TY - JOUR
T1 - Mapping the mAb 383C epitope to α2(187-199) of the Torpedo acetylcholine receptor on the three-dimensional model
AU - Fairclough, Robert H
AU - Twaddle, George M.
AU - Gudipati, Eswari
AU - Stone, Remington J S
AU - Richman, David P
AU - Burkwall, David A.
AU - Josephs, Robert
PY - 1998/9/18
Y1 - 1998/9/18
N2 - Monoclonal antibody 383C is an anti-acetylcholine receptor antibody whose binding to the receptor is blocked by α-bungarotoxin and by carbamylcholine. Monoclonal antibody 383C binds to the α 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 α subunit, 383C binds uniquely to three overlapping peptides; α(184-196), α(187-199) and α(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 Å above the synaptic side phosphate head groups. The electron micrographic images display extra stain exclusion from the antibody at a site adjacent to the α2 subunit on the periphery of the rosette clockwise to the α2 vertex. This mapping localizes several residues of the ACh receptor a subunit involved in the binding of acetylcholine. Despite these residues being present in both α subunits, only the α2 subunit is decorated with this monoclonal antibody.
AB - Monoclonal antibody 383C is an anti-acetylcholine receptor antibody whose binding to the receptor is blocked by α-bungarotoxin and by carbamylcholine. Monoclonal antibody 383C binds to the α 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 α subunit, 383C binds uniquely to three overlapping peptides; α(184-196), α(187-199) and α(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 Å above the synaptic side phosphate head groups. The electron micrographic images display extra stain exclusion from the antibody at a site adjacent to the α2 subunit on the periphery of the rosette clockwise to the α2 vertex. This mapping localizes several residues of the ACh receptor a subunit involved in the binding of acetylcholine. Despite these residues being present in both α subunits, only the α2 subunit is decorated with this monoclonal antibody.
KW - 3-D epitope mapping
KW - Acetylcholine binding residues
KW - Acetylcholine receptor
KW - Anti-acetylcholine receptor monoclonal antibody
KW - mAb epitope mapping
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U2 - 10.1006/jmbi.1998.2000
DO - 10.1006/jmbi.1998.2000
M3 - Article
C2 - 9735289
AN - SCOPUS:0032544333
VL - 282
SP - 301
EP - 315
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
SN - 0022-2836
IS - 2
ER -