Skip to main content
SpringerLink
Log in

N-glycan analysis of human α1-antitrypsin produced in Chinese hamster ovary cells

  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

Abstract

Human alpha-1-antitrypsin (α1AT) is a glycoprotein with protease inhibitor activity protecting tissues from degradation. Patients with inherited α1AT deficiency are treated with native α1AT (nAT) purified from human plasma. In the present study, recombinant α1AT (rAT) was produced in Chinese hamster ovary (CHO) cells and their glycosylation patterns, inhibitory activity and in vivo half-life were compared with those of nAT. A peptide mapping analysis employing a deglycosylation reaction confirmed full occupancy of all three glycosylation sites and the equivalency of rAT and nAT in terms of the protein level. N-glycan profiles revealed that rAT contained 10 glycan structures ranging from bi-antennary to tetra-antennary complex-type glycans while nAT displayed six peaks comprising majorly bi-antennary glycans and a small portion of tri-antennary glycans. In addition, most of the rAT glycans were shown to have only core α(1 - 6)-fucose without terminal fucosylation, whereas only minor portions of the nAT glycans contained core or Lewis X-type fucose. As expected, all sialylated glycans of rAT were found to have α(2 - 3)-linked sialic acids, which was in sharp contrast to those of nAT, which had mostly α(2 - 6)-linked sialic acids. However, the degree of sialylation of rAT was comparable to that of nAT, which was also supported by an isoelectric focusing gel analysis. Despite the differences in the glycosylation patterns, both α1ATs showed nearly equivalent inhibitory activity in enzyme assays and serum half-lives in a pharmacokinetic experiment. These results suggest that rAT produced in CHO cells would be a good alternative to nAT derived from human plasma.

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

Similar content being viewed by others

References

  1. Wurm, F.M.: Production of recombinant protein therapeutics in cultivated mammalian cells. Nat. Biotechnol. 22(11), 1393–1398 (2004)

    Article  PubMed  CAS  Google Scholar 

  2. Omasa, T., Onitsuka, M., Kim, W.D.: Cell engineering and cultivation of chinese hamster ovary (CHO) cells. Curr. Pharm. Biotechnol. 11(3), 233–240 (2010)

    Article  PubMed  CAS  Google Scholar 

  3. Chung, C.H., Mirakhur, B., Chan, E., Le, Q.T., Berlin, J., Morse, M., Murphy, B.A., Satinover, S.M., Hosen, J., Mauro, D., Slebos, R.J., Zhou, Q., Gold, D., Hatley, T., Hicklin, D.J., Platts-Mills, T.A.: Cetuximab-induced anaphylaxis and IgE specific for galactose-alpha-1,3-galactose. N. Engl. J. Med. 358(11), 1109–1117 (2008)

    Article  PubMed  CAS  Google Scholar 

  4. Bosques, C.J., Collins, B.E., Meador 3rd, J.W., Sarvaiya, H., Murphy, J.L., Dellorusso, G., Bulik, D.A., Hsu, I.H., Washburn, N., Sipsey, S.F., Myette, J.R., Raman, R., Shriver, Z., Sasisekharan, R., Venkataraman, G.: Chinese hamster ovary cells can produce galactose-alpha-1,3-galactose antigens on proteins. Nat. Biotechnol. 28(11), 1153–1156 (2010)

    Article  PubMed  CAS  Google Scholar 

  5. Kolarich, D., Turecek, P.L., Weber, A., Mitterer, A., Graninger, M., Matthiessen, P., Nicolaes, G.A., Altmann, F., Schwarz, H.P.: Biochemical, molecular characterization, and glycoproteomic analyses of alpha(1)-proteinase inhibitor products used for replacement therapy. Transfusion 46(11), 1959–1977 (2006)

    Article  PubMed  CAS  Google Scholar 

  6. Johansen, H., Sutiphong, J., Sathe, G., Jacobs, P., Cravador, A., Bollen, A., Rosenberg, M., Shatzman, A.: High-level production of fully active human alpha 1-antitrypsin in Escherichia coli. Mol. Biol. Med. 4(5), 291–305 (1987)

