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Generation of Properly Folded Epidermal Growth Factor-Like (EGF) Repeats and Glycosyltransferases Enables In Vitro O-Glycosylation

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Notch Signaling Research

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2472))

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Abstract

The epidermal growth factor-like (EGF) domain is an evolutionarily conserved motif found widely distributed among numerous secreted and membrane-anchored proteins, including the Notch receptors. Notch receptors include numerous EGF repeats tandemly connected in the extracellular domain. These EGF repeats must be properly folded in order for them to undergo the three different types of O-glycosylation associated with these extracellular proteins: O-fucose, O-glucose, and O-N-acetylglucosamine via glycosyltransferases POFUT1, POGLUT1, and EOGT. The O-glycosylation of the EGF repeats in the Notch receptors regulates the activation of Notch signaling and mutations in POFUT1, POGLUT1, and EOGT have been linked to specific human diseases. A large amount of EGF repeat and glycosyltransferase protein is required to construct an in vitro O-glycosylation system. Here, we describe how we prepared properly folded EGF repeats using two different bacterial expression vectors, generated recombinant glycosyltransferases, and performed in vitro O-glycosylation and subsequent product analysis by mass spectrometry. The methods described here are useful for investigating the enzymatic activities of mutated glycosyltransferases, revealing the structural basis of the O-glycosylation mechanism by co-crystallization of the glycosyltransferase-EGF repeat complexes, or identifying potential inhibitors of these glycosyltransferases.

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Acknowledgments

This work is partly supported by the JSPS KAKENHI grants JP19H03176, JP19KK0195, and 19K22490 (to H.T.). We would like to thank Prof. Tetsuya Okajima at Nagoya University for the critical comments on our research project. Chenyu Ma and Yohei Tsukamoto contributed equally to this work.

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Author notes

  1. Hideyuki Takeuchi

    Present address: Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan

  2. Chenyu Ma and Yohei Tsukamoto contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biochemistry, Nagoya University Graduate School of Medicine, Nagoya, Japan

    Chenyu Ma, Yohei Tsukamoto & Hideyuki Takeuchi

  2. Institute for Glyco-Core Research (iGCORE), Nagoya University, Nagoya, Japan

    Hideyuki Takeuchi

Authors
  1. Chenyu Ma
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  2. Yohei Tsukamoto
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  3. Hideyuki Takeuchi
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Corresponding author

Correspondence to Hideyuki Takeuchi .

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Editors and Affiliations

  1. Department of Biology, Georgia Southern University, Statesboro, GA, USA

    Dongyu Jia

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Ma, C., Tsukamoto, Y., Takeuchi, H. (2022). Generation of Properly Folded Epidermal Growth Factor-Like (EGF) Repeats and Glycosyltransferases Enables In Vitro O-Glycosylation. In: Jia, D. (eds) Notch Signaling Research. Methods in Molecular Biology, vol 2472. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2201-8_3

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  • DOI: https://doi.org/10.1007/978-1-0716-2201-8_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-2200-1

  • Online ISBN: 978-1-0716-2201-8

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