Skip to main content
SpringerLink
Log in

Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

The peptide-N 4-(N-acetyl-β-d-glucosaminyl) asparagine amidase F (PNGase F) catalyzes the cleavage of N-linked oligosaccharides between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides from glycoproteins. The PNGase F has broad substrate specificity and thus is extensively used for the structural and functional studies of the glycoproteins. In this study, we tried to produce active recombinant PNGase F as secreted and intracellular-expressed forms using baculovirus expression vector system (BEVS) through silkworm larvae or cultured cells. PNGase F itself contains potential N-linked glycosylation sites and we found that it was N-glycosylated when PNGase F secreted from silkworm cells. Intriguingly, the secreted recombinant PNGase F has the lower catalytic activity and self-digests its N-linked glycans and therefore this secreted form of this enzyme produced from BEVS is not appropriate for carbohydrate chain analysis. Instead, we successfully mass-produced (2.1 mg/20 silkworm larvae) and purified active recombinant PNGase F as an intracellular protein without N-glycosylations. Besides, we confirmed by directed mutagenesis that several amino acid residues are crucial for the function of PNGase F. Our results provide an alternative method for the mass production of active enzymes involved in the study of glycoproteins.

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
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Imperiali, B., & O’Connor, S. E. (1999). Effect of N-linked glycosylation on glycopeptides and glycoprotein structure. Current Opinion in Chemical Biology, 3, 643–649.

    Article  CAS  Google Scholar 

  2. Apweiler, R., Hermjakob, H., & Sharon, N. (1999). On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochimica et Biophysica Acta, 1473, 4–8.

    Article  CAS  Google Scholar 

  3. Plummer, T. H, Jr, Elder, J. H., Alexander, S., Phelan, A. W., & Tarentino, A. L. (1984). Demonstration of peptide: N-glycosidase F activity in endo-beta-N-acetylglucosaminidase F preparations. The Journal of Biological Chemistry, 259, 10700–10704.

    CAS  Google Scholar 

  4. Elder, J. H., & Alexander, S. (1982). Endo-beta-N-acetylglucosaminidase F: Endoglycosidase from Flavobacterium meningosepticum that cleaves both high-mannose and complex glycoproteins. Proceedings of the National Academy of Sciences of the United States of America, 79, 4540–4544.

    Article  CAS  Google Scholar 

  5. Chu, F. K. (1986). Requirements of cleavage of high mannose oligosaccharides in glycoproteins by peptide N-glycosidase F. The Journal of Biological Chemistry, 261, 172–177.

    CAS  Google Scholar 

  6. Tretter, V., Altmann, F., & März, L. (1991). Peptide-N 4-(N-acetyl-beta-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached alpha 1-3 to the asparagine-linked N-acetylglucosamine residue. European Journal of Biochemistry, 199, 647–652.

    Article  CAS  Google Scholar 

  7. Mussar, K. J., Murray, G. J., Martin, B. M., & Viswanatha, T. (1989). Peptide: N-glycosidase F: studies on the glycoprotein aminoglycan amidase from Flavobacterium meningosepticum. Journal of Biochemical and Biophysical Methods, 20, 53–68.

    Article  CAS  Google Scholar 

  8. Norris, G. E., Stillman, T. J., Anderson, B. F., & Baker, E. N. (1994). The three-dimensional structure of PNGase F, a glycosylasparaginase from Flavobacterium meningosepticum. Structure, 2, 1049–1059.

    Article  CAS  Google Scholar 

  9. Barsomian, G. D., Johnson, T. L., Borowski, M., Denman, J., Ollington, J. F., Hirani, S., et al. (1990). Cloning and expression of peptide-N 4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F in Escherichia coli. The Journal of Biological Chemistry, 265, 6967–6972.

    CAS  Google Scholar 

  10. Loo, T., Patchett, M. L., Norris, G. E., & Lott, J. S. (2002). Using secretion to solve a solubility problem: high-yield expression in Escherichia coli and purification of the bacterial glycoamidase PNGase F. Protein Expression and Purification, 24, 90–98.

    Article  CAS  Google Scholar 

  11. Hua, L., Gao, X., Yang, X., Wan, D., He, C., Cao, J., et al. (2014). Highly efficient production of peptides: N-Glycosidase F for N-glycomics analysis. Protein Expression and Purification, 97, 17–22.

    Article  CAS  Google Scholar 

  12. O’Reilly, D. R., Miller, L., & Luckow, V. A. (1992). Baculovirus expression vectors: A laboratory manual (pp. 216–234). New York: Oxford University Press.

