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B M Conti-Tronconi

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

At least 55 records · Page 3Linked to original sources
PubMed1991

Alpha-bungarotoxin and the competing antibody WF6 interact with different amino acids within the same cholinergic subsite.

In the nicotinic acetylcholine receptors (AChRs), the sequence segment surrounding two invariant vicinal cysteinyl residues at positions 192 and 193 of the alpha subunit contains important structural component(s) of the binding site for acetylcholine and high molecular weight cholinergic antagonists, like snake alpha-neurotoxins. At least a second sequence region contributes to the formation of the cholinergic site. Studying the binding of alpha-bungarotoxin and three different monoclonal antibodies, able to compete with alpha-neurotoxins and cholinergic ligands, to a panel of synthetic peptides as representative structural elements of the AChR from Torpedo, we recently identified the sequence segments alpha 181-200 and alpha 55-74 as contributing to form the cholinergic site (Conti-Tronconi et al., 1990). As a first attempt to elucidate the structural requirements for ligand binding to the subsite formed by the sequence alpha 181-200, we have now studied the binding of alpha-bungarotoxin and of antibody WF6 to the synthetic peptide alpha 181-200, and to a panel of peptide analogues differing from the parental sequence alpha 181-200 by substitution of a single amino acid residue. CD spectral analysis of the synthetic peptide analogues indicated that they all have comparable structures in solution, and they can therefore be used to analyze the influence of single amino acid residues on ligand binding. Distinct clusters of amino acid residues, discontinuously positioned along the sequence 181-200, seem to serve as attachment points for the two ligands studied, and the residues necessary for binding of alpha-bungarotoxin are different from those crucial for binding of antibody WF6. In particular, residues at positions 188-190 (VYY) and 192-194 (CCP) were necessary for binding of alpha-bungarotoxin, while residues W187, T191, and Y198 and the three residues at positions 193-195 (CPD) were necessary for binding of WF6. Comparison of the CD spectra of the toxin/peptide complexes, and those obtained for the same peptides and alpha-bungarotoxin in solution, indicates that structural changes of the ligand(s) occur upon binding, with a net increase of the beta-structure component. The cholinergic binding site is therefore a complex surface area, formed by discontinuous clusters of amino acid residues from different sequence regions. Such complex structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody/antigen complexes [reviewed in Davies et al. (1988)]. Within this relatively large structure, cholinergic ligands bind with multiple points of attachment, and ligand-specific patterns of the attachment points exist.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Experimental myasthenia gravis in congenic mice. Sequence mapping and H-2 restriction of T helper epitopes on the alpha subunits of Torpedo californica and murine acetylcholine receptors.

Immunization of mice with nicotinic acetylcholine receptor from Torpedo electric organ (TAChR) causes a disease similar to human myasthenia gravis (experimental autoimmune myasthenia gravis, EAMG). Susceptibility to EAMG correlates with the H-2 haplotype. In this study we used overlapping synthetic peptide corresponding to the complete sequences of the alpha subunits from TAChR and murine muscle AChR (MAChR) to map T helper epitopes in congenic murine strains of different H-2 haplotype. C57BL/6 and BALB/B mice (highly susceptible to EAMG) and BALB/c and CB17 mice (less susceptible to EAMG), immunized with TAChR, developed similar anti-TAChR antibody titers and L3T4+ (T helper) cell sensitization. Different sequence segments of the TAChR alpha subunit were recognized by L3T4+ cells from strains of H-2b and H-2d haplotype. The sequence segments recognized by the H-2d strains have the highest predicted propensity to form amphipatic alpha helices, while those recognized by the H-2b strains do not. We investigated whether in EAMG T helper cells cross-react with autologous AChR sequences, and a true break of the tolerance occurs. Overlapping synthetic peptides, corresponding to the complete sequence of MAChR alpha subunit, were used to test L3T4+ cell from mice immunized with TAChR. L3T4+ cell strains did not cross-react with any murine peptide sequence, while L3T4+ cells from H-2d mice were strongly stimulated by the peptide sequence Ma alpha 304-322, which is very similar to the homologous Torpedo peptide.

Amino Acid Sequence↗
PubMed1991

T cells in myasthenia gravis specific for embryonic acetylcholine receptor.

In myasthenia gravis (MG) there is an autoimmune response against muscle acetylcholine receptor (AChR). Embryonic and adult muscles express different AChRs; embryonic AChR contains a gamma subunit, instead of the homologous epsilon subunit that contributes to form adult AChR. We report propagation from the blood of MG patients of T helper (TH) cell lines specific for human embryonic AChR, by cycles of stimulation with a pool of synthetic peptides corresponding to the complete sequence of the gamma subunit (gamma pool). The TH lines strongly recognized AChR from embryonic mammalian muscle, and reacted less or not at all with adult muscle AChR. The existence of TH cells specific for embryonic AChR strongly suggests that the primary anti-AChR sensitization in MG occurs in a tissue other than the innervated skeletal muscle. This may be within the thymus, which expresses an AChR similar or identical to embryonic muscle AChR.

