Myasthenia gravis. Residues of the alpha and gamma subunits of muscle acetylcholine receptor involved in formation of immunodominant CD4+ epitopes.

We propagated from myasthenia gravis (MG) patients by stimulation in vitro with synthetic sequences (alpha 48-67, alpha 304-322, gamma 75-94 and gamma 321-340) of the human muscle acetylcholine receptor (AChR), CD4+ lines against four 20-residue sequence regions of the AChR alpha and gamma subunits that are recognized by Th cells of most MG patients. Most lines secreted IL-2 and not IL-4, suggesting that they comprise Th1 cells. For three lines we verified that, as reported previously, AChR epitopes are presented by DR molecules: their response to the relevant peptide was abolished by anti-DR Abs.

Residues within the alpha subunit sequence 304-322 of muscle acetylcholine receptor forming autoimmune CD4+ epitopes in BALB/c mice.

BALB/c mice develop myasthenic symptoms after immunization with rodent acetylcholine receptor (AChR). After immunization with Torpedo AChR (TAChR), their CD4+ cells become strongly sensitized against a conserved region of the TAChR alpha subunit sequence (residues alpha 304-322), and cross-react vigorously with the homologous sequences of mouse and human AChR, which are almost identical. Therefore AChR-specific potentially autoreactive CD4+ cells exist in this strain.

Human acetylcholine receptor presentation in myasthenia gravis. DR restriction of autoimmune T epitopes and binding of synthetic receptor sequences to DR molecules.

Autoimmune Th cells in myasthenia gravis recognize several sequence regions of the human muscle acetylcholine receptor (AChR). Most AChR Th epitopes are presented by HLA class II DR molecules (DR). Four sequence regions of the AChR alpha-subunit form Th epitopes recognized by most myasthenic patients, irrespective of their DR haplotype.

Monoclonal antibodies FK1 and WF6 define two neighboring ligand binding sites on Torpedo acetylcholine receptor alpha-polypeptide.

Previous studies have identified the sequence region flanking the invariant vicinal cysteinyl residues at positions 192 and 193 of the nicotinic acetylcholine receptor alpha-subunit as containing major elements of the binding site for acetylcholine and its agonists and antagonists, including antibody WF6 (Conti-Tronconi, B. M., Diethelm, B.

Preferential pairing of T and B cells for production of antibodies without covalent association of T and B epitopes.

T cell from H-2b mice recognize at least 12 sequence regions on the Torpedo acetylcholine receptor (TAChR) alpha, gamma and delta subunits. Immunization of C57BL/6 mice with individual synthetic TAChR sequences known to contain CD4+ epitopes resulted in most cases (10 out of 12 peptides) in anti-peptide antibody (Ab) production, indicating that short TAChR sequences contain both CD4+ and B epitopes. Immunization of C57BL/6 mice with a mixture of a CD4+ epitope peptide, from the TAChR or from an unrelated protein, plus another TAChR sequence forming a "pure" B epitope (T alpha 63-80), induced in most cases anti-peptide Ab and CD4+ cell sensitization only against the peptide containing the CD4+ epitope.

An alpha-bungarotoxin-binding sequence on the Torpedo nicotinic acetylcholine receptor alpha-subunit: conservative amino acid substitutions reveal side-chain specific interactions.

In the alpha subunit of the Torpedo nicotinic cholinergic receptor (AChR), a sequence region surrounding a pair of adjacent cysteinyl residues at positions 192 and 193 contributes to a binding site for cholinergic ligands, including the snake alpha-neurotoxins. Synthetic and biosynthetic peptides corresponding to this region bind alpha-bungarotoxin (alpha-BTX) in the absence of other structural components of the AChR and, therefore, represent a "prototope" for alpha-BTX. Using synthetic peptides corresponding to the complete AChR alpha subunits of Torpedo electroplax and mammalian muscle, we previously defined a sequence segment corresponding to a universal prototope for alpha-BTX binding between amino acid residues 181 and 200 [Conti-Tronconi, B.

