Suppression of beta-N-acetylglucosaminidase in the N-glycosylation pathway for complex glycoprotein formation in Drosophila S2 cells.
Glycobiology
; 19(3): 301-8, 2009 Mar.
Article
em En
| MEDLINE
| ID: mdl-19054802
ABSTRACT
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Acetilglucosaminidase
/
Biossíntese de Proteínas
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Proteínas de Drosophila
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Drosophila
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
2009
Tipo de documento:
Article