Glyco-engineering of Biotherapeutic Proteins in Plants

  • Ko, Kisung (Department of Biological Science, College of Natural Sciences, Wonkwang University) ;
  • Ahn, Mi-Hyun (Department of Biological Science, College of Natural Sciences, Wonkwang University) ;
  • Song, Mira (Department of Biological Science, College of Natural Sciences, Wonkwang University) ;
  • Choo, Young-Kug (Department of Biological Science, College of Natural Sciences, Wonkwang University) ;
  • Kim, Hyun Soon (Plant Cell Biotechnology Lab., Korea Research Institute of Bioscience and Biotechnology) ;
  • Ko, Kinarm (Max Planck Institute for Molecular Biomedicine, Department of Cell and Developmental Biology) ;
  • Joung, Hyouk (Plant Cell Biotechnology Lab., Korea Research Institute of Bioscience and Biotechnology)
  • Received : 2007.09.28
  • Accepted : 2008.01.24
  • Published : 2008.06.30

Abstract

Many therapeutic glycoproteins have been successfully generated in plants. Plants have advantages regarding practical and economic concerns, and safety of protein production over other existing systems. However, plants are not ideal expression systems for the production of biopharmaceutical proteins, due to the fact that they are incapable of the authentic human N-glycosylation process. The majority of therapeutic proteins are glycoproteins which harbor N-glycans, which are often essential for their stability, folding, and biological activity. Thus, several glyco-engineering strategies have emerged for the tailor-making of N-glycosylation in plants, including glycoprotein subcellular targeting, the inhibition of plant specific glycosyltranferases, or the addition of human specific glycosyltransferases. This article focuses on plant N-glycosylation structure, glycosylation variation in plant cell, plant expression system of glycoproteins, and impact of glycosylation on immunological function. Furthermore, plant glyco-engineering techniques currently being developed to overcome the limitations of plant expression systems in the production of therapeutic glycoproteins will be discussed in this review.

Keywords

Acknowledgement

Supported by : Rural Development Administration

References

  1. AS Insights (2005). Monoclonal antibody therapeutics-current market dynamics & future outlook. pp. 42
  2. Bakker, H., Bardor, M., Molthoff, J.W., Gomord, V., Elbers, I., Stevens, L.H., Jordi, W., Lommen, A., Faye, L., Lerouge, P., et al. (2001a). Galactose-extended glycans of antibodies produced by transgenic plants. Proc. Natl. Acad. Sci. USA 98, 2899-2904
  3. Bakker, H., Schijlen, E., de Vries, T., Schiphorst, W.E., Jordi, W., Lommen, A., Bosch, D., and van Die, I. (2001b). Plant members of the alpha1 ${\to}$ 3/4-fucosyltransferase gene family encode an alpha1 ${\to}$ 4-fucosyltransferase, potentially involved in Lewis (a) biosynthesis, and two core alpha1 ${\to}$ 3-fucosyltransferases. FEBS Lett. 507, 307-312 https://doi.org/10.1016/S0014-5793(01)02999-4
  4. Bardor, M., Loutelier-Bourhis, C., Paccalet, T., Cosette, P., Fitchette, A.C., Vézina, L.P., Trepanier, S., Dargis, M., Lemieux, R., Lange, C., et al. (2003). Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N-glycosylation that is homogenous and suitable for glyco-engineering into human-compatible structures. Plant Biotechnol. J. 1, 451-462 https://doi.org/10.1046/j.1467-7652.2003.00041.x
