title: Characterization of glycoproteins expressing the blood group H type 1 epitope on human induced pluripotent stem (hiPS) cells.
abstract: Recently, we established two mouse monoclonal antibodies (R-10G and R-17F). The R-17F antibody (IgG1 subtype) exhibited a strong cytotoxic effect on hiPS/ES cells. The R-17F antigen isolated from a total lipid extract of hiPS (Tic) cells was identified as LNFP I (Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc). In the present study, R-17F binding proteins were isolated from hiPS (Tic) cell lysates with an affinity column of R-17F. They gave one major R-17F positive band around 250 kDa, and several minor bands between 150 kDa and 25 kDa. The former band was identified as podocalyxin by LC/MS/MS after SDS-PAGE. Hapten inhibition studies on R-17F binding to R-17F column-purified proteins with various synthetic oligosaccharides revealed that the blood group H type 1 triaose structure (Fucα1-2Galβ1-3GlcNAc) was the predominant epitope on all the R-17F binding proteins. These bands disappeared completely on digestion with α1-2 fucosidase, but not with α1-3/4 fucosidase. Upon PNGase F digestion, the R-17F positive band around and above 250 kDa did not show any change, while the minor bands between 150 kDa and 25 kDa disappeared completely, suggesting that the epitope is expressed on N-glycans in the latter and probably on O-glycans in the former. These results, together with those obtained in our previous studies on R-10G (Kawabe et al. Glycobiology, 23, 322-336 (2013)), indicated that both R-10G and R-17F epitopes are carried on the same podocalyxin molecule. The R-17F epitopes on these glycoproteins expressed on hiPS cells could be associated with the molecular mechanism underlying the carbohydrate-mediated cytotoxic activity of R-17F.
authors.affiliation: Research Center for Glycobiotechnology, Ritsumeikan University, Noji-Higashi, 1-1-1, Kusatsu, Shiga, 525-8577, Japan.
authors.forename: Hiromi
authors.initials: H
authors.lastname: Nakao
authors.affiliation: Research Center for Glycobiotechnology, Ritsumeikan University, Noji-Higashi, 1-1-1, Kusatsu, Shiga, 525-8577, Japan.
authors.forename: Shogo
authors.initials: S
authors.lastname: Matsumoto
authors.affiliation: Laboratory of Bio-analytical Chemistry, College of Pharmaceutical Sciences, Ritsumeikan University, Shiga, 525-8577, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nagai
authors.affiliation: Laboratory of Bio-analytical Chemistry, College of Pharmaceutical Sciences, Ritsumeikan University, Shiga, 525-8577, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Kojima
authors.affiliation: Laboratory of Bio-analytical Chemistry, College of Pharmaceutical Sciences, Ritsumeikan University, Shiga, 525-8577, Japan.
authors.forename: Hidenao
authors.initials: H
authors.lastname: Toyoda
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, 158-8501, Japan.
authors.forename: Noritaka
authors.initials: N
authors.lastname: Hashii
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, 158-8501, Japan.|Department of Medical Life Science, Yokohama City University, Kanagawa, 230-0045, Japan.
authors.forename: Daisuke
authors.initials: D
authors.lastname: Takakura
authors.affiliation: Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, 158-8501, Japan.|Department of Medical Life Science, Yokohama City University, Kanagawa, 230-0045, Japan.
authors.forename: Nana
authors.initials: N
authors.lastname: Kawasaki
authors.affiliation: Laboratory of Stem Cell Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, 567-0085, Japan.
authors.forename: Tomoko
authors.initials: T
authors.lastname: Yamaguchi
authors.affiliation: Laboratory of Stem Cell Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, 567-0085, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kawabata
authors.affiliation: Research Center for Glycobiotechnology, Ritsumeikan University, Noji-Higashi, 1-1-1, Kusatsu, Shiga, 525-8577, Japan.
authors.forename: Nobuko
authors.initials: N
authors.lastname: Kawasaki
authors.affiliation: Research Center for Glycobiotechnology, Ritsumeikan University, Noji-Higashi, 1-1-1, Kusatsu, Shiga, 525-8577, Japan. tkawasak@fc.ritsumei.ac.jp.
authors.forename: Toshisuke
authors.identifier: 0000-0003-0993-0274
authors.initials: T
authors.lastname: Kawasaki
mesh_terms: D000017:ABO Blood-Group System / Q000737:chemistry / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology; D002460:Cell Line; D000939:Epitopes / Q000276:immunology*; D006023:Glycoproteins / Q000737:chemistry / Q000276:immunology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000276:immunology*
chemical_list: D000017:ABO Blood-Group System; D000911:Antibodies, Monoclonal; D000939:Epitopes; D006023:Glycoproteins
keywords: Cytotoxic antibody; Induced pluripotent stem (iPS) cells; Lacto-N-fucopentaose I; Monoclonal antibody R-17F; Podocalyxin