N-Glycan Modification of a Recombinant Protein via Coexpression of Human Glycosyltransferases in Silkworm Pupae.

Recombinant proteins produced in insect cells and insects, unlike those produced in mammalian cells, have pauci-mannose-type N-glycans. In this study, we examined complex-type N-glycans on recombinant proteins via coexpression of human ß-1,2-N-acetylglucosaminyltransferase II (hGnT II) and human ß1,4-galactosyltransferase (hGalT I) in silkworm pupae, by using the Bombyx mori nucleopolyhedrovirus (BmNPV) bacmid system. The actin A3 promoter from B. mori and the polyhedrin promoter from Autographa californica multiple nucleopolyhedroviruses (AcMNPVs) were used to coexpress hGnT II and hGalT I. These recombinant BmNPVs were coexpressed with human IgG (hIgG), hGnT II and hGalT I in silkworm pupae. When hIgG was coexpressed with hGnT II, approximately 15% of all N-glycans were biantennary, with both arms terminally modified with N-acetylglucosamine (GlcNAc). In contrast, when hIgG was coexpressed with both hGnT II and hGalT I under the control of the polyhedrin promoter, 27% of all N-glycans were biantennary and terminally modified with GlcNAc, with up to 5% carrying one galactose and 11% carrying two. The obtained N-glycan structure was dependent on the promoters used for coexpression of hGnT II or hGalT I. This is the first report of silkworm pupae producing a biantennary, terminally galactosylated N-glycan in a recombinant protein. These results suggest that silkworms can be used as alternatives to insect and mammalian hosts to produce recombinant glycoproteins with complex N-glycans.

Alteration of a recombinant protein N-glycan structure in silkworms by partial suppression of N-acetylglucosaminidase gene expression.

OBJECTIVE: To synthesize complex type N-glycans in silkworms, shRNAs against the fused lobe from Bombyx mori (BmFDL), which codes N-acetylglucosaminidase (GlcNAcase) in the Golgi, was expressed by recombinant B. mori nucleopolyhedrovirus (BmNPV) in silkworm larvae. RESULTS: Expression was under the control of the actin promoter of B. mori or the U6-2 and i.e.-2 promoters from Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV). The reduction of specific GlcNAcase activity was observed in Bm5 cells and silkworm larvae using the U6-2 promoter. In silkworm larvae, the partial suppression of BmFDL gene expression was observed. When shRNA against BmFDL was expressed under the control of U6-2 promoter, the Man3GlcNAc(Fuc)GlcNAc structure appeared in a main N-glycans of recombinant human IgG. These results suggested that the control of BmFDL expression by its shRNA in silkworms caused the modification of its N-glycan synthetic pathway, which may lead to the alteration of N-glycans in the expressed recombinant proteins. CONCLUSIONS: Suppression of BmFDL gene expression by shRNA is not sufficient to synthesize complex N-glycans in silkworm larvae but can modify the N-glycan synthetic pathway.