Glycosylation-modified erythropoietin with improved half-life and biological activity
Rent the article at a discountRent now
* Final gross prices may vary according to local VAT.Get Access
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.
- Nissenson AR. Erythropoietin overview. Blood Purif. 1994;12(1):6–13. CrossRef
- Krantz SB. Erythropoietin. Blood. 1991;77(3):419–34.
- Lacombe C, Mayeux P. Biology of erythropoietin. Haematologica. 1998;83:724–32.
- 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.
- 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.
- 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.
- 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.
- 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. CrossRef
- 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.
- 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. CrossRef
- 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. CrossRef
- 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. CrossRef
- 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. CrossRef
- 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.
- 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.
- 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). CrossRef
- Marshall RD. The nature and metabolism of the carbohydrate-peptide linkages of glycoproteins. Biochem Soc Symp. 1974;40:17–26.
- Egrie JC, Browne JK. Development and characterization of novel erythropoiesis stimulating protein (NESP). Nephrol Dial Transplant. 2001;16(Suppl 3):3–13.
- Glycosylation-modified erythropoietin with improved half-life and biological activity
International Journal of Hematology
Volume 91, Issue 2 , pp 238-244
- Cover Date
- Print ISSN
- Online ISSN
- Springer Japan
- Additional Links
- Human EPO
- Sialic acid
- Industry Sectors
- Author Affiliations
- 1. School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No. 3 A1 Road 10, Shenyang Economy and Technology Development Zone, Shenyang, 110027, China
- 2. R&D Department, Shenyang Sunshine Pharmaceutical Co., Ltd, No. 3 A1 Road 10, Shenyang Economy and Technology Development Zone, Shenyang, 110027, China
- 3. School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, No. 103 Wenhua Road, Shenyang, 110016, China