title: Parallel quantification of lectin-glycan interaction using ultrafiltration.
abstract: Using ultrafiltration membrane, a simple method for screening protein-ligand interaction was developed. The procedure comprises three steps: mixing ligand with protein, ultrafiltration of the solution, and quantification of unbound ligands by HPLC. By conducting analysis with variable protein concentrations, affinity constants were easily obtained. Multiple ligands can be analyzed simultaneously as a mixture, when concentration of ligands was controlled. Feasibility of this method for lectin-glycan interaction analysis was examined using fluorescently labeled high-mannose-type glycans and recombinant intracellular lectins or endo-α-mannosidase mutants. Estimated Ka values of malectin and VIP36 were in good agreement indeed with those evaluated by conventional methods such as isothermal titration calorimetry (ITC) or frontal affinity chromatography (FAC). Finally, several mutants of endo-α-mannosidase were produced and their affinities to monoglucosylated glycans were evaluated.
authors.affiliation: Japan Science and Technology Agency (JST), ERATO, Ito Glycotrilogy Project, Wako, Saitama, Japan. yotakeda@riken.jp
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Takeda
authors.forename: Akira
authors.initials: A
authors.lastname: Seko
authors.forename: Masafumi
authors.initials: M
authors.lastname: Sakono
authors.forename: Masakazu
authors.initials: M
authors.lastname: Hachisu
authors.forename: Akihiko
authors.initials: A
authors.lastname: Koizumi
authors.forename: Kohki
authors.initials: K
authors.lastname: Fujikawa
authors.forename: Yukishige
authors.initials: Y
authors.lastname: Ito
mesh_terms: D002851:Chromatography, High Pressure Liquid; D037102:Lectins / Q000032:analysis* / Q000737:chemistry*; D015394:Molecular Structure; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry*; D014462:Ultrafiltration
chemical_list: D037102:Lectins; D011134:Polysaccharides