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. 1993 Sep;2(9):1441–1451. doi: 10.1002/pro.5560020908

Recombinant human erythropoietin (rHuEPO): cross-linking with disuccinimidyl esters and identification of the interfacing domains in EPO.

M Haniu 1, L O Narhi 1, T Arakawa 1, S Elliott 1, M F Rohde 1
PMCID: PMC2142457  PMID: 8401229

Abstract

Several amino groups of recombinant human erythropoietin are selectively cross-linked by specific cross-linkers including disuccinimidyl suberate or dithiobis(succinimidyl propionate). Intramolecular cross-linkings are obtained without significant change of the protein conformation using appropriate concentrations (0.2 mM) of the cross-linkers, which possess an 11-12-A length of a spacer between two reacting groups. Intramolecularly cross-linked peptides obtained suggest that several amino groups in erythropoietin (EPO) are positioned at a distance of near 12 A in the solution state. These interfacing amino groups include Lys 20-Lys 154, Lys 45-Lys 140, Lys 52-Lys 154, Lys 52-Lys 140, and Ala 1-Lys 116. A comparison of the cross-linking results between nonglycosylated EPO and glycosylated EPO suggests that both proteins retain high similarity regarding protein conformation. These results fit a structural model similar to that of human growth hormone, in which four alpha-helical bundles and a long stretch of beta-sheet structure are involved in the active protein.

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Selected References

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  1. Abdel-Meguid S. S., Shieh H. S., Smith W. W., Dayringer H. E., Violand B. N., Bentle L. A. Three-dimensional structure of a genetically engineered variant of porcine growth hormone. Proc Natl Acad Sci U S A. 1987 Sep;84(18):6434–6437. doi: 10.1073/pnas.84.18.6434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bazan J. F. Genetic and structural homology of stem cell factor and macrophage colony-stimulating factor. Cell. 1991 Apr 5;65(1):9–10. doi: 10.1016/0092-8674(91)90401-j. [DOI] [PubMed] [Google Scholar]
  3. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  4. Eridani S. Erythropoietin. Chapter for the monograph on haemopoietic growth factors. Biotherapy. 1990;2(4):291–298. doi: 10.1007/BF02170078. [DOI] [PubMed] [Google Scholar]
  5. Espada J., Gutnisky A. Purificación de eritropoyetina urinaria humana. Acta Physiol Lat Am. 1970;20(2):122–129. [PubMed] [Google Scholar]
  6. Goldwasser E., Kung C. K., Eliason J. On the mechanism of erythropoietin-induced differentiation. 13. The role of sialic acid in erythropoietin action. J Biol Chem. 1974 Jul 10;249(13):4202–4206. [PubMed] [Google Scholar]
  7. Jacobs K., Shoemaker C., Rudersdorf R., Neill S. D., Kaufman R. J., Mufson A., Seehra J., Jones S. S., Hewick R., Fritsch E. F. Isolation and characterization of genomic and cDNA clones of human erythropoietin. 1985 Feb 28-Mar 6Nature. 313(6005):806–810. doi: 10.1038/313806a0. [DOI] [PubMed] [Google Scholar]
  8. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  9. Lai P. H., Everett R., Wang F. F., Arakawa T., Goldwasser E. Structural characterization of human erythropoietin. J Biol Chem. 1986 Mar 5;261(7):3116–3121. [PubMed] [Google Scholar]
  10. Lin F. K., Suggs S., Lin C. H., Browne J. K., Smalling R., Egrie J. C., Chen K. K., Fox G. M., Martin F., Stabinsky Z. Cloning and expression of the human erythropoietin gene. Proc Natl Acad Sci U S A. 1985 Nov;82(22):7580–7584. doi: 10.1073/pnas.82.22.7580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Mason-Garcia M., Beckman B. S. Signal transduction in erythropoiesis. FASEB J. 1991 Nov;5(14):2958–2964. doi: 10.1096/fasebj.5.14.1752362. [DOI] [PubMed] [Google Scholar]
  12. Miyake T., Kung C. K., Goldwasser E. Purification of human erythropoietin. J Biol Chem. 1977 Aug 10;252(15):5558–5564. [PubMed] [Google Scholar]
  13. Sasaki H., Ochi N., Dell A., Fukuda M. Site-specific glycosylation of human recombinant erythropoietin: analysis of glycopeptides or peptides at each glycosylation site by fast atom bombardment mass spectrometry. Biochemistry. 1988 Nov 15;27(23):8618–8626. doi: 10.1021/bi00423a017. [DOI] [PubMed] [Google Scholar]
  14. Satake R., Kozutsumi H., Takeuchi M., Asano K. Chemical modification of erythropoietin: an increase in in vitro activity by guanidination. Biochim Biophys Acta. 1990 Mar 29;1038(1):125–129. doi: 10.1016/0167-4838(90)90020-g. [DOI] [PubMed] [Google Scholar]
  15. Schiffer M., Edmundson A. B. Use of helical wheels to represent the structures of proteins and to identify segments with helical potential. Biophys J. 1967 Mar;7(2):121–135. doi: 10.1016/S0006-3495(67)86579-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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. 1989 Oct;86(20):7819–7822. doi: 10.1073/pnas.86.20.7819. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Wojchowski D. M., Caslake L. Biotinylated recombinant human erythropoietins: bioactivity and utility as receptor ligand. Blood. 1989 Aug 15;74(3):952–958. [PubMed] [Google Scholar]
  18. de Vos A. M., Ultsch M., Kossiakoff A. A. Human growth hormone and extracellular domain of its receptor: crystal structure of the complex. Science. 1992 Jan 17;255(5042):306–312. doi: 10.1126/science.1549776. [DOI] [PubMed] [Google Scholar]

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