title: Sialylation of N-glycans on the recombinant proteins expressed by a baculovirus-insect cell system under beta-N-acetylglucosaminidase inhibition.
abstract: We investigated the ability of a baculovirus-insect cell system to produce sialylated glycoproteins. Despite the presence of enzymes for synthesizing complex-type N-glycans, the most frequent structure of insect N-glycan is the paucimannosidic type, Man(3)GlcNAc(2)(+/-Fuc). The reason for the overwhelming assembly of paucimannosidic N-glycans is not yet well understood. We hypothesized that this predominance might be due to insect-specific, Golgi-associated beta-N-acetylglucosaminidase (GlcNAcase)-mediated removal of N-acetylglucosamine residues from the precursor N-glycan, thereby preventing its galactosylation and terminal sialylation. As we expected, the suppression of intrinsic GlcNAcase activity with a specific inhibitor, 2-acetamido-1,2-dideoxynojirimycin, allowed the accumulation of sialylated glycoproteins in the supernatants of insect cell cultures after baculoviral infection. Our observation indicates that GlcNAcase-dependent depletion of N-acetylglucosamine residues from intermediate N-glycans is critical for the assembly of paucimannosidic N-glycans in insect cells and, more importantly, that insect cells (under specific conditions) retain the ability to construct sialylated N-glycans like those in mammalian cells.
authors.affiliation: Department of Immunology, National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan. satochan@affrc.go.jp
authors.forename: Satoko
authors.initials: S
authors.lastname: Watanabe
authors.forename: Takehiro
authors.initials: T
authors.lastname: Kokuho
authors.forename: Hitomi
authors.initials: H
authors.lastname: Takahashi
authors.forename: Masashi
authors.initials: M
authors.lastname: Takahashi
authors.forename: Takayuki
authors.initials: T
authors.lastname: Kubota
authors.forename: Shigeki
authors.initials: S
authors.lastname: Inumaru
mesh_terms: D000118:Acetylglucosaminidase / Q000037:antagonists & inhibitors*; D000818:Animals; D016367:Baculoviridae; D001665:Binding Sites; D015153:Blotting, Western; D002417:Cattle; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006023:Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology; D007313:Insecta; D037102:Lectins / Q000378:metabolism; D051379:Mice; D008956:Models, Chemical; D019158:N-Acetylneuraminic Acid / Q000737:chemistry* / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism*
chemical_list: D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D006023:Glycoproteins; D037102:Lectins; D011134:Polysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D000118:Acetylglucosaminidase; D019158:N-Acetylneuraminic Acid