Glycoconjugate Journal

, 25:753 | Cite as

Detection of a high affinity binding site in recombinant Aleuria aurantia lectin

  • Johan OlaussonEmail author
  • Lena Tibell
  • Bengt-Harald Jonsson
  • Peter Påhlsson


Lectins are carbohydrate binding proteins that are involved in many recognition events at molecular and cellular levels. Lectin-oligosaccharide interactions are generally considered to be of weak affinity, however some mushroom lectins have unusually high binding affinity towards oligosaccharides with K d values in the micromolar range. This would make mushroom lectins ideal candidates to study protein–carbohydrate interactions. In the present study we investigated the properties of a recombinant form of the mushroom lectin Aleuria aurantia (AAL). AAL is a fucose-binding lectin composed of two identical 312-amino acid subunits. Each subunit contains five binding sites for fucose. We found that one of the binding sites in rAAL had unusually high affinities towards fucose and fucose-containing oligosaccharides with K d values in the nanomolar range. This site could bind to oligosaccharides with fucose linked α1-2, α1-3 or α1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with α1-6 linked fucose. This binding site is not detected in native AAL (nAAL) one possible explanation may be that this site is blocked with free fucose in nAAL. Recombinant AAL was produced in E. coli as a His-tagged protein, and purified in a one-step procedure. The resulting protein was analyzed by electrophoresis, enzyme-linked lectin assay and circular dichroism spectroscopy, and compared to nAAL. Binding properties were measured using tryptophan fluorescence and surface plasmon resonance. Removal of the His-tag did not alter the binding properties of recombinant AAL in the enzyme-linked lectin assay. Our study forms a basis for understanding the AAL-oligosaccharide interaction and for using molecular techniques to design lectins with novel specificities and high binding affinities towards oligosaccharides.


Aleuria aurantia lectin Fucose Surface plasmon resonance Tryptophan fluorescence Oligosaccharides 



Aleuria aurantia lectin


Bovine serum albumin


Circular dichroism


Enzyme-linked lectin assay


Dissociation constants


Minimum inhibiting concentration


Native aleuria aurantia lectin


Phosphate buffer saline


His-tagged recombinant aleuria aurantia lectin


Surface plasmon resonance



This study was supported by grants from the Health Research Council of Southeast of Sweden. We thank Dr. Maria Bergström, Kalmar University, for critically reading the manuscript and valuable discussions.


