RESUMO
Galectins are a family of lectins characterized by their carbohydrate recognition domains containing eight conserved amino acid residues, which allows the binding of galectin to ß-galactoside sugars such as Galß1-4GlcNAc. Since galectin-glycan interactions occur extracellularly, recombinant galectins are often used for the functional analysis of these interactions. Although it is relatively easy to purify galectins via affinity to Galß1-4GlcNAc using affinity adsorbents such as asialofetuin-Sepharose, it could be difficult to do so with mutated galectins, which may have reduced affinity towards their endogenous ligands. However, this is not the case with Caenorhabditis elegans galectin LEC-6; binding to its endogenous recognition unit Galß1-4Fuc, a unique disaccharide found only in invertebrates, is not necessarily affected by point mutations of the eight well-conserved amino acids. In this study, we constructed mutants of mouse galectin-1 carrying substitutions of each of the eight conserved amino acid residues (H44F, N46D, R48H, V59A, N61D, W68F, E71Q, and R73H) and examined their affinity for Galß1-4GlcNAc and Galß1-4Fuc. These mutants, except W68F, had very low affinity for asialofetuin-Sepharose; however, most of them (with the exception of H44F and R48H) could be purified using Galß1-4Fuc-Sepharose. The affinity of the purified mutant galectins for glycans containing Galß1-4Fuc or Galß1-4GlcNAc moieties was quantitatively examined by frontal affinity chromatography, and the results indicated that the mutants retained the affinity only for Galß1-4Fuc. Given that other mammalian galectins are known to bind Galß1-4Fuc, our data suggest that immobilized Galß1-4Fuc ligands could be generally used for easy one-step affinity purification of mutant galectins.
