Antibodies to synthetic peptides as probes of acetylcholine receptor structure

Abstract

Peptides containing the C-terminus of the alpha-chain of the nicotinic acetylcholine receptor (nAChR), as deduced from cDNA data, were synthesised and shown to bind to antibodies to denatured nAChR. Conversely, peptide-specific antibodies bound both to native and to denatured nAChR. Binding was exclusively to the alphachain.Trypsinization experiments and the use of the unique C-terminal hexapeptide of the alpha-chain demonstrated that the proposed C-terminus does exist on the mature alpha-chain, and that post-translational cleavage can be discounted as an explanation of the discrepancy of the molecular masses of the alpha-chain deduced from SDS gel electrophoresis (40 kDa) and from the DNA sequencing (50 kDa).Cleavage of the alpha-chain in the membrane occurs at two closely linked sites, resulting in the formation of a large fragment (approximately 35 kDa) and the remainder of the chain (approximately 9 -10 kDa).No signs of experimental myasthenia gravis were observed in rabbits immunised with C-terminal peptide coupled to carrier protein.The nicotinic acetylcholine receptor (nAChR) purified from the electric ray, Torpedo, shows four bands (CI, b, y, S) on SDS-gel electrophoresis (reviewed in [1,2]) with apparent molecular masses of 40, 50, 60 and 65 kDa, all of which bear carbohydrate [3]. The cDNAs for all chains of T. californica [4 -71 and that for the alpha-chain of T. marmorata [8,9] have been cloned and the amino acid sequences deduced. However, the calculated molecular masses are 50, 54, 57 and 58 kDa respectively, neglecting the additional contribution of carbohydrate residues. This poses the question whether the chains, and in particular the alpha-chain, undergo post-translational cleavage. The N-terminus of the derived sequence is already known to be present on the mature alpha-chain [lo]. Cleavage would therefore result in the generation of a new C-terminus. Models proposed for the insertion of the receptor chains in the membrane place the C-terminus outside [5,7,9] or inside [Ill the cell. If cleavage does occur, the new C-terminus would be internal on either model. We have synthesised the C-terminal peptide of the deduced alpha-chain sequence and raised antisera against the peptide. We here report that this proposed C-terminus is clearly present on the mature alpha-chain. MATERIALS AND METHODS Materials

Mapping the Main Immunogenic Region and Toxin-Binding Site of the Nicotinic Acetylcholine Receptor

“…2a. Immunoblotting was used to assess the reactivity of the purified material with monoclonal antibodies to the MIR (6) and with a-bungarotoxin (aBTX) as described (7). Certain fusion proteins bound anti-MIR antibodies and toxin (X4, X8, Xll), whereas others bound only anti-MIR antibodies (Xl, XIO) or only toxin (X7, X12, X13) [ Fig.…”

A modified nicotinic acetylcholine receptor lacking the ‘ion channel amphipathic helices’

“…Their apparent molecular masses ranged from 36 to 40 kDa for tnAChR compared with from 41 to 68 kDa for nAChR ( fig.la). The band at 36 kDa reacted with polyclonal antibodies to peptide Pl (a-chain residues 151-169), with mAbs to the MIR and with aBTX, as described [8]. In addition, a high molecular mass band was always present which reacted with these same reagents ( fig.lb).…”

“…Results shown are means + SE of 2-5 determinations. Antibodies are from rabbit 1 (left) and rabbit 2 (right fig.lc) showed that the anti-P9 antibodies bound to the same 10 kDa fragment previously described as containing the C-terminal peptide, P3 [8]. Anti-P9 antibodies bound to purified nAChR coated on microtitre plates; the binding was completely abolished by competition with an excess of soluble nAChR ( fig.2a).…”

“…Before use, the closed vesicles were opened by 3 cycles of freeze-thaw and 2 sonication steps; the effectiveness of this process was monitored by the increased sensitivity of the cychain to trypsin degradation [8]. An aliquot (30 ~1) was mixed with an equal volume of '2SI-~BTX (52 nM) and incubated for 30 min.…”

“…Antisera to P9 conjugated to KLH were raised as described for antisera to Pl and P3 [7,8]. Anti-P9 antibodies were purified by affinity chromatography on P9 conjugated to Affigel 10 (Biorad).…”