Kim Yeon Kyu, Kim Kyoung Ro, Kang Dong Gyun, Jang So Young, Kim Young Hwan, Cha Hyung Joon
National Research Laboratory of Molecular Biotechnology, Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.
Glycobiology. 2009 Mar;19(3):301-8. doi: 10.1093/glycob/cwn138. Epub 2008 Dec 2.
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 beta-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase in the Golgi membrane 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.
大多数昆虫细胞具有简单的N-糖基化过程，因此寡甘露糖型或简单核心聚糖占主导地位。此前，我们已经表明寡甘露糖型N-聚糖结构在果蝇S2细胞中占主导。有人提出，β-N-乙酰氨基葡萄糖苷酶（GlcNAcase），一种高尔基体膜中的己糖胺酶，可去除末端N-乙酰氨基葡萄糖（GlcNAc），可能在除S2细胞外的几种昆虫和昆虫衍生细胞中促成简单的N-糖基化。在本研究中，我们使用两种GlcNAcase抑制策略研究了GlcNAcase对果蝇S2细胞中N-聚糖模式的实质影响：一种是使用RNA干扰（RNAi）的mRNA靶向方法，另一种是使用特异性化学抑制剂2-乙酰氨基-1,2-二脱氧野尻霉素（2-ADN）的蛋白质靶向方法。通过高效液相色谱（HPLC）和基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）分析，我们发现，在抑制GlcNAcase后，稳定转染的S2细胞分泌的人促红细胞生成素（hEPO）的N-糖基化模式更复杂，产生了带有末端GlcNAc和/或半乳糖的N-聚糖结构。这些数据表明，GlcNAcase可能是果蝇S2细胞中寡甘露糖型核心N-聚糖形成的重要因素，并表明通过抑制N-糖基化途径中的GlcNAcase，有可能在工程化的果蝇S2细胞中表达复杂糖蛋白。