    Google Scholar 

  13. Kato, T., Kajikawa, M., Maenaka, K., & Park, E. Y. (2010). Silkworm expression system as a platform technology in life science. Applied Microbiology and Biotechnology, 85, 459–470.

    Article  CAS  Google Scholar 

  14. Mitsudome, T., Xu, J., Nagata, Y., Masuda, A., Iiyama, K., Morokuma, D., et al. (2014). Expression, purification, and characterization of endo-β-N-acetylglucosaminidase H using baculovirus-mediated silkworm protein expression system. Applied Biochemistry and Biotechnology, 172, 3978–3988.

    Article  CAS  Google Scholar 

  15. Ono, C., Nakatsukasa, T., Nishijima, Y., Asano, S., Sahara, K., & Bando, H. (2007). Construction of the BmNPV T3 bacmid system and its application to the functional analysis of BmNPV he65. Journal of Insect Biotechnology and Sericology, 76, 161–167.

    CAS  Google Scholar 

  16. Soejima, Y., Lee, J., Nagata, Y., Mon, H., Iiyama, K., Kitano, H., et al. (2013). Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system. Central European Journal of Biology, 8, 1–7.

    CAS  Google Scholar 

  17. Kuhn, P., Tarentino, A. L., Plummer, T. H, Jr, & Van Roey, P. (1994). Crystal structure of peptide-N 4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F at 2.2-A resolution. Biochemistry, 33, 11699–11706.

    Article  CAS  Google Scholar 

  18. Kuhn, P., Guan, C., Cui, T., Tarentino, A. L., Plummer, T. H., & Van Roey, P. (1995). Active site and oligosaccharide recognition residues of peptide-N 4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase F. The Journal of Biological Chemistry, 270, 29493–29497.

    Article  CAS  Google Scholar 

  19. Lemp, D., Haselbeck, A., & Klebl, F. (1990). Molecular cloning and heterologous expression of N-glycosidase F from Flavobacterium meningosepticum. The Journal of Biological Chemistry, 265, 15606–15610.

    CAS  Google Scholar 

  20. Tarentino, A. L., Quinones, G., Trumble, A., Changchien, L. M., Duceman, B., Maley, F., et al. (1990). Molecular cloning and amino acid sequence of peptide-N 4-(N-acetyl-beta-D-glucosaminyl)asparagine amidase from Flavobacterium meningosepticum. The Journal Biological Chemistry, 265, 6961–6966.

    CAS  Google Scholar 

  21. Lee, J., Kawakami, N., Mon, H., Mitsunobu, H., Iiyama, K., Ninaki, S., et al. (2012). Establishment of a Bombyx mori nucleopolyhedrovirus (BmNPV) hyper-sensitive cell line from the silkworm e21 strain. Biotechnology Letters, 34, 1773–1779.

    Article  Google Scholar 

  22. Motohashi, T., Shimojima, T., Fukagawa, T., Maenaka, K., & Park, E. Y. (2005). Efficient large-scale protein production of larvae and pupae of silkworm by Bombyx mori nuclear polyhedrosis virus bacmid system. Biochemical and Biophysical Research Communications, 326, 564–569.

    Article  CAS  Google Scholar 

  23. Mon, H., Lee, J., Fukushima, M., Nagata, Y., Fujii, M., Xu, J., et al. (2013). Production and characterization of the celery mismatch endonuclease CEL II using baculovirus/silkworm expression system. Applied Microbiology and Biotechnology, 97, 6813–6822.

    Article  CAS  Google Scholar 

  24. Kawakami, N., Lee, J., Mon, H., Kubo, Y., Banno, Y., Kawaguchi, Y., et al. (2008). Efficient protein expression in Bombyx mori larvae of the strain d17 highly sensitive to B. mori nucleopolyhedrovirus. Molecular Biotechnology, 40, 180–185.

    Article  CAS  Google Scholar 

  25. Green, E. D., Adelt, G., Baenziger, J. U., Wilson, S., & Van Halbeek, H. (1988). The asparagine-linked oligosaccharides on bovine fetuin. Structural analysis of N-glycanase-released oligosaccharides by 500-megahertz 1H NMR spectroscopy. The Journal of Biological Chemistry, 263, 18253–18268.

    CAS  Google Scholar 

  26. Corradi Da Silva, M. L., Stubbs, H. J., Tamura, T., & Rice, K. G. (1995). 1H NMR characterization of a hen ovalbumin tyrosinamide N-linked oligosaccharide library. Archives of Biochemistry and Biophysics, 318, 465–475.

