Bianca M. Conti-Tronconi

The Nicotinic Acetylcholine Receptor: Structure and Autoimmune Pathology

The nicotinic acetylcholine receptors (AChR) are presently the best-characterized neurotransmitter receptors. They are pentamers of homologous or identical subunits, symmetrically arranged to form a transmembrane cation channel. The AChR subunits form a family of homologous proteins, derived from a common ancestor. An autoimmune response to muscle AChR causes the disease myasthenia gravis. This review summarizes recent developments in the understanding of the AChR structure and its molecular recognition by the immune system in myasthenia.

Amphipathic segment of the nicotinic receptor alpha subunit contains epitopes recognized by T lymphocytes in myasthenia gravis.

Autoimmune helper T lymphocytes were selected from the blood of two myasthenic patients of different HLA-DR type, using acetylcholine receptor (AChR) from Torpedo californica. These polyclonal T cell lines were tested for reactivity with three synthetic peptides corresponding to the NH2-terminal region of the human AChR alpha subunit. This segment is a good candidate for T cell epitopes since it has a propensity to form an amphipathic alpha helix. The peptides elicited 10-30% of the response induced by native Torpedo AChR. Different peptides were recognized by the autoreactive T cells of the two patients. These results suggest that the NH2-terminal region of the AChR alpha chain contains T cell-stimulating epitopes, and that the T cell autoimmune response in myasthenia gravis, like the B cell response, is heterogeneous.

Cellular immune response against acetylcholine receptor in myasthenia gravis I. Relevance to clinical course and pathogenesis

The cellular immune response to acetylcholine receptor from Torpedo electric organ was studied in 100 myasthenic patients and 41 healthy subjects. The mean stimulation index (SI) was 2 ± 0.15 for the patients, and 1.06 ± 0.08 for the controls. Stimulation was significantly greater when the test medium contained autologous serum rather than a standard universal serum (AB serum). Young patients were generally good responders (SI, 2.39 ± 0.26), but older patients usually did not respond (mean SI, 1.18 ± 0.13). Among the younger patients, men had higher responses than women (mean SI, 3.13 ± 0.63 and 2.05 ± 0.23, respectively). There was no correlation between degree of lymphocytic reactivity and duration or severity of symptoms.

Epitope mapping of polyclonal and monoclonal antibodies against two α-bungarotoxin-binding α subunits from neuronal nicotinic receptors

Abstract Recently, cDNAs for α subunits of two different neuronal α-bungarotoxin-binding proteins (αBgtBP) were isolated from chick brain, designated αBgtBP α1 and αBgtBP α2. These are now also referred to as subunits α7 and α8, respectively. Expression studies in Xenopus oocytes have indicated that α7 subunits are able to form cation channels that are sensitive to nicotinic ligands, and therefore represent bona fide nicotinic acetylcholine receptor subunits. Polyclonal and monoclonal antibodies (mAbs) have been produced against: (i) affinity-purified chick brain αBgtBP; and (ii) fusion proteins containing the unique cytoplasmic sequences α7(327–412) and α8(293–435). Here, synthetic overlapping peptides corresponding to their deduced amino acid sequence are used to map the epitopes recognized by the different antibodies. The polyclonal response to affinity-purified αBgtBPs and the fusion proteins indicates that sequence segments 290–420 of both subunits contain several major and minor epitopes. mAbs selected for their ability to bind both native and denatured αBgtBPs isolated from chick brain also recognise subunit-specific sequential epitopes within the sequence segment 290–420. The epitopes recognized by the mAbs correspond to the minor epitopes defined using antisera. The mAbss characterized in these studies will provide useful probes for further studies of αBgtBP structure and histological localization.

Main immunogenic region of Torpedo electroplax and human muscle acetylcholine receptor: localization and microheterogeneity revealed by the use of synthetic peptides

Abstract: Most anti‐nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR α‐subunit. Thirty‐two synthetic peptides, corresponding to the complete Torpedoα‐subunit sequence and to a segment of human muscle α‐subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti‐AChR mAbs directed against epitopes on the α‐subunit other than the MIR. A main constituent loop of the MIR was localized within residues α67‐76. Residues 70 and 75, which are different in the Torpedo and human α‐subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop α67‐76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment α67‐76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68‐71. The most stable structure predicted for this segment, in both the Torpedo and human α‐subunits, is a hairpin loop, whose apex is a type I β‐turn and whose arms are β‐strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non‐MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues α331‐351.

CD4+ T‐epitope repertoire on the human acetylcholine receptor α subunit in severe myasthenia gravis: A study with synthetic peptides

The α subunit of the nicotinic acetylcholine receptor (AChR) seems crucial in the pathogenesis of the autoimmune paralysis myasthenia gravis (MG) because it contains both the epitopes that dominate the antibody response against the AChR and those recognized by CD4+ AChR-specific T helper (Th) cells. To define the repertoire of anti-AChR Th cells, we investigated the response of unselected blood CD4+ cells or total lymphocytes, or both, from 22 MG patients to 20-residue overlapping synthetic peptides, screening the complete sequence of human-muscle AChR α subunit. Several epitopes were identified. Only the most severely affected patients recognized α subunit epitopes, and they were mainly young women. Detection of in vitro AChR-specific CD4+ response was facilitated by removal of the CD8+ cells because in two patients a clear response to several α subunit peptide sequences could be detected when CD+-depleted cells were used, while their total peripheral blood mononuclear cell population did not respond to any α subunit peptide. Although each patient had a unique pattern of peptide recognition, four immunodominant regions recognized by long-term AChR-specific CD4+ T-cell lines, or flanking peptide sequences, were recognized most frequently (residues 48–67, 101–137, 293–337, and 308–437).