  5. Bardor, M., Cabrera, G., Rudd, P.M., Dwek, R.A., Cremata, J.A., and Lerouge, P. (2006). Analytical strategies to investigate plant N-glycan profiles in the context of plant-made pharmaceuticals. Curr. Opin. Struct. Biol. 16, 576-583 https://doi.org/10.1016/j.sbi.2006.08.009
  6. Brodzik, R., Glogowska, M., Bandurska, K., Okulicz, M., Deka, D., Ko, K., van der Linden, J., Leusen, J.H.W., Pogrebnyak, N., Golovkin, M., et al. (2006). Plant-derived anti-Lewis Y mAb exhibits biological activities for efficient immunotherapy against human cancer cells. Proc. Natl. Acad. Sci. USA 103, 8804-8809
  7. 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 https://doi.org/10.1093/glycob/9.4.365
  8. Chargeleque, D., Vine, N.D., van Dolleweerd, C.J., Drake, P.M.W., and Ma, J.K. (2000). A murine monoclonal antibody produced in transgenic plants with plant-specific glycans is not immunogenic in mice. Transgenic Res. 9, 187-194 https://doi.org/10.1023/A:1008976219939
  9. Conrad, U., and Fiedler, U. (1998). Compartment-specific accumulation of recombinant immunoglobulins in plant cells: an essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Mol. Biol. 38, 101-109 https://doi.org/10.1023/A:1006029617949
  10. Deisenhofer, J. (1981). Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry 20, 2361-2370 https://doi.org/10.1021/bi00512a001
  11. Dorai, H., Mueller, B.M., Reisfeld, R.A., and Gillies, S.D. (1991). Aglycosylated chimeric mouse/human IgG1 antibody retains some effector function. Hybridoma 10, 211-217 https://doi.org/10.1089/hyb.1991.10.211
  12. 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 Nglycan heterogeneity of transgenic immunoglobulin G and endogenous proteins in tobacco leaves. Plant Physiol. 126, 1314-1322 https://doi.org/10.1104/pp.126.3.1314
  13. Faye, L., Johnson, K.D., and Chrispeels, M.J. (1986a). Oligosaccharide side chains of glycoproteins that remain in the high-mannose form are not accessible to glycosidases. Plant Physiol. 81, 206-211 https://doi.org/10.1104/pp.81.1.206
  14. Faye, L., Sturm, A., Bollini, R., Vitale, A., and Chrispeels, M.J. (1986b). The position of the oligosaccharide side-chains of phytohemaglutinin and their accessibility to glycosidases determines their subsequent processing in the Golgi. Eur. J. Biochem. 158, 655-661 https://doi.org/10.1111/j.1432-1033.1986.tb09803.x
  15. Faye, L., Gomord, V., Fitchette-Lainé, A.C., and Chrispeels, M.J. (1993). Affinity purification of antibodies specific for Asnlinked glycans containing alpha 1 ${\to}$ 3 fucose or beta 1 ${\to}$ 2 xylose. Anal. Biochem. 209, 104-108 https://doi.org/10.1006/abio.1993.1088
  16. Faye, L., Boulaflous, A., Benchabane, M., Gomord, V., and Michaud, D. (2005). Protein modifications in the plant secretory pathway: current status and practical implications in molecular pharming. Vaccine 23, 1770-1778 https://doi.org/10.1016/j.vaccine.2004.11.003
  17. Fitchette-Lainé, A.C., Gomord, V., Cabanes, M., Michalski, J.C., Saint Macary, M., Foucher, B., Cavelier, B., Hawes, C., Lerouge, P., and Faye, L. (1997). N-glycans harboring the Lewis a epitope are expressed at the surface of plant cells. Plant J. 12, 1411-1417 https://doi.org/10.1046/j.1365-313x.1997.12061411.x
  18. Fitchette, A.C., Cabanes-Macheteau, M., Marvin, L., Martin, B., Satiat-Jeunemaitre, B., Gomord, V., Crooks, K., Lerouge, P., Faye, L., and Hawes, C. (1999). Biosynthesis and immunolocalization of Lewis a-containing N-glycans in the plant cell. Plant Physiol. 121, 333-344 https://doi.org/10.1104/pp.121.2.333
