Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors.

The baculovirus-insect cell expression system is well-suited for recombinant glycoprotein production because baculovirus vectors can provide high levels of expression and insect cells can modify newly synthesized proteins in eucaryotic fashion. However, the N-glycosylation pathway of baculovirus-infected insect cells differs from the pathway found in higher eucaryotes, as indicated by the fact that glycoproteins produced in the baculovirus system typically lack complex biantennary N-linked oligosaccharide side chains containing penultimate galactose and terminal sialic acid residues. We recently developed a new type of baculovirus vector that can express foreign genes immediately after infection under the control of the viral ie1 promoter. These immediate early baculovirus expression vectors can be used to modify the insect cell N-glycosylation pathway and produce a foreign glycoprotein with more extensively processed N-linked oligosaccharides. These vectors can also be used to study the influence of the late steps in N-linked oligosaccharide processing on glycoprotein function. Further development could lead to baculovirus-insect cell expression systems that can produce recombinant glycoproteins with complex biantennary N-linked oligosaccharides structurally identical to those produced by higher eucaryotes.

Biochemical analysis of the N-glycosylation pathway in baculovirus-infected lepidopteran insect cells.

The baculovirus-insect cell system is used routinely for foreign glycoprotein production, but the precise nature of the N-glycosylation pathway in this system remains unclear. Some studies indicate that these cells cannot process N-linked oligosaccharides to complex forms containing outer-chain galactose and sialic acid, while others indicate that they can. In this study, we used the major virion envelope glycoprotein of the baculovirus Autographa california multicapsid nuclear polyhedrosis virus (AcMNPV) to probe the N-glycosylation pathway in baculovirus-infected lepidopteran insect cells. The results showed that gp64 contained mannose, fucose, and probably N-acetylglucosamine, but no detectable galactose or sialic acid. These same results were observed with gp64 produced in any one of three different lepidopteran insect cell lines derived from Spodoptera frugiperda, Trichoplusia ni, or Estigmene acrea, whether it was produced at relatively earlier or later times after infection. These results indicated that the gp64 produced in AcMNPV-infected lepidopteran insect cells lacks complex N-linked oligosaccharides containing outer-chain galactose and sialic acid. By contrast, gp64 produced in mammalian cells contained both galactose and sialic acid, and endoglycosidase digestions revealed that these sugars were constituents of N-linked, not O-linked, oligosaccharides. This showed that at least one N-linked side chain on gp64 has the potential to be processed to a complex form. Together, these results suggest either that AcMNPV-infected lepidopteran insect cells are unable to convert any of the N-linked side chains on gp64 to complex structures or that outer-chain galactose and sialic acid residues are added to gp64 and then removed by cellular or viral exoglycosidases.