    PubMed  CAS  Google Scholar 

  7. Kwon, K.S., Song, M., Yu, M.H.: Purification and characterization of alpha 1-antitrypsin secreted by recombinant yeast Saccharomyces diastaticus. J. Biotechnol. 42(3), 191–195 (1995)

    Article  PubMed  CAS  Google Scholar 

  8. Chill, L., Trinh, L., Azadi, P., Ishihara, M., Sonon, R., Karnaukhova, E., Ophir, Y., Golding, B., Shiloach, J.: Production, purification, and characterization of human alpha1 proteinase inhibitor from Aspergillus niger. Biotechnol. Bioeng. 102(3), 828–844 (2009)

    Article  PubMed  CAS  Google Scholar 

  9. Blanchard, V., Liu, X., Eigel, S., Kaup, M., Rieck, S., Janciauskiene, S., Sandig, V., Marx, U., Walden, P., Tauber, R., Berger, M.: N-glycosylation and biological activity of recombinant human alpha1-antitrypsin expressed in a novel human neuronal cell line. Biotechnol. Bioeng. 108(9), 2118–2128 (2011)

    Article  PubMed  CAS  Google Scholar 

  10. Nakagawa, T., Uozumi, N., Nakano, M., Mizuno-Horikawa, Y., Okuyama, N., Taguchi, T., Gu, J., Kondo, A., Taniguchi, N., Miyoshi, E.: Fucosylation of N-glycans regulates the secretion of hepatic glycoproteins into bile ducts. J. Biol. Chem. 281(40), 29797–29806 (2006)

    Article  PubMed  CAS  Google Scholar 

  11. Hagglund, P., Matthiesen, R., Elortza, F., Hojrup, P., Roepstorff, P., Jensen, O.N., Bunkenborg, J.: An enzymatic deglycosylation scheme enabling identification of core fucosylated N-glycans and O-glycosylation site mapping of human plasma proteins. J. Proteome Res. 6(8), 3021–3031 (2007)

    Article  PubMed  Google Scholar 

  12. Packer, N.H., Lawson, M.A., Jardine, D.R., Redmond, J.W.: A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj. J. 15(8), 737–747 (1998)

    Article  PubMed  CAS  Google Scholar 

  13. Mun, J.Y., Lee, K.J., Kim, Y.J., Kwon, O., Kim, S.J., Lee, S.G., Park, W.S., Heo, W.D., Oh, D.B.: Development of fluorescent probes for the detection of fucosylated N-glycans using an Aspergillus oryzae lectin. Appl. Microbiol. Biotechnol. 93(1), 251–260 (2012)

    Article  PubMed  Google Scholar 

  14. Goetz, J.A., Novotny, M.V., Mechref, Y.: Enzymatic/chemical release of O-glycans allowing MS analysis at high sensitivity. Anal. Chem. 81(23), 9546–9552 (2009)

    Article  PubMed  CAS  Google Scholar 

  15. Bigge, J.C., Patel, T.P., Bruce, J.A., Goulding, P.N., Charles, S.M., Parekh, R.B.: Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Anal. Biochem. 230(2), 229–238 (1995)

    Article  PubMed  CAS  Google Scholar 

  16. Doucet, A., Bouchard, D., Janelle, M.F., Bellemare, A., Gagne, S., Tremblay, G.M., Bourbonnais, Y.: Characterization of human pre-elafin mutants: full antipeptidase activity is essential to preserve lung tissue integrity in experimental emphysema. Biochem. J. 405(3), 455–463 (2007)

    Article  PubMed  CAS  Google Scholar 

  17. Kolarich, D., Weber, A., Turecek, P.L., Schwarz, H.P., Altmann, F.: Comprehensive glyco-proteomic analysis of human alpha1-antitrypsin and its charge isoforms. Proteomics 6(11), 3369–3380 (2006)

