

  • bisecting GlcNAc
  • FDL
  • GnT-III
  • lepidopteran insect
  • N-glycan

Fingerprint Dive into the research topics of 'N-Glycosylation engineering of lepidopteran insect cells by the introduction of the β1,4-N-acetylglucosaminyltransferase III gene'. Together they form a unique fingerprint.

  • Cite this