Abstract
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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 301-315 |
| Number of pages | 15 |
| Journal | Journal of Molecular Biology |
| Volume | 282 |
| Issue number | 2 |
| DOIs | |
| State | Published - Sep 18 1998 |
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Keywords
- 3-D epitope mapping
- Acetylcholine binding residues
- Acetylcholine receptor
- Anti-acetylcholine receptor monoclonal antibody
- mAb epitope mapping
ASJC Scopus subject areas
- Virology
Cite this
Mapping the mAb 383C epitope to α2(187-199) of the Torpedo acetylcholine receptor on the three-dimensional model. / Fairclough, Robert H; Twaddle, George M.; Gudipati, Eswari; Stone, Remington J S; Richman, David P; Burkwall, David A.; Josephs, Robert.
In: Journal of Molecular Biology, Vol. 282, No. 2, 18.09.1998, p. 301-315.Research output: Contribution to journal › Article
}
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
UR - http://www.scopus.com/inward/record.url?scp=0032544333&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0032544333&partnerID=8YFLogxK
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 -