Glycoconjugate Journal

, Volume 29, Issue 7, pp 467–479 | Cite as

Expression analysis of a type S2 EUL-related lectin from rice in Pichia pastoris

  • Bassam Al Atalah
  • Pierre Rougé
  • David F. Smith
  • Paul Proost
  • Yi Lasanajak
  • Els J. M. Van Damme


Rice (Oryza sativa) expresses different putative carbohydrate-binding proteins belonging to the class of lectins containing an Euonymus lectin (EUL)-related domain, one of them being OrysaEULS2. The OrysaEULS2 sequence consists of a 56 amino acid N-terminal domain followed by the EUL sequence. In this paper the original sequence of the EUL domain of OrysaEULS2 and some mutant forms have been expressed in Pichia pastoris. Subsequently, the recombinant proteins were purified and their carbohydrate binding properties determined. Analysis of the original protein on the glycan array revealed interaction with mannose containing structures and to a lesser extent with glycans containing lactosamine related structures. It was shown that mutation of tryptophan residue 134 into leucine resulted in an almost complete loss of carbohydrate binding activity of OrysaEULS2. Our results show that the EUL domain in OrysaEULS2 interacts with glycan structures, and hence can be considered as a lectin. However, the binding of the protein with the array is much weaker than that of other EUL-related lectins. Furthermore, our results indicate that gene divergence within the family of EUL-related lectins lead to changes in carbohydrate binding specificity.


Lectin Carbohydrate-binding Mutant EUL protein Glycan array 



This work was funded primarily by the Fund for Scientific Research – Flanders (FWO grants G.0022.08 and KAN, the Research Council of Ghent University (projects BOF2005 ⁄ GOA ⁄ 008 and BOF2007 ⁄ GOA ⁄ 0017). Bassam Al Atalah is recipient of a doctoral grant from the Special Research Council of Ghent University. The authors want to thank the Consortium for Functional Glycomics funded by the NIGMS GM62116 for the glycan array analysis. The authors also want to thank the Rice Genome Resource Center, National Institute of Agrobiological Sciences, Japan for providing the cDNA clone encoding OrysaEULS2.


