Production and characterization of a monomeric form and a single-site form of Aleuria aurantia lectin.
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.
Olausson
Johan
J
Department of Clinical and Experimental Medicine, Division of Cell Biology, Linköping University, Linköping, Sweden.
Aström
Eva
E
Jonsson
Bengt-Harald
BH
Tibell
Lena A E
LA
Påhlsson
Peter
P
D001665:Binding Sites; D002846:Chromatography, Affinity; D005643:Fucose / Q000737:chemistry / Q000378:metabolism; D006386:Hemagglutination Tests; D037102:Lectins / Q000737:chemistry* / Q000302:isolation & purification; D020349:Surface Plasmon Resonance
D037102:Lectins; C064549:lectin, Aleuria aurantia; D005643:Fucose