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Analysis of specificity of antibodies against synthetic fragments of different neuronal nicotinic acetylcholine receptor subunits

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Abstract

We have compared specificity of a panel of polyclonal antibodies against synthetic fragments of the α7 subunit of homooligomeric acetylcholine receptor (AChR) and some subunits of heteromeric AChRs. The antibody interaction with extracellular domain of α7 subunit of rat AChR (residues 7-208) produced by heterologous expression in E. coli and rat adrenal membranes was investigated by the ELISA method. For comparison, membranes from the Torpedo californica ray electric organ enriched in muscle-type AChR and polyclonal antibodies raised against the extracellular domain (residues 1-209) of the T. californica AChR α1 subunit were also used. Antibody specificity was also characterized by Western blot analysis using rat AChR extracellular domain α7 (7-208) and the membrane-bound T. californica AChR. Epitope localization was analyzed within the framework of AChR extracellular domain model based on the crystal structure of acetylcholine-binding protein available in the literature. According to this analysis, the 179–190 epitope is located on loop C, which is exposed and mobile. Use of antibodies against α7 (179–190) revealed the presence of α7 AChR in rat adrenal membranes.

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Abbreviations

AChBP:

acetylcholine binding protein

AChR:

acetylcholine receptor

mAB:

monoclonal antibodies

ELISA:

enzyme linked immunosorbent assay

References

  1. Karlin, A. (2002) Nat. Rev. Neurosci., 3, 102–114.

    Article  PubMed  CAS  Google Scholar 

  2. Le Novere, N., Corringer, P.J., and Changeux, J. P. (2002) J. Neurobiol., 53, 447–456.

    Article  PubMed  CAS  Google Scholar 

  3. Utkin, Yu. N., Tsetlin, V. I., and Hucho, F. (2000) Membr. Cell Biol., 13, 143–164.

    PubMed  Google Scholar 

  4. Unwin, N. (2005) J. Mol. Biol., 346, 967–989.

    Article  PubMed  CAS  Google Scholar 

  5. Lindstrom, J. (2002) J. Neurobiol., 53, 656–665.

    Article  PubMed  CAS  Google Scholar 

  6. Vincent, A., Beeson, D., and Lang, B. (2000) Eur. J. Biochem., 267, 6717–6728.

    Article  PubMed  CAS  Google Scholar 

  7. Paterson, D., and Nordberg, A. (2000) Progr. Neurobiol., 61, 75–111.

    Article  PubMed  CAS  Google Scholar 

  8. Skok, M. V., Koval, L. N., and Bachinskaya, N. Yu. (2003) Dokl. Ukr. Nats. Akad. Nauk, 9, 188–191.

    Google Scholar 

  9. Fabian-Fine, R., Skehel, P., Errington, M. L., Davies, H. A., Sher, E., Stewart, M. G., and Fine, A. (2001) J. Neurosci., 21, 7993–8003.

    PubMed  CAS  Google Scholar 

  10. Dominguez del Toro, E., Juiz, J. M., Peng, X., Lindstrom, J., and Criado, M. (1994) J. Comp. Neurol., 349, 325–342.

    Article  PubMed  CAS  Google Scholar 

  11. Schoepfer, R., Conroy, W. G., Whiting, P., Gore, M., and Lindstrom, J. (1990) Neuron, 5, 35–48.

    Article  PubMed  CAS  Google Scholar 

  12. Orr-Urtreger, A., Goldner, F. M., Saeki, M., Lorenzo, I., Goldberg, L., De Biasi, M., Dani, J. A., Patrick, J. W., and Beaudet, A. L. (1997) J. Neurosci., 17, 9165–9171.

    PubMed  CAS  Google Scholar 

  13. Chen, D., and Patrick, J. W. (1997) J. Biol. Chem., 272, 24024–24029.

    Article  PubMed  CAS  Google Scholar 

  14. Moretti, M., Vailati, S., Zoli, M., Lippi, G., Riganti, L., Longhi, R., Viegi, A., Clementi, F., and Gotti, C. (2004) Mol. Pharmacol., 66, 85–96.

    Article  PubMed  CAS  Google Scholar 

  15. Skok, M. V., Voitenko, L. P., Voitenko, S. V., Lykhmus, E. Y., Kalashnik, E. N., Litvin, T. I., Tzartos, S. J., and Skok, V. I. (1999) Neurosci., 93, 1427–1436.