Epitopes on the beta subunit of human muscle acetylcholine receptor recognized by CD4+ cells of myasthenia gravis patients and healthy subjects.

We investigated the sequence regions of the human muscle acetylcholine receptor (AChR) beta subunit forming epitopes recognized by T helper cells in myasthenia gravis (MG), using overlapping synthetic peptides, 20 residues long, which screened the sequence of the AChR beta subunit. Since CD4+ lymphocytes from MG patients' blood did not respond to the peptides, we attempted propagation of beta subunit-specific T lines from six MG patients and seven healthy controls by cycles of stimulation of blood lymphocytes with the pooled peptides corresponding to the beta subunit sequence. CD4+ T lines were obtained from four patients and three controls.

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.

A high-affinity site for acetylcholine occurs close to the alpha-gamma subunit interface of Torpedo nicotinic acetylcholine receptor.

Affinity labeling techniques have been used to investigate the location of high-affinity binding sites for cholinergic agonists on the Torpedo acetylcholine receptor and the extent of overlap of these sites with those for long alpha-neurotoxins. Following reduction of the receptor by dithiothreitol, reaction with [3H]bromoacetylcholine leads to covalent incorporation into each of the two alpha subunits. At high concentrations of [3H]bromoacetylcholine (240 microM) and with prolonged incubation times (1-2 h), this labeling was not inhibited by either alpha-bungarotoxin or alpha-najatoxin.

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.

Cryptic epitopes on the nicotinic acetylcholine receptor are recognized by autoreactive CD4+ cells.

Experimental autoimmune myasthenia gravis is induced in C57BL/6 mice by injection of Torpedo nicotinic acetylcholine receptor (TAChR). We investigated here the presence of cryptic CD4+ epitopes on the TAChR molecule, and their relationship with potentially autoreactive CD4+ cells, which survived clonal deletion. CD4+ cells from C57BL/6 mice immunized with native or denatured TAChR were challenged in vitro with overlapping synthetic peptides, 20-residue long, screening the sequences of TAChR alpha, gamma, and delta subunits.

Homologous kappa-neurotoxins exhibit residue-specific interactions with the alpha 3 subunit of the nicotinic acetylcholine receptor: a comparison of the structural requirements for kappa-bungarotoxin and kappa-flavitoxin binding.

kappa-Flavotoxin (kappa-FTX), a snake neurotoxin that is a selective antagonist of certain neuronal nicotinic acetylcholine receptors (AChRs), has recently been isolated and characterized [Grant, G. A., Frazier, M.

Myasthenia gravis: recognition of a human autoantigen at the molecular level.

The symptoms of myasthenia gravis are primarily or exclusively due to an autoimmune response against the muscle nicotinic acetylcholine receptor (AChR) and this has been the object of intensive investigations for almost 20 years. A detailed picture at the molecular level of the interaction of this autoantigen with the key elements involved in the autoimmune response, such as anti-AChR antibodies, the T-cell receptor and restricting major histocompatibility complex molecules, is now emerging for both human myasthenia gravis and its experimental model, experimental autoimmune myasthenia gravis. Here, Maria Pia Protti and colleagues focus on the molecular interactions occurring in human myasthenia gravis and summarize recent information on pathogenic mechanisms of the autoimmune response, and the structure of epitopes recognized by B cells and CD4+ T cells of myasthenic patients on the AChR molecule.

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.

An assay for simultaneous multiple determinations of peptide binding to MHC class II molecules.

Binding of positively charged radiolabeled synthetic peptides to human major histocompatibility complex class II DR molecules, purified by affinity chromatography from lymphoblastoid B cell lines of different haplotypes, is rapidly, quantitatively, and specifically assayed by selective adsorption of the complexes between peptide and DR molecules onto DEAE-cellulose paper disks. This assay can be used as a revealing system of the ability of unlabeled test peptides to competitively inhibit the binding between the radiolabeled peptide and the DR molecules, thus measuring the binding of the competitor peptides, irrespective of their charge properties, to different DR molecules.

