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

Tatsuya Kato,Koichi Kato,Takatsugu Miyazaki,Hirokazu Yagi,Natsumi Kako,Sachiko Kondo,Kotaro Kikuta,Enoch Y Park

doi:10.1038/s41598-017-01630-6

Abstract

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 β-1,2-N-acetylglucosaminyltransferase II (hGnT II) and human β1,4-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.

Highlights

The post-translational modifications that occur in insect cells are similar to those in mammalian cells; the N-glycans of proteins expressed in insect cells are mainly of the pauci-mannose type, whereas the N-glycans of proteins expressed in mammalian cells are of the complex type[5, 6]
When human immunoglobulin G (hIgG) was coexpressed with human GnT II (hGnT II) under the control of the actin A3 promoter (PAct-hGnT II) and with hGnT II under the control of the polyhedrin promoter (PPol-hGnT), 14.2% and 15.4% of the N-glycans, respectively, were GlcNAc terminated (N8: GlcNAc2Man3GlcNAc2, N11: GlcNAc2Man3GlcNAc(Fuc)GlcNAc)
To terminally galactosylate the N-glycans of hIgG in silkworm pupae, hIgG was coexpressed with hGnT II and human GalT I (hGalT I) in the following combinations: hGnT II under the control of the actin A3 promoter (PAct-hGnT II) and hGalT I under the control of the polyhedrin promoter (PPol-hGalT I); hGnT II under the control of the polyhedrin promoter (PPol-hGnT II) and hGalT I under the control of the actin A3 promoter (PAct-hGalT I); and hGnT II under the control of the polyhedrin promoter (PPol-hGnT II) and hGalT I under the control of the polyhedrin promoter (PPol-hGalT I)

Summary

Introduction

The post-translational modifications that occur in insect cells are similar to those in mammalian cells; the N-glycans of proteins expressed in insect cells are mainly of the pauci-mannose type, whereas the N-glycans of proteins expressed in mammalian cells are of the complex type[5, 6]. Bombyx mori (Silkworm) has been used for the production of recombinant proteins To those in insect cells, most of the N-glycans on glycoproteins expressed in silkworms are of the pauci-mannose type[19, 20]. In the posterior silk gland (PSG), up to 17% of N-glycans are terminally galactosylated in transgenic silkworm larvae coexpressing human GnT II and bovine GalT I under the control of a PSG-specific promoter[21]. This result indicates that mammalian-like N-glycans can be produced in the PSG of silkworms via the expression of mammalian glycosyltransferases. The expression system developed in this study is promising for the production of galactosylated recombinant proteins in silkworm pupae

Methods

Results

Conclusion