Biotechnology and Bioprocess Engineering:BBE

The Korean Society for Biotechnology and Bioengineering (한국생물공학회)
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Analysis of Erythropoietin Glycoform Produced by Recombinant CHO Cells Using the Lectin-Blotting Technique

  • Published : 1998.06.01
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Abstract

The glycosylation pattern of Erythropoietin (EPO), produced by recombinant CHO cells, was studied using the simple and rapid technique of 'Lectin-blotting'. In this experiment we used three different kinds of lectins, MAA(Maackia amurensis agglutinine), RCA(Ricinus communis agglutinine), and DSA(Datura stramonium agglutinine), which bind to the terminal sialic acid, galactose, and the N-acetyllactosamine chain respectively. The lectin-blotting technique was used to analyze the carbohydrate structure of EPO produced in the presence of two physiologically active chemical compounds, ammonium and chloroquine. The effect of the ammonium ion on the glycosylation of EPO was studied because it accumulated in the medium mainly as a by-product of glutamine matabolism. Ammonium chloride significantly inhibited the sialylation of the terminal galactose residue at concentrations of 8mM or more. Chloroquine, a potent inhibitor of glycosylation, inhibited terminal sialylation at concentrations of 100 and 200 $\mu$M, and at a concentration of 300 $\mu$M, also inhibited Nacetyllactosamine chain synthesis.

Keywords

References

  1. Bio/Technol v.8 Environmental effects on protein glycosylation Goochee, C. F.;T. Monica
  2. Bio/Technol v.9 The oligo-saccharides of glycoproteins: bioprocess factors affecting oligosaccharide structure and their effect on glycoprotein properties Goochee, C. F.;M. J. Gramer;D.C. Andersen;J. B. Bahr;J. R. Rasmussen
  3. Biotechnol. Bioeng v.43 Ammoium affects the glycosylation patterns of recombinant placental lactogen-Ⅰ by Chinese hamster ovary cells in a pH-dependent manner Borys, M.;D. I. H. Linzer;E. T. Papoutsakis
  4. Nature v.313 Isolation and characterization of genomic and cDNA clones of human erythropoietin Jacobs, K.;C. Shoemaker;R. Rundersdorf;S. Neill;R. Hewick;E. F. Fritsch;M. Kawkita;T. Shimizu;T. Miyake
  5. Proc. Natl. Acad. Sci. USA v.82 Cloning and expression of the human erythropoietin gene Lin, F. K.;S. Suggs;C. H. Lin;J. K. Browne;R. Smalling;J. C. Egrie;K. K. Chen;G. M. Fox;F. Martin;Z. Stabinsky;S. M. Badrawi;P. Lai;E. Goldwasser
  6. J. Biol. Chem v.261 Structural characterization of human erythroprotein Lai, P.;R. Everett;F. F. Wang;T. Arakawa;E. Goldwasser
  7. J. Biol. Chem v.266 Effects of site-directed removal of N-glycosylation sites in human erythropoietin on its production and biological properties Yamaguchi, K.;K. Akai;G. Kawanish;M. Ueda;S. Masuda;R. Sasaki
  8. Biochem. Biophys. Acta v.910 Active human erythropoietin expressed in insect cells using a baculovirus vector: a role for N-linked oligosaccharide Wojchowski, D. M.;S. H. Orkin;A. J. Sytkowski
  9. Proc. Natl. Acad. Sci. USA v.86 Relationship between sugar chain structure and biological activity of recombinant human erythropoietin produced in Chinese hamster ovary cells Takeuchi, M.;N. Inoue;T. W. Strickland;M. Kubota;M. Wada;R. Shimizu;S. Hoshi;H. Kozutsumi;S. Takasaki;A. Kobata
  10. Eur. J. Biochem v.188 The role of carbohydrate in recombinant human erythropoietin Tsuda, E.;G. Kawanishi, M. Ueda;S. Masuda;R. Sasaki
  11. Nature v.227 Cleavage of structural proteins during the assembly of the head of bacteriophage T4 Laemmeli, U. K.
  12. J. Biol. Chem v.267 Role of sugar chains in the expression of the biological activity of human erythropoietin Higuchi, M.;M. Oh-eda;H. Kuboniwa;K. Tomonoh;Y. Shimonaka;N. Ochi
  13. Anal. Biochem v.191 Structural characterization of glycoprotein carbohydrate chain by using Digoxigenin-labeled lectins on blots Haselbeck, A.;E. Schickaneder;H. Eltz;W. Hosel
  14. Glycoprotein analysis in biomedicine Immunological detection of glycoproteins on blots base on labeling with Digoxigenin Haselbeck, A.;W. Hosel;Hounsell, E. F.(eds.)