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 alpha1-2, alpha1-3 or alpha1-4, but in contrast to the other binding sites in AAL it could not bind oligosaccharides with alpha1-6 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.

Lectins have widely been used in structural and functional studies of complex carbohydrates. They usually bind carbohydrates with relatively low affinity, but compensate for this by multivalency. This multivalent nature of lectins can sometimes produce unwanted reactions such as agglutination or precipitation of target glycoproteins, when using them in different biological and analytical assays. The mushroom lectin Aleuria aurantia binds to fucose-containing oligosaccharides. It is composed of two identical subunits, and each subunit contains five binding sites for fucose. In this study, two forms of recombinant AAL were produced using site-directed mutagenesis. A monomeric form of AAL was produced by exchanging Tyr6 with Arg6, and a single-site fragment of AAL was produced by insertion of an NdeI restriction enzyme cleavage site and a stop codon in the coding sequence. The AAL forms were expressed as His-tagged proteins in Escherichia coli and purified by affinity chromatography. Binding properties of the two AAL forms were performed using surface plasmon resonance, enzyme-linked lectin assay analyses and isothermal titration calorimetry. Both the monomeric AAL (mAAL) and the single-site AAL (S2-AAL) forms retained their capacity to bind fucosylated oligosaccharides. However, both constructs exhibited properties that differed from the intact recombinant AAL (rAAL). mAAL showed similar binding affinities to fucosylated oligosaccharides as rAAL, but had less hemagglutinating capacity. S2-AAL showed a lower binding affinity to fucosylated oligosaccharides and, in contrast to rAAL and mAAL, S2-AAL did not bind to sialylated fuco-oligosaccharides. The study shows that molecular engineering is a highly useful tool for producing lectins with more defined properties such as decreased valency and defined specificities and affinities. Thus, this approach has high potential in developing reliable diagnostic and biological assays for carbohydrate analysis.

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