Mapping the mAb 383C epitope to α2(187-199) of the Torpedo acetylcholine receptor on the three-dimensional model

Robert H Fairclough, George M. Twaddle, Eswari Gudipati, Remington J S Stone, David P Richman, David A. Burkwall, Robert Josephs

Research output: Contribution to journalArticle

7 Scopus citations

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 languageEnglish (US)
Pages (from-to)301-315
Number of pages15
JournalJournal of Molecular Biology
Volume282
Issue number2
DOIs
StatePublished - Sep 18 1998

Keywords

  • 3-D epitope mapping
  • Acetylcholine binding residues
  • Acetylcholine receptor
  • Anti-acetylcholine receptor monoclonal antibody
  • mAb epitope mapping

ASJC Scopus subject areas

  • Virology

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