Expression of β-1,4-galactosyltransferase and suppression of β- N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells

• Published in: Journal of biotechnology Vol. 153; no. 3; pp. 145 - 152
• Main Authors: Kim, Yeon Kyu, Kim, Kyoung Ro, Kang, Dong Gyun, Jang, So Young, Kim, Young Hwan, Cha, Hyung Joon
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 20.05.2011; Elsevier
• Subjects: Acetylglucosaminidase - antagonists & inhibitors; Acetylglucosaminidase - biosynthesis; Acetylglucosaminidase - genetics; Acetylglucosaminidase - metabolism; Animals; Biological and medical sciences; Biotechnology; Blotting, Western; Cell Line; CHO Cells; Chromatography, High Pressure Liquid; Cricetinae; Cricetulus; Drosophila; Drosophila melanogaster - genetics; Drosophila melanogaster - metabolism; Drosophila S2 cells; erythropoietin; Erythropoietin - chemistry; Erythropoietin - genetics; Erythropoietin - metabolism; Fundamental and applied biological sciences. Psychology; galactose; glycosyltransferases; Human erythropoietin; Humans; insects; Microscopy, Fluorescence; N-acetylglucosamine; N-Acetyllactosamine Synthase - biosynthesis; N-Acetyllactosamine Synthase - genetics; N-Acetyllactosamine Synthase - metabolism; N-Glycosylation; Polysaccharides - biosynthesis; Polysaccharides - chemistry; Polysaccharides - metabolism; Protein Engineering - methods; Recombinant Proteins; RNA Interference; synergism; β- N-Acetylglucosaminidase; β-1,4-Galactosyltransferase; FucT; GlcNAcT II; Human erythropoietin; GalT; Sia; N-Glycosylation; SiaT; Gal; Fuc; β-1,4-Galactosyltransferase; GlcNAc; Drosophila S2 cells; Man; β- N-Acetylglucosaminidase; GlcNAcase; Glc; Human; β-N-Acetylglucosaminidase; Erythropoietin; Enzyme; Transferases; Drosophila; Insecta; Suppression; Glycosyltransferases; Glycosylation; Complexes; Glycan; Drosophilidae; N-Acetyllactosaminide α-1,3-galactosyltransferase; Arthropoda; β-1.4-Galactosyltransferase; Hexosyltransferases; Invertebrata; Diptera
• ISSN: 0168-1656; 1873-4863
• DOI: 10.1016/j.jbiotec.2011.03.021

Previously, we have shown that simple paucimannosidic N-glycan structures in insect Drosophila S2 cells arise mainly because of β- N-acetylglucosaminidase (GlcNAcase) action. Thus, in an earlier report, we suppressed GlcNAcase activity and clearly demonstrated that more complex N-glycans with two terminal N-acetylglucosamine (GlcNAc) residues were then synthesized. In the present work, we investigated the synergistic effects of β-1,4-galactosyltransferase (GalT) expression and GlcNAcase suppression on N-glycan patterns. We found that the N-glycan pattern of human erythropoietin secreted by engineered S2 cells expressing GalT but not GlcNAcase was complete, even in small portion, except for sialylation; the N-glycan structures had two terminal galactose (Gal) residues. When GalT was expressed but GlcNAcase was not inhibited, N-glycan with GlcNAc and Gal at only one branch end was synthesized. Therefore, it will be possible to express a complete functional human glycoprotein in engineered Drosophila S2 cells by suppressing GlcNAcase and co-expressing additional glycosyltransferases of N-glycosylation pathway.