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B M Conti-Tronconi

Publications and source records attributed to B M Conti-Tronconi.

At least 73 records · Page 4Linked to original sources
PubMed1988

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.

Adult↗
PubMed1987

Human T-helper lymphocytes in myasthenia gravis recognize the nicotinic receptor alpha subunit.

Myasthenia gravis is a human disease caused by an autoimmune response against the nicotinic acetylcholine receptor (AcChoR). Since the molecular structure of AcChoR is well known, myasthenia gravis is an excellent system for studying the recognition of a complex membrane antigen in the human immune system. Human T-helper (TH) cell lines reactive to the AcChoR were isolated from four myasthenic patients by selection with native AcChoR from Torpedo californica. The selected TH cells could efficiently recognize native and fully denatured AcChoR. The vast majority of the TH-stimulating AcChoR epitopes were located on the denatured alpha subunit of AcChoR. Antibody competition experiments using a panel of rat anti-AcChoR monoclonal antibodies showed that 39-45% of the autoantibodies present in the sera of these same patients bound to the conformation-sensitive "main immunogenic region" (MIR), also located on the alpha subunit. However, AcChoR-induced stimulation of the T cells could not be inhibited with up to 20-fold molar excess of different rat anti-MIR monoclonal antibodies. These results suggest that the Torpedo AcChoR alpha subunit contains conformation-insensitive epitopes that play a role in the autosensitization of TH cells and that seem to be physically separated from the MIR. The specificity of the TH cell response may contribute to directing the B-cell response to other alpha-subunit determinants, such as the MIR itself.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

Nicotinic acetylcholine receptor contains multiple binding sites: evidence from binding of alpha-dendrotoxin.

We have studied the stoichiometry of the binding of the long alpha-neurotoxins from the venom of Dendroaspis viridis (alpha-dendrotoxin) and Naja naja siamensis (alpha-cobratoxin) to the membrane-bound acetylcholine receptor (AcChoR) from Torpedo californica electric organ. The number of toxin molecules bound to one AcChoR molecule was determined by simultaneous-quantitative gas-phase microsequencing of all the amino acid sequences present in AcChoR-alpha-neurotoxin complexes. This method permits the use of homogeneous (nonradiolabeled) preparations of native toxins to obtain molar ratios of neurotoxin-receptor complexes. The stoichiometry obtained for alpha-cobratoxin was 2.1 +/- 0.2 (n = 4), in agreement with the accepted view that alpha-cobratoxin, like alpha-bungarotoxin, binds to the two alpha subunits, which are constituent polypeptides of the AcChoR molecule. alpha-Dendrotoxin gave a stoichiometry of 4.1 +/- 0.5 (n = 12); therefore, the AcChoR molecule contains four binding sites for this alpha-neurotoxin, two of which are recognized by alpha-cobratoxin. In support of this contention we have also found that when the AcChoR is saturated with alpha-bungarotoxin, addition of alpha-dendrotoxin markedly accelerates the dissociation of the bound alpha-bungarotoxin, demonstrating that the occupancy of the additional two sites by the latter toxin influences and decreases the affinity of the former toxin for its two binding sites. The fact that the AcChoR molecule is a pseudosymmetric complex of five highly homologous peptides suggests the possibility that as many as five binding sites for cholinergic ligand could be present, one on each subunit.

Animals↗
PubMed1985

Structural and functional aspects of the nicotinic acetylcholine receptor.

