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. 2011 Feb 20;2(1):41–47. doi: 10.1007/s13238-011-1007-4

Generation of glyco-engineered BY2 cell lines with decreased expression of plant-specific glycoepitopes

Bo-jiao Yin 1,2, Ting Gao 1, Nuo-yan Zheng 1,2, Yin Li 2, San-yuan Tang 1, Li-ming Liang 2, Qi Xie 1,✉
PMCID: PMC4875288  PMID: 21337008

Abstract

Plants are known to be efficient hosts for the production of mammalian therapeutic proteins. However, plants produce complex N-glycans bearing β1,2-xylose and core α1,3-fucose residues, which are absent in mammals. The immunogenicity and allergenicity of plant-specific Nglycans is a key concern in mammalian therapy. In this study, we amplified the sequences of 2 plant-specific glycosyltransferases from Nicotiana tabacum L. cv Bright Yellow 2 (BY2), which is a well-established cell line widely used for the expression of therapeutic proteins. The expression of the endogenous xylosyltranferase (XylT) and fucosyltransferase (FucT) was downregulated by using RNA interference (RNAi) strategy. The xylosylated and core fucosylated N-glycans were significantly, but not completely, reduced in the glycoengineered lines. However, these RNAi-treated cell lines were stable and viable and did not exhibit any obvious phenotype. Therefore, this study may provide an effective and promising strategy to produce recombinant glycoproteins in BY2 cells with humanized N-glycoforms to avoid potential immunogenicity.

Keywords: BY2 cells, N-glycosylation, glycosyltransferase, RNA interference

Footnotes

These authors contributed equally to this work.

