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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
Article

Abstract

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.

Keywords

Aleuria aurantia lectin Fucose Surface plasmon resonance Tryptophan fluorescence Oligosaccharides 

Abbreviations

AAL

Aleuria aurantia lectin

BSA

Bovine serum albumin

CD

Circular dichroism

ELLA

Enzyme-linked lectin assay

Kd

Dissociation constants

MIC

Minimum inhibiting concentration

nAAL

Native aleuria aurantia lectin

PBS

Phosphate buffer saline

rAAL

His-tagged recombinant aleuria aurantia lectin

SPR

Surface plasmon resonance

Notes

Acknowledgements

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.

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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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