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

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 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.

Entomotoxic and nematotoxic lectins and protease inhibitors from fungal fruiting bodies

“…Each of the AAL protomers contains six potential binding sites for terminal fucose residues, but their affinities have been shown not to be equivalent. One binding site is not functional at all, while another has a much higher affinity than the others and was occupied by free fucose when the hololectin was isolated from fruiting bodies (Olausson et al 2008). The primary sequence and fold of AFL are very similar to those of AAL, but, in contrast to AAL, all six binding sites, although not equivalent, are functional in that they bind fucose (Houser et al 2015).…”

“…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;…”

Optimization of the enzyme-linked lectin assay for enhanced glycoprotein and glycoconjugate analysis

“…BSA is the most commonly used blocking agents used in ELISA methods and its use in ELLA's has also been reported (24,35). We evaluated the suitability of BSA for use as a blocking agent in broad spectrum lectin screens (6,36).…”

Development of recombinant Aleuria aurantia lectins with altered binding specificities to fucosylated glycans

“…The Aleuria aurantia lectin (AAL) detects various fucose linkages (α-1,2; α-1,3; α-1,4; α-1,6) attached to either a N -acetylglucosamine (GlcNAc) or a galactose residue. The crystal structure for AAL has been solved by several groups and reveals that AAL monomers display a six-bladed β-propeller fold with five conserved fucose binding sites located between each blade [17-19]. The sixth site does not contain the conserved amino acids residues required to form the fucose binding pocket and it is proposed that the sixth site forms closure contacts between blades 1 and 6 to help stabilize the overall β-propeller structure [18].…”