  19. Freeze, H.H., and Aebi, M. (2005). Altered glycan structures: the molecular basis of congenital disorders of glycosylation. Curr. Opin. Struct. Biol. 15, 490-498 https://doi.org/10.1016/j.sbi.2005.08.010
  20. Gomord, V., and Faye, L. (2004). Posttranslational modification of therapeutic protein in plants. Curr. Opin. Plant Biol. 7, 171-181 https://doi.org/10.1016/j.pbi.2004.01.015
  21. Gomord, V., Sourrouille, C., Fitchette, A.C., Bardor, M., Pagny, S., Lerouge, P., and Faye, L. (2004). Production and glycosylation of plant-made pharmaceuticals: the antibodies as a challenge. Plant Biotechnol. J. 2, 83-100 https://doi.org/10.1111/j.1467-7652.2004.00062.x
  22. Gomord, V., Chamberlain, P., Jefferis, R., and Faye, L. (2005). Biopharmaceutical production in plants: problems, solutions and opportunities. Trends Biotechnol. 23, 559-565 https://doi.org/10.1016/j.tibtech.2005.09.003
  23. Helenius, A., and Aebi, M. (2001). Intracellular functions of N-linked glycans. Science 291, 2364-2369 https://doi.org/10.1126/science.291.5512.2364
  24. Hiatt, A., Cafferkey, R., and Bowdish, K. (1989). Production of antibodies in transgenic plants. Nature 342, 76-78 https://doi.org/10.1038/342076a0
  25. Jobling, S.A., Jarman, C., Teh, M.M., Holmberg, N., Blake, C., and Verhoeyen, M.E. (2003). Immunomodulation of enzyme function in plants by single-domain antibody fragments. Nat. Biotechnol. 21, 77-80 https://doi.org/10.1038/nbt772
  26. Kaneko, M., and Nighorn, A. (2003). Interaxonal Eph-ephrin signaling may mediate sorting of olfactory sensory axons in Manduca sexta. J. Neurosci. 23, 11523-11538 https://doi.org/10.1523/JNEUROSCI.23-37-11523.2003
  27. Kanwar, Y.S., Hascall, V.C., Jakubowski, M.L., and Gibbons, J.T. (1984). Effect of beta-D-xyloside on the glomerular proteoglycans. I. Biochemical studies. J. Cell Biol. 99, 715-722 https://doi.org/10.1083/jcb.99.2.715
  28. Kelm, S., and Schauer, R. (1997). Sialic acids in molecular and cellular interactions. Int. Rev. Cytol. 175, 137-240 https://doi.org/10.1016/S0074-7696(08)62127-0
  29. Kim, S.M., Lee, J.S., Lee, Y.H., Kim, W.J., Do, S.I., Choo, Y.K., and Park, Y.I. (2007). Increased ${\alpha}$(2,3)-Sialylation and hyperglycosylation of N-glycans in embryonic rat cortical neurons during Camptothecin-induced apoptosis. Mol. Cells 24, 416-423
  30. Ko, K., and Koprowski, H. (2005). Plant biopharming of monoclonal antibodies. Virus Res. 111, 93-100 https://doi.org/10.1016/j.virusres.2005.03.016
  31. Ko, K., Tekoah, Y., Rudd, P.M., Harvey, D.J., Dwek, R.A., Spitsin, S., Hanlon, C.A., Rupprecht, C., Dietzschold, B., Golovkin, M., et al. (2003). Function and glycosylation of plant-derived antiviral monoclonal antibody. Proc. Natl. Acad. Sci. USA 100, 8013-8018
  32. Ko, K., Steplewski, Z., Glogowska, M., and Koprowski, H. (2005). Inhibition of tumor growth by plant-derived mAb. Proc. Natl. Acad. Sci. USA 102, 7026-7030
  33. Koprivova, A., Stemmer, C., Altmann, F., Hoffmann, A., Kopriva, S., Gorr, G., Reski, R., and Decker, E.L. (2004). Targeted knockouts of Physcomitrella lacking plant specific immunogenic N-glycans. Plant Biotech. J. 2, 517-523 https://doi.org/10.1111/j.1467-7652.2004.00100.x
