TY - JOUR
T1 - Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the native structure
AU - Igawa, Tatsuhiro
AU - Kishikawa, Shuhei
AU - Abe, Yoshito
AU - Yamashita, Tomohiro
AU - Nagai, Saki
AU - Shiroishi, Mitsunori
AU - Shinozaki, Chinatsu
AU - Tanaka, Hiroyuki
AU - Tozaki-Saitoh, Hidetoshi
AU - Tsuda, Makoto
AU - Inoue, Kazuhide
AU - Ueda, Tadashi
N1 - Funding Information:
Funding information 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.
Funding Information:
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
Publisher Copyright:
© 2019, Springer Nature B.V.
PY - 2019/3/15
Y1 - 2019/3/15
N2 - 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.
AB - 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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U2 - 10.1007/s11302-019-09646-5
DO - 10.1007/s11302-019-09646-5
M3 - Article
C2 - 30684150
AN - SCOPUS:85060618757
SN - 1573-9538
VL - 15
SP - 27
EP - 35
JO - Purinergic Signalling
JF - Purinergic Signalling
IS - 1
ER -