Suppression of beta-N-acetylglucosaminidase in the N-glycosylation pathway for complex glycoprotein formation in Drosophila S2 cells.
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.
Kim
Yeon Kyu
YK
National Research Laboratory of Molecular Biotechnology, Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Korea.
Kim
Kyoung Ro
KR
Kang
Dong Gyun
DG
Jang
So Young
SY
Kim
Young Hwan
YH
Cha
Hyung Joon
HJ
D000118:Acetylglucosaminidase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004330:Drosophila / Q000166:cytology* / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004921:Erythropoietin / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006031:Glycosylation; D008954:Models, Biological; D014176:Protein Biosynthesis; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014162:Transfection
D029721:Drosophila Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004921:Erythropoietin; D000118:Acetylglucosaminidase