    Article  CAS  Google Scholar 

  27. Harvey, D. J., Wing, D. R., Küster, B., & Wilson, I. B. (2000). Composition of N-linked carbohydrates from ovalbumin and co-purified glycoproteins. Journal of American Society for Mass Spectrometry, 11, 564–571.

    Article  CAS  Google Scholar 

  28. Fu, D., Chen, L., & O’Neill, R. A. (1994). A detailed structural characterization of ribonuclease B oligosaccharides by 1H NMR spectroscopy and mass spectrometry. Carbohydrate Research, 261, 173–186.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The NIAS-Bm-oyanagi2 cell line for propagation of recombinant BmNPVs was kindly provided by Dr. Imanihi (National Institute of Agrobiological Sciences, Japan). We also thank Dr. Chisa Aoki (Kyushu University Graduate School) for providing the Bme21 cell line for the expression of recombinant protein. The MALDI-TOF MS was kindly supported by Center for Advanced Instrumental and Educational Supports (Faculty of Agriculture, Kyushu University).

Conflict of interest

None.

Author information

Authors and Affiliations

  1. Laboratory of Silkworm Science, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Hakozaki 6-10-1, Higashi-ku, Fukuoka, 812-8581, Japan

    Atsushi Masuda, Jian Xu, Takumi Mitsudome, Yudai Nagata, Daisuke Morokuma, Hiroaki Mon, Takahiro Kusakabe & Jae Man Lee

  2. Laboratory of Silkworm Genetic Resources, Institute of Genetic Resources, Graduate School of Bio Resources and Bioenvironmental Science, Kyushu University, Higashi-ku, Fukuoka, 812-8581, Japan

    Yutaka Banno

Authors
  1. Atsushi Masuda
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jian Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takumi Mitsudome
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Yudai Nagata
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Daisuke Morokuma
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hiroaki Mon
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yutaka Banno
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Takahiro Kusakabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Jae Man Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jae Man Lee.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplemental Fig. 1

Diagram of substrate specificities of PNGase F. The reaction results in release of the oligosaccharide and the aspartic acid-containing polypeptide. PNGase F is not able to cleave N-linked glycans from glycoproteins when the innermost GlcNAc residue is linked to an α(1-3) fucose residue. Black arrow represents the cleavage site of PNGase F. Gray circles: mannose, Black squares: GlcNAc, Black triangle: fucose, X: any sugar. Supplementary material 1 (JPEG 420 kb)

Supplemental Fig. 2

Comparison of the enzymatic activity of 30K-PNGase F before and after the self-digestion. 30K-PNGase F (self-digested) was generated by incubating 30K-PNGase F under the reaction condition at 37 °C for 1 h. 200 ng of 30K-PNGase F (with N-glycan) (lane 2, 3), 30K-PNGase F (self-digested) (lane 4, 5) and NoSP-PNGase F (lane 6, 7) which were incubated with denatured 15 μg fetuin (lane 3, 5, 7) at 37 °C for 1 h and that before the incubation (lane 2, 4, 6) were analyzed by CBB-stained 10 % SDS-PAGE. The arrows indicate the bands of rPNGase F. Mock (lane 1): fetuin without enzyme treatment. M: molecular mass markers. Supplementary material 2 (JPEG 525 kb)

Supplemental Fig. 3

Purification of rPNGase F by nickel affinity chromatography. The NoSP-PNGase F proteins purified from (a) the Bme21 cells, and (b) the silkworm fat bodies by nickel affinity chromatography. M molecular mass markers, IP input, FT flow-through, WS wash fraction, lanes 1–9 elution fractions by 500 mM imidazole. All samples were analyzed by Coomassie-stained 10 % SDS-PAGE, and the arrows indicate the bands of rPNGase F. Supplementary material 3 (JPEG 652 kb)

Supplemental Fig. 4

Structure of N-linked oligosaccharides of RNase B. The gray circles and black squares represent mannose and GlcNAc, respectively. Black arrow represents the cleavage site of PNGase F. Supplementary material 4 (JPEG 524 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Masuda, A., Xu, J., Mitsudome, T. et al. Mass Production of an Active Peptide-N-Glycosidase F Using Silkworm-Baculovirus Expression System. Mol Biotechnol 57, 735–745 (2015). https://doi.org/10.1007/s12033-015-9866-1

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-015-9866-1

Keywords

Search

Navigation