References

  1. Bakker H., Rouwendal G.J., Karnoup A.S., Florack D.E., Stoopen G.M., Helsper J.P., van Ree R., van Die I., Bosch D. An antibody produced in tobacco expressing a hybrid beta-1,4-galactosyltransferase is essentially devoid of plant carbohydrate epitopes. Proc Natl Acad Sci U S A. 2006;103:7577–7582. doi: 10.1073/pnas.0600879103. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bardor M., Faveeuw C., Fitchette A.C., Gilbert D., Galas L., Trottein F., Faye L., Lerouge P. Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha(1,3)-fucose and core xylose. Glycobiology. 2003;13:427–434. doi: 10.1093/glycob/cwg024. [DOI] [PubMed] [Google Scholar]
  3. Cabanes-Macheteau M., Fitchette-Lainé A.C., Loutelier-Bourhis C., Lange C., Vine N.D., Ma J.K., Lerouge P., Faye L. N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants. Glycobiology. 1999;9:365–372. doi: 10.1093/glycob/9.4.365. [DOI] [PubMed] [Google Scholar]
  4. Catherine R., Patrice L., Loic F. The protein N-glycosylation in plants. J Exp Bot. 1998;49:1463–1472. doi: 10.1093/jxb/49.326.1463. [DOI] [Google Scholar]
  5. Cox K.M., Sterling J.D., Regan J.T., Gasdaska J.R., Frantz K.K., Peele C.G., Black A., Passmore D., Moldovan-Loomis C., Srinivasan M., et al. Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol. 2006;24:1591–1597. doi: 10.1038/nbt1260. [DOI] [PubMed] [Google Scholar]
  6. Dirnberger D., Steinkellner H., Abdennebi L., Remy J.J., van de Wiel D. Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants. Eur J Biochem. 2001;268:4570–4579. doi: 10.1046/j.1432-1327.2001.02384.x. [DOI] [PubMed] [Google Scholar]
  7. Doran P.M. Foreign protein production in plant tissue cultures. Curr Opin Biotechnol. 2000;11:199–204. doi: 10.1016/S0958-1669(00)00086-0. [DOI] [PubMed] [Google Scholar]
  8. Elbers I.J., Stoopen G.M., Bakker H., Stevens L.H., Bardor M., Molthoff J.W., Jordi W.J., Bosch D., Lommen A. Influence of growth conditions and developmental stage on N-glycan heterogeneity of transgenic immunoglobulin G and endogenous proteins in tobacco leaves. Plant Physiol. 2001;126:1314–1322. doi: 10.1104/pp.126.3.1314. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fischer R., Emans N., Schuster F., Hellwig S., Drossard J. Towards molecular farming in the future: using plant-cell-suspension cultures as bioreactors. Biotechnol Appl Biochem. 1999;30:109–112. [PubMed] [Google Scholar]
  10. Gomord V., Chamberlain P., Jefferis R., Faye L. Biopharmaceutical production in plants: problems, solutions and opportunities. Trends Biotechnol. 2005;23:559–565. doi: 10.1016/j.tibtech.2005.09.003. [DOI] [PubMed] [Google Scholar]
  11. Gomord V., Faye L. Posttranslational modification of therapeutic proteins in plants. Curr Opin Plant Biol. 2004;7:171–181. doi: 10.1016/j.pbi.2004.01.015. [DOI] [PubMed] [Google Scholar]
  12. Hellwig S., Drossard J., Twyman R.M., Fischer R. Plant cell cultures for the production of recombinant proteins. Nat Biotechnol. 2004;22:1415–1422. doi: 10.1038/nbt1027. [DOI] [PubMed] [Google Scholar]
  13. Lerouge P., Cabanes-Macheteau M., Rayon C., Fischette-Lainé A. C., Gomord V., Faye L. N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol Biol. 1998;38:31–48. doi: 10.1023/A:1006012005654. [DOI] [PubMed] [Google Scholar]
  14. Matsumoto S., Ikura K., Ueda M., Sasaki R. Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells. Plant Mol Biol. 1995;27:1163–1172. doi: 10.1007/BF00020889. [DOI] [PubMed] [Google Scholar]
  15. Niwa R., Hatanaka S., Shoji-Hosaka E., Sakurada M., Kobayashi Y., Uehara A., Yokoi H., Nakamura K., Shitara K. Enhancement of the antibody-dependent cellular cytotoxicity of low-fucose IgG1 Is independent of FcgammaRIIIa functional polymorphism. Clin Cancer Res. 2004;10:6248–6255. doi: 10.1158/1078-0432.CCR-04-0850. [DOI] [PubMed] [Google Scholar]
  16. Pagny S., Bouissonnie F., Sarkar M., Follet-Gueye M.L., Driouich A., Schachter H., Faye L., Gomord V. Structural requirements for Arabidopsis beta1,2-xylosyltransferase activity and targeting to the Golgi. Plant J. 2003;33:189–203. doi: 10.1046/j.0960-7412.2002.01604.x. [DOI] [PubMed] [Google Scholar]
  17. Palacpac N.Q., Yoshida S., Sakai H., Kimura Y., Fujiyama K., Yoshida T., Seki T. Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns. Proc Natl Acad Sci U S A. 1999;96:4692–4697. doi: 10.1073/pnas.96.8.4692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Rempel H.C., Nelson L. M. Analysis of conditions for Agrobacterium-mediated transformation of tobacco cells in suspension. Transgenic Res. 1995;4:199–207. doi: 10.1007/BF01968785. [DOI] [Google Scholar]
  19. Sourrouille C., Marquet-Blouin E., D’Aoust M.A., Kiefer-Meyer M. C., Seveno M., Pagny-Salehabadi S., Bardor M., Durambur G., Lerouge P., Vezina L., et al. Down-regulated expression of plant-specific glycoepitopes in alfalfa. Plant Biotechnol J. 2008;6:702–721. doi: 10.1111/j.1467-7652.2008.00353.x. [DOI] [PubMed] [Google Scholar]
  20. Strasser R., Altmann F., Mach L., Glössl J., Steinkellner H. Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose. FEBS Lett. 2004;561:132–136. doi: 10.1016/S0014-5793(04)00150-4. [DOI] [PubMed] [Google Scholar]
  21. Strasser R., Stadlmann J., Schähs M., Stiegler G., Quendler H., Mach L., Glössl J., Weterings K., Pabst M., Steinkellner H. Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnol J. 2008;6:392–402. doi: 10.1111/j.1467-7652.2008.00330.x. [DOI] [PubMed] [Google Scholar]
  22. Swiatek A., Lenjou M., Van Bockstaele D., Inzé D., Van Onckelen H. Differential effect of jasmonic acid and abscisic acid on cell cycle progression in tobacco BY-2 cells. Plant Physiol. 2002;128:201–211. doi: 10.1104/pp.010592. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Triguero A., Cabrera G., Cremata J.A., Yuen C.T., Wheeler J., Ramírez N.I. Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is Nglycosylated homogeneously throughout the plant with mostly high-mannose-type N-glycans. Plant Biotechnol J. 2005;3:449–457. doi: 10.1111/j.1467-7652.2005.00137.x. [DOI] [PubMed] [Google Scholar]
  24. Twyman R.M., Stoger E., Schillberg S., Christou P., Fischer R. Molecular farming in plants: host systems and expression technology. Trends Biotechnol. 2003;21:570–578. doi: 10.1016/j.tibtech.2003.10.002. [DOI] [PubMed] [Google Scholar]
  25. van Ree R., Cabanes-Macheteau M., Akkerdaas J., Milazzo J.P., Loutelier-Bourhis C., Rayon C., Villalba M., Koppelman S., Aalberse R., Rodriguez R., et al. Beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J Biol Chem. 2000;275:11451–11458. doi: 10.1074/jbc.275.15.11451. [DOI] [PubMed] [Google Scholar]
  26. Wilson I.B., Rendić D., Freilinger A., Dumić J., Altmann F., Mucha J., Müller S., Hauser M.T. Cloning and expression of cDNAs encoding alpha1,3-fucosyltransferase homologues from Arabidopsis thaliana. Biochim Biophys Acta. 2001;1527:88–96. doi: 10.1016/S0304-4165(01)00151-9. [DOI] [PubMed] [Google Scholar]
  27. Yin B., Zheng N., Li Y., Tang S., Liang L., Xie Q. Growth phase-dependent expression of proteins with decreased plant-specific N-glycans and immunogenicity in tobacco BY2 cells. Sci China C Life Sci. 2009;52:739–746. doi: 10.1007/s11427-009-0093-5. [DOI] [PubMed] [Google Scholar]

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