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

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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.

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References

  • Altmann F, Staudacher E, Wilson IB, März L (1999) Insect cells as hosts for the expression of recombinant glycoproteins. Glycoconj J 16:109–123

    Article  CAS  PubMed  Google Scholar 

  • Aoki K, Perlman M, Lim JM, Cantu R, Wells L, Tiemeyer M (2007) Dynamic developmental elaboration of N-linked glycan complexity in the Drosophila melanogaster embryo. J Biol Chem 282:9127–9142

    Article  CAS  PubMed  Google Scholar 

  • Aumiller JJ, Mabashi-Asazuma H, Hillar A, Shi X, Jarvis DL (2012) A new glycoengineered insect cell line with an inducibly mammalianized protein N-glycosylation pathway. Glycobiology 22:417–428

    Article  CAS  PubMed  Google Scholar 

  • Bieniossek C, Imasaki T, Takagi T, Berger I (2012) MultiBac: expanding the research toolbox for multiprotein complexes. Trend Biochem Sci 37:49–57

    Article  CAS  PubMed  Google Scholar 

  • Egloff S, O’Reilly D, Murphy S (2008) Expression of human snRNA genes from beginning to end. Biochem Soc Trans 36:590–594

    Article  CAS  PubMed  Google Scholar 

  • Felberbaum RS (2015) The baculovirus expression vector system: a commercial manufacturing platform for viral vaccines. Biotechnol J 10:701–714

    Article  Google Scholar 

  • Francis BR, Paquin L, Weinkauf C, Jarvis DL (2002) Biosynthesis and processing of Spodoptera frugiperda alpha-mannosidase III. Glycobiology 12:369–377

    Article  CAS  PubMed  Google Scholar 

  • Geisler C, Jarvis DL (2010) Identification of genes encoding N-glycan processing beta-N-acetylglucosaminidases in Trichoplusia ni and Bombyx mori: implications for glycoengineering of baculovirus expression systems. Biotechnol Prog 26:34–44

    CAS  PubMed  PubMed Central  Google Scholar 

  • Geisler C, Jarvis DL (2012a) Innovative use of a bacterial enzyme involved in sialic acid degradation to initiate sialic acid biosynthesis in glycoengineered insect cells. Metab Eng 14:642–652

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Geisler C, Jarvis DL (2012b) Substrate specificities and intracellular distribution of three N-glycan processing enzymes functioning at a key branch point in the insect N-glycosylation pathway. J Biol Chem 287:7084–7097

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Geisler C, Aumiller JJ, Jarvis DL (2008) A fused lobes gene encodes the processing beta-N-acetylglucominidase in Sf9 cells. J Biol Chem 283:11330–11339

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Giering JC, Grimm D, Storm TA, Kay MA (2008) Expression of shRNA from a tissue-specific polII promoter is an effective and safe RNAi therapeutic. Mol Ther 16:1630–1636

    Article  CAS  PubMed  Google Scholar 

  • Hassinen A, Rivinoja A, Kauppila A, Kellokumpu S (2010) Golgi N-glycosyltransferases form both homo- and heterodimeric enzyme complexes in living cells. J Biol Chem 285:17771–17777

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang HH, Stanley P (2010) A testis-specific regulator of complex and hybrid N-glycan synthesis. J Cell Biol 190:893–910

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang HH, Hassinen A, Sundaram S, Spiess AN, Kellokumpu S, Stanley P (2015) GnT1IP-L specifically inhibits MGAT1 in the Golgi via its luminal domain. Elife 4:e08916

    PubMed Central  Google Scholar 

  • Jarvis DL, Finn EE (1996) Modifying the insect cell N-glycosylation pathway with immediate early baculovirus expression vectors. Nat Biotechnol 14:1288–1292

    Article  CAS  PubMed  Google Scholar 

  • Kato T, Manoha SL, Tanaka S, Park EY (2009) High-titer preparation of Bombyx mori nucleopolyhedrovirus (BmNPV) displaying recombinant protein in silkworm larvae by size exclusion chromatography and its characterization. BMC Biotechnol 9:55