    Article  CAS  Google Scholar 

  16. Neff, S., Dineley-Miller, K., Char, D., Quik, M., and Patrick, J. (1995) J. Neurochem., 64, 332–339.

    Article  PubMed  CAS  Google Scholar 

  17. Graus, Y., Meng, F., Vincent, A., van Breda Vriesman, P., and de Baets, M. (1995) J. Immunol., 154, 6382–6396.

    PubMed  CAS  Google Scholar 

  18. Tzartos, S. J., Rand, D. E., Einarson, B. L., and Lindstrom, J. M. (1981) J. Biol. Chem., 256, 8635–8645.

    PubMed  CAS  Google Scholar 

  19. Psaridi-Linardaki, L., Mamalaki, A., Remoundos, M., and Tzartos, S. J. (2002) J. Biol. Chem., 277, 26980–26986.

    Article  PubMed  CAS  Google Scholar 

  20. Herber, D. L., Severance, E. G., Cuevas, J., Morgan, D., and Gordon, M. N. (2004) J. Histochem. Cytochem., 52, 1367–1376.

    Article  PubMed  CAS  Google Scholar 

  21. Hucho, F., and Weise, C. (2001) Angew. Chem. Int. Ed., 40, 3100–3116.

    Article  CAS  Google Scholar 

  22. Vailati, S., Hanke, W., Bejan, A., Barabino, B., Longhi, R., Balestra, B., Moretti, M., Clementi, F., and Gotti, C. (1999) Mol. Pharmacol., 56, 11–19.

    PubMed  CAS  Google Scholar 

  23. Di Angelantonio, S., Matteoni, C., Fabretti, E., and Nistri, A. (2003) Eur. J. Neurosci., 17, 2313–2322.

    Article  PubMed  Google Scholar 

  24. Di Angelantonio, S., Nistri, A., Moretti, M., Clementi, F., and Gotti, C. (2000) Br. J. Pharmacol., 129, 1771–1779.

    Article  PubMed  Google Scholar 

  25. Mousavi, M., Hellstrom-Lindahl, E., Guan, Z. Z., Bednar, L., and Nordberg, A. (2001) Life Sci., 70, 577–590.

    Article  PubMed  CAS  Google Scholar 

  26. Gershkovich, A. A., and Kibirev, V. K. (1992) Chemical Synthesis of Peptides [in Russian], Naukova Dumka, Kiev.

    Google Scholar 

  27. Dean, C. J. (1994) in Basic Protein and Peptide Protocols (Walker, J. M., ed.) Humana Press, Totowa, New Jersey, p. 367.

    Google Scholar 

  28. Alexeev, T., Krivoshein, A., Shevalier, A., Kudelina, I., Telyakova, O., Vincent, A., Utkin, Y., Hucho, F., and Tsetlin, V. (1999) Eur. J. Biochem., 259, 310–319.

    Article  PubMed  CAS  Google Scholar 

  29. Korotina, A. S., Kryukova, E. V., Azeeva, E. A., Sheval’e, A. F., Utkin, Yu. N., and Tsetlin, V. I. (2003) Bioorg. Khim., 29, 391–396.

    PubMed  CAS  Google Scholar 

  30. Maeda, T., Balakrishnan, K., and Mehdi, S. Q. (1983) Biochim. Biophys. Acta, 731, 115–120.

    Article  PubMed  CAS  Google Scholar 

  31. Peterson, G. L. (1977) Analyt. Biochem., 83, 346–356.

    Article  PubMed  CAS  Google Scholar 

  32. Guex, N., and Peitsch, M. C. (1997) Electrophoresis, 18, 2714–2723.

    Article  PubMed  CAS  Google Scholar 

  33. Kao, P. N., Dwork, A. J., Kaldany, R. R., Silver, M. L., Wideman, J., Stein, S., and Karlin, A. (1984) J. Biol. Chem., 259, 11662–11665.

    PubMed  CAS  Google Scholar 

  34. Neumann, D., Gershoni, J. M., Fridkin, M., and Fuchs, S. (1985) Proc. Natl. Acad. Sci. USA, 82, 3490–3493.

    Article  PubMed  CAS  Google Scholar 

  35. Scherf, T., Kasher, R., Balass, M., Fridkin, M., Fuchs, S., and Katchalski-Katzir, E. (2001) Proc. Natl. Acad. Sci. USA, 98, 6629–6634.

