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Characterization of glycoproteins expressing the blood group H type 1 epitope on human induced pluripotent stem (hiPS) cells

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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.

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Abbreviations

hES:

Human embryonic stem

hiPS:

Human induced pluripotent stem

HRP:

Horseradish peroxidase

KS:

Keratan sulfate

LC/MS/MS:

High-performance liquid chromatography with tandem mass spectrometry

PBS:

Phosphate-buffered saline

All of the sugar residues have the D-configuration except fucose, which has the L-configuration

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Acknowledgments

We wish to thank Saori Kamo for the secretarial assistance.

Author information

Author notes

  1. Daisuke Takakura & Nana Kawasaki

    Present address: Department of Medical Life Science, Yokohama City University, Kanagawa, 230-0045, Japan

Authors and Affiliations

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

    Hiromi Nakao, Shogo Matsumoto, Nobuko Kawasaki & Toshisuke Kawasaki

  2. Laboratory of Bio-analytical Chemistry, College of Pharmaceutical Sciences, Ritsumeikan University, Shiga, 525-8577, Japan

    Yuko Nagai, Aya Kojima & Hidenao Toyoda

  3. Division of Biological Chemistry and Biologicals, National Institute of Health Sciences, Tokyo, 158-8501, Japan

    Noritaka Hashii, Daisuke Takakura & Nana Kawasaki

  4. Laboratory of Stem Cell Regulation, National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, 567-0085, Japan

    Tomoko Yamaguchi & Kenji Kawabata

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  1. Hiromi Nakao
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  2. Shogo Matsumoto
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  12. Toshisuke Kawasaki
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Corresponding author

Correspondence to Toshisuke Kawasaki.

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Funding

This work was supported by a Grant-in-Aid for Scientific Research, C- 24,570,171 (to T.K.), from the Japan Society for the Promotion of Science (JSPS), a Grant-in-Aid for Scientific Research on Innovative Areas 24,110,517 (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.) from the Japan Science and Technology Agency (JST), the R-GIRO (Ritsumeikan Global Innovation Research Organization) Program (to H.T.), and the Mizutani Foundation Research Grant 160,204 (to T.K.).

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Nakao, H., Matsumoto, S., Nagai, Y. et al. Characterization of glycoproteins expressing the blood group H type 1 epitope on human induced pluripotent stem (hiPS) cells. Glycoconj J 34, 779–787 (2017). https://doi.org/10.1007/s10719-016-9710-2

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  • DOI: https://doi.org/10.1007/s10719-016-9710-2

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