  1. 1.
    Peumans, W.J., Van Damme, E.J.M.: Lectins as plant defense proteins. Plant Physiol. 109, 347–352 (1995)PubMedCrossRefGoogle Scholar
  2. 2.
    Van Damme, E.J.M., Lannoo, N., Fouquaert, E., Peumans, W.J.: The identification of inducible cytoplasmic/nuclear carbohydrate-binding proteins urges to develop novel concepts about the role of plant lectins. Glycoconj. J. 20, 449–460 (2004)PubMedCrossRefGoogle Scholar
  3. 3.
    Van Damme, E.J.M., Lannoo, N., Peumans, W.J.: Plant lectins. Adv. Bot. Res. 48, 107–209 (2008)Google Scholar
  4. 4.
    Fouquaert, E., Peumans, W.J., Smith, D.F., Proost, P., Savvides, S., Van Damme, E.J.M.: The old Euonymus europaeus agglutinin represents a novel family of ubiquitous plant proteins. Plant Physiol. 147, 1316–1324 (2008)PubMedCrossRefGoogle Scholar
  5. 5.
    Fouquaert, E., Peumans, W.J., Vandekerckhove, T.T.M., Ongenaert, M., Van Damme, E.J.M.: Proteins with an Euonymus lectin-like domain are ubiquitous in Embryophyta. BMC Plant Biol. 9, 136 (2009)PubMedCrossRefGoogle Scholar
  6. 6.
    Moons, A., Gielen, J., Vandekerckhove, J., Van Der Straeten, D., Gheysen, G., Van Montagu, M.: An abscisic-acid and salt-stress-responsive rice cDNA from a novel plant gene family. Planta 202, 443–454 (1997)PubMedCrossRefGoogle Scholar
  7. 7.
    Cereghino, J.L., Cregg, J.M.: Heterologous protein, expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 24, 45–66 (2000)PubMedCrossRefGoogle Scholar
  8. 8.
    Macauley-Patrick, S., Fazenda, M.L., McNeil, B., Harvey, L.M.: Heterologous protein production using the Pichia pastoris expression system. Yeast 22, 249–270 (2005)PubMedCrossRefGoogle Scholar
  9. 9.
    Daly, R., Hearn, M.T.W.: Expression of heterologous proteins in Pichia pastoris: a useful experimental tool in protein engineering and production. J. Mol. Recognit. 18, 119–138 (2005)PubMedCrossRefGoogle Scholar
  10. 10.
    Elias, S.B., Brust, P.F., Koutz, P.J., Waters, A.F., Harpold, M.M., Gingeras, T.R.: Isolation of alcohol oxidase and two other methanol regulatable genes from the yeast Pichia pastoris. Mol. Cell. Biol. 5, 1111–1121 (1985)Google Scholar
  11. 11.
    Hartner, F.S., Glieder, A.: Regulation of methanol utilisation pathway genes in yeasts. Microb. Cell Fact. 5, 39 (2006)PubMedCrossRefGoogle Scholar
  12. 12.
    Lannoo, N., Vervecken, W., Proost, P., Rougé, P., Van Damme, E.J.M.: Expression of the nucleocytoplasmic tobacco lectin in the yeast Pichia pastoris. Protein Expr. Purif. 53, 275–282 (2007)PubMedCrossRefGoogle Scholar
  13. 13.
    Fouquaert, E., Smith, D.F., Peumans, W.J., Proost, P., Balzarini, J., Savvides, S.N., Van Damme, E.J.M.: Related lectins from snowdrop and maize differ in their carbohydrate- binding specificity. Biochem. Biophys. Res. Commun. 380, 260–265 (2009)PubMedCrossRefGoogle Scholar
  14. 14.
    Al Atalah, B., Fouquaert, E., Vanderschaeghe, D., Proost, P., Balzarini, J., Smith, D.F., Rougé, P., Lasanajak, Y., Callewaert, N., Van Damme, E.J.M.: Expression analysis of the nucleocytoplasmic lectin “Orysata” from rice in Pichia pastoris. FEBS J. 278, 2064–2079 (2011)PubMedCrossRefGoogle Scholar
  15. 15.
    Van Hove, J., Fouquaert, E., Smith, D.F., Proost, P., Van Damme, E.J.M.: Lectin activity of the nucleocytoplasmic EUL protein from Arabidopsis thaliana. Biochem. Biophys. Res. Commun. 414, 101–105 (2011)PubMedCrossRefGoogle Scholar
  16. 16.
    Treiber, N., Reinert, D.J., Carpusca, I., Aktories, K., Schulz, G.E.: Structure and mode of action of a mosquitocidal holotoxin. J. Mol. Biol. 381, 150–159 (2008)PubMedCrossRefGoogle Scholar
  17. 17.
    Krieger, E., Koraimann, G., Vriend, G.: Increasing the precision of comparative models with YASARA NOVA - a self-parameterizing force field. Proteins 47, 393–402 (2002)PubMedCrossRefGoogle Scholar
  18. 18.
