title: 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.
authors: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan. Electronic address: t-take@josai.ac.jp.
authors: Tomoharu
authors: T
authors: Takeuchi
authors: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors: Mayumi
authors: M
authors: Tamura
authors: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors: Nobuaki
authors: N
authors: Ishii
authors: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors: Hiroki
authors: H
authors: Ishikida
authors: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors: Saori
authors: S
authors: Sugimoto
authors: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors: Daichi
authors: D
authors: Suzuki
authors: Laboratory of Synthetic Organic and Medicinal Chemistry, School of Pharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors: Kazusa
authors: K
authors: Nishiyama
authors: Laboratory of Synthetic Organic and Medicinal Chemistry, School of Pharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors: Hideyo
authors: H
authors: Takahashi
authors: Laboratory of Synthetic Organic and Medicinal Chemistry, School of Pharmaceutical Sciences, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo 173-8605, Japan.
authors: Hideaki
authors: H
authors: Natsugari
authors: Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Josai University, 1-1 Keyakidai, Sakado, Saitama 350-0295, Japan.
authors: Yoichiro
authors: Y
authors: Arata
mesh_terms: D000818:Animals; D017173:Caenorhabditis elegans / Q000737:chemistry* / Q000235:genetics; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000379:methods*; D004187:Disaccharides / Q000737:chemistry*; D005643:Fucose / Q000737:chemistry*; D005690:Galactose / Q000737:chemistry*; D037161:Galectins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D009154:Mutation
chemical_list: D029742:Caenorhabditis elegans Proteins; D004187:Disaccharides; D037161:Galectins; C534045:LEC-6 protein, C elegans; D005643:Fucose; D005690:Galactose
keywords: Affinity purification; Frontal affinity chromatography; Galactoseβ1–4Fucose; Galectin; Galectin-1