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Glycobiology Advance Access published online on December 2, 2008
Glycobiology, doi:10.1093/glycob/cwn138
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© The Author 2008. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Suppression of β-N-acetylglucosaminidase in N-glycosylation pathway for complex glycoprotein formation in Drosophila S2 cells

Yeon Kyu Kim1, Kyoung Ro Kim1, Dong Gyun Kang1, So Young Jang2, Young Hwan Kim2 and Hyung Joon Cha1,*

1 National Research Laboratory of Molecular Biotechnology, Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790–784, Korea
2 Mass Spectrometry Research Team, Korea Basic Science Institute, Ochang 363–883, Korea

* To whom correspondence should be addressed; Tel: (82–54) 279–2280. e-mail: hjcha{at}postech.ac.kr

Received on September 11, 2008; accepted on November 27, 2008

Most insect cells have a simple N-glycosylation process and consequently paucimannosidic or simple core glycans predominate. Previously, we have shown that paucimannosidic N-glycan structures are dominant in Drosophila S2 cells. It has been proposed that β-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane and which removes a terminal N-acetylglucosamine (GlcNAc), might contribute to simple N-glycosylation in several insects and insect-derived cells except S2 cells. In the present work, we investigated the substantial effects of GlcNAcase on N-glycan patterns in Drosophila S2 cells using two GlcNAcase suppression strategies: an mRNA-targeting approach using RNA interference (RNAi) and a protein-targeting approach using the specific chemical inhibitor 2-acetamido-1,2-dideoxynojirimycin (2-ADN). Using high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analyses, we found that the N-glycosylation patterns of human erythropoietin (hEPO) secreted by stably transfected S2 cells were more complex following GlcNAcase suppression, which generated N-glycan structures with a terminal GlcNAc and/or galactose. These data demonstrate that GlcNAcase may be an important factor in the formation of paucimannosidic core N-glycans in Drosophila S2 cells, and suggest that it may be possible to express complex glycoproteins in engineered Drosophila S2 cells by suppressing GlcNAcase in the N-glycosylation pathway.

Key words: Drosophila S2 cells / N-glycosylation / β-N-acetylglucosaminidase / suppression / glycoprotein / human erythropoietin


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