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. 1985 May;82(10):3490–3493. doi: 10.1073/pnas.82.10.3490

Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.

D Neumann, J M Gershoni, M Fridkin, S Fuchs
PMCID: PMC397802  PMID: 2582416

Abstract

Synthetic peptides and their respective antibodies were used in an attempt to localize and identify the ligand-binding site of the nicotinic acetylcholine receptor. Two peptides of the receptor alpha subunit were synthesized, the first corresponding to the NH2-terminal domain (positions 1-20) and the other, to a segment (residues 126-143) that contains the first two cysteine residues. Specific antipeptide antibodies were elicited in rabbits after immunization with the peptides conjugated to bovine serum albumin. The antipeptide antibodies thus obtained cross-reacted with the receptor and bound specifically to its alpha subunit. The antipeptide antibodies were used to test whether the peptide sequences corresponded to the alpha-bungarotoxin (alpha-BTX)-binding site. Staphylococcus aureus V8-protease digestion of the isolated receptor alpha subunit generated several fragments. Antipeptide (1-20) and antipeptide (126-143) both bound a 26-kDa fragment, whereas only antipeptide (126-143) bound a 17-kDa fragment. None of these fragments were found to bind alpha-BTX. On the other hand, alpha-BTX bound to an 18-kDa fragment that did not react with either of the antipeptide antibodies. Moreover, the 26-kDa and 17-kDa fragments were also found to contain the endoglycosidase H-susceptible oligosaccharide chain. Our results indicate that the toxin-binding site lies beyond the first possible V8 protease cleavage site after residues 126-143: i.e., Asp-152. This location is in agreement with the possibility that cysteine residues 192 and/or 193 are in close proximity to or contiguous with the ligand-binding site.