  34. Koprowski, H., and Croce, C. (1980). Hybridomas revisited. Science 210, 248 https://doi.org/10.1126/science.210.4467.248-a
  35. Kurosaka, A., Yano, A., Itoh, N., Kuroda, Y., Nakagawa, T., and Kawasaki, T. (1991). The structure of a neural specific carbohydrate epitope of horseradish peroxidase recognized by antihorseradish peroxidase antiserum. J. Biol. Chem. 266, 4168-4172
  36. Li, L., Yan, J., and Zhao, M.P. (2006). Improvement of the performance of an immunoaffinity extraction method via region-specific immobilization of IgG. J. Chromatogr. A. 1103, 350-355
  37. Ma, J.K., Hikmat, B.Y., Wycoff, K., Vine, N.D., Chargelegue, D., Yu, L., Hein, M.B., and Lehner, T. (1998). Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat. Med. 4, 601-606 https://doi.org/10.1038/nm0598-601
  38. Ma, J.K., Drake, P.M., and Christou, P. (2003). The production of recombinant pharmaceutical proteins in plants. Nat. Rev. Genet. 4, 794-805 https://doi.org/10.1038/nrg1177
  39. Mason, H.S., Lam, D.M., and Arntzen, C.J. (1992). Expression of hepatitis B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. USA 89, 11745-11749
  40. Mayfield, S.P., Franklin, S.E., and Lerner, R.A. (2003). Expression and assembly of a fully active antibody in algae. Proc. Natl. Acad. Sci. USA 100, 438-442
  41. Misaki, R., Fujiyama, K., and Seki, T. (2006). Expression of human CMP-N-acetylneuraminic acid synthetase and CMP-sialic acid transporter in tobacco suspension-cultured cell. Biochem. Biophys. Res. Commun. 339, 1184-1189 https://doi.org/10.1016/j.bbrc.2005.11.130
  42. 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 Fcgamma RIIIa functional polymorphism. Clin. Cancer Res. 10, 6248-6255 https://doi.org/10.1158/1078-0432.CCR-04-0850
  43. Okazaki, A., Shoji-Hosaka, E., Nakamura, K., Wakitani, M., Uchida, K., Kakita, S., Tsumoto, K., Kumagai, I., and Shitara, K. (2004). Fucose depletion from human IgG1 oligosaccharide enhances binding enthalpy and association rate between IgG1 and FcgammaRIIIa. J. Mol. Biol. 336, 1239-1249 https://doi.org/10.1016/j.jmb.2004.01.007
  44. Paccalet, T., Bardor, M., Rihouey, C., Delmas, F., Chevalier, C., D'Aoust, M.A., Faye, L., Vézina, L., Gomord, V., and Lerouge, P. (2007). Engineering of a sialic acid synthesis pathway in transgenic plants by expression of bacterial Neu5Ac-synthesizing enzymes. Plant Biotechnol. J. 5, 16-25 https://doi.org/10.1111/j.1467-7652.2006.00211.x
  45. Pagny, S., Cabanes-Macheteau, M., Gillikin, J.W., Leborgne- Castel, N., Lerouge, P., Boston, R.S., Faye, L., and Gomord, V. (2000). Protein recycling from the Golgi apparatus to the endoplasmic reticulum in plants and its minor contribution to calreticulin retention. Plant Cell 12, 739-756 https://doi.org/10.1105/tpc.12.5.739
  46. Peeters, K., De Wilde, C., and Depicker, A. (2001). Highly efficient targeting and accumulation of a F(ab) fragment within the secretory pathway and apoplast of Arabidopsis thaliana. Eur. J. Biochem. 268, 4251-4260 https://doi.org/10.1046/j.1432-1327.2001.02340.x
  47. Perlman, S., van den Hazel, B., Christiansen, J., Gram-Nielsen, S., Jeppesen, C.B., Andersen, K.V., Halkier, T., Okkels, S., and Schambye, H.T. (2003). Glycosylation of an N-terminal extension prolongs the half-life and increases the in vivo activity of follicle stimulating hormone. J. Clin. Endocrinol. Metab. 88, 3227-3235 https://doi.org/10.1210/jc.2002-021201