The 'embryonic' gamma subunit of the nicotinic acetylcholine receptor is expressed in adult extraocular muscle.

We have PCR-amplified cDNA sequence of the "embryonic" gamma subunit of muscle acetylcholine receptor (AChR) from adult bovine extraocular muscle (EOM). We cloned and sequenced this product and used it to probe Northern blots. We detected the gamma subunit in EOM mRNA, but not in control skeletal muscle.

Monoclonal antibodies against synthetic sequences of the nicotinic receptor cross-react fully with the native receptor and reveal the transmembrane disposition of their epitopes.

Monoclonal antibodies (mAbs) were derived from mice immunized with synthetic peptide sequence regions of the alpha subunit of the nicotinic acetylcholine receptor from Torpedo electric tissue (TAChR). Sequence-specific mAbs were obtained against the following peptides: alpha 1-20, alpha 291-308, alpha 304-322, alpha 332-350, alpha 346-364, alpha 360-378, alpha 376-393, alpha 390-409, and alpha 420-437. The ability of mAbs to recognize native TAChR was quantitated by immunoprecipitation of TAChR solubilized in the nondenaturing detergent Triton X-100.

Amino acid residues within the sequence region alpha 55-74 of Torpedo nicotinic acetylcholine receptor interacting with antibodies to the main immunogenic region and with snake alpha-neurotoxins.

The sequence region 55-74 of the alpha-subunit of the acetylcholine receptor (AChR) from Torpedo californica electroplax comprises the amino-terminal end of a sequence segment--residues alpha 67-76--forming the main immunogenic region (MIR), which is most frequently recognized by anti-AChR autoantibodies in myasthenia gravis. The synthetic sequence alpha 55-74 of Torpedo AChR binds alpha-bungarotoxin (alpha BTX), suggesting that amino acid residues within this sequence region may contribute to formation of an alpha BTX binding site. Using single-residue substituted synthetic analogues of the sequence alpha 55-74 of Torpedo AChR, in which each residue was sequentially substituted by either glycine or alanine, we sought identification of the amino acids involved in interaction with alpha-neurotoxins and with three different anti-MIR monoclonal antibodies (mAbs 6, 22, and 198).

Myasthenia gravis: effect on antibody binding of conservative substitutions of amino acid residues forming the main immunogenic region of the nicotinic acetylcholine receptor.

In Myasthenia Gravis most anti-acetylcholine receptor (AChR) antibodies are against a highly conserved area of the AChR alpha-subunit called the Main Immunogenic Region (MIR). Amino acid residues critical for MIR formation have been located within the sequence alpha 67-76. In the present study, binding of anti-AChR monoclonal antibodies (mAbs) to synthetic peptide analogues of the sequence alpha 67-76 of human and Torpedo AChRs containing conservative single-residue substitutions identified the amino acid residues most important to the antigenicity of the MIR sequence, and offered clues to its tridimensional structure.

Scanning tunneling microscopy imaging of Torpedo acetylcholine receptor.

The synaptic surface of the acetylcholine receptor in membranes from Torpedo californica electric organ has been imaged by scanning tunneling microscopy. The molecule appears pentameric, with one major and four minor protrusions rising above the surface, and these protrusions encompass a large central cavity. The outer diameter of the molecule is 69 +/- 10 A, while the diameter of the cavity, measured at the widest complete contour line delimiting the opening, is 26 +/- 7 A.

Myasthenia gravis. CD4+ T epitopes on the embryonic gamma subunit of human muscle acetylcholine receptor.

In myasthenia gravis (MG) an autoimmune response against muscle acetylcholine receptor (AChR) occurs. Embryonic muscle AChR contains a gamma subunit, substituted in adult muscle by a homologous epsilon subunit. Antibodies and CD4+ cells specific for embryonic AChR have been demonstrated in MG patients.

CD4+ T-epitope repertoire on the human acetylcholine receptor alpha subunit in severe myasthenia gravis: a study with synthetic peptides.

The alpha 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 alpha subunit. Several epitopes were identified.