    Gaboriaud, C., Bissery, V., Benchetrit, T., Mornon, J.P.: Hydrophobic cluster analysis: an efficient new way to compare and analyse amino acid sequences. FEBS Lett. 224, 149–155 (1987)PubMedCrossRefGoogle Scholar
  19. 19.
    Laskowski, R.A., MacArthur, M.W., Moss, D.S., Thornton, J.M.: PROCHECK: a program to check the stereochemistry of protein structures. J. Appl. Cryst. 26, 283–291 (1993)CrossRefGoogle Scholar
  20. 20.
    Nicholls, A., Sharp, K.A., Honig, B.: Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins Struc. Func. Genet. 11, 281–296 (1991)CrossRefGoogle Scholar
  21. 21.
    Gilson, M.K., Honig, B.H.: Calculation of electrostatic potential in an enzyme active site. Nature 330, 84–86 (1987)PubMedCrossRefGoogle Scholar
  22. 22.
    Blixt, O., Head, S., Mondala, T., Scanlan, C., Huflejt, M.E., Alvarez, R., Bryan, M.C., Fazio, F., Calarese, D., Stevens, J., Skehel, J., van Die, I., Burton, R., Wilson, A., Cummings, R., Bovin, N., Wong, C.-H., Paulson, C.: Printed covalent glycan array for ligand profiling of diverse glycan binding proteins. Proc. Natl. Acad. Sci. U.S.A. 101, 17033–17038 (2004)PubMedCrossRefGoogle Scholar
  23. 23.
    Bradford, M.M.: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976)PubMedCrossRefGoogle Scholar
  24. 24.
    Laemmli, U.K.: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685 (1970)PubMedCrossRefGoogle Scholar
  25. 25.
    Van Damme, E.J.M., Hao, Q., Chen, Y., Barre, A., Vandenbussche, F., Desmyter, S., Rougé, P., Peumans, W.J.: Ribosome-inactivating proteins: a family of plant proteins that do more than inactivate ribosomes. Crit. Rev. Plant Sci. 20, 395–465 (2001)Google Scholar
  26. 26.
    Arndt, J.W., Gu, J., Jaroszewski, L., Schwarzenbacher, R., Hanson, M.A., Lebeda, F.J., Stevens, R.C.: The structure of the neurotoxin-associated protein HA33/A from Clostridium botulinum suggests a reoccurring β-trefoil fold in the progenitor toxin complex. J. Mol. Biol. 346, 1083–1093 (2005)PubMedCrossRefGoogle Scholar
  27. 27.
    Van Damme, E.J.M., Rougé, P., Peumans, W.J.: Carbohydrate-protein interactions: plant lectins. In: Kamerling, J.P., Boons, G.J., Lee, Y.C., Suzuki, A., Taniguchi, N., Voragen, A.G.J. (eds.) Comprehensive glycoscience - from chemistry to systems biology, pp. 563–599. Elsevier, New York (2007)Google Scholar
  28. 28.
    Smith, D.F., Song, X., Cummings, R.D.: Use of glycan microarrays to explore specificity of glycan-binding proteins. In: Fukuda, M. (ed.) Meth. Enzymol, vol. 480, pp. 417–444. Academic, Burlington (2010)Google Scholar
  29. 29.
    Van Damme, E.J.M., Barre, A., Rougé, P., Peumans, W.J.: Cytoplasmic/ nuclear plant lectins: a new story. Trends Plant Sci. 9, 484–489 (2004)PubMedCrossRefGoogle Scholar
  30. 30.
    Oliveira, C., Felix, W., Moreira, R.A., Teixeira, J.A., Domingues, L.: Expression of frutalin, an α-D-galactose-binding jacalin-related lectin, in the yeast Pichia pastoris. Protein Expr. Purif. 60, 188–193 (2008)PubMedCrossRefGoogle Scholar
  31. 31.
    Sreekrishna, K., Brankamp, R.G., Kropp, K.E., Blankenship, D.T., Tsay, J.T., Smith, P.L., Wierschke, J.D., Subramaniam, A., Birkenberger, L.A.: Strategies for the optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris. Gene 190, 55–62 (1997)PubMedCrossRefGoogle Scholar
  32. 32.
    Rutenber, E., Katzin, B.J., Ernst, S., Collins, E.J., Mlsna, D., Ready, M.P., Robertus, J.D.: Crystallographic refinement of ricin to 2.5-Å. Proteins 10, 240–250 (1991)PubMedCrossRefGoogle Scholar
  33. 33.
    Fujimoto, Z., Kuno, A., Kaneko, S., Kobayashi, H., Kusakabe, I., Mizuno, H.: Crystal structures of the sugar complexes of Streptomyces olivaceoviridis E-86 xylanase: sugar binding structure of the family 13 carbohydrate binding module. J. Mol. Biol. 316, 65–78 (2002)PubMedCrossRefGoogle Scholar
  34. 34.