    Article  PubMed  CAS  Google Scholar 

  18. Llop, E., Gutierrez-Gallego, R., Segura, J., Mallorqui, J., Pascual, J.A.: Structural analysis of the glycosylation of gene-activated erythropoietin (epoetin delta, Dynepo). Anal. Biochem. 383(2), 243–254 (2008)

    Article  PubMed  CAS  Google Scholar 

  19. Lee, E.U., Roth, J., Paulson, J.C.: Alteration of terminal glycosylation sequences on N-linked oligosaccharides of Chinese hamster ovary cells by expression of beta-galactoside alpha 2,6-sialyltransferase. J. Biol. Chem. 264(23), 13848–13855 (1989)

    PubMed  CAS  Google Scholar 

  20. Matsumura, K., Higashida, K., Ishida, H., Hata, Y., Yamamoto, K., Shigeta, M., Mizuno-Horikawa, Y., Wang, X., Miyoshi, E., Gu, J., Taniguchi, N.: Carbohydrate binding specificity of a fucose-specific lectin from Aspergillus oryzae: a novel probe for core fucose. J. Biol. Chem. 282(21), 15700–15708 (2007)

    Article  PubMed  CAS  Google Scholar 

  21. Misaizu, T., Matsuki, S., Strickland, T.W., Takeuchi, M., Kobata, A., Takasaki, S.: Role of antennary structure of N-linked sugar chains in renal handling of recombinant human erythropoietin. Blood 86(11), 4097–4104 (1995)

    PubMed  CAS  Google Scholar 

  22. Takeuchi, M., Inoue, N., Strickland, T.W., Kubota, M., Wada, M., Shimizu, R., Hoshi, S., Kozutsumi, H., Takasaki, S., Kobata, A.: Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells. Proc. Natl. Acad. Sci. U. S. A. 86(20), 7819–7822 (1989)

    Article  PubMed  CAS  Google Scholar 

  23. Yuen, C.T., Storring, P.L., Tiplady, R.J., Izquierdo, M., Wait, R., Gee, C.K., Gerson, P., Lloyd, P., Cremata, J.A.: Relationships between the N-glycan structures and biological activities of recombinant human erythropoietins produced using different culture conditions and purification procedures. Br. J. Haematol. 121(3), 511–526 (2003)

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We would like to thank Heung-Soo Cho and Seung-Bum Yoo for the help with the animal study. This study was supported by grants (to D.-B. Oh) from the Next-Generation BioGreen 21 Program (SSAC-PJ008001) of the Rural Development Administration and from the Korea Research Council of Fundamental Science and Technology (KRCF) and by a grant (to S. Park) from the Korea Small and Medium Business Administration (SA113037).

Author information

Authors and Affiliations

  1. Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahak-ro, Yuseong-gu, Daejeon, 305-806, Korea

    Kyung Jin Lee, Jin Young Gil, Ohsuk Kwon & Doo-Byoung Oh

  2. Alteogen Inc., Bioventure town, Daejeon, 305-812, Korea

    Sang Mee Lee, Soon Jae Park & Hye-Shin Chung

  3. Department of Biotechnology, Hannam University, Daejeon, 305-811, Korea

    Jin Young Gil & Hye-Shin Chung

  4. Korea Basic Science Institute, Ochang-eup, Cheongwon-gun, Chungbuk, 363-883, Korea

    Jin Young Kim

  5. Department of Biological Science, Wonkwang University, Iksan, 570-749, Korea

    Kyung Jin Lee

Authors
  1. Kyung Jin Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sang Mee Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jin Young Gil
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ohsuk Kwon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jin Young Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Soon Jae Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hye-Shin Chung
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Doo-Byoung Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Hye-Shin Chung or Doo-Byoung Oh.

Additional information

Kyung Jin Lee and Sang Mee Lee contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 513 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, K.J., Lee, S.M., Gil, J.Y. et al. N-glycan analysis of human α1-antitrypsin produced in Chinese hamster ovary cells. Glycoconj J 30, 537–547 (2013). https://doi.org/10.1007/s10719-012-9453-7

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10719-012-9453-7

Keywords

Search

Navigation