Purification of galectin-1 mutants using an immobilized Galactoseβ1–4Fucose affinity adsorbent

Abstract

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 β-galactoside sugars such as Galβ1–4GlcNAc. 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 Galβ1–4GlcNAc 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 Galβ1–4Fuc, 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 Galβ1–4GlcNAc and Galβ1–4Fuc. 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 Galβ1–4Fuc–Sepharose. The affinity of the purified mutant galectins for glycans containing Galβ1–4Fuc or Galβ1–4GlcNAc moieties was quantitatively examined by frontal affinity chromatography, and the results indicated that the mutants retained the affinity only for Galβ1–4Fuc. Given that other mammalian galectins are known to bind Galβ1–4Fuc, our data suggest that immobilized Galβ1–4Fuc ligands could be generally used for easy one-step affinity purification of mutant galectins.