International Journal of Hematology

, Volume 91, Issue 2, pp 238–244 | Cite as

Glycosylation-modified erythropoietin with improved half-life and biological activity

Original Article

Abstract

Erythropoietin (EPO) controls the production of red blood cells, so it is important to maintain high levels of EPO activity and half-life. Here, we modified glycosylation sites in human erythropoietin (HuEPO) gene, resulting in proteins with addition of 1–4 glycosylation sites. The modified gene was introduced into CHO cells. The expressed EPO analogs were analyzed by SDS-PAGE. Half-life of the analogs was determined by sialic acid content test. In vivo potencies of analogs were evaluated by reticulocyte count and haematocrit level. The metabolic clearance of recombinant human erythropoietin (rHuEPO) and its analogs were determined by EPO immunoradiometrics assay. We have shown that the carbohydrate content in modified EPO molecules is increased. The modified EPO, [Val3Asn4Thr6Asn30Thr32Val87Asn88Thr90]EPO, increases 3.3 times in elimination half-life, 2.1 times in activity and prolongs 2 days functional time in vivo in comparison to rHuEPO. These findings suggest that the addition of glycosylation sites in EPO enhances half-life and biological activity of EPO, duration of action of EPO anlogues positively correlated with the number of glycosylated sites, while addition of 4 glycosylation sites does not further enhance the erythropoietic potency.

Keywords

Human EPO Glycosylation Sialic acid Erythropoiesis Reticulocytes 

References

  1. 1.
    Nissenson AR. Erythropoietin overview. Blood Purif. 1994;12(1):6–13.CrossRefPubMedGoogle Scholar
  2. 2.
    Krantz SB. Erythropoietin. Blood. 1991;77(3):419–34.PubMedGoogle Scholar
  3. 3.
    Lacombe C, Mayeux P. Biology of erythropoietin. Haematologica. 1998;83:724–32.PubMedGoogle Scholar
  4. 4.
    Louise CW, Gregg T, Patricia M, John S, Andrew JD, Jaime C, et al. The importance of N- and O-linked oligosaccharides for the biosynthesis and in vitro and in vivo biologic activities of erythropoietin[J]. Blood. 1991;77(12):2624–32.Google Scholar
  5. 5.
    Egrie JC, Strickland TW, Lane J, Aoki K, Cohen AM, Smalling R, et al. Characterization and biological effects of recombinant human erythropoietin. Immunobiology. 1986;172:213–24.PubMedGoogle Scholar
  6. 6.
    Browne JK, Cohen AM, Egrie JC, Lai PH, Lin FK, Strickland T, et al. Erythropoietin: gene cloning, protein structure, and biological properties. Cold Spring Harb Symp Quant Biol. 1986;51:693–702.PubMedGoogle Scholar
  7. 7.
    Recny MA, Scoble HA, Kim Y. Structural characterization of natural human urinary and recombinant DNA-derived erythropoietin, Identification of des-arginine 166 erythropoietin. J Biol Chem. 1987;262:17156–63.PubMedGoogle Scholar
  8. 8.
    Eschbach JW, Egrie JC, Downing MR, Browne JK, Adamson JW. Correction of the anemia of end-stage renal disease with recombinant human erythropoietin[J]. N Engl J Med. 1987;316(2):73–8.PubMedCrossRefGoogle Scholar
  9. 9.
    Abels RI. Use of recombinant human erythropoietin in the treatment of anemia in patients who have cancer[J]. Semin Oncol. 1992;19(3 Suppl 8):29–35.PubMedGoogle Scholar
  10. 10.
    Elliott S, Lorenzini T, Asher S, Aoki K, Brankow D, Buck L, et al. Enhancement of therapeutic protein in vivo activities through glycoengineering. Nat Biotechnol. 2003;21:414–21.CrossRefPubMedGoogle Scholar
  11. 11.
    Nimtz M, Martin W, Wray V, Klöppel KD, Augustin J, Conradt HS. Structures of sialylated oligosaccharides of human erythropoietin expressed in recombinant BHK-21 cells. Eur J Biochem. 1998;213:39–56.CrossRefGoogle Scholar
  12. 12.
    Imai N, Higuchi M, Kawamura A, Tomonoh K, Oh-Eda M, Fujiwara M, et al. Physicochemical and biological characterization of asialoerythropoietin. Eur J Biochem. 1990;194:457–61.CrossRefPubMedGoogle Scholar
  13. 13.
    Elliott S, Chang D, Delorme E, Eris T, Lorenzini T. Structural requirements for additional N-linked carbohydrate on recombinant human erythropoietin. J Biol Chem. 2004;279:16854–62.CrossRefPubMedGoogle Scholar
  14. 14.
    Zhou Y, Wang Q-Z, Wang J-Z, Huang P-T. The research and development of long-lasting erythropoietin and the current status of its quality control [J]. Chin J Biochem Pharm. 2008;29(1):69–71.Google Scholar
  15. 15.
    Elliott S, Lorenzini T, Chang D, Barzilay J, Delorme E, Giffin J, et al. Fine-structure epitope mapping of antierythropoietin monoclonal antibodies reveals a model of recombinant human erythropoietin structure. Blood. 1996;87:2702–13.PubMedGoogle Scholar
  16. 16.
    Egrie JC, Grant JR, Gillies DK, Aoki KH, Strickland TW. The role of carbohydrate on the biological activity of erythropoietin [abstract]. Glycoconj J. 1993;10:263. (abstract S7.7).CrossRefGoogle Scholar
  17. 17.
    Marshall RD. The nature and metabolism of the carbohydrate-peptide linkages of glycoproteins. Biochem Soc Symp. 1974;40:17–26.PubMedGoogle Scholar
  18. 18.
    Egrie JC, Browne JK. Development and characterization of novel erythropoiesis stimulating protein (NESP). Nephrol Dial Transplant. 2001;16(Suppl 3):3–13.PubMedGoogle Scholar

Copyright information

© The Japanese Society of Hematology 2010

Authors and Affiliations

  1. 1.School of Life Science and BiopharmaceuticsShenyang Pharmaceutical UniversityShenyangChina
  2. 2.R&D DepartmentShenyang Sunshine Pharmaceutical Co., LtdShenyangChina
  3. 3.School of Life Science and BiopharmaceuticsShenyang Pharmaceutical UniversityShenyangChina

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