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Selected References

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  1. Bartfeld D., Fuchs S. Active acetylcholine receptor fragment obtained by tryptic digestion of acetylcholine receptor from Torpedo californica. Biochem Biophys Res Commun. 1979 Jul 27;89(2):512–519. doi: 10.1016/0006-291x(79)90659-4. [DOI] [PubMed] [Google Scholar]
  2. Bartfeld D., Fuchs S. Immunological characterization of an irreversibly denatured acetylcholine receptor. FEBS Lett. 1977 May 15;77(2):214–218. doi: 10.1016/0014-5793(77)80237-8. [DOI] [PubMed] [Google Scholar]
  3. Cahill S., Schmidt J. An immunochemical approach to the identification of the MBTA binding site of the nicotinic acetylcholine receptor of Torpedo californica. Biochem Biophys Res Commun. 1984 Jul 31;122(2):602–608. doi: 10.1016/s0006-291x(84)80075-3. [DOI] [PubMed] [Google Scholar]
  4. Changeux J. P., Devillers-Thiéry A., Chemouilli P. Acetylcholine receptor: an allosteric protein. Science. 1984 Sep 21;225(4668):1335–1345. doi: 10.1126/science.6382611. [DOI] [PubMed] [Google Scholar]
  5. Changeux J. P. The acetylcholine receptor: an "allosteric" membrane protein. Harvey Lect. 1979 1980;75:85–254. [PubMed] [Google Scholar]
  6. Chou P. Y., Fasman G. D. Empirical predictions of protein conformation. Annu Rev Biochem. 1978;47:251–276. doi: 10.1146/annurev.bi.47.070178.001343. [DOI] [PubMed] [Google Scholar]
  7. Conti-Tronconi B. M., Hunkapiller M. W., Raftery M. A. Molecular weight and structural nonequivalence of the mature alpha subunits of Torpedo californica acetylcholine receptor. Proc Natl Acad Sci U S A. 1984 May;81(9):2631–2634. doi: 10.1073/pnas.81.9.2631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Conti-Tronconi B. M., Raftery M. A. The nicotinic cholinergic receptor: correlation of molecular structure with functional properties. Annu Rev Biochem. 1982;51:491–530. doi: 10.1146/annurev.bi.51.070182.002423. [DOI] [PubMed] [Google Scholar]
  9. Damle V. N., McLaughlin M., Karlin A. Bromoacetylcholine as an affinity label of the acetylcholine receptor from Torpedo californica. Biochem Biophys Res Commun. 1978 Oct 30;84(4):845–851. doi: 10.1016/0006-291x(78)91661-3. [DOI] [PubMed] [Google Scholar]
  10. Devillers-Thiery A., Giraudat J., Bentaboulet M., Changeux J. P. Complete mRNA coding sequence of the acetylcholine binding alpha-subunit of Torpedo marmorata acetylcholine receptor: a model for the transmembrane organization of the polypeptide chain. Proc Natl Acad Sci U S A. 1983 Apr;80(7):2067–2071. doi: 10.1073/pnas.80.7.2067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gershoni J. M., Davis F. E., Palade G. E. Protein blotting in uniform or gradient electric fields. Anal Biochem. 1985 Jan;144(1):32–40. doi: 10.1016/0003-2697(85)90080-6. [DOI] [PubMed] [Google Scholar]
  12. Gershoni J. M., Hawrot E., Lentz T. L. Binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of Torpedo californica: quantitative analysis with protein blots. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4973–4977. doi: 10.1073/pnas.80.16.4973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gullick W. J., Tzartos S., Lindstrom J. Monoclonal antibodies as probes of acetylcholine receptor structure. 1. Peptide mapping. Biochemistry. 1981 Apr 14;20(8):2173–2180. doi: 10.1021/bi00511a016. [DOI] [PubMed] [Google Scholar]
  14. Haggerty J. G., Froehner S. C. Restoration of 125I-alpha-bungarotoxin binding activity to the alpha subunit of Torpedo acetylcholine receptor isolated by gel electrophoresis in sodium dodecyl sulfate. J Biol Chem. 1981 Aug 25;256(16):8294–8297. [PubMed] [Google Scholar]
  15. Kao P. N., Dwork A. J., Kaldany R. R., Silver M. L., Wideman J., Stein S., Karlin A. Identification of the alpha subunit half-cystine specifically labeled by an affinity reagent for the acetylcholine receptor binding site. J Biol Chem. 1984 Oct 10;259(19):11662–11665. [PubMed] [Google Scholar]
  16. Karlin A., Cowburn D. The affinity-labeling of partially purified acetylcholine receptor from electric tissue of Electrophorus. Proc Natl Acad Sci U S A. 1973 Dec;70(12):3636–3640. doi: 10.1073/pnas.70.12.3636. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Mochly-Rosen D., Fuchs S. Monoclonal anti-acetylcholine-receptor antibodies directed against the cholinergic binding site. Biochemistry. 1981 Sep 29;20(20):5920–5924. doi: 10.1021/bi00523a041. [DOI] [PubMed] [Google Scholar]
  18. Müller G. M., Shapira M., Arnon R. Anti-influenza response achieved by immunization with a synthetic conjugate. Proc Natl Acad Sci U S A. 1982 Jan;79(2):569–573. doi: 10.1073/pnas.79.2.569. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Neumann D., Fridkin M., Fuchs S. Anti-acetylcholine receptor response achieved by immunization with a synthetic peptide from the receptor sequence. Biochem Biophys Res Commun. 1984 Jun 15;121(2):673–679. doi: 10.1016/0006-291x(84)90234-1. [DOI] [PubMed] [Google Scholar]
  20. Noda M., Takahashi H., Tanabe T., Toyosato M., Furutani Y., Hirose T., Asai M., Inayama S., Miyata T., Numa S. Primary structure of alpha-subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA sequence. Nature. 1982 Oct 28;299(5886):793–797. doi: 10.1038/299793a0. [DOI] [PubMed] [Google Scholar]
  21. Oblas B., Boyd N. D., Singer R. H. Analysis of receptor-ligand interactions using nitrocellulose gel transfer: application to Torpedo acetylcholine receptor and alpha-bungarotoxin. Anal Biochem. 1983 Apr 1;130(1):1–8. doi: 10.1016/0003-2697(83)90641-3. [DOI] [PubMed] [Google Scholar]
  22. Smythies J. R. An hypothesis concerning the molecular structure of the nicotinic acetylcholine receptor. Med Hypotheses. 1980 Sep;6(9):943–950. doi: 10.1016/0306-9877(80)90046-8. [DOI] [PubMed] [Google Scholar]
  23. Souroujon M. C., Mochly-Rosen D., Gordon A. S., Fuchs S. Interaction of monoclonal antibodies to Torpedo acetylcholine receptor with the receptor of skeletal muscle. Muscle Nerve. 1983 May;6(4):303–311. doi: 10.1002/mus.880060410. [DOI] [PubMed] [Google Scholar]
  24. Sumikawa K., Houghton M., Smith J. C., Bell L., Richards B. M., Barnard E. A. The molecular cloning and characterisation of cDNA coding for the alpha subunit of the acetylcholine receptor. Nucleic Acids Res. 1982 Oct 11;10(19):5809–5822. doi: 10.1093/nar/10.19.5809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Suter M. A modified ELISA technique for anti-hapten antibodies. J Immunol Methods. 1982 Aug 27;53(1):103–108. doi: 10.1016/0022-1759(82)90244-7. [DOI] [PubMed] [Google Scholar]
  26. Tzartos S. J., Changeux J. P. High affinity binding of alpha-bungarotoxin to the purified alpha-subunit and to its 27,000-dalton proteolytic peptide from Torpedo marmorata acetylcholine receptor. Requirement for sodium dodecyl sulfate. EMBO J. 1983;2(3):381–387. doi: 10.1002/j.1460-2075.1983.tb01434.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wilson P. T., Gershoni J. M., Hawrot E., Lentz T. L. Binding of alpha-bungarotoxin to proteolytic fragments of the alpha subunit of Torpedo acetylcholine receptor analyzed by protein transfer on positively charged membrane filters. Proc Natl Acad Sci U S A. 1984 Apr;81(8):2553–2557. doi: 10.1073/pnas.81.8.2553. [DOI] [PMC free article] [PubMed] [Google Scholar]

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