Expression and Characterization of Human N-Acetylglucosaminyltransferases and ${\alpha}$2,3-Sialyltransferase in Insect Cells for In Vitro Glycosylation of Recombinant Erythropoietin

  • Published : 2008.02.29

Abstract

The glycans linked to the insect cell-derived glycoproteins are known to differ from those expressed in mammalian cells, partly because of the low level or lack of glycosyltransferase activities. GnT II, GnT IV, GnT V, and ST3Gal IV, which play important roles in the synthesis of tetraantennarytype complex glycan structures in mammalian cells, were overexpressed in Trichoplusia ni cells by using a baculovirus expression vector. The glycosyltransferases, expressed as a fusion form with the IgG-binding domain, were secreted into the culture media and purified using IgG sepharose resin. The enzyme assay, performed using a pyridylaminated-sugar chain as an acceptor, indicated that the purified glycosyltransferases retained their enzyme activities. Human erythropoietin expressed in T. ni cells (rhEPO) was subjected to in vitro glycosylation by using recombinant glycosyltransferases and was converted into complex-type glycan with terminal sialic acid. The presence of Nacetylglucosamine, galactose, and sialic acid on the rhEPO moiety was detected by a lectin blot analysis, and the addition of galactose and sialic acid to rhEPO was confirmed by autoradiography using $UDP-^{14}C-Gal\;and\;CMP-^{14}C-Sia$ as donors. The in vitro glycosylated rhEPO was injected into mice, and the number of reticulocytes among the ed blood cells was counted using FACS. A significant increase in the number of reticulocytes was not observed in the mice injected with in vitro glycosylated rhEPO as compared with those injected with rhEPO.

