Skip to main content
SpringerLink
Log in

Highly efficient transglycosylation of sialo-complex-type oligosaccharide using Coprinopsis cinerea endoglycosidase and sugar oxazoline

  • Original Research Paper
  • Published:
Biotechnology Letters Aims and scope Submit manuscript

Abstract

Objectives

To establish an efficient method of chemoenzymatic modification for making N-linked oligosaccharide chains of glycoproteins structurally homogeneous, which crucially affects their bioactivities.

Results

Deglycosylated-RNase B (GlcNAc-RNase B; acceptor), sialylglyco (SG)-oxazoline (donor) and an N180H mutant of Coprinopsis cinerea endo-β-N-acetylglucosaminidase (Endo-CCN180H) were employed. pH 7.5 was ideal for both SG-oxazoline’s stability and Endo-CC’s transglycosylation reaction. The most efficient reaction conditions for producing glycosylated-RNase B, virtually modified completely with sialo-biantennary-type complex oligosaccharide, were: 80 μg GlcNAc-RNase B, 200 μg SG-oxazoline and 3 μg Endo-CCN180H in 20 μl 20 mM Tris/HCl pH 7.5 at 30 °C for 30–60 min.

Conclusions

This transglycosylation method using SG-oxazoline and Endo-CCN180H is beneficial for producing pharmaceutical glycoproteins modified with homogenous biantennary-complex-type oligosaccharides.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Eshima Y, Higuchi Y, Kinoshita T, Nakakita S, Takegawa K (2015) Transglycosylation activity of glycosynthase mutants of endo-β-N-acetylglucosaminidase from Coprinopsis cinerea. PLoS One 10:e0132859

    Article  PubMed  PubMed Central  Google Scholar 

  • Fujita K, Kobayashi K, Iwamatsu A, Takeuchi M, Kumagai H, Yamamoto K (2004) Molecular cloning of Mucor hiemalis endo-conclusions β-N-acetylglucosaminidase and some properties of the recombinant enzyme. Arch Biochem Biophys 432:41–49

    Article  CAS  PubMed  Google Scholar 

  • Huang W, Li C, Li B, Umekawa M, Yamamoto K, Zhang X, Wang LX (2009) Glycosynthases enable a highly efficient chemoenzymatic synthesis of N-glycoproteins carrying intact natural N-glycans. J Am Chem Soc 131:2214–2223

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Huang W, Yang Q, Umekawa M, Yamamoto K, Wang LX (2010) Arthrobacter endo-β-N-acetylglucosaminidase shows transglycosylation activity on complex-type N-glycan oxazolines: one-pot conversion of ribonuclease B to sialylated ribonuclease C. ChemBioChem 11:1350–1355

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kadowaki S, Yamamoto K, Fujisaki M, Izumi K, Tochikura T, Yokoyama T (1990) Purification and characterization of a novel fungal endo-β-N-acetylglucosaminidase acting on complex oligosaccharides of glycoproteins. Agric Biol Chem 54:97–106

    CAS  PubMed  Google Scholar 

  • Kadowaki S, Yamamoto K, Fujisaki M, Tochikura T (1991) Microbial endo-β-N-acetylglucosaminidases acting on complex-type sugar chains of glycoproteins. J Biochem 110:17–21

    CAS  PubMed  Google Scholar 

  • Matsumiya S, Yamaguchi Y, Saito J, Nagano M, Sasakawa H, Otaki S, Satoh M, Shitara K, Kato K (2007) Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1. J Mol Biol 368:767–779

    Article  CAS  PubMed  Google Scholar 

  • Noguchi M, Fujieda T, Huang WC, Ishihara M, Kobayashi A, Shoda SI (2012) A practical one-step synthesis of 1,2-oxazoline derivatives from unprotected sugars and its application to chemoenzymatic β-N-acetylglucosaminidation of disialo-oligosaccharide. Helv Chim Acta 95:1928–1936

    Article  CAS  Google Scholar 

  • Takegawa K, Inami M, Yamamoto K, Kumagai H, Tochikura T, Mikami B, Morita Y (1988a) Elucidation of the role of sugar chains in glucoamylase using endo-β-N-acetylglucosaminidase from Flavobacterium sp. Biochim Biophys Acta 955:187–193

    Article  CAS  Google Scholar 

  • Takegawa K, Inami M, Yamamoto K, Kumagai H, Tochikura T, Mikami B, Morita Y (1988b) Deglycosylated glucoamylase from Rhizopus niveus is precipitated by Flavobacterium sp. endo-β-N-acetylglucosaminidase. Agric Biol Chem 52:2941–2942

    CAS  Google Scholar 

  • Takegawa K, Nakoshi M, Iwahara S, Yamamoto K, Tochikura T (1989) Induction and purification of endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae grown in ovalbumin. Appl Environ Microbiol 55:3107–3112

