Gregor Fels

Antibodies against preselected peptides to map functional sites on the acetylcholine receptor

Rabbit immune sera and mouse monoclonal antibodies were raised against the synthetic peptide Tyr‐Cys‐Glu‐Ile‐Ile‐Val matching in sequence residues 127–132 of the α‐subunit of all nicotinic acetylcholine receptors sequenced so far. Representative cholinergic ligands did not interfere with the binding of these antibodies to the receptor from Torpedo marmorala, indicating that this sequence is not part of the binding sites for cholinergic ligands. The applicability of antigenic sites analysis to the mapping of functional sites on receptor proteins is discussed.

Use of Synthetic Peptides and High Affinity Protein Ligands for Structural Studies of Central and Peripheral Nicotinic Receptors

Neurotransmitter receptors are key components of neuronal function. A major breakthrough in their study has been provided by cloning and sequencing of their genes and deduction of the amino acid sequence of their precursors.

Antibodies as Probes of the Structure and Function of the Nicotinic Acetylcholine Receptor

Structural and functional studies of the nicotinic acetylcholine receptor from Torpedo marmorata are discussed as examples for the distinct differences in properties of antibodies raised against a native antigen as compared to antibodies raised against short or long synthetic peptides.

Antibodies Directed against Functional Sites on the Acetylcholine Receptor from Torpedo Marmorata

Antibodies directed against functional sites on the acetylcholine receptor from Torpedo marmorata have been obtained by the following two procedures: (i) Our library of monoclonal antibodies raised against the whole receptor protein was screened for antibodies competing with cholinergic agonists, antagonists and local anesthetics for receptor binding, (ii) antibodies were raised against short peptides matching the sequence of predetermined sites on the receptor protein. In this way, a topographic map of the functional sites on the receptor surface can be constructed.

The Limited Sequence Specificity of Anti-Peptide Antibodies may Introduce Ambiguity in Topological Studies

Our present view of the membrane topology of nicotinic acetylcholine receptors (nAChR) heavily depends on hydropathy plots (Kyte kath Doolittle, 1982; Noda et al., 1982; Claudio et al., 1983) and epitope mapping (Lindstrom et al., 1984; Maelicke et al., 1984; Neumann et al., 1984; Ratnam kath Lindstrom, 1984; Plumer et al., 1984; Young et al., 1985; Fuchs kath Safran, 1986; Ratnam et al., 1986a,b). The latter is usually performed with antibodies raised against rather long synthetic peptides. We have criticized this approach (Maelicke et al., 1984; Plumer et al., 1984) as it may lead to ambiguous results: Recent structural data have established that the antigen recognition site of antibodies is quite large and that antibody-antigen interaction probably involves multipoint attachment (Amit et al., 1986). Thus, only under limiting conditions will the charge density pattern defining an antibody binding site be directly correlated to the primary structure of this particular region of the antigen. The following results of a topological study of the nAChR from Torpedo electric organ (Maelicke et al., 1989) may exemplify this problem.

Evidence for Multipoint Attachment of Ligands to the Nicotinic Acetylcholine Receptor

Binding of 125I-labelled α-bungarotoxin (αBTX) and of several 3H-labelled αBTX-competitive anti-nAChR antibodies suggest that the cholinergic binding site(s) are “discontinuous”, i.e. they are formed by several sequence segments which are well separated along the primary structure of the nAChR α-subunit (Conti-Tronconi et al., 1989). Presumably, these non-continuous segments are brought together, by the tertiary folding of the α-subunit, to form the area of the nAChR surface recognized by the neurotoxin and the antibodies. Our data agree with previous reports (Neumann et al., 1986; Gotti et al., 1987; Ralston et al., 1987; Gershoni 1987; Gotti et al., 1989; Gershoni et al., 1989) in that the region around cysteines 192 and 193 forms a major component of the toxin binding site. They result in a different model of the cholinergic binding site, however, in that they suggest a rather intricately structured binding region, with particular sensitivity to con-formational changes and the capability of selective binding of classes of ligands (Conti-Tronconi et al., unpublished).

Antibodies as Probes of the Structure of the Nicotinic Acetylcholine Receptor

Antibodies were raised against eight synthetic peptides matching preselected portions of the amino acid sequence of the nicotinic acetylcholine receptor (AChR) from Torpedo marmorata. To avoid ambiguity as to the exact sequence location of the antibody epitopes, synthetic peptides of only 5–7 amino acids in length were employed.

Specific immunosorbents in diagnosis and management of myasthenia gravis.

It would certainly be helpful for both our basic understanding and the clinical management of myasthenia gravis if sensitive procedures were available to monitor (and, if advisable, to extract) the anti-AChR antibodies circulating in myasthenic patients and animals. To develop antibody-trapping devices for this purpose, we have immobilized various peptides and proteins on nylon surfaces and have tested the chromatographic properties of these “affinity tubes.” We find that (i) high coupling densities of the affinity ligand can be obtained; (ii) the biospecific properties of the affinity ligands tested are preserved after immobilization at the nylon matrix; and (iii) depending on the specific purpose, simple and reusable devices for titer determinations and antibody extraction can be developed. In all of these respects, nylon affinity tubes appear to favorably compare with classical column materials.’.* Nylon is a solid matrix with structural similarities to polypeptides: the backbones of both polymers are formed by secondary amide bonds; these are spaced by either one (polypeptides) or several (nylon) methylene groups. Both polymers form hydrogen-bonded structures. To covalently immobilize proteins at the nylon surface’-’ some of the nylon amide bonds are hydrolyzed, and the resulting amino and carboxy groups are cross-linked or directly coupled to amino or carboxy groups of the protein. To test the properties of these affinity matrices, we have mainly worked with tubular nylon supports and purified AChR, BSA, and a-cobratoxin as affinity ligands. We have limited experience with nylon hollow fibers as support and antibodies as affinity ligands.

Monoclonal Antibodies as Probes of Acetylcholine Receptor Function

So far, monoclonal antibodies (mAb) to the AChR have mainly been employed in the structural analysis of the AChR (see related papers by Lindstrom et al., Fuchs, et al., Tzartos et al., Barkas et al., Froehner, et al. in this volume; Young et al. 1985). The existence of only a few reports on functional studies (Moshly-Rosen and Fuchs, 1981; Watters and Maelicke, 1983; Mikovilovic and Richman, 1984; Lindstrom, 1984) is mainly due to the fact that antibodies interfering with ligand binding or channel gating of the AChR are rather uncommon in mAb libraries obtained by conventional immunization techniques (Gomez and Richman, 1983; Lindstrom, 1984). Recently, several methods have been developed to overcome the limited variation of the common immune response (Matthew and Patterson, 1983; Maelicke et al., in preparation). In addition, suitable assays for the selection of function-affecting antibodies have been developed (Watters and Maelicke, 1982, 1983; Fels et al., 1985, 1986). Here we summarize and discuss some recent data from our laboratory obtained with such antibodies. As the key results, (i) we have identified antibodies which affect receptor function indirectly, i.e. by allosteric interactions with ligand binding sites and channel gating sites, and (ii) channel gating probably requires the concerted action of two molecules of bound agonist.