The molecular structure of nicotinic acetylcholine receptors (AcChR) from different peripheral tissues and from different brain areas has been studied. AcChRs from Torpedo and Electrophorus electric organ and from piscine, avian, and mammalian muscle have been shown to be highly conserved proteins composed of four types of homologous subunits (alpha, beta, gamma, and delta) which associate in a stoichiometry alpha 2 beta 1 gamma 1 delta 1 to form a pseudosymmetric pentameric complex molecule. Genealogical analysis suggests that all the subunits of these AcChR derive from a common ancestral gene and that the divergence occurred very early in the evolution of the receptor. This shared ancestry and the very early divergence of the four subunits, as well as their highly conserved structures along the animal evolution, suggests that each of the subunits evolved to perform specific crucial roles in the function of the AcChR complex. In Torpedo the two alpha-subunits present in the AcChR molecule are glycosylated to a different extent. This may be the reason why the binding sites for cholinergic ligands that are located on the alpha-subunits are non-equivalent. The pseudosymmetric pentameric complex has all the properties of a physiological receptor with respect to known parameters leading to postsynaptic depolarization, and therefore contains the molecular structure/structures that constitute a cation selective channel for transport across the membrane. The complex also contains recognition sites for acetylcholine, cholinergic antagonists, polypeptide neurotoxins, small molecule neurotoxins, and local anesthetics. Therefore a multiplicity of receptor-ligand associations are possible. This leads to more complex models of such interactions than previously considered.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1985

Brain and muscle nicotinic acetylcholine receptors are different but homologous proteins.

An alpha-bungarotoxin-binding protein was purified from chick optic lobe and brain by an improved method. Previous and present observations justify its designation as a brain nicotinic acetylcholine receptor (AcChoR). It contains subunits whose apparent molecular weights are somewhat larger than those of subunits of peripheral AcChoRs. The size of the optic lobe AcChoR complex is greater than that of the peripheral receptor when estimated from its sedimentation behavior. Brain AcChoR subunits can be specifically precipitated by a monoclonal antibody directed against chick muscle AcChoR. Amino-terminal amino acid sequence analysis was performed on AcChoR preparations and isolated subunits from the optic lobe and from the rest of the chick brain. The sequences obtained demonstrate that, at least for the lowest molecular weight component, the AcChoRs from different brain areas are identical and they are highly homologous to muscle AcChoR. It is concluded that the brain alpha-bungarotoxin-binding protein is indeed a nicotinic AcChoR and is encoded by a set of genes that is different from, but strongly related to, that for the muscle AcChoR.

Amino Acid Sequence↗
PubMed1984

Molecular weight and structural nonequivalence of the mature alpha subunits of Torpedo californica acetylcholine receptor.

A discrepancy of about 20% exists between the molecular weight of the alpha subunit of Torpedo californica electroplax acetylcholine receptor as determined by gel electrophoresis of the mature protein (Mr 40,000 +/- 2000) and by nucleotide sequence analysis of cDNA (Mr approximately equal to 50,000). We demonstrate by amino acid sequence analysis that post-translational processing does not occur and that the mature subunit has a Mr of approximately equal to 50,000. The functional acetylcholine receptor contains two copies of this alpha subunit in addition to one each of related beta, gamma, and delta subunits. The binding sites for cholinergic ligands that are located on the alpha subunits have been shown to be nonequivalent. Amino acid sequence analysis of peptides obtained by proteolytic cleavage of the alpha subunit reveals that N-asparagine glycosylation at a single site (residue 141) occurs to a different extent in the two copies of this polypeptide in the mature protein and provides an explanation for nonequivalence of their binding sites.

Amino Acid Sequence↗
PubMed1984

Multisubunit structure and amino-terminal sequences of piscine muscle acetylcholine receptor.

The nicotinic acetylcholine receptor (AcChR) has been purified from both the electric organ and the muscle of the fish Electrophorus electricus. Upon SDS gel electrophoresis muscle AcChRs appeared to contain four main polypeptides whose molecular weights were similar but not identical to the molecular weights of the four peptides present in the electric organ AcChR. Each of these peptides has been isolated and their amino-terminal sequences have been determined. The AcChRs from muscle were found to be composed of four homologous proteins of apparent molecular weight 40,500, 50,000, 56,000 and 63,000, respectively. The subunit of Mr 40,500 is present in two copies for each AcChR molecule, while the other three components are present in one copy. No difference was found between the sequenced segments of corresponding subunits from muscle and from electric organ AcChR, suggesting that AcChRs in different tissues of the same animal are products of identical genes. The Electrophorus AcChR subunits are highly homologous with the corresponding subunits of Torpedo californica AcChR.

