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

D. Neumann, J. M. Gershoni, M. Fridkin, S. Fuchs

Research output: Contribution to journalArticlepeer-review

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 α 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 α subunit. The antipeptide antibodies were used to test whether the peptide sequences corresponded to the α-bungarotoxin (α-BTX)-binding site. Staphylococcus aureus V8-protease digestion of the isolated receptor α 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 α-BTX. On the other hand, α-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 Vi 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.

Original languageEnglish
Pages (from-to)3490-3493
Number of pages4
JournalProceedings of the National Academy of Sciences of the United States of America
Volume82
Issue number10
DOIs
StatePublished - 1985
Externally publishedYes

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