Adult thymus expresses an embryonic nicotinic acetylcholine receptor-like protein

The subunit composition of acetylcholine receptor-like protein(s) (AChR-LP) expressed by normal thymus was investigated. In skeletal muscle, the AChR exists in two forms, an embryonic form which contains the gamma-subunit and an adult form where the gamma-subunit is substituted by a different, homologous subunit called epsilon. Antibodies against unique sequence segments of the embryonic gamma-subunit and of the adult epsilon-subunit of bovine muscle AChR, in addition to antibodies specific for the alpha-, beta-, and delta-subunits of bovine muscle AChR, were used to probe immunoblots of AChR-LP(s) from bovine thymus. Subunits of approximate Mr 41 kDa, 48-54 kDa, 57 kDa and 67-72 kDa were recognized by anti-alpha, anti-beta, anti-gamma and anti-delta antibodies respectively. Anti-epsilon antibodies did not recognize any protein band from bovine thymus. AChR-LP similar or identical to the embryonic muscle AChR is therefore expressed in normal thymus.

T helper cell recognition of muscle acetylcholine receptor in myasthenia gravis. Epitopes on the gamma and delta subunits.

We tested the response of CD4+ cells and/or total lymphocytes from the blood of 22 myasthenic patients and 10 healthy controls to overlapping synthetic peptides, 20 residues long, to screen the sequence of the gamma and delta subunits of human muscle acetylcholine receptor (AChR). The gamma subunit is part of the AChR expressed in embryonic muscle and is substituted in the AChRs of most adult muscles by an epsilon subunit. The delta subunit is present in both embryonic and adult AChRs. Adult extrinsic ocular muscles, which are preferentially and sometimes uniquely affected by myasthenic symptoms, and thymus, which has a still obscure but important role in the pathogenesis of myasthenia gravis, express the embryonic gamma subunit. Anti-AChR CD4+ responses were more easily detected after CD8+ depletion. All responders recognized epitopes on both the gamma and delta subunits and had severe symptoms. In four patients the CD4+ cell response was tested twice, when the symptoms were severe and during a period of remission. Consistently, the response was only detectable, or larger, when the patients were severely affected.

T‐Helper Epitopes on Human Nicotinic Acetylcholine Receptor in Myasthenia Gravis

The synthesis of AChR antibodies requires intervention of AChR-specific Th cells. Because of the paucity of anti-AChR Th cells in the blood of myasthenia gravis (MG) patients, direct studies of these autoimmune cells in the blood are seldom possible. Propagation in vitro of anti-AChR T cells from MG patients by cycles of stimulation with AChR antigens selectively enriches and expands the autoimmune T-cell clones, allowing investigation of their function and epitope specificity. Torpedo electroplax AChR was initially used for propagation of anti-AChR T-cell lines. Those studies demonstrated the feasibility of in vitro propagation of AChR-specific T cells. These are bona fide CD4+ Th cells, which stimulate production in vitro of anti-AChR antibodies by B cells of myasthenic patients and recognize equally well denatured and native AChR, suggesting the usefulness of synthetic human AChR sequences as antigens for propagation of the autoimmune Th cells. We used pools of overlapping synthetic peptides, corresponding to the complete sequences of the human AChR alpha-, beta-, gamma-, and delta-subunits, to propagate AChR-specific Th cells from the blood of MG patients. The AChR sequence regions forming epitopes recognized by the autoimmune T cells were determined by challenging the lines with individual synthetic peptides, 20 residues long, screening the AChR subunit sequences. Although each line had an individual pattern of epitope recognition--as expected from their different HLA-DR haplotype--some peptides were recognized by most of all the CD4+ T-cell lines, irrespective of their DR haplotype. The existence of immunodominant regions of the AChR sequence was verified by investigating the response of unselected CD4+ cells from the blood of a relatively large number of MG patients to the individual peptides screening the human alpha-, gamma-, and delta-subunit sequences. Those studies confirmed that each patient has an individual pattern of peptide recognition. The studies also identified a large number of T epitopes of the human AChR and verified the existence of sequence regions immunodominant for T-helper sensitization, because a limited number of sequence regions, including all those immunodominant for the T-helper lines, were recognized by most patients. Anti-AChR CD4+ T lines could be propagated from some healthy controls only for a brief period of time. They recognized AChR sequences poorly, suggesting a low affinity of their T-cell receptors for the corresponding AChR epitopes.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylcholine receptor dimers are stabilized by extracellular disulfide bonding.

Torpedo acetylcholine receptor (AcChR) exists predominantly as dimers, formed by two monomers held together by a disulfide bridge(s). The dimers are easily cleaved to monomers by reducing agents. 2-mercaptoethanesulfonic acid is shown to be a membrane-impermeant reducing agent which cleaves receptor dimers when it is present only on the outside of intact membrane vesicles. There is no increase in the extent of cleavage when 2-mercaptoethanesulfonic acid is also loaded inside the vesicles. Therefore the disulfide bond(s) involved in the dimerization of the Torpedo acetylcholine receptor is (are) formed by cysteine residues which are exposed on the extracellular side of the membrane.