  48. Petruccelli, S., Otegui, M.S., Lareu, F., Tran Dinh, O., Fitchette, A.C., Circosta, A., Rumbo, M., Bardor, M., Carcamo, R., Gomord, V., et al. (2006). A KDEL-tagged monoclonal antibody is efficiently retained in the endoplasmic reticulum in leaves, but is both partially secreted and sorted to protein storage vacuoles in seeds. Plant Biotechnol. J. 4, 511-527
  49. Rayon, C., Cabanes-Macheteau, M., Loutelier-Bourhis, C., Salliot-Maire, I., Lemoine, J., Reiter, W.D., Lerouge, P., and Faye, L. (1999). Characterization of N-glycans from Arabi-dopsis. Application to a fucose-deficient mutant. Plant Physiol. 119, 725-734 https://doi.org/10.1104/pp.119.2.725
  50. Rudd, P.M., Wormald, M.R., and Dwek, R.A. (2004). Sugar-mediated ligand-receptor interactions in the immune system. Trends Biotechnol. 22, 524-530 https://doi.org/10.1016/j.tibtech.2004.07.012
  51. Schahs, M., Strasser, R., Stadlmann, J., Kunert, R., Rademacher, T., and Steinkellner, H. (2007). Production of a monoclonal antibody in plants with a humanized N-glycosylation pattern. Plant Biotechnol. J. 5, 657-663 https://doi.org/10.1111/j.1467-7652.2007.00273.x
  52. Schouten, A., Roosien, J., van Engelen, F.A., de Jong, G.A., Borst-Vrenssen, A.W., Zilverentant, J.F., Bosch, D., Stiekema, W.J., Gommers, F.J., Schots, A., et al. (1996). The C-terminal KDEL sequence increases the expression level of a single-chain antibody designed to be targeted to both the cytosol and the secretory pathway in transgenic tobacco. Plant Mol. Biol. 30, 781-793 https://doi.org/10.1007/BF00019011
  53. Seveno, M., Bardor, M., Paccalet, T., Gomord, V., Lerouge, P., Faye, L. (2004). Glycoprotein sialylation in plants? Nat. Biotechnol. 22, 1351-1352 https://doi.org/10.1038/nbt1104-1351
  54. Shah, N., Kuntz, D.A., and Rose, D.R. (2003). Comparison of kifunensine and 1-deoxymannojirimycin binding to class I and II alpha-mannosidases demonstrates different saccharide distortions in inverting and retaining catalytic mechanisms. Biochemistry 42, 13812-13816 https://doi.org/10.1021/bi034742r
  55. Sharp, J.M., and Doran, P.M. (2001). Characterization of monoclonal antibody fragments produced by plant cells. Biotechnol. Bioeng. 73, 338-346 https://doi.org/10.1002/bit.1067
  56. Shields, R.L., Lai, J., Keck, R., O'Connell, L.Y., Hong, K., Meng, Y.G., Weikert, S.H., and Presta, L.G. (2002). Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fcgamma RIII and antibody-dependent cellular toxicity. J. Biol. Chem. 277, 26733-26740 https://doi.org/10.1074/jbc.M202069200
  57. Shin, D.J., Kang, J.Y., Kim, Y.U., Yoon, J.S., Choy, H.E., Maeda, Y., Kinoshita, T., and Hong, Y. (2006). Isolation of new CHO cell mutants defective in CMP-Sialic Acid biosynthesis and transport. Mol. Cells 22, 343-352
  58. Shinkawa, T., Nakamura, K., Yamane, N., Shoji-Hosaka, E., Kanda, Y., Sakurada, M., Uchida, K., Anazawa, H., Satoh, M., Yamasaki, M., et al. (2003). The absence of fucose but not the presence of galactose or bisecting N-acetylglucosamine of human IgG1 complex-type oligosaccharides shows the critical role of enhancing antibody-dependent cellular cytotoxicity. J. Biol. Chem. 278, 3466-3473 https://doi.org/10.1074/jbc.M210665200
  59. Sriraman, R., Bardor, M., Sack, M., Vaquero, C., Faye, L., Fischer, R., Finnern, R., and Lerouge, P. (2004). Recombinant antihCG antibodies retained in the endoplasmic reticulum of transformed plants lack core-xylose and core-alpha(1,3)-fucose residues. Plant Biotechnol. J. 2, 279-287 https://doi.org/10.1111/j.1467-7652.2004.00078.x