    Article  PubMed  PubMed Central  Google Scholar 

  • Kim YK, Kim KR, Kang DG, Jang SY, Kim YH, Cha HJ (2011) Expression of β-1,4-galactosyltransferase and suppression of β-N-acetylglucosaminidase to aid synthesis of complex N-glycans in insect Drosophila S2 cells. J Biotechnol 153:145–152

    Article  CAS  PubMed  Google Scholar 

  • Kokuho T, Yasukochi Y, Watanabe S, Inumaru S (2010) Molecular cloning and expression of profile analysis of a novel beta-d-acetylglucosaminidase of domestic silkworm (Bombyx mori). Genes Cells 15:525–536

    CAS  PubMed  Google Scholar 

  • Léonard R, Rendic D, Rabouille C, Wilson IB, Préat T, Altmann F (2006) The Drosophila fused lobes gene encodes an N-acetylglucosaminidase involved in N-glycan processing. J Biol Chem 281:4867–4875

    Article  PubMed  Google Scholar 

  • Mabashi-Asazuma H, Shi X, Geisler C, Kuo CW, Khoo KH, Jarvis DL (2013) Impact of a human CMP-sialic acid transporter on recombinant glycoprotein sialylation in glycoengineered insect cells. Glycobiology 23:199–210

    Article  CAS  PubMed  Google Scholar 

  • Nakagawa H, Kawamura Y, Kato K, Shimada I, Arata Y, Takahashi N (1995) Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography. Anal Biochem 226:130–138

    Article  CAS  PubMed  Google Scholar 

  • Nilsson T, Rabouille C, Hui N, Watson R, Warren G (1996) The role of the membrane-spanning domain and stalk region of N-acetylglucosaminyltransferase I in retention, kin recognition and structural maintenance of the Golgi apparatus in HeLa cells. J Cell Sci 109:1975–1989

    CAS  PubMed  Google Scholar 

  • Nomura T, Ikeda M, Ishiyama S, Mita K, Tamura T, Okada T, Fujiyama K, Usami A (2010) Cloning and characterization of a β-N-acetylglucosaminidase (BmFDL) from silkworm Bombyx mori. J Biosci Bioeng 110:386–391

    Article  CAS  PubMed  Google Scholar 

  • Nomura T, Suganuma H, Higa Y, Kataoka Y, Funaguma S, Okazaki H, Suzuki T, Kobayashi I, Sezutsu H, Fujiyama K (2015) Improvement of glycosylation structure by suppression of β-N-acetylglucosminidase in silkworm. J Biosci Bioeng 119:131–139

    Article  CAS  PubMed  Google Scholar 

  • Okada T, Ishiyama S, Sezutsu H, Usami A, Tamura T, Mita K, Fujiyama K, Seki T (2007) Molecular cloning and expression of two novel beta-d-acetylglucosminidase from silkworm Bombyx mori. Biosci Biotechnol Biochem 71:1626–1635

    Article  CAS  PubMed  Google Scholar 

  • Palmberger D, Wilson IB, Berger I, Grabherr R, Rendic D (2012) SweetBac: a new approach for the production of mammalianised glycoproteins in insect cells. PLoS ONE 7:e34226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Park EY, Ishikiriyama M, Nishina T, Kato T, Yagi H, Kato K, Ueda H (2007) Human IgG1 expression in silkworm larval hemolymph using BmNPV bacmids and its N-linked glycan structure. J Biotechnol 139:108–114

    Article  Google Scholar 

  • Park EY, Abe T, Kato T (2008) Improved expression of fusion protein using a cysteine-protease- and chitinase-deficient Bombyx mori (silkworm) multiple nucleopolyhedrovirus bacmid in silkworm larvae. Biotechnol Appl Biochem 49:135–140

    Article  CAS  PubMed  Google Scholar 

  • Schanen BC, Li X (2011) Transcriptional regulation of mammalian miRNA genes. Genomics 97:1–6

    Article  CAS  PubMed  Google Scholar 

  • Schoberer J, Liebminger E, Botchway SW, Strasser R, Hawes C (2013) Time-resolved fluorescence imaging reveals differential interaction s of N-glycan processing enzymes across the Golgi stack in planta. Plant Physiol 161:1737–1754

