Recombinant Proteins From Plants pp 239-264

Part of the Methods in Molecular Biology™ book series (MIMB, volume 483) | Cite as

N-Glycosylation of Plant Recombinant Pharmaceuticals

  • Muriel Bardor
  • Gleysin Cabrera
  • Johannes Stadlmann
  • Patrice Lerouge
  • José A. Cremata
  • Véronique Gomord
  • Anne-Catherine Fitchette

Summary

N-glycosylation is a maturation event necessary for the correct function, efficiency, and stability of a high number of biopharmaceuticals. This chapter presented here proposes various methods to determine whether, how, and where a plant pharmaceutical is N-glycosylated. These methods rely on blot detection with glycan-specific probes, specific deglycosylation of glycoproteins followed by mass spectrometry, N-glycan profile analysis, and glycopeptide identification by LC-MS.

Keywords

Plant pharmaceuticals N-glycosylation Western Blotting Immunodetection Affinodetection Glycosidase Mass spectrometry N-glycan profiling HPLC Glycopeptide LC-MS MALDI-TOF 

References

  1. 1.
    Saint-Jore-Dupas, C., Faye, L. and Gomord, V. (2007) From planta to pharma with glycosylation in the toolbox. Trends Biotechnol. 25, 317–323.CrossRefGoogle Scholar
  2. 2.
    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.CrossRefPubMedGoogle Scholar
  3. 3.
    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.CrossRefPubMedGoogle Scholar
  4. 4.
    Faye, L. and Chrispeels, M.J. (1985) Characterization of N -linked oligosaccharides by affinoblotting with concanavalin A-peroxidase and treatment of the blots with glycosidases. Anal. Biochem. 149, 218 – 224.CrossRefPubMedGoogle Scholar
  5. 5.
    Shibuya, N., Goldstein, I.J., van Damme, E.J.M. and Peumans, W.J. (1988) Binding properties of a mannose-specific lectin from the snowdrop (Galanthus nivealis) bulb. J. Biol. Chem. 263, 728 – 734.PubMedGoogle Scholar
  6. 6.
    Animashaun, T., Mahmood, N., Hay, A.J. and Hughes, R.C. (1993) Inhibitory effect of novel mannose-binding lectins on HIV-infectivity and syncitium formation. Antiv. Chem. Chemother. 4, 145 – 153.Google Scholar
  7. 7.
    Nagata, Y. and Burger, M.M. (1974) Wheat germ agglutinin. Molecular characteristics and specificity for sugar binding. J. Biol. Chem. 249, 3116 – 3122.PubMedGoogle Scholar
  8. 8.
    Faye, L., Gomord, V., Fitchette-Lainé, A.-C. and Chrispeels, M.J. (1993) Affinity purification of antibodies specific for Asn-linked glycans containing α1- > 3 fucose or β1- > 2 xylose. Anal. Biochem. 209, 104 – 108.CrossRefPubMedGoogle Scholar
  9. 9.
    Tomiya, N., Kurono, M., Ishihara, H., Tejima, S., Endo, S., Arata, Y. and Takahashi, N. (1987) Structural analysis of N -linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and highperformance liquid chromatography. Anal. Biochem. 163, 489 – 499.CrossRefPubMedGoogle Scholar
  10. 10.
    Ogawa, H., Hijikata, A., Amano, M., Fukushima, H., Ishizuka, I., Kurihara, Y. and Matsumoto, I. (1996) Structures and contribution to the antigenicity of oligosaccharides of Japanese cedar (Cryptomeria japonica) pollen allergen Cry j I: relationship between the structures and antigenic epitopes of plant N -linked complex-type glycans. Glycoconj. J. 13, 555 – 566.CrossRefPubMedGoogle Scholar
  11. 11.
    Séveno, M., Cabrera, G., Trigero, A., Burel, C., Leprince, J., Rihouey, C., Vézina, L.-P., D'aoust, M.-A., Rudd, P.M., Royle, L., Dwek, R.A., Harvey, D.J., Lerouge, P., Cremata, J.A. and Bardor, M. (2008) Plant N-glycan profiling of minute amounts of material. Anal. Biochem. 379(1), 66–72.CrossRefPubMedGoogle Scholar
  12. 12.
    Bakker, H., Bardor, M., Molthoff, J.W.,Gomord, V., Elbers, I., Stevens, L.H., Jordi, W., Lommen, A., Faye, L., Lerouge, P. and Bosch, D. (2001) Galactose-extended glycans of antibodies produced by transgenic plants. Proc. Natl. Acad. Sci. USA 98 (5), 2899 – 2904.CrossRefPubMedGoogle Scholar
  13. 13.
    Rayon, C., Cabanes-Macheteau, M., Loutelier-Bourrhis, C., Saliot-Maire, I., Lemoine, J., Reiter, W.D., Lerouge, P. and Faye, L. (1999) Characterization of N -glycans from Arabidopsis thaliana. Application to a fucose-deficient mutant. Plant Physiol. 119, 725 – 733.CrossRefPubMedGoogle Scholar
  14. 14.
    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.CrossRefGoogle Scholar
  15. 15.
