Detection of a high affinity binding site in recombinantAleuria aurantia lectin
2008 (English)In: Glycoconjugate Journal, ISSN 0282-0080, Vol. 25, no 8, 753-762 p.Article in journal (Refereed) Published
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 Kd 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 fucosecontaining oligosaccharides with Kd 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.
Place, publisher, year, edition, pages
Springer Netherlands , 2008. Vol. 25, no 8, 753-762 p.
Aleuria aurantia lectin, Fucose, Surface plasmon resonance, Tryptophan fluorescence, Oligosaccharides
Cell and Molecular Biology
IdentifiersURN: urn:nbn:se:liu:diva-20306DOI: 10.1007/s10719-008-9135-7OAI: oai:DiVA.org:liu-20306DiVA: diva2:233889
ProjectsStudies of recombinant forms of Aleuria aurantia lectin