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Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the native structure

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Abstract

P2X purinergic receptors are ATP-driven ionic channels expressed as trimers and showing various functions. A subtype, the P2X4 receptor present on microglial cells is highly involved in neuropathic pain. In this study, in order to prepare antibodies recognizing the native structure of rat P2X4 (rP2X4) receptor, we immunized mice with rP2X4’s head domain (rHD, Gln111–Val167), which possesses an intact structure stabilized by S-S bond formation (Igawa and Abe et al. FEBS Lett. 2015), as an antigen. We generated five monoclonal antibodies with the ability to recognize the native structure of its head domain, stabilized by S-S bond formation. Site-directed mutagenesis revealed that Asn127 and Asp131 of the rHD, in which combination of these amino acid residues is only conserved in P2X4 receptor among P2X family, were closely involved in the interaction between rHD and these antibodies. We also demonstrated the antibodies obtained here could detect rP2X4 receptor expressed in 1321N1 human astrocytoma cells.

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Abbreviations

CD:

Circular dichroism

CFA:

Complete Freund’s adjuvant

ECD:

Extracellular domain of P2X4 receptor

IFA:

Incomplete Freund’s adjuvant

KLH:

Keyhole limpet hemocyanin

PBS:

Phosphate-buffered saline

rHD:

Rat head domain of P2X4 receptor

TAPS:

Trimethylammonium propyl sulfonate

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Acknowledgements

We thank Dr. Jose Caaveiro of Kyshu University for critical readership of the manuscript. ITC measurements were supported by the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Funding

This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan to UT (23390155) and YA (18K06597) and by a grant from the Japan Foundation for Applied Enzymology.

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Author notes

  1. Tatsuhiro Igawa, Shuhei Kishikawa and Yoshito Abe contributed equally to this work.

Authors and Affiliations

  1. Department of Protein Structure, Function and Design, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, 812-8582, Japan

    Tatsuhiro Igawa, Shuhei Kishikawa, Yoshito Abe, Saki Nagai, Mitsunori Shiroishi, Chinatsu Shinozaki & Tadashi Ueda

  2. Department of Global Health-Care, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

    Tomohiro Yamashita

  3. Department of Pharmacognosy Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

    Hiroyuki Tanaka

  4. Department of Life Innovation, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan

    Hidetoshi Tozaki-Saitoh & Makoto Tsuda

  5. Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, Higashi-ku, Fukuoka, 812-8582, Japan

    Kazuhide Inoue

Authors
  1. Tatsuhiro Igawa
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  2. Shuhei Kishikawa
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  3. Yoshito Abe
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  8. Hiroyuki Tanaka
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  9. Hidetoshi Tozaki-Saitoh
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  10. Makoto Tsuda
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  11. Kazuhide Inoue
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  12. Tadashi Ueda
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Corresponding authors

Correspondence to Kazuhide Inoue or Tadashi Ueda.

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Conflicts of interest

Tatsuhiro Igawa declares that he has no conflict of interest.

Shuhei Kishikawa declares that he has no conflict of interest.

Yoshito Abe declares that he has no conflict of interest.

Tomohiro Yamashita declares that he has no conflict of interest.

Saki Nagai declares that she has no conflict of interest.

Mitsunori Shiroishi declares that he has no conflict of interest.

Chinatsu Shinozaki declares that he has no conflict of interest.

Hiroyuki Tanaka declares that he has no conflict of interest.

Hidetoshi Tozaki-Saitoh declares that he has no conflict of interest.

Makoto Tsuda declares that he has no conflict of interest.

Kazuhide Inoue declares that he has no conflict of interest.

Tadashi Ueda declares that he has no conflict of interest.

Ethical approval

All animal experiments were conducted according to the relevant national and international guidelines in the Act on Welfare and Management of Animals (Ministry of Environment of Japan) and the Regulation of Laboratory Animals (Kyushu University), and under the protocols approved by the Institutional Animal Care and Use Committee review panels at Kyushu University.

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Igawa, T., Kishikawa, S., Abe, Y. et al. Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the native structure. Purinergic Signalling 15, 27–35 (2019). https://doi.org/10.1007/s11302-019-09646-5

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  • DOI: https://doi.org/10.1007/s11302-019-09646-5

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