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The baculovirus-insect cell expression system is in widespread use for expressing post-translationally modified proteins. As a result, it is potentially applicable for the production of glycoproteins for therapeutic and diagnostic purposes. For practical use, however, remodeling of the biosynthetic pathway of host-cell N-glycosylation is required because insect cells produce paucimannosidic glycoforms, which are different from the typical mammalian glycoform, due to trimming of the non-reducing terminal beta1,2-GlcNAc residue of the core structure by a specific beta-N-acetylglucosaminidase. In order to establish a cell line which could be used as a host for the baculovirus-based production of glycoproteins with mammalian-type N-glycosylation, we prepared and characterized Spodoptera frugiperda Sf21 cells that had been transfected with the rat cDNA for beta1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the addition of a bisecting GlcNAc. As evidenced by structural analyses of N-glycans prepared from whole cells and the expressed recombinant glycoproteins, the introduction of GnT-III led to the production of bisected hybrid-type N-glycans in which the beta1,2-GlcNAc residue at the alpha1,3-mannosyl branch is completely retained and which has the potential to be present in mammalian cells. These results and other related findings suggest that bisected oligosaccharides are highly resistant to beta-N-acetylglucosaminidase activity of the S. frugiperda fused lobes gene product, or other related enzymes, which was confirmed in Sf21 cells. Our present study demonstrates that GnT-III transfection has the potential to be an effective approach in humanizing the N-glycosylation of lepidopteran insect cells, thereby providing a possible preliminary step for the generation of complex-type glycoforms if the presence of a bisecting GlcNAc can be tolerated.

FUT8, a eukaryotic alpha1,6-fucosyltransferase, catalyzes the transfer of a fucosyl residue from guanine nucleotide diphosphate-beta-l-fucose to the innermost GlcNAc of an asparagine-linked oligosaccharide (N-glycan). The catalytic domain of FUT8 is structurally similar to that of NodZ, a bacterial alpha1,6-fucosyltransferase, which acts on a chitooligosaccharide in the synthesis of Nod factor. While the substrate specificities for the nucleotide sugar and the N-glycan have been determined, it is not known whether FUT8 is able to fucosylate other sugar chains such as chitooligosaccharides. The present study was conducted to investigate the action of FUT8 on chitooligosaccharides that are not generally thought to be a substrate in mammals, and the results indicate that FUT8 is able to fucosylate such structures in a manner comparable to NodZ. Surprisingly, structural analyses of the fucosylated products by high performance liquid chromatography, mass spectrometry and nuclear magnetic resonance indicated that FUT8 does not utilize the reducing terminal GlcNAc for fucose transfer but shows a preference for the third GlcNAc residue from the nonreducing terminus of the acceptor. These findings suggest that FUT8 catalyzes the fucosylation of chitooligosaccharide analogous to NodZ, but that a nonreducing terminal chitotriose structure is required for the reaction. The substrate recognition by which FUT8 selects the position to fucosylate might be distinct from that for NodZ and could be due to structural factor requirements which are inherent in FUT8.

beta-1,4-N-Acetylglucosaminyltransferase III (GnT-III) catalyzes the formation of the bisecting GlcNAc and plays a regulatory role in the biosynthesis of the N-linked oligosaccharide. In this study, we examined whether the glycosyl transfer catalyzed by GnT-III is reversible, and, in addition, investigated the equilibrium of the GnT-III-catalyzed reaction. Incubation of the agalactosyl-bisected biantennary oligosaccharide with GnT-III in the presence of the sufficiently high concentration of uridine diphosphate (UDP) resulted in conversion of the bisected oligosaccharide into the nonbisected one. This reaction was accompanied by the stoichiometric formation of UDP-GlcNAc, which appeared to result from the transfer of GlcNAc from the oligosaccharide to UDP. Thus, these results indicate that GnT-III is capable of perceivably catalyzing the reverse reaction in vitro, as found in some glycosyltransferases. When the equilibrium of the reaction was kinetically analyzed, it was found that the state of the equilibrium is greatly displaced toward the formation of the bisecting GlcNAc. In terms of free energy change, as estimated, the reaction by GnT-III can be comparable to the hydrolysis of ATP. Although GnT-III catalyzes bidirectional transfer of GlcNAc between the oligosaccharide and UDP, the removal of the bisecting GlcNAc is unlikely in vivo, due to the displacement of the equilibrium. It is known that equilibria of certain glycosyltransferase reactions are not biased as greatly as the case of GnT-III, and thus it seems likely that there are a variety of equilibrium states in glycosyltransferase reactions. In living cells, the assembly of oligosaccharides could be regulated by not only rate control but also equilibrium control.