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

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 N-glycans 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 glyco-engineered 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

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., and Bosch, D. (2006). 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 103, 7577–7582 .16675551
[2] Bardor, M., Faveeuw, C., Fitchette, A.C., Gilbert, D., Galas, L., Trottein, F., Faye, L., and Lerouge, P. (2003). Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha(1,3)-fucose and core xylose. Glycobiology 13, 427–434 .12626420
[3] Cabanes-Macheteau, M., Fitchette-Lainé, A.C., Loutelier-Bourhis, C., Lange, C., Vine, N.D., Ma, J.K., Lerouge, P., and Faye, L. (1999). N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants. Glycobiology 9, 365–372 .10089210
[4] Catherine, R., Patrice, L., Loic F. (1998). The protein N-glycosylation in plants. J Exp Bot 49, 1463–1472 .
[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., (2006). Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol 24, 1591–1597 .17128273
[6] Dirnberger, D., Steinkellner, H., Abdennebi, L., Remy, J.J., and van de Wiel, D. (2001). Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants. Eur J Biochem 268, 4570–4579 .11502219
[7] Doran, P.M. (2000). Foreign protein production in plant tissue cultures. Curr Opin Biotechnol 11, 199–204 .10753768
[8] Elbers, I.J., Stoopen, G.M., Bakker, H., Stevens, L.H., Bardor, M., Molthoff, J.W., Jordi, W.J., Bosch, D., and Lommen, A. (2001). Influence of growth conditions and developmental stage on N-glycan heterogeneity of transgenic immunoglobulin G and endogenous proteins in tobacco leaves. Plant Physiol 126, 1314–1322 .11457982
[9] Fischer, R., Emans, N., Schuster, F., Hellwig, S., and Drossard, J. (1999). Towards molecular farming in the future: using plant-cell-suspension cultures as bioreactors. Biotechnol Appl Biochem 30, 109–112 .10512788
[10] Gomord, V., Chamberlain, P., Jefferis, R., and Faye, L. (2005). Biopharmaceutical production in plants: problems, solutions and opportunities. Trends Biotechnol 23, 559–565 .16168504
[11] Gomord, V., and Faye, L. (2004). Posttranslational modification of therapeutic proteins in plants. Curr Opin Plant Biol 7, 171–181 .15003218
[12] Hellwig, S., Drossard, J., Twyman, R.M., and Fischer, R. (2004). Plant cell cultures for the production of recombinant proteins. Nat Biotechnol 22, 1415–1422 .15529167
[13] Lerouge, P., Cabanes-Macheteau, M., Rayon, C., Fischette-Lainé, A.C., Gomord, V., and Faye, L. (1998). N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol Biol 38, 31–48 .9738959
[14] Matsumoto, S., Ikura, K., Ueda, M., and Sasaki, R. (1995). Characterization of a human glycoprotein (erythropoietin) produced in cultured tobacco cells. Plant Mol Biol 27, 1163–1172 .7766897
[15] Niwa, R., Hatanaka, S., Shoji-Hosaka, E., Sakurada, M., Kobayashi, Y., Uehara, A., Yokoi, H., Nakamura, K., and Shitara, K. (2004). Enhancement of the antibody-dependent cellular cytotoxicity of low-fucose IgG1 Is independent of FcgammaRIIIa functional polymorphism. Clin Cancer Res 10, 6248–6255 .15448014
[16] Pagny, S., Bouissonnie, F., Sarkar, M., Follet-Gueye, M.L., Driouich, A., Schachter, H., Faye, L., and Gomord, V. (2003). Structural requirements for Arabidopsis beta1,2-xylosyltransferase activity and targeting to the Golgi. Plant J 33, 189–203 .12943552
[17] Palacpac, N.Q., Yoshida, S., Sakai, H., Kimura, Y., Fujiyama, K., Yoshida, T., and Seki, T. (1999). Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns. Proc Natl Acad Sci U S A 96, 4692–4697 .10200324
[18] Rempel, H.C., Nelson L. M. (1995). Analysis of conditions for Agrobacterium-mediated transformation of tobacco cells in suspension. Transgenic Res 4, 199–207 .
[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., (2008). Down-regulated expression of plant-specific glycoepitopes in alfalfa. Plant Biotechnol J 6, 702–721 .18498310
[20] Strasser, R., Altmann, F., Mach, L., Gl?ssl, J., and Steinkellner, H. (2004). Generation of Arabidopsis thaliana plants with complex N-glycans lacking beta1,2-linked xylose and core alpha1,3-linked fucose. FEBS Lett 561, 132–136 .15013764
[21] Strasser, R., Stadlmann, J., Sch?hs, M., Stiegler, G., Quendler, H., Mach, L., Gl?ssl, J., Weterings, K., Pabst, M., and Steinkellner, H. (2008). Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnol J 6, 392–402 .18346095
[22] Swiatek, A., Lenjou, M., Van Bockstaele, D., Inzé, D., and Van Onckelen, H. (2002). Differential effect of jasmonic acid and abscisic acid on cell cycle progression in tobacco BY-2 cells. Plant Physiol 128, 201–211 .11788766
[23] Triguero, A., Cabrera, G., Cremata, J.A., Yuen, C.T., Wheeler, J., and Ramírez, N.I. (2005). Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is N-glycosylated homogeneously throughout the plant with mostly high-mannose-type N-glycans. Plant Biotechnol J 3, 449–457 .17173632
[24] Twyman, R.M., Stoger, E., Schillberg, S., Christou, P., and Fischer, R. (2003). Molecular farming in plants: host systems and expression technology. Trends Biotechnol 21, 570–578 .14624867
[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., (2000). Beta(1,2)-xylose and alpha(1,3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens. J Biol Chem 275, 11451–11458 .10753962
[26] Wilson, I.B., Rendi?, D., Freilinger, A., Dumi?, J., Altmann, F., Mucha, J., Müller, S., and Hauser, M.T. (2001). Cloning and expression of cDNAs encoding alpha1,3-fucosyltransferase homologues from Arabidopsis thaliana. Biochim Biophys Acta 1527, 88–96 .11420147
[27] Yin, B., Zheng, N., Li, Y., Tang, S., Liang, L., and Xie, Q. (2009). Growth phase-dependent expression of proteins with decreased plant-specific N-glycans and immunogenicity in tobacco BY2 cells. Sci China C Life Sci 52, 739–746 .19727592