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Su J, Zhu Z, Wang Y, Xiong F, Zou J (2008) The cytomegalovirus promoter-driven short hairpin RNA constructs mediate effective RNA interference in zebrafish in vivo. Mar Biotechnol (NY) 10:262–269

    Article  CAS  Google Scholar 

  • Takahashi N, Kato K (2003) GALAXY (glycoanalysis by the three axes of MS and chromatography): a web application that assists structural analysis of N-glycans. Trend Glycosci Glycotechnol 15:235–251

    Article  CAS  Google Scholar 

  • Takahashi N, Nakagawa H, Fujikawa K, Kawamura Y, Tomiya N (1995) Three-dimensional elution mapping of pyridylaminated N-linked neutral and sialyl oligosaccharides. Anal Biochem 226:139–146

    Article  CAS  PubMed  Google Scholar 

  • Tanaka H, Fujita K, Sagisaka A, Tomimoto K, Imanishi S, Yamanaka M (2009) shRNA expression plasmids generated by a novel method efficiently induce gene-specific knockdown in a silkworm cell line. Mol Biotechnol 41:173–179

    Article  CAS  PubMed  Google Scholar 

  • Tomiya N, Kurono M, Ishihara H, Tejima S, Endo S, Arata Y, Takahashi N (1987) Structural analysis of N-linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and high-performance liquid chromatography. Anal Biochem 163:489–499

    Article  CAS  PubMed  Google Scholar 

  • Tomiya N, Betenbaugh MJ, Lee YC (2003a) Humanization of lepidopteran insect-cell-produced glycoproteins. Acc Chem Res 36:613–620

    Article  CAS  PubMed  Google Scholar 

  • Tomiya N, Howe D, Aumiller JJ, Pathak M, Park J, Palter KH, Jarvis DL, Betenbaugh MJ, Lee YC (2003b) Complex-type biantennary N-glycans of recombinant human transferrin from Trichoplusia ni insect cells expressing mammalian β-1,4-galactosyltransferase and β-1,2-N-acetylglucosaminyltransferase II. Glycobiology 13:23–34

    Article  CAS  PubMed  Google Scholar 

  • Tomiya N, Narang S, Park J, Abdul-Rahman B, Choi O, Singh S, Hiratake J, Sakata K, Betenbaugh MJ, Palter KB, Lee YC (2006) Purification, characterization, and cloning of a Spodoptera frugiperda Sf9 beta-N-acetylglucosaminidase that hydrolyzes terminal N-acetylglucosamine on the N-glycan core. J Biol Chem 281:19545–19560

    Article  CAS  PubMed  Google Scholar 

  • Toth AM, Kuo CW, Khoo KH, Jarvis DL (2014) A new cell glycoengineering approach provides baculovirus-inducible glycogene expression and increases human-type glycosylation efficiency. J Biotechnol 182–183:19–29

    Article  PubMed  Google Scholar 

  • Wolff MW, Murhammer DW, Jarvis DL, Linhardt RJ (1999) Electrophoresis analysis of glycoprotein glycans produced by lepidopteran insect cells infected with an immediate early recombinant baculovirus encoding mammalian beta1,4-galactosyltransferase. Glycoconj J 16:753–756

    Article  CAS  PubMed  Google Scholar 

  • Yagi H, Takahashi N, Yamaguchi Y, Kimura N, Uchimura K, Kannagi R, Kato K (2005) Development of structural analysis of sulfated N-glycans by multidimentional high performance liquid chromatography mapping methods. Glycobiology 15:1051–1060

    Article  CAS  PubMed  Google Scholar 

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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.

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Correspondence to Enoch Y. Park.

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Kato, T., Kikuta, K., Kanematsu, A. et al. Alteration of a recombinant protein N-glycan structure in silkworms by partial suppression of N-acetylglucosaminidase gene expression. Biotechnol Lett 39, 1299–1308 (2017). https://doi.org/10.1007/s10529-017-2361-y

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  • DOI: https://doi.org/10.1007/s10529-017-2361-y

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