title: Mutated Leguminous Lectin Containing a Heparin-Binding like Motif in a Carbohydrate-Binding Loop Specifically Binds to Heparin.
abstract: We previously introduced random mutations in the sugar-binding loops of a leguminous lectin and screened the resulting mutated lectins for novel specificities using cell surface display. Screening of a mutated peanut agglutinin (PNA), revealed a mutated PNA with a distinct preference for heparin. Glycan microarray analyses using the mutated lectin fused to the Fc region of human immunoglobulin, revealed that a particular sulfated glycosaminoglycan (GAG), heparin, had the highest binding affinity for mutated PNA among 97 glycans tested, although wild-type PNA showed affinity towards Galβ1-3GalNAc and similar galactosylated glycans. Further analyses of binding specificity using an enzyme-linked immunoadsorbent assay demonstrated that the mutated PNA specifically binds to heparin, and weakly to de-2-O-sulfated heparin, but not to other GAG chains including de-6-O-sulfated and de-N-sulfated heparins. The mutated PNA had six amino acid substitutions within the eight amino acid-long sugar-binding loop. In this loop, the heparin-binding like motif comprised three arginine residues at positions 124, 128, and 129, and a histidine at position 125 was present. Substitution of each arginine or histidine residue to alanine reduced heparin-binding ability, indicating that all of these basic amino acid residues contributed to heparin binding. Inhibition assay demonstrated that heparin and dextran sulfate strongly inhibited mutated PNA binding to heparin in dose-dependent manner. The mutated PNA could distinguish between CHO cells and proteoglycan-deficient mutant cells. This is the first report establishing a novel leguminous lectin that preferentially binds to highly sulfated heparin and may provide novel GAG-binding probes to distinguish between heterogeneous GAG repeating units.
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, 277-8562 Chiba, Japan.
authors.forename: Hirohito
authors.initials: H
authors.lastname: Abo
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, 277-8562 Chiba, Japan.
authors.forename: Keisuke
authors.initials: K
authors.lastname: Soga
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, 277-8562 Chiba, Japan.
authors.forename: Atsuhiro
authors.initials: A
authors.lastname: Tanaka
authors.affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tateno
authors.affiliation: Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki 305-8568, Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Hirabayashi
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, 277-8562 Chiba, Japan.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D009154:Mutation; D019887:Peanut Agglutinin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D015202:Protein Engineering; D013379:Substrate Specificity
chemical_list: D019887:Peanut Agglutinin; D006493:Heparin