Antibodies to synthetic peptides as probes for the binding site on the alpha subunit of the acetylcholine receptor.
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.
Neumann
D
D
Gershoni
J M
JM
Fridkin
M
M
Fuchs
S
S
D000109:Acetylcholine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002038:Bungarotoxins; D000939:Epitopes; D007163:Immunosorbent Techniques; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000276:immunology*
D002038:Bungarotoxins; D000939:Epitopes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D000109:Acetylcholine