| N-Glycosylation engineering of lepidopteran insect cells by the introduction of the beta1,4-N-acetylglucosaminyltransferase III gene. | |
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MedLine Citation:
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PMID: 20554946 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Takahiro Okada; Hideyuki Ihara; Ritsu Ito; Miyako Nakano; Kana Matsumoto; Yoshiki Yamaguchi; Naoyuki Taniguchi; Yoshitaka Ikeda |
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Publication Detail:
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Type: Evaluation Studies; Journal Article; Research Support, Non-U.S. Gov't Date: 2010-06-16 |
Journal Detail:
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Title: Glycobiology Volume: 20 ISSN: 1460-2423 ISO Abbreviation: Glycobiology Publication Date: 2010 Sep |
Date Detail:
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Created Date: 2010-08-05 Completed Date: 2010-12-02 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 9104124 Medline TA: Glycobiology Country: England |
Other Details:
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Languages: eng Pagination: 1147-59 Citation Subset: IM |
Affiliation:
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Division of Molecular Cell Biology, Department of Biomolecular Sciences, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga 849-5801, Japan. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Carbohydrate Sequence Cells, Cultured Glycosylation 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* Rats Recombinant Proteins / chemistry, genetics, metabolism Spodoptera Transfection |
| Chemical | |
Reg. No./Substance:
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0/Oligosaccharides; 0/Recombinant Proteins; EC 2.4.1.-/N-Acetylglucosaminyltransferases; EC 2.4.1.144/beta-1,4-mannosyl-glycoprotein beta-1,4-N-acetylglucosaminyltransferase |
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