title: Expression of β-1,4-galactosyltransferase and suppression of β-N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells.
abstract: 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.
authors.affiliation: Department of Chemical Engineering, Pohang University of Science and Technology, Pohang 790-784, Republic of Korea.
authors.forename: Yeon Kyu
authors.initials: YK
authors.lastname: Kim
authors.forename: Kyoung Ro
authors.initials: KR
authors.lastname: Kim
authors.forename: Dong Gyun
authors.initials: DG
authors.lastname: Kang
authors.forename: So Young
authors.initials: SY
authors.lastname: Jang
authors.forename: Young Hwan
authors.initials: YH
authors.lastname: Kim
authors.forename: Hyung Joon
authors.initials: HJ
authors.lastname: Cha
mesh_terms: D000118:Acetylglucosaminidase / Q000037:antagonists & inhibitors* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D003412:Cricetulus; D004331:Drosophila melanogaster / Q000235:genetics / Q000378:metabolism*; D004921:Erythropoietin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D009237:N-Acetyllactosamine Synthase / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D034622:RNA Interference; D011994:Recombinant Proteins
chemical_list: D011134:Polysaccharides; D011994:Recombinant Proteins; D004921:Erythropoietin; D009237:N-Acetyllactosamine Synthase; D000118:Acetylglucosaminidase