title: Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides.
abstract: Recently, we established a mouse monoclonal antibody specific to hiPS/ hES cells, R-10G, which recognizes a type of keratan sulfate. Keratan sulfates (KS) comprise a family of glycosaminoglycans consisting of the repeating unit of [Gal-GlcNAc(6S)]. However, there is a diversity in the degree of sulfation at Gal and GlcNAc residues, and also in the mode of linkage, Galβ1 - 3GlcNAc (type 1) or Galβ1 - 4GlcNAc (type 2). To gain more insight into the binding specificity of R-10G, we carried out an ELISA test on avidin-coated plates using polyethylene glycol (PEG)3-biotinylated derivatives of a series of N-acetyllactosamine tetrasaccharides (keratan sulfates (KSs)). The results suggested that the minimum epitope structure is Galβ1 - 4GlcNAc(6S)β1 - 3Galβ1 - 4GlcNAc(6S)β1 (type 2- type 2 keratan sulfate). Removal of sulfate from GlcNAc(6S) or addition of sulfate to Gal abolished the binding activity almost completely. We also examined the binding specificity of TRA-1-60/81 in the same assay system. The minimum epitope structure was shown to be Galβ1 - 3GlcNAcβ1 - 3Galβ1 - 4GlcNAcβ1 in agreement with the previous study involving glycan arrays (Natunen et al., Glycobiology, 21, 1125-1130 (2011)). Interestingly, however, TRA-1-60/81 was shown to bind to Galβ1 - 3GlcNAc(6S)β1 - 3Galβ1 - 4GlcNAc(6S)β1 (type 1- type 2 keratan sulfate) dose-dependently, being more than one-third the binding activity toward Galβ1 - 3GlcNAcβ1 - 3Galβ1 - 4GlcNAcβ1 than in the case of TRA-1-60. In addition, a substrate specificity study on keratanase II revealed that keratanase II degraded not only "type 2-type 2 keratan sulfate" but also "type 1-type 2 keratan sulfate", significantly.
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, Shiga, 525-8577, Japan.
authors.forename: Yuko
authors.initials: Y
authors.lastname: Nagai
authors.affiliation: Research Center for Glycobiotechnology, Ritsumeikan University, Shiga, 525-8577, Japan.
authors.forename: Aya
authors.initials: A
authors.lastname: Kojima
authors.affiliation: Research Center for Glycobiotechnology, Ritsumeikan University, Shiga, 525-8577, Japan.
authors.forename: Hidenao
authors.initials: H
authors.lastname: Toyoda
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: D000118:Acetylglucosaminidase / Q000378:metabolism*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology*; D000918:Antibody Specificity; D006801:Humans; D007632:Keratan Sulfate / Q000138:chemical synthesis / Q000737:chemistry / Q000276:immunology*; D013379:Substrate Specificity
chemical_list: D000911:Antibodies, Monoclonal; D007632:Keratan Sulfate; C086950:keratanase II; D000118:Acetylglucosaminidase
keywords: Induced pluripotent stem (iPS) cells; Keratan sulfates; Keratanase II; N-acetyllactosamine tetrasaccharides; R-10G; TRA-1-60