title: Engineering of the glycan-binding specificity of Agrocybe cylindracea galectin towards α(2,3)-linked sialic acid by saturation mutagenesis.
abstract: Sialic acid represents a critical sugar component located at the outermost position of glycoconjugates, playing important roles in extensive biological processes. To date, however, there have been only few probes which show affinity to α(2,3)-linked sialic acid-containing glycoconjugates. Agrocybe cylindracea galectin is known to have a relatively high affinity towards Neu5Acα(2,3)Galβ(1,4)Glc (3'-sialyl lactose), but it significantly recognizes various β-galactosides, such as Galβ(1,4)GlcNAcβ (LacNAc) and Galβ(1,3)GalNAcα (T-antigen). To eliminate this background specificity, we focused an acidic amino acid residue (Glu86), which interacts with the glucose unit of 3'-sialyl lactose and substituted it with all other amino acids. Carbohydrate-binding specificity of the derived 14 mutants was analysed by surface plasmon resonance, and it was found that E86D mutant (Glu86 substituted with Asp) substantially lost the binding ability to LacNAc and T-antigen, while it retained the high affinity for 3'-sialyl lactose. Further, frontal affinity chromatography analysis using 132 pyridylaminated oligosaccharides confirmed that the E86D mutant had a strong preference for α(2,3)-disialo biantennary N-linked glycan. However, it showed the large decrease in the affinity for any of the asialo complex-type N-glycans and the glycolipid-type glycans. Thus, the developed mutant E86D will be of practical use in various fields relevant to cell biology and glycotechnology.
authors.affiliation: Research & Development Division, Glyence Co., Ltd., Nagoya, Aichi, Japan. imamura.koji@mbl.co.jp
authors.forename: Koji
authors.initials: K
authors.lastname: Imamura
authors.forename: Hideaki
authors.initials: H
authors.lastname: Takeuchi
authors.forename: Rikio
authors.initials: R
authors.lastname: Yabe
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tateno
authors.forename: Jun
authors.initials: J
authors.lastname: Hirabayashi
mesh_terms: D055362:Agrocybe / Q000235:genetics / Q000378:metabolism*; D037161:Galectins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D016296:Mutagenesis / Q000235:genetics / Q000502:physiology*; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism*; D011134:Polysaccharides / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*
chemical_list: D037161:Galectins; D011134:Polysaccharides; D019158:N-Acetylneuraminic Acid