    Bardor, M., Cabanes-Macheteau, M., Faye, L. and Lerouge, P. (2000) Monitoring the N -glycosylation by fluorophore-assisted carbohydrate electrophoresis. Electrophoresis 21, 2550 – 2556.CrossRefPubMedGoogle Scholar
  16. 16.
    Ko, K., Tekoah, Y., Rudd, P.M., Harvey, D.J., Dwek, R.A., Spitsin, S., Hanlon, C.A., Rupprecht, C., Dietzschold, B., Golovkin, M. and Koprowski, H. (2003) Function and glycosylation of plant-derived antiviral monoclonal antibody. Proc. Natl. Acad. Sci. USA 100, 8013 – 8018.CrossRefPubMedGoogle Scholar
  17. 17.
    Triguero, A., Cabrera, G., Cremata, J.A., Yuen, C.-T., Wheeler, J. and Ramirez, 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 Biotech. J. 3, 449 – 457.CrossRefGoogle Scholar
  18. 18.
    Guile, G.R., Rudd, P.M., Wing, D.R., Prime, S.B. and Dwek, R.A. (1996) A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem. 240, 210 – 226.CrossRefPubMedGoogle Scholar
  19. 19.
    Rudd, P.M., Colominas, C., Royle, L., Murphy, N., Hart, E., Merry, A.H., Hebestreit, H.F., Dwek, R.A. (2001) A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N -linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands. Proteomics 1, 285 – 294.CrossRefPubMedGoogle Scholar
  20. 20.
    Harvey, D.J., Dwek, R.A. and Rudd, P.M. (2006) Determining the structure of glycan moieties by mass spectrometry, in “Current Protocols in Protein Science”, (Coligan, J.E., Dunn, B.M., Speicher, D.W. and Wingfield, P. T. eds.), John Wiley and Sons, New York, Unit 12, pp. 12.7.1 – 12.7.18.Google Scholar
  21. 21.
    Royle, L., Radcliffe, C.M., Dwek, R.A. and Rudd, P.M. (2006) Detailed structural analysis of N -glycans released from glycoproteins in SDS-PAGE gel bands using HPLC combined with exoglycosidase array digestions, in “Methods in Molecular Biology, Glycobiology Protocols”, (Brockhausen-Schutzbach, I. ed.), Humana Press, vol. 347, pp. 125–144.Google Scholar
  22. 22.
    Bardor, M., Loutelier-Bourhis, C., Marvin, L., et-al. (1999) Analysis of plant glycoproteins by matrix-assisted laser desorption ionisation mass spectrometry: application to the N -glycosylation of bean phytohemagglutinin. Plant Physiol. Biochem. 37, 319 – 325.CrossRefGoogle Scholar
  23. 23.
    Bardor, M., Faye, L. and Lerouge, P. (1999) Analysis of the N -glycosylation of recombinant glycoproteins produced in transgenic plants. Trends Plant Sci. 4, 376 – 380.CrossRefPubMedGoogle Scholar
  24. 24.
    Bird, C.R., Gearing, A.J.H. and Thorpe, R. (1988) The use of Tween-20 alone as a blocking agent for the immunoblotting can cause artefactual results. J. Immunol. Methods 106, 175 – 179.CrossRefPubMedGoogle Scholar
  25. 25.
    Lainé, A.-C. and Faye, L. (1988) Significant immunological cross-reactivity of plant glycoproteins. Electrophoresis 9, 841 – 844.CrossRefPubMedGoogle Scholar
  26. 26.
    Kubelka, V., Altmann, F., Staudacher, E., Trotter, V., März, L., Hard, K., Kamerling, J.P. and Vliegenthert, J.F.G. (1993) Primary structures of the N -linked carbohydrate chains from honeybee venom phospholipase A2. Eur. J. Biochem. 213, 1193 – 1204.CrossRefPubMedGoogle Scholar
  27. 27.
    Vitale, A., Warner, T.G. and Chrispeels, M.J. (1984) Phaseolus vulgaris phytohemagglutinin contains high-mannose and modified oligoasaccharide chains. Planta 160, 256 – 263.CrossRefGoogle Scholar
  28. 28.
    Bardor, M., Loutelier-Bourhis, C., Paccalet, T., Cosette, P., Fitchette, A.-C., Vézina, L.P., Trepanier, S., Dargis, M., Lemieux, R., Lange, C., Faye, L. and Lerouge, P. (2003) Monoclonal C5-1 antibody produced in transgenic alfalfa plants exhibits a N -glycosylation that is homogeneous and suitable for glyco-engineering into a human-compatible structure. Plant Biotech. J. 1, 451 – 462.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • Muriel Bardor
    • 1
  • Gleysin Cabrera
    • 2
  • Johannes Stadlmann
    • 3
  • Patrice Lerouge
    • 3
  • José A. Cremata
    • 2
  • Véronique Gomord
    • 4
  • Anne-Catherine Fitchette
    • 1
  1. 1.Faculté des SciencesUniversité de RouenFrance
  2. 2.Department of Carbohydrate ChemistryCenter for Genetic Engineering and BiotechnologyHavanaCuba
  3. 3.Glycobiology Division, Department of ChemistryUniversity of Natural Resources and Applied Life SciencesViennaAustria
  4. 4.CNRS, Université de RouenFrance

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