Production and characterization of a monomeric form and a single-site form of Aleuria aurantia lectin

Abstract

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.

Reverse lectin ELISA for detecting fucosylated forms of α1-acid glycoprotein associated with hepatocellular carcinoma

“…In contrast to AAL, S2 will not bind sialylated/fucosylated epitopes such as SLe x and SLe a and has strongest affinity towards α1–6 fucosylated (core-fucosylated) and multifucosylated oligosaccharide structures [17]. Since S2 also binds to fucosylated structures with a lower overall affinity than AAL, it is possible that only multifucosylated glycoforms of AGP will have affinity enough to bind to S2.…”

“…To overcome some of these problems we have previously produced a recombinant form of AAL comprising only binding site 2, (S2) [17]. This monovalent lectin showed a more restricted binding towards fucosylated oligosaccharides with reduced binding towards sialylated/fucosylated oligosaccharides compared to AAL.…”

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

“…Homodimer 4 Gal na Entomotoxic (Hamshou et al 2013;Hamshou et al 2010b;Hamshou et al 2012;Skamnaki et al 2013) Bleuler-Martinez et al 2011;Fujihashi et al 2003;Fukumori et al 1990;Kochibe and Furukawa 1980;Olausson et al 2010;Olausson et al 2008;Tateno et al 2009;Wimmerova et al 2003) GlcNAc-β1,4-X na nd (Cioci et al 2006;Ueda et al 1999a;Ueda et al 2002;Ueda et al 1999b;Ueda et al 2003) Entomotoxic (Birck et al 2004;Bleuler-Martinez et al 2011;Damian et al 2005;Francis et al 2003;Jaber et al 2008;Jaber et al 2007;…”

Structural insights intoAspergillus fumigatuslectin specificity: AFL binding sites are functionally non-equivalent

“…The average affinity for fucose is in the micromolar range (Wimmerova et al, 2003), but it has been proposed that some of the individual sites display much higher affinities (Olausson et al, 2008). Several efforts have been made to understand the structure-function relationship at the binding-site level in order to correlate the composition of each binding site with its specificity and affinity (Amano et al, 2004;Olausson et al, 2011;Romano et al, 2011). The studies of bacterial structural homologues (Audfray et al, 2011;Kostlánová et al, 2005) are beneficial but, as these lectins are much smaller and bear only two types of binding sites, they cannot completely explain the observed variability in AAL.…”