    Arndt, J.W., Gu, J., Jaroszewski, L., Schwarzenbacher, R., Hanson, M.A., Lebeda, F.J., Stevens, R.C.: The structure of the neurotoxin-associated protein HA33/A from Clostridium botulinum suggests a reoccurring β-trefoil fold in the progenitor toxin complex. J. Mol. Biol. 346, 1083–1093 (2005)PubMedCrossRefGoogle Scholar
  35. 35.
    Schouppe, D., Rougé, P., Lasanajak, Y., Barre, A., Smith, D.F., Proost, P., Van Damme, E.J.M.: Mutational analysis of the carbohydrate binding activity of the tobacco lectin. Glycoconj. J. 27, 613–623 (2010)PubMedCrossRefGoogle Scholar
  36. 36.
    Loris, R., Hamelryck, T., Bouckaert, J., Wyns, L.: Legume lectin structure. Biochim. Biophys. Acta 1383, 9–36 (1998)PubMedCrossRefGoogle Scholar
  37. 37.
    Rougé, P., Peumans, W.J., Barre, A., Van Damme, E.J.M.: A structural basis for the difference in specificity between the two jacalin-related lectins from mulberry (Morus nigra) bark. Biochem. Biophys. Res. Commun. 304, 91–97 (2003)PubMedCrossRefGoogle Scholar
  38. 38.
    Hao, Q., Van Damme, E.J.M., Hause, B., Barre, A., Chen, Y., Rougé, P., Peumans, W.J.: Iris bulbs express type 1 and type 2 ribosome inactivating proteins with unusual properties. Plant Physiol. 125, 866–876 (2001)PubMedCrossRefGoogle Scholar
  39. 39.
    Notenboom, V., Boraston, A.B., Williams, S.J., Kilburn, D.G., Rose, D.R.: High-resolution crystal structures of the lectin-like xylan binding domain from Streptomyces lividans xylanase 10A with bound substrates reveal a novel mode of xylan binding. Biochemistry 41, 4246–4254 (2002)PubMedCrossRefGoogle Scholar
  40. 40.
    Stanley, P., Cummings, R.D.: Structures common to different glycans. In: Varki, A., Cummings, R.D., Esko, J.D., Freeze, H.H., Stanley, P., Bertozzi, C.R., Hart, G.W., Etzler, M.E. (eds.) Essentials of glycobiology, 2nd edition, Chapter 13, pp. 175–198. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2009)Google Scholar
  41. 41.
    Preston, A., Mandrell, R.E., Gibson, B.W., Apicella, M.A.: The lipooligosaccharides of pathogenic gram-negative bacteria. Crit. Rev. Microbiol. 22, 139–180 (1996)PubMedCrossRefGoogle Scholar
  42. 42.
    Wang, G., Ge, Z., Rasko, D.A., Taylor, D.E.: Lewis antigens in Helicobacter pylori: biosynthesis and phase variation. Mol. Microbiol. 36, 1187–1196 (2000)PubMedCrossRefGoogle Scholar
  43. 43.
    Monzavi-Karbassi, B., Luo, P., Cunto-Amesty, G., Jousheghany, F., Pashov, A., Weissman, D., Kieber-Emmons, T.: Fucosylated lactosamines participate in adhesion of HIV-1 envelope glycoprotein to dendritic cells. Arch. Virol. 149, 75–91 (2004)PubMedCrossRefGoogle Scholar
  44. 44.
    Leonard, R.: The presence of Lewis A epitopes in Arabidopsis thaliana glycoconjugates depends on an active α4-fucosyltransferase gene. Glycobiology 12, 299–306 (2002)PubMedCrossRefGoogle Scholar
  45. 45.
    Melo, N.: Identification of the human Lewis A carbohydrate motif in a secretory peroxidase from a plant cell suspension culture (Vaccinium myrtillus L.). FEBS Lett. 415, 186–191 (1997)PubMedCrossRefGoogle Scholar
  46. 46.
    Dam, T.K.: Fine specificities of two lectins from Cymbosema roseum seeds: a lectin specific for high-mannose oligosaccharides and a lectin specific for blood group H type II trisaccharide. Glycobiology 21, 925–933 (2011)PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Bassam Al Atalah
    • 1
  • Pierre Rougé
    • 2
  • David F. Smith
    • 3
  • Paul Proost
    • 4
  • Yi Lasanajak
    • 3
  • Els J. M. Van Damme
    • 1
  1. 1.Laboratory of Biochemistry and Glycobiology, Department of Molecular BiotechnologyGhent UniversityGhentBelgium
  2. 2.Signaux et Messages Cellulaires chez les Végétaux, UMR CNRS-UPS 5546, Pole de Biotechnologie végétaleCastanet-TolosanFrance
  3. 3.Department of BiochemistryEmory University School of MedicineAtlantaUSA
  4. 4.Laboratory of Molecular Immunology, Rega Institute for Medical ResearchKatholieke Universiteit LeuvenLeuvenBelgium

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