title: Generation of monoclonal antibodies against the Galβ1-4Gal epitope: a key tool in studies of species-specific glycans expressed in fish, amphibians and birds.
abstract: Whereas the Galβ1-4Gal epitope is rarely found in mammalian glycans, it has been found in glycans of various species of non-mammalian vertebrates, such as fish, amphibians and birds. Although glycans containing Galβ1-4Gal in these vertebrates were detected by precise structural analysis of the glycans using mass spectrometry and/or NMR spectrometry, there are no convenient methods to detect Galβ1-4Gal from various samples. To analyze systematically the distribution of Galβ1-4Gal in nature, we generated mouse monoclonal antibodies (mAbs) specific for Galβ1-4Gal using extracts of medaka eggs as an immunogen. Four mAbs (two immunoglobulin (Ig)Ms and two IgG1s) were obtained by enzyme-linked immunosorbent assay-based screening. The specificities of these mAbs were evaluated by frontal affinity chromatography using 142 kinds of 2-aminopyridine (PA)-derivatized oligosaccharides. While all mAbs interacted with (Galβ1-4Gal)-containing oligosaccharides at their non-reducing termini with dissociation constants (K(d)) ranging from 1.0 x 10⁻⁵ to 2.8 x 10⁻⁴ M, no apparent interaction was observed with any other glycans. The number of branches containing Galβ1-4Gal on N-glycans did not significantly affect K(d) of mAbs of IgG1 subclasses, but those of IgM mAbs were decreased by ∼1 order of magnitude, in increments of the number of branches present. Using the mAbs, we established that Galβ1-4Gal is also expressed on glycoproteins in various tissues from the African clawed frog. Immunohistochemical staining of medaka sections revealed that Galβ1-4Gal epitopes were expressed in the endothelium, epithelium and epidermis, which directly contact the external environment or invading organisms. Thus, these mAbs are useful for systematically investigating the species-specific expression of glycans, which may act as a barrier against infection.
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan. nrsuzuki@k.u-tokyo.ac.jp
authors.forename: Noriko
authors.initials: N
authors.lastname: Suzuki
authors.forename: Daisuke
authors.initials: D
authors.lastname: Nawa
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tateno
authors.forename: Takako
authors.initials: T
authors.lastname: Yasuda
authors.forename: Shoji
authors.initials: S
authors.lastname: Oda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mitani
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Nishimaki
authors.forename: Takafumi
authors.initials: T
authors.lastname: Katsumura
authors.forename: Hiroki
authors.initials: H
authors.lastname: Oota
authors.forename: Tsunehiko
authors.initials: T
authors.lastname: Hanihara
authors.forename: Atsunori
authors.initials: A
authors.lastname: Oga
authors.forename: Jun
authors.initials: J
authors.lastname: Hirabayashi
authors.forename: Kazuo
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D001717:Birds; D004187:Disaccharides / Q000737:chemistry / Q000276:immunology; D000939:Epitopes / Q000737:chemistry / Q000276:immunology*; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D009844:Oligosaccharides / Q000737:chemistry / Q000276:immunology; D009928:Organ Specificity; D009990:Oryzias; D011134:Polysaccharides / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D013045:Species Specificity; D014982:Xenopus laevis; D015027:Zebrafish
chemical_list: D000911:Antibodies, Monoclonal; D004187:Disaccharides; D000939:Epitopes; D006023:Glycoproteins; D009844:Oligosaccharides; D011134:Polysaccharides