Document Detail

N-Glycosylation engineering of lepidopteran insect cells by the introduction of the beta1,4-N-acetylglucosaminyltransferase III gene.
MedLine Citation:
PMID:  20554946     Owner:  NLM     Status:  MEDLINE    
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 beta1,2-GlcNAc residue of the core structure by a specific beta-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 beta1,4-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 beta1,2-GlcNAc residue at the alpha1,3-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 beta-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.
Takahiro Okada; Hideyuki Ihara; Ritsu Ito; Miyako Nakano; Kana Matsumoto; Yoshiki Yamaguchi; Naoyuki Taniguchi; Yoshitaka Ikeda
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Publication Detail:
Type:  Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-06-16
Journal Detail:
Title:  Glycobiology     Volume:  20     ISSN:  1460-2423     ISO Abbreviation:  Glycobiology     Publication Date:  2010 Sep 
Date Detail:
Created Date:  2010-08-05     Completed Date:  2010-12-02     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9104124     Medline TA:  Glycobiology     Country:  England    
Other Details:
Languages:  eng     Pagination:  1147-59     Citation Subset:  IM    
Division of Molecular Cell Biology, Department of Biomolecular Sciences, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-5801, Japan.
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MeSH Terms
Carbohydrate Sequence
Cells, Cultured
Lepidoptera / enzymology,  genetics*,  metabolism
Molecular Sequence Data
N-Acetylglucosaminyltransferases / genetics*,  metabolism
Oligosaccharides / chemistry,  metabolism
Organisms, Genetically Modified
Protein Engineering / methods*
Protein Processing, Post-Translational / genetics*
Recombinant Proteins / chemistry,  genetics,  metabolism
Reg. No./Substance:
0/Oligosaccharides; 0/Recombinant Proteins; EC 2.4.1.-/N-Acetylglucosaminyltransferases; EC,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase

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