  60. Tekoah, Y., Ko, K., Koprowski, H., Harvey, D.J., Wormald, M.R., Dwek, R.A., and Rudd, P.M. (2004). Controlled glycosylation of therapeutic antibodies in plants. Arch. Biochem. Biophys. 426, 266-278 https://doi.org/10.1016/j.abb.2004.02.034
  61. Umana, P., Jean-Mairet, J., and Bailey, J.E. (1999). Tetracycline-regulated overexpression of glycosyltransferases in Chinese hamster ovary cells. Biotechnol. Bioeng. 65, 542-549 https://doi.org/10.1002/(SICI)1097-0290(19991205)65:5<542::AID-BIT7>3.0.CO;2-Z
  62. 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. (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 https://doi.org/10.1074/jbc.275.15.11451
  63. Vitale, A., and Chrispeels, M.J. (1984). Transient N-acetylglucosamine in the biosynthesis of phytohemagglutinin: attachment in the Golgi apparatus and removal in protein bodies. J. Cell Biol. 99(1 Pt 1), 133-140 https://doi.org/10.1083/jcb.99.1.133
  64. Walker, M.R., Lund, J., Thompson, K.M., and Jefferis, R. (1989). A glycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing Fc gamma RI and/or Fc gamma RII receptors. Biochem. J. 259, 347-353 https://doi.org/10.1042/bj2590347
  65. Wee, E.G., Sherrier, D.J., Prime, T.A., and Dupree, P. (1998). Targeting of active sialyltransferase to the plant Golgi apparatus. Plant Cell 10, 1759-1768 https://doi.org/10.1105/tpc.10.10.1759
  66. Wenderoth, I., and von Schaewen, A. (2000). Isolation and characterization of plant- N-acetyl glucosaminyltransferase I (GntI) cDNA sequences. Functional analyses in the Arabidopsis cgl mutant and in antisense plants. Plant Physiol. 123, 1097-1108 https://doi.org/10.1104/pp.123.3.1097
  67. Werner, R.G., Kopp, K., and Schlueter, M. (2007). Glycosylation of therapeutic proteins in different production systems. Acta Paediatr. Suppl. 96, 17-22
  68. Wide, L. (1986). The regulation of metabolic clearance rate of human FSH in mice by variation of the molecular structure of the hormone. Acta Endocrinologica 112, 519-529
  69. Wright, A., and Morrison, S.L. (1997). Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol. 15, 26-32 https://doi.org/10.1016/S0167-7799(96)10062-7
  70. Wright, K.E., Prior, F., Sardana, R., Altosaar, I., Dudani, A.K., Ganz, P.R., and Tackaberry, E.S. (2001). Sorting of glycoprotein B from human cytomegalovirus to protein storage vesicles in seeds of transgenic tobacco. Transgenic Res. 10, 177-181 https://doi.org/10.1023/A:1008912305913
  71. Zeitlin, L., Olmsted, S.S., Moench, T.R., Co, M.S., Martinell, B.J., Paradkar, V.M., Russell, D.R., Queen, C., Cone, R.A., and Whaley, K.J. (1998). A humanized monoclonal antibody produced in transgenic plants for immunoprotection of the vagina against genital herpes. Nat. Biotechnol. 16, 1361-1364 https://doi.org/10.1038/4344
  72. Zeleny, R., Kolarich, D., Strasser, R., and Altmann, F. (2006a). Sialic acid concentrations in plants are in the range of inadvertent contamination. Planta 224, 222-227 https://doi.org/10.1007/s00425-005-0206-8
  73. Zeleny, R., Leonard, R., Dorfner, G., Dalik, T., Kolarich, D., and Altmann, F. (2006b). Molecular cloning and characterization of a plant alpha1, 3/4-fucosidase based on sequence tags from almond fucosidase I. Phytochemistry 67, 641-648 https://doi.org/10.1016/j.phytochem.2006.01.021