Mapping the mAb 383C epitope to alpha 2(187-199) of the Torpedo acetylcholine receptor on the three-dimensional model.
Monoclonal antibody 383C is an anti-acetylcholine receptor antibody whose binding to the receptor is blocked by alpha-bungarotoxin and by carbamylcholine. Monoclonal antibody 383C binds to the alpha subunit of the Torpedo acetylcholine (ACh) receptor as well as to its V8-protease 20 kDa fragment that possesses the affinity alkylatable Cys192/193. In an epitope scanning experiment spanning the N-terminal 211 amino acid residues of the alpha subunit, 383C binds uniquely to three overlapping peptides; alpha(184-196), alpha(187-199) and alpha(190-202). These peptides span a cluster of amino acid residues implicated in the binding of acetylcholine, including Cys192/193. To map the location of these residues on the three-dimensional model of the ACh receptor, we have employed a combination of X-ray diffraction from oriented complexes of 383C with ACh receptor-enriched membrane vesicles and electron microscopy of negatively stained tubular arrays of 383C/receptor complexes. The X-ray diffraction study finds extra electron density in the presence of 383C centered 35 A above the synaptic side phosphate head groups. The electron micrographic images display extra stain exclusion from the antibody at a site adjacent to the alpha2 subunit on the periphery of the rosette clockwise to the alpha2 vertex. This mapping localizes several residues of the ACh receptor alpha subunit involved in the binding of acetylcholine. Despite these residues being present in both alpha subunits, only the alpha2 subunit is decorated with this monoclonal antibody.
Fairclough
R H
RH
Department of Neurology, University of California Davis, Davis, CA, 95616, USA.
Twaddle
G M
GM
Gudipati
E
E
Stone
R J
RJ
Richman
D P
DP
Burkwall
D A
DA
Josephs
R
R
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D018360:Crystallography, X-Ray; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes / Q000032:analysis* / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry; D008958:Models, Molecular; D011940:Receptor Aggregation; D011950:Receptors, Cholinergic / Q000737:chemistry*; D014101:Torpedo
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008051:Lipid Bilayers; D011950:Receptors, Cholinergic