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Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides

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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.

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Acknowledgements

We wish to thank Saori Kamo for the secretarial assistance.

We greatly thank Tokyo Chemical Industry Co., Ltd. for providing the PEG3-biotinylated derivatives of a series of seven N-acetyllactosamine tetrasaccharides (keratan sulfate homologues (KS1 to KS7)). We also thank Dr. Ten Feizi (Imperial College of London, UK) for her valuable suggestions in the writing of the manuscript.

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Authors and Affiliations

  1. Research Center for Glycobiotechnology, Ritsumeikan University, Noji-Higashi, 1-1-1, Kusatsu, Shiga, 525-8577, Japan

    Hiromi Nakao, Nobuko Kawasaki & Toshisuke Kawasaki

  2. Research Center for Glycobiotechnology, Ritsumeikan University, Shiga, 525-8577, Japan

    Yuko Nagai, Aya Kojima & Hidenao Toyoda

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  1. Hiromi Nakao
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  2. Yuko Nagai
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  3. Aya Kojima
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  4. Hidenao Toyoda
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  5. Nobuko Kawasaki
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  6. Toshisuke Kawasaki
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Correspondence to Toshisuke Kawasaki.

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Funding

This work was supported by a Grant-in-Aid for Scientific Research, C- 24570171 (to T.K.), from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Scientific Research on Innovative Areas, 24110517 (to T.K.), from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT), Adaptable and Seamless Technology Transfer Programs through Target-driven R&D (A-STEP), AS242Z01520P (to T.K.) and AS251Z01560P (to H.T.), of the Japan Science and Technology Agency (JST), the R-GIRO (Ritsumeikan Global Innovation Research Organization) Program (to H.T.), and Mizutani Foundation Research Grant 160204 (to T.K.).

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None declared.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Nakao, H., Nagai, Y., Kojima, A. et al. Binding specificity of R-10G and TRA-1-60/81, and substrate specificity of keratanase II studied with chemically synthesized oligosaccharides. Glycoconj J 34, 789–795 (2017). https://doi.org/10.1007/s10719-017-9765-8

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  • DOI: https://doi.org/10.1007/s10719-017-9765-8

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