Sialylation of N-Glycans on the Recombinant Proteins Expressed by a Baculovirus-Insect Cell System under β-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-typeN-glycans, the most frequent structure of insectN-glycan is the paucimannosidic type, Man3GlcNAc2(±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 β-N-acetylglucosaminidase (GlcNAcase)-mediated removal ofN-acetylglucosamine residues from the precursorN-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 ofN-acetylglucosamine residues from intermediateN-glycans is critical for the assembly of paucimannosidicN-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.