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

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...

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Autores principales:	Tatsuya Kato, Natsumi Kako, Kotaro Kikuta, Takatsugu Miyazaki, Sachiko Kondo, Hirokazu Yagi, Koichi Kato, Enoch Y. Park
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/15262b0d71334e178790fac4080f4ecc
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spelling	oai:doaj.org-article:15262b0d71334e178790fac4080f4ecc2021-12-02T16:06:10ZN-Glycan Modification of a Recombinant Protein via Coexpression of Human Glycosyltransferases in Silkworm Pupae10.1038/s41598-017-01630-62045-2322https://doaj.org/article/15262b0d71334e178790fac4080f4ecc2017-05-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-01630-6https://doaj.org/toc/2045-2322Abstract 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.Tatsuya KatoNatsumi KakoKotaro KikutaTakatsugu MiyazakiSachiko KondoHirokazu YagiKoichi KatoEnoch Y. ParkNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-10 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Tatsuya Kato Natsumi Kako Kotaro Kikuta Takatsugu Miyazaki Sachiko Kondo Hirokazu Yagi Koichi Kato Enoch Y. Park N-Glycan Modification of a Recombinant Protein via Coexpression of Human Glycosyltransferases in Silkworm Pupae
description	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.
format	article
author	Tatsuya Kato Natsumi Kako Kotaro Kikuta Takatsugu Miyazaki Sachiko Kondo Hirokazu Yagi Koichi Kato Enoch Y. Park
author_facet	Tatsuya Kato Natsumi Kako Kotaro Kikuta Takatsugu Miyazaki Sachiko Kondo Hirokazu Yagi Koichi Kato Enoch Y. Park
author_sort	Tatsuya Kato
title	N-Glycan Modification of a Recombinant Protein via Coexpression of Human Glycosyltransferases in Silkworm Pupae
title_short	N-Glycan Modification of a Recombinant Protein via Coexpression of Human Glycosyltransferases in Silkworm Pupae
title_full	N-Glycan Modification of a Recombinant Protein via Coexpression of Human Glycosyltransferases in Silkworm Pupae
title_fullStr	N-Glycan Modification of a Recombinant Protein via Coexpression of Human Glycosyltransferases in Silkworm Pupae
title_full_unstemmed	N-Glycan Modification of a Recombinant Protein via Coexpression of Human Glycosyltransferases in Silkworm Pupae
title_sort	n-glycan modification of a recombinant protein via coexpression of human glycosyltransferases in silkworm pupae
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/15262b0d71334e178790fac4080f4ecc
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N-Glycan Modification of a Recombinant Protein via Coexpression of Human Glycosyltransferases in Silkworm Pupae

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