    CAS  PubMed  PubMed Central  Google Scholar 

  • Takegawa K, Tabuchi M, Yamaguchi S, Kondo A, Kato I, Iwahara S (1995) Synthesis of neoglycoproteins using oligosaccharide-transfer activity with endo-β-N-acetylglucosaminidase. J Biol Chem 270:3094–3099

    Article  CAS  PubMed  Google Scholar 

  • Takegawa K, Yamabe K, Fujita K, Tabuchi M, Mita M, Izu H, Watanabe A, Asada Y, Sano M, Kondo A, Kato I, Iwahara S (1997) Cloning, sequencing, and expression of Arthrobacter protophormiae endo-β-N-acetylglucosaminidase in Escherichia coli. Arch Biochem Biophys 338:22–28

    Article  CAS  PubMed  Google Scholar 

  • Umekawa M, Huang W, Li B, Fujita K, Ashida H, Wang LX, Yamamoto K (2008) Mutants of Mucor hiemalis endo-β-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities. J Biol Chem 283:4469–4479

    Article  CAS  PubMed  Google Scholar 

  • Umekawa M, Higashiyama T, Koga Y, Tanaka T, Noguchi M, Kobayashi A, Shoda S, Huang W, Wang LX, Ashida H, Yamamoto K (2010a) Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline. Biochim Biophys Acta 1800:1203–1209

    Article  CAS  PubMed  Google Scholar 

  • Umekawa M, Li C, Higashiyama T, Huang W, Ashida H, Yamamoto K, Wang LX (2010b) Efficient glycosynthase mutant derived from Mucor hiemalis endo-β-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan. J Biol Chem 285:511–521

    Article  CAS  PubMed  Google Scholar 

  • Wang LX (2008) Chemoenzymatic synthesis of glycopeptides and glycoproteins through endoglycosidase-catalyzed transglycosylation. Carbohydr Res 343:1509–1522

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang LX (2011) The amazing transglycosylation activity of endo-β-N-acetylglucosaminidases. Trends Glycosci Glycotechnol 23:33–52

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang LX, Amin MN (2014) Chemical and chemoenzymatic synthesis of glycoproteins for deciphering functions. Chem Biol 21:51–66

    Article  PubMed  PubMed Central  Google Scholar 

  • Wang LX, Lomino JV (2012) Emerging technologies for making glycan-defined glycoproteins. ACS Chem Biol 7:110–122

    Article  CAS  PubMed  Google Scholar 

  • Yamaguchi Y, Nishimura M, Nagano M, Yagi H, Sasakawa H, Uchida K, Shitara K, Kato K (2006) Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy. Biochim Biophys Acta 1760:693–700

    Article  CAS  PubMed  Google Scholar 

  • Yamamoto K, Kadowaki S, Fujisaki M, Kumagai H, Tochikura T (1994) Novel specificities of Mucor hiemalis endo-β-N-acetylglucosaminidase acting complex asparagine-linked oligosaccharides. Biosci Biotechnol Biochem 58:72–77

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

We would like to thank Dr. Jun Hirabayashi (AIST Japan) and Masahito Dozaki (Fushimi Pharm. Co. Ltd) for numerous discussions. This study was supported in part by JSPS KAKENHI Grant Number 26292054 and Mizutani Foundation for Glycoscience Grant Number 160190 (K.T.).

Supporting information

Supplementary Fig. 1—Transglycosylation of deglycosylated RNase B under several reaction conditions.Supplementary Fig. 2—Images of original SDS-PAGE gels used in this study to be used in conjunction with Figs. 2, 3 and 4 and also Supplementary Fig. 1.

Author information

Authors and Affiliations

  1. Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Fukuoka, 812-8581, Japan

    Yujiro Higuchi, Yasunari Eshima, Yibo Huang & Kaoru Takegawa

  2. Fushimi Pharmaceutical Co. Ltd., Marugame, Kagawa, 763-8605, Japan

    Takashi Kinoshita & Wataru Sumiyoshi

  3. Department of Functional Glycomics, Life Science Research Center, Kagawa University, Miki-Cho, Kagawa, 761-0793, Japan

    Shin-ichi Nakakita

Authors
  1. Yujiro Higuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yasunari Eshima
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yibo Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Takashi Kinoshita
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Wataru Sumiyoshi
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Shin-ichi Nakakita
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Kaoru Takegawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Kaoru Takegawa.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 702 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Higuchi, Y., Eshima, Y., Huang, Y. et al. Highly efficient transglycosylation of sialo-complex-type oligosaccharide using Coprinopsis cinerea endoglycosidase and sugar oxazoline. Biotechnol Lett 39, 157–162 (2017). https://doi.org/10.1007/s10529-016-2230-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10529-016-2230-0

Keywords

Search

Navigation