Amino Acid Sequence↗
PubMed1984

Purification and characterization of a bungarotoxin polypeptide which blocks nicotinic receptor function in primary culture of adrenal chromaffin cells.

In primary cultures of bovine chromaffin cells, commercially available preparations of alpha-bungarotoxin inhibit the acetylcholine (ACh)- or nicotine-evoked release of endogenous catecholamines. The potency of different lots of alpha-bungarotoxin is not related to the alpha-bungarotoxin peptide content but to that of another peptide (termed P-4 bungarotoxin) present as an impurity in the alpha-bungarotoxin preparations. P-4 Bungarotoxin was isolated and purified to homogeneity by high-pressure liquid chromatography (HPLC). Homogeneity was established by a variety of means, including polyacrylamide gel electrophoresis, HPLC, end carboxy group analysis and NH2-terminal amino acid sequence. Purified P-4 bungarotoxin contains approximately 121 amino acid residues, and it is different in its amino composition, molecular weight, and amino acid sequence from alpha-bungarotoxin and beta-bungarotoxin. P-4 Bungarotoxin (IC50 congruent to 1 nM) blocked the ACh-induced release of endogenous catecholamines but failed to block the KCl-induced catecholamine release. Although P-4 bungarotoxin is endowed with phospholipase A2 activity, its effect on ACh-evoked catecholamine release persists when the phospholipase activity is blocked (99.9%) by treatment of the toxin with p-bromophenacyl bromide. P-4 Bungarotoxin may represent a useful tool with which to study nicotinic receptor function in sympathetic and central nervous system neurons.

Acetylcholine↗
PubMed1983

Separate sites of low and high affinity for agonists on Torpedo californica acetylcholine receptor.

We have studied alkylation of the membrane-bound acetylcholine receptor (AcChR) from Torpedo californica electric organ by the cholinergic agonist bromo-acetylcholine (BrAcCh). Following reduction of the AcChR with dithiothreitol (DTT) under strictly controlled conditions, a single class of binding sites was covalently labeled by BrAcCh. The extent of alkylation was dependent on the concentration of both DTT and BrAcCh and reached a maximum when a number of sites equivalent to the number of alpha-bungarotoxin (alpha-BTx) binding sites were labeled. The reaction with BrAcCh was completely inhibited by saturating concentrations of alpha-BTx. On the contrary, complete alkylation of the AcChR with [3H]BrAcCh consistently inhibited only approximately 50% of alpha-BTx binding. The effects of DTT reduction and subsequent BrAcCh alkylation on the cation-gating properties of the AcChR were investigated in rapid kinetic experiments. DTT reduction resulted in a slight decrease in the maximum cation flux and a small shift in the effective dissociation constant to higher acetylcholine (AcCh) concentration. The flux response was completely inhibited by maximal alkylation of the membrane vesicles by BrAcCh. A low-affinity binding site for AcCh, which is likely to be important in AcChR activation, has been revealed for T. californica AcChR by studying the effects of cholinergic ligands on the fluorescence of a probe, 4-[(iodoacetoxy)ethylmethylamino]-7-nitro-2,1,3-benzoxadiazole (IANBD), covalently bound to the AcChR protein. Maximal labeling by BrAcCh did not affect the binding of AcCh to the low-affinity binding site, as monitored by changes in the fluorescence of this probe. This low-affinity binding site must therefore be distinct from the site labeled by BrAcCh. The results strongly support the notion that the nicotinic AcChR contains multiple binding sites for cholinergic ligands.

Acetylcholine↗
PubMed1983

Specific involvement of peripheral T lymphocytes against acetylcholine receptors in myasthenia gravis.

A specific sensitisation of blood T lymphocytes against the nicotinic acetylcholine receptor has been demonstrated in myasthenic patients. After thymectomy the T lymphocytes response was quickly decreased and in most patients it reached normal values. The T response remained at low or normal levels for at least two years. In patients who had undergone thymectomy three or more years before, a T lymphocyte response against acetylcholine receptors was frequently detected.

Adolescent↗