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      Sialylation of N-glycans on the recombinant proteins expressed by a baculovirus-insect cell system under beta-N-acetylglucosaminidase inhibition.

      The Journal of Biological Chemistry
      Acetylglucosaminidase, antagonists & inhibitors, Animals, Baculoviridae, Binding Sites, Blotting, Western, Cattle, Cell Line, DNA, Complementary, metabolism, Enzyme Inhibitors, pharmacology, Glycoproteins, chemistry, Golgi Apparatus, enzymology, Insects, Lectins, Mice, Models, Chemical, N-Acetylneuraminic Acid, Polysaccharides, Protein Binding, RNA, Messenger, Recombinant Proteins

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          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.

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