  1. 1.
    Liljeblad, M., Lundblad, A., Pahlsson, P.: Analysis of glycoproteins in cell culture supernatants using a lectin immunosensor technique. Biosens. Bioelectron. 17, 883–891 (2002)PubMedCrossRefGoogle Scholar
  2. 2.
    Rudiger, H., Gabius, H.J.: Plant lectins: occurrence, biochemistry, functions and applications. Glycoconj. J. 18, 589–613 (2001)PubMedCrossRefGoogle Scholar
  3. 3.
    Yamashita, K., Kochibe, N., Ohkura, T., Ueda, I., Kobata, A.: Fractionation of L-fucose-containing oligosaccharides on immobilized Aleuria aurantia lectin. J. Biol. Chem. 260, 4688–4693 (1985)PubMedGoogle Scholar
  4. 4.
    Kochibe, N., Furukawa, K.: Purification and properties of a novel fucose-specific hemagglutinin of Aleuria aurantia. Biochemistry. 19, 2841–2846 (1980)PubMedCrossRefGoogle Scholar
  5. 5.
    Debray, H., Montreuil, J.: Aleuria aurantia agglutinin. A new isolation procedure and further study of its specificity towards various glycopeptides and oligosaccharides. Carbohydr. Res. 185, 15–26 (1989)PubMedCrossRefGoogle Scholar
  6. 6.
    Fukumori, F., Takeuchi, N., Hagiwara, T., Ito, K., Kochibe, N., Kobata, A., Nagata, Y.: Cloning and expression of a functional fucose-specific lectin from an orange peel mushroom, Aleuria aurantia. FEBS Lett. 250, 153–156 (1989)PubMedCrossRefGoogle Scholar
  7. 7.
    Nagata, Y., Fukumori, F., Sakai, H., Hagiwara, T., Hiratsuka, Y., Kochibe, N., Kobata, A.: Crystallization and characterization of a lectin obtained from a mushroom, Aleuria aurantia. Biochim. Biophys. Acta. 1076, 187–190 (1991)PubMedGoogle Scholar
  8. 8.
    Wimmerova, M., Mitchell, E., Sanchez, J.F., Gautier, C., Imberty, A.: Crystal structure of fungal lectin: six-bladed beta-propeller fold and novel fucose recognition mode for Aleuria aurantia lectin. J. Biol. Chem. 278, 27059–27067 (2003)PubMedCrossRefGoogle Scholar
  9. 9.
    Kobata, A., Endo, T.: Immobilized lectin columns: useful tools for the fractionation and structural analysis of oligosaccharides. J. Chromatogr. 597, 111–122 (1992)PubMedCrossRefGoogle Scholar
  10. 10.
    Ryden, I., Lundblad, A., Pahlsson, P.: Lectin ELISA for analysis of alpha(1)-acid glycoprotein fucosylation in the acute phase response. Clin. Chem. 45, 2010–2012 (1999)PubMedGoogle Scholar
  11. 11.
    Ryden, I., Pahlsson, P., Lindgren, S.: Diagnostic accuracy of alpha(1)-acid glycoprotein fucosylation for liver cirrhosis in patients undergoing hepatic biopsy. Clin. Chem. 48, 2195–2201 (2002)PubMedGoogle Scholar
  12. 12.
    Ryden, I., Pahlsson, P., Lundblad, A., Skogh, T.: Fucosylation of alpha1-acid glycoprotein (orosomucoid) compared with traditional biochemical markers of inflammation in recent onset rheumatoid arthritis. Clin. Chim. Acta. 317, 221–229 (2002)PubMedCrossRefGoogle Scholar
  13. 13.
    Hashimoto, S., Asao, T., Takahashi, J., Yagihashi, Y., Nishimura, T., Saniabadi, A.R., Poland, D.C., van Dijk, W., Kuwano, H., Kochibe, N., Yazawa, S.: alpha1-acid glycoprotein fucosylation as a marker of carcinoma progression and prognosis. Cancer. 101, 2825–2836 (2004)PubMedCrossRefGoogle Scholar
  14. 14.
    Fujihashi, M., Peapus, D.H., Kamiya, N., Nagata, Y., Miki, K.: Crystal structure of fucose-specific lectin from Aleuria aurantia binding ligands at three of its five sugar recognition sites. Biochemistry. 42, 11093–11099 (2003)PubMedCrossRefGoogle Scholar
  15. 15.
    Amano, K., Takase, M., Ando, A., Nagata, Y.: Production of functional lectin in Pichia pastoris directed by cloned cDNA from Aleuria aurantia. Biosci. Biotechnol. Biochem. 67, 2277–2279 (2003)PubMedCrossRefGoogle Scholar
  16. 16.
    Imberty, A., Mitchell, E.P., Wimmerova, M.: Structural basis of high-affinity glycan recognition by bacterial and fungal lectins. Curr. Opin. Struck. Biol. 15, 525–534 (2005)CrossRefGoogle Scholar
  17. 17.
    Kostlanova, N., Mitchell, E.P., Lortat-Jacob, H., Oscarson, S., Lahmann, M., Gilboa-Garber, N., Chambat, G., Wimmerova, M., Imberty, A.: The fucose-binding lectin from Ralstonia solanacearum. A new type of beta-propeller architecture formed by oligomerization and interacting with fucoside, fucosyllactose, and plant xyloglucan. J Biol. Chem. 280, 27839–27849 (2005)PubMedCrossRefGoogle Scholar
  18. 18.
    Tateno, H., Winter, H.C., Goldstein, I.J.: Cloning, expression in Escherichia coli and characterization of the recombinant Neu5Acalpha2,6Galbeta1,4GlcNAc-specific high-affinity lectin and its mutants from the mushroom Polyporus squamosus. Biochem. J. 382, 667–675 (2004)PubMedCrossRefGoogle Scholar
  19. 19.
    Amano, K., Fujihashi, M., Ando, A., Miki, K., Nagata, Y.: Involvement of tyrosines at fucose-binding sites of Aleuria aurantia lectin: non-equal response to site-directed mutagenesis among five sites. Biosci. Biotechnol. Biochem. 68, 841–847 (2004)PubMedCrossRefGoogle Scholar
  20. 20.
    Streicher, H., Sharon, N.: Recombinant plant lectins and their mutants. Methods Enzymol. 363, 47–77 (2003)PubMedCrossRefGoogle Scholar
  21. 21.
    Sawardeker, J.S., Sloneker, J.H., Jeanes, A.: Quantitative determination of monosaccharides as their alditol acetates by gas liquid chromatography. Anal. Biochem. 37, 1602–1604 (1965)Google Scholar
  22. 22.
    Haselhorst, T., Weimar, T., Peters, T.: Molecular recognition of sialyl Lewis(x) and related saccharides by two lectins. J. Am. Chem. Soc. 123, 10705–10714 (2001)PubMedCrossRefGoogle Scholar
  23. 23.
    Glaudemans, P.J., Miller, E., Eugenia, M.: The measurement of affinity between ligand and antibody using ligand-induced antibody fluorescence change. Carbohydr. Res. 300, 169–170 (1997)PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Johan Olausson
    • 1
    Email author
  • Lena Tibell
    • 1
  • Bengt-Harald Jonsson
    • 2
  • Peter Påhlsson
    • 1
  1. 1.Department of Clinical and Experimental Medicine, Division of Cell BiologyLinköping UniversityLinköpingSweden
  2. 2.Molecular Biotechnology/IFMLinköping UniversityLinköpingSweden

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