title: Engineering of a 3'-sulpho-Galβ1-4GlcNAc-specific probe by a single amino acid substitution of a fungal galectin.
abstract: Among sulphated glycans, little is known about 3'-sulphation because of the lack of useful probes. In the course of molecular engineering of a fungal galectin from Agrocybe cylindracea, we found that a single substitution of Glu86 with alanine resulted in acquisition of specific binding for the 3'-sulpho-Galβ1-4GlcNAc structure. Extensive glyco-technological analysis revealed that this property was obtained in a 'loss-of-function' manner. Though this mutant (E86A) had low total affinity, it showed substantial binding to a naturally occurring N-glycan, of which the terminal galactose is 3-sulphated. Moreover, E86A specifically bound to HeLa cells, in which galactose-3-O-sulfotransferases (Gal3ST2 or Gal3ST3) were over-expressed.
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China; Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; Division of Functional Glycomics, Life Science Research Center, Institute of Research Promotion, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-Gun, Kagawa 761-0793, Japan; and Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China; Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; Division of Functional Glycomics, Life Science Research Center, Institute of Research Promotion, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-Gun, Kagawa 761-0793, Japan; and Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Dan
authors.initials: D
authors.lastname: Hu
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China; Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; Division of Functional Glycomics, Life Science Research Center, Institute of Research Promotion, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-Gun, Kagawa 761-0793, Japan; and Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Hang
authors.initials: H
authors.lastname: Huang
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China; Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; Division of Functional Glycomics, Life Science Research Center, Institute of Research Promotion, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-Gun, Kagawa 761-0793, Japan; and Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tateno
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China; Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; Division of Functional Glycomics, Life Science Research Center, Institute of Research Promotion, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-Gun, Kagawa 761-0793, Japan; and Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Nakakita
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China; Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; Division of Functional Glycomics, Life Science Research Center, Institute of Research Promotion, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-Gun, Kagawa 761-0793, Japan; and Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Sato
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China; Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; Division of Functional Glycomics, Life Science Research Center, Institute of Research Promotion, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-Gun, Kagawa 761-0793, Japan; and Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Hisashi
authors.initials: H
authors.lastname: Narimatsu
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China; Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; Division of Functional Glycomics, Life Science Research Center, Institute of Research Promotion, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-Gun, Kagawa 761-0793, Japan; and Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan tyaoxs@jnu.edu.cn.
authors.forename: Xinsheng
authors.initials: X
authors.lastname: Yao
authors.affiliation: Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou 510632, People's Republic of China; Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan; Division of Functional Glycomics, Life Science Research Center, Institute of Research Promotion, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-Gun, Kagawa 761-0793, Japan; and Research Center for Medical Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Central 2, 1-1-1 Umezono, Tsukuba, Ibaraki 305-8568, Japan jun-hirabayashi@aist.go.jp.
authors.forename: Jun
authors.initials: J
authors.lastname: Hirabayashi
mesh_terms: D019943:Amino Acid Substitution / Q000235:genetics; D005434:Flow Cytometry; D005658:Fungi / Q000737:chemistry*; D037161:Galectins / Q000737:chemistry* / Q000235:genetics*; D005818:Genetic Engineering; D015335:Molecular Probes / Q000737:chemistry / Q000235:genetics; D011509:Proteoglycans / Q000737:chemistry
chemical_list: D037161:Galectins; D015335:Molecular Probes; D011509:Proteoglycans
keywords: frontal affinity chromatography; galectin; glycan microarry; glycoengineering; molecular evolution; sulphated glycan