Elucidating the selectivity of recombinant forms of Aleuria aurantia lectin using weak affinity chromatography.
Aberrant glycosylation is connected to several pathological conditions and lectins are useful tools to characterize glycosylated biomarkers. The Aleuria aurantia lectin (AAL) is of special interest since it interacts with all types of fucosylated saccharides. AAL has been expressed in Escherichia coli as a fully functional recombinant protein. Engineered variants of AAL have been developed with the aim of creating monovalent lectins with more homogenous binding characteristics. Four different forms of AAL were studied in the present work: native AAL purified from A. aurantia mushrooms, recombinant AAL dimer, recombinant AAL monomer and recombinant AAL site 2 (S2-AAL). The affinities of these AAL forms toward a number of saccharides were determined with weak affinity chromatography (WAC). Disaccharides with fucose linked α1-3 to GlcNAc interacted with higher affinity compared to fucose linked α1-6 or α1-4 and the obtained dissociation constants (K(d)) were in the range of 10μM for all AAL forms. Tetra- and pentasaccharides with fucose in α1-2, α1-3 or α1-4 had K(d) values ranging from 0.1 to 7mM while a large α1-6 fucosylated oligosaccharide had a K(d) of about 20μM. The recombinant multivalent AAL forms and native AAL exhibited similar affinities toward all saccharides, but S2-AAL had a lower affinity especially regarding a sialic acid containing fucosylated saccharide. It was demonstrated that WAC is a valuable technique in determining the detailed binding profile of the lectins. Specific advantages with WAC include a low consumption of non-labeled saccharides, possibility to analyze mixtures and a simple procedure using standard HPLC equipment.
School of Natural Sciences, Linnæus University, SE-39182 Kalmar, Sweden. maria.bergstrom@lnu.se
Maria
M
Bergström
Eva
E
Aström
Peter
P
Påhlsson
Sten
S
Ohlson
D000117:Acetylglucosamine; D001667:Binding, Competitive; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005643:Fucose / Q000378:metabolism; D055767:Immobilized Proteins / Q000737:chemistry / Q000378:metabolism; D037102:Lectins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004718:Saccharomycetales / Q000235:genetics
D055767:Immobilized Proteins; D037102:Lectins; D009844:Oligosaccharides; D011994:Recombinant Proteins; C064549:lectin, Aleuria aurantia; D005643:Fucose; D000117:Acetylglucosamine