References

  1. Altmann, F., G. Kornfeld, T. Dalik, E. Staudacher, and J. Glossl. 1993. Processing of asparagines-linked oligosaccharides in insect cells. N-Acetylglucosaminyltransferase I and II activities in cultured lepidopteran cells. Glycobiology 3: 619-625 https://doi.org/10.1093/glycob/3.6.619
  2. Altmann, F., E. Staudacher, I. B. H. Wilson, and L. Marz. 1999. Insect cells as host for the expression of recombinant glycoproteins. Glycoconj. J. 16: 109-123 https://doi.org/10.1023/A:1026488408951
  3. Butters, T. D., R. C. Hughes, and P. Vischer. 1981. Steps in the biosynthesis of mosquito cell membrane glycoproteins and the effects of tunicamycin. Biochim. Biophys. Acta 640: 672-686 https://doi.org/10.1016/0005-2736(81)90097-3
  4. Chang, K. H., N. I. Back, J. M. Yang, J. M. Lee, J. H. Bo, and I. S. Chung. 2005. Expression and characterization of recombinant $\beta-secretase$ from Trichoplusia ni BTI Tn5B1-4 cells transformed with cDNAs encoding human $\beta 1,4- galactosyltransferase$ and $Gal\beta1,4-GlcNAc\alpha2,6-sialyltransferase$. Protein Expr. Purif. 44: 87-93 https://doi.org/10.1016/j.pep.2005.08.010
  5. Demir, I. and Z. Demirbag. 2006. A productive replication of Hyphantria cunea nucleopolyhedrovirus in Lymantria dispar cell line. J. Microbiol. Biotechnol. 16: 1485-1490
  6. Frederick, W. Q., L. F. Caslake, R. E. Burkert, and D. M. Wojchowski. 1989. High-level expression and purification of a recombinant human erythropoietin produced using a baculovirus vector. Blood 74: 652-657
  7. Goldwasser, E. and C. K. Kung. 1968. Progress in the purification of erythropoietin. Ann. N. Y. Acad. Sci. 149: 49-53 https://doi.org/10.1111/j.1749-6632.1968.tb15135.x
  8. Gunasekaran, K., B. Ma, B. Ramakrishnan, P. K. Oasba, and R. Nussinov. 2003. Interdependence of backbone flexibility, residue conservation, and enzyme function: A case study on $\beta1,4-galactosyltransferase-I$. Biochemistry 42: 3674-3687 https://doi.org/10.1021/bi034046r
  9. Hollister, J. R., J. H. Shaper, and D. L. Jarvis. 1998. Stable expression of mammalian $\beta1,4-galactosyltransferase$ extends the N-glycosylation pathway in insect cells. Glycobiology 8: 473-480 https://doi.org/10.1093/glycob/8.5.473
  10. Jarvis, D. L. and E. E. Finn. 1995. Biochemical analysis of the N-glycosylation pathway in baculovirus-infected lepidopteran insect cells. Virology 212: 500-511 https://doi.org/10.1006/viro.1995.1508
  11. Jarvis, D. L. and E. E. Finn. 1996. Modifying the insect cell Nglycosylation pathway with immediate early baculovirus expression vectors. Nature Biotechnol. 14: 1288-1292 https://doi.org/10.1038/nbt1096-1288
  12. Jarvis, D. L., Z. S. Kawar, and J. R. Hollister. 1998. Engineering N-glycosylation pathway in the baculovirus-insect cell system. Curr. Opin. Biotechnol. 9: 528-533 https://doi.org/10.1016/S0958-1669(98)80041-4
  13. Jarvis, D. L., D. Home, and J. J. Aumiller. 2001. Novel baculovirus expression vectors that provide sialylation of recombinant glycoproteins in lepidopteran insect cells. J. Virol. 75: 6223-6227 https://doi.org/10.1128/JVI.75.13.6223-6227.2001
  14. Jarvis, D. L. 2003. Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production. Virology 310: 1-7 https://doi.org/10.1016/S0042-6822(03)00120-X
  15. Jason, R. H. and D. L. Jarvis. 2001. Engineering lepidopteran insect cells for sialoglycoprotein production by genetic transformation with mammalian $\beta1,4-galactosyltransferase$ and $\alpha2,6-sialyltransferase$. Glycobiology 11: 1-9 https://doi.org/10.1093/glycob/11.1.1
  16. Kang, C. S., S. Y. Son, and I. S. Bang. 2006. High-level expression T4 endonuclease V in insect cells as biologically active form. J. Microbiol. Biotechnol. 16: 1583-1590
  17. Kim, H. G., S. M. Yang, Y. C. Lee, S. I. Do, I. S. Chung, and J. M. Yang. 2003. High-level expression of human glycosyltransferases in insect cells as biochemically active form. Biochem. Biophys. Res. Commun. 305: 488-493 https://doi.org/10.1016/S0006-291X(03)00795-2
  18. Kim, J. S., J. Y. Choi, J. Y. Roh, H. Y. Lee, S. S. Jang, and Y. H. Je. 2007. Production of recombinant polyhedral containing Cry1Ac fusion protein in insect cell lines. J. Microbiol. Biotechnol. 17: 739-744
  19. Klingmuller, U., H. Wu, J. G. Hsiao, A. Toker, B. C. Duckworth, L. C. Cantley, and H. F. Lodish. 1997. Identification of a novel pathway important for proliferation and differentiation of primary erythroid progenitors. Pro. Natl. Acad. Sci. USA 94: 3016-3021
  20. Kornfeld, R. and S. Kornfeld. 1985. Assembly of asparaginelinked oligosaccharides. Annu. Rev. Biochem. 54: 631-664 https://doi.org/10.1146/annurev.bi.54.070185.003215
  21. Krantz, S. B. 1991. Erythropoietin. Blood 77: 419-434
  22. Lee, K.Y., H. G. Kim, M. R. Hwang, J. I. Chae, J. M. Yang, Y. C. Lee, Y. K. Choo, Y. I. Lee, S. S. Lee, and S. I. Do. 2002. The hexapeptide inhibitor of $Gal\beta1,3GalNAc- specific$$\alpha2,3-sialyltransferase$ as a generic inhibitor of sialyltransferases. J. Biol. Chem. 277: 49341-49351 https://doi.org/10.1074/jbc.M209618200
  23. Li, M. S., J. Y. Choi, J. Y. Roh, H. J. Shim, J. N. Kang, Y. S. Kim, Y. Wang, Z. N. Yu, B. R. Jin, and Y. H. Je. 2007. Identification and molecular characterization of novel cry1-type toxin genes from Bacillus thuringiensis K1 isolated in Korea. J. Microbiol. Biotechnol. 17: 15-20
  24. Martin, L., H. Wang, H. Zhihong, and J. A. Jehle. 2004. Towards a molecular identification and classification system of lepidopteran-specific baculovirus. Virology 325: 36-47 https://doi.org/10.1016/j.virol.2004.04.023
  25. Miller, L. K. 1988. Baculoviruses for foreign gene expression in insect cells. Biotechnology 10: 457-465
  26. Noboru, T., D. Howe, J. J. Aumiller, M. Pathak, J. Park, K. B. Palter, D. L. Jarvis, M. J. Betenbaugh, and Y. C. Lee. 2003. Complex-type biantennary N-glycans of recombinant human transferrin from Trichoplusia ni insect cells expressing mammalian $\beta1,4-galactosyltransferase$ and $\beta1,2-Nacetylglucosaminyltransferase$ II. Glycobiology 13: 23-34 https://doi.org/10.1093/glycob/cwg012
  27. Nomura, T., M. Takizawa, J. Aoki, H. Arai, K. Inoue, E. Wakisaka, N. Yoshizuka, G. Imokawa, N. Dohmae, K. Takio, M. Hattori, and N. Matsuo. 1998. Purification, cDNA cloning, and expression of $UDP-Gal:Glucosylceramide\beta -1,4-galactosyltransferase$ from rat brain. J. Biol. Chem. 273: 13570-13577 https://doi.org/10.1074/jbc.273.22.13570
  28. Park, E. I., M. Stephen, M. Manzella, and J. U. Baenziger. 2003. Rapid clearance of sialylated glycoproteins by the asialoglycoprotein receptor. J. Biol. Chem. 278: 4597-4602 https://doi.org/10.1074/jbc.M210612200
  29. Paulson, J. C., and K. J. Colley. 1989. Glycosyltransferases: Structure, localization, and control of cell type-specific glycosylation. J. Biol. Chem. 264: 17615-17618
  30. Sasaki, H., B. Bothner, A. Dell, and M. Fukuda. 1987. Carbohydrate structure of erythropoietin expressed in Chinese hamster ovary cells by a human erythropoietin cDNA. J. Biol. Chem. 262: 12059-12076
  31. Steve, E., T. Lorenzini, D. Chang, J. Barzilay, and E. Delorme. 1997. Mapping of the active site of recombinant human erythropoietin. Blood 89: 493-502
  32. Stollar, V., B. D. Stollar, R. Koo, K. A. Harrap, and R. W. Schlesinger. 1976. Sialic acid contents of Sindbis virus from vertebrate and mosquito cells. Equivalence of biological and immunological viral properties. Virology 69: 104-115 https://doi.org/10.1016/0042-6822(76)90198-7
  33. Strasser, R., H. Steinkellner, M. Boren, F. Altman, L. Mach, J. Glossl, and J. Mucha. 1999. Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana. Glycoconj. J. 16: 787-791 https://doi.org/10.1023/A:1007127815012
  34. Varki, A. 1993. Biological roles of oligosaccharides: All of the theories are correct. Glycobiology 3: 97-130 https://doi.org/10.1093/glycob/3.2.97