    Article  PubMed  CAS  Google Scholar 

  36. Basus, V.J., Song, G., and Hawrot, E. (1993) Biochemistry, 32, 12290–12298.

    Article  PubMed  CAS  Google Scholar 

  37. Brejc, K., van Dijk, W. J., Klaassen, R. V., Schuurmans, M., van der Oost, J., Smit, A. B., and Sixma, T. K. (2001) Nature, 411, 269–276.

    Article  PubMed  CAS  Google Scholar 

  38. Smit, A. B., Syed, N. I., Schaap, D., van Minnen, J., Klumperman, J., Kits, K. S., Lodder, H., van der Schors, R. C., van Elk, R., Sorgedrager, B., Brejc, K., Sixma, T. K., and Geraerts, W. P. (2001) Nature, 411, 261–268.

    Article  PubMed  CAS  Google Scholar 

  39. Hibbs, R. E., Talley, T. T., and Taylor, P. (2004) J. Biol. Chem., 279, 28483–28491.

    Article  PubMed  CAS  Google Scholar 

  40. Bouzat, C., Gumilar, F., Spitzmaul, G., Wang, H. L., Rayes, D., Hansen, S. B., Taylor, P., and Sine, S. M. (2004) Nature, 430, 896–900.

    Article  PubMed  CAS  Google Scholar 

  41. Grutter, T., Prado de Carvalho, L., Virginie, D., Taly, A., Fischer, M., and Changeux, J. P. (2005) Comptes Rendus Biologies, 328, 223–234.

    Article  PubMed  CAS  Google Scholar 

  42. Celie, P. H., van Rossum-Fikkert, S. E., van Dijk, W. J., Brejc, K., Smit, A. B., and Sixma, T. K. (2004) Neuron, 41, 907–914.

    Article  PubMed  CAS  Google Scholar 

  43. Celie, P. H., Kasheverov, I. E., Mordvintsev, D. Y., Hogg, R. C., van Nierop, P., van Elk, R., van Rossum-Fikkert, S. E., Zhmak, M. N., Bertrand, D., Tsetlin, V., Sixma, T. K., and Smit, A. B. (2005) Nat. Struct. Mol. Biol., 12, 582–588.

    Article  PubMed  CAS  Google Scholar 

  44. Dutertre, S., and Lewis, R. J. (2004) Eur. J. Biochem., 271, 2327–2334.

    Article  PubMed  CAS  Google Scholar 

  45. Ellison, M., Gao, F., Wang, H. L., Sine, S. M., McIntosh, J. M., and Olivera, B. M. (2004) Biochemistry, 43, 16019–16026.

    Article  PubMed  CAS  Google Scholar 

  46. Skok, M. V., Kalashnik, E. N., Koval, L. N., Tsetlin, V. I., Utkin, Y. N., Changeux, J. P., and Grailhe, R. (2003) Mol. Pharmacol., 64, 885–889.

    Article  PubMed  CAS  Google Scholar 

  47. Tsetlin, V. I., and Hucho, F. (2004) FEBS Lett., 557, 9–13.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia

    I. V. Shelukhina, E. V. Kryukova, M. N. Zhmak, D. Yu. Mordvintsev, I. E. Kasheverov & V. I. Tsetlin

  2. Palladin Institute of Biochemistry, Ukrainian Academy of Sciences, ul. Leontovicha 9, 01601, Kiev, Ukraine

    M. V. Skok & E. Yu. Lykhmus

Authors
  1. I. V. Shelukhina
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  2. E. V. Kryukova
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  3. M. V. Skok
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  4. E. Yu. Lykhmus
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  5. M. N. Zhmak
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  6. D. Yu. Mordvintsev
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  7. I. E. Kasheverov
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  8. V. I. Tsetlin
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Additional information

Original Russian Text © I. V. Shelukhina, E. V. Kryukova, M. V. Skok, E. Yu. Lykhmus, M. N. Zhmak, D. Yu. Mordvintsev, I. E. Kasheverov, V. I. Tsetlin, 2006, published in Biokhimiya, 2006, Vol. 71, No. 7, pp. 924–935.

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Shelukhina, I.V., Kryukova, E.V., Skok, M.V. et al. Analysis of specificity of antibodies against synthetic fragments of different neuronal nicotinic acetylcholine receptor subunits. Biochemistry (Moscow) 71, 749–758 (2006). https://doi.org/10.1134/S0006297906070078

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  • DOI: https://doi.org/10.1134/S0006297906070078

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