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Biotechnology Letters

, Volume 39, Issue 9, pp 1299–1308 | Cite as

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

  • Tatsuya Kato
  • Kotaro Kikuta
  • Ayumi Kanematsu
  • Sachiko Kondo
  • Hirokazu Yagi
  • Koichi Kato
  • Enoch Y. Park
Original Research Paper

Abstract

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.

Keywords

BmNPV N-Glycan IgG MALDI-TOF-MS RNAi Silkworm 

Notes

Acknowledgements

The authors wish to thank Prof. Hiroshi Ueda of Tokyo Institute of Technology for providing the human IgG gene.

Supporting information

Supplementary Table 1—Primers used.

Supplementary material

10529_2017_2361_MOESM1_ESM.docx (14 kb)
Supplementary material 1 (DOCX 13 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  1. 1.Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of AgricultureShizuoka UniversityShizuokaJapan
  2. 2.Laboratory of Biotechnology, Research Institute of Green Science and TechnologyShizuoka UniversityShizuokaJapan
  3. 3.Graduate School of Pharmaceutical SciencesNagoya City UniversityNagoyaJapan
  4. 4.Medical & Biological Laboratories Co., Ltd.Naka-KuJapan
  5. 5.Institute for Molecular Science and Okazaki Institute for Integrative BioscienceNational Institutes of Natural SciencesOkazakiJapan

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