title: Binding assay between murine Dectin-1 and β-glucan/DNA complex with quartz-crystal microbalance.
abstract: A β-glucan called schizophyllan (SPG) forms a stoichiometric complex with polynucleotides with its two main chain glucoses interacting with one nucleotide base. This complex can be used as a Dectin-1 targeting delivery for therapeutic oligonucleotides (ODN), where Dectin-1 is a membrane receptor of immunocyte cell that can recognize β-glucans. Our in vivo and in vitro assays phenomenologically implied that such a targeting is indeed achieved. However, we do not know whether SPG/ODN complexes are recognized by Dectin-1. In this study, we examined the binding affinity between SPG/poly(dA) complex and a constructed protein representing the extracellular carbohydrate-recognition domain of murine Dectin-1 by use of quartz-crystal microbalance (QCM). It was shown that the SPG/dA60 complex made form phosphodiester was recognized in the same manner as SPG, while its dissociation constant (Kd) was much larger than SPG itself, that is, less affinity than SPG. When the phosphodiester linkage of dA60 was changed to phosphorothioate (denoted by dA60(S)), the QCM frequency decrease was dramatically enhanced. There seemed to be multiple binding sites; the same site as SPG and SPG/dA60, and an additional site (or sites) for which phosphate anion specific electrostatic interactions were mainly involved. Interestingly, this new site showed a comparable affinity with that between SPG and its original binding site.
authors.affiliation: Department of Chemistry and Biochemistry, The University of Kitakyushu, 1-1, Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan.
authors.forename: Shinichi
authors.initials: S
authors.lastname: Mochizuki
authors.affiliation: Department of Chemistry and Biochemistry, The University of Kitakyushu, 1-1, Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Morishita
authors.affiliation: Laboratory for Immunopharmacology of Microbial Products, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Adachi
authors.affiliation: Structural Glycobiology Team, Systems Glycobiology Research Group, Chemical Biology Department, RIKEN, Advanced Science Institute, 2-1 Hirosawa Wako, Saitama 351-0198, Japan.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Yamaguchi
authors.affiliation: Department of Chemistry and Biochemistry, The University of Kitakyushu, 1-1, Hibikino, Wakamatsu-ku, Kitakyushu, Fukuoka 808-0135, Japan; CREST, Japan Science and Technology Agency, 4-1-8, Honcho, Kawaguchi-shi, Saitama 332-0012, Japan. Electronic address: sakurai@kitakyu-u.ac.jp.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Sakurai
mesh_terms: D000818:Animals; D001665:Binding Sites; D004247:DNA / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D037181:Lectins, C-Type / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D016297:Mutagenesis, Site-Directed; D058525:Quartz Crystal Microbalance Techniques; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D047071:beta-Glucans / Q000737:chemistry*; D000099181:Dectin-1
chemical_list: D004247:DNA; D037181:Lectins, C-Type; D011994:Recombinant Proteins; D047071:beta-Glucans; D000099181:Dectin-1
keywords: Dectin-1; Quartz-crystal microbalance; Schizophyllan