Adult↗
PubMed1991

Nicotinic acetylcholine receptors have ligand-specific attachment point patterns.

Employing a panel of synthetic peptides as representative structural elements of the nicotinic acetylcholine receptor from Torpedo electric organ, we recently identified three sequence regions of the receptor (alpha 55-74, alpha 134-153 and alpha 181-200) serving as subsites for the binding of high molecular weight antagonists of acetylcholine (Conti-Tronconi et al. 1990). The relative binding affinities to these subsites of alpha-bungarotoxin and three competitive antibodies varied in a ligand-specific fashion. Employing a set of homologous synthetic peptides differing from alpha 181-200 by the exchange of single amino acid residues along the sequence, we now find that ligand binding crucially depends on the presence of particular amino acids within the subsite while others influence binding only marginally if at all. The existence of ligand-specific attachment points may account for the wide range in binding and kinetic parameters, pharmacological specificity and distinct mean open times of the receptor-integral cation channel observed for cholinergic ligands.

Amino Acid Sequence↗
PubMed1990

Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and alpha-bungarotoxin.

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].

Alkylation↗
PubMed1990

Identification of a brain acetylcholine receptor alpha subunit able to bind alpha-bungarotoxin.

Peptides corresponding to sequence segments homologous to an alpha-bungarotoxin (alpha-BGT) binding region on the alpha subunit of the Torpedo nicotinic cholinergic receptor (nAChR) were synthesized for each identified nAChR alpha subunit of the rat nervous system (alpha 1, which is expressed in muscle, and alpha 2, alpha 3, alpha 4, and alpha 5, which are expressed by neurons). The peptides were tested for their ability to directly bind 125I-alpha-BGT and to compete for 125I-alpha-BGT with Torpedo nAChR and with the alpha-BGT-binding component expressed by PC12, a sympathetic neuronal cell line. In addition to peptides of the muscle alpha 1 subunit, peptides corresponding to the sequence of a neuronal subunit, alpha 5, were able to bind 125I-alpha-BGT. Peptides containing the sequence segments 182-201 of the alpha 1 subunit and 180-199 of the alpha 5 subunit competed with Torpedo nAChR for 125I-alpha-BGT binding with IC50 values of 0.5 and 3.5 microM, respectively. Both of these peptides were also able to compete for 125I-alpha-BGT binding with native Torpedo nAChR and with the alpha-BGT-binding protein(s) expressed on PC12 cells. To determine if other sequence segments contribute to form the neuronal alpha-BGT-binding site, overlapping peptides corresponding to the putative extracellular domain of the alpha 5 subunit were synthesized and used both in direct binding assays and in competition experiments. Peptides corresponding to amino acids 16-35 and 180-199 of the alpha 5 subunit directly bound 125I-alpha-BGT and inhibited the binding of toxin to both Torpedo nAChR and PC12 cells. The results of these studies strongly support identification of the alpha 5 subunit as a component of a neuronal alpha-BGT-binding nAChR.

Amino Acid Sequence↗
PubMed1990

Use of synthetic peptides to establish anti-human acetylcholine receptor CD4+ cell lines from myasthenia gravis patients.

Acetylcholine receptor-(AcChR) specific T cell lines were propagated from the PBL of six myasthenia gravis (MG) patients by the use of a pool of synthetic peptides (alpha-pool) corresponding to the complete sequence of the alpha-subunit of the human AcChR. All the lines had CD4+ phenotype and strongly recognized the alpha-pool. Four lines cross-reacted with native Torpedo AcChR. Five lines showed, at certain stages of their propagation, some degree of reactivity to autologous or DR-matched APC. One of the CD4+ T lines was challenged with each one of the peptides present in the alpha-pool. Several peptides, corresponding to the sequence segments 48-67, 101-120, 304-322, 320-337, and 419-437 of the human alpha-subunit were recognized, indicating that different epitopes and multiple T cell clones are involved in the recognition of the autoantigen in MG. Human AcChR-specific CD4+ T cell lines will be useful to identify the repertoire of epitopes recognized by the autoreactive Th cells in MG, to investigate the TCR genes utilized by autoreactive Th cells and to develop specific immunosuppressive treatments using anti-T cell vaccination.

Amino Acid Sequence↗
PubMed1990

CD4+ T cell response to the human acetylcholine receptor alpha subunit in myasthenia gravis. A study with synthetic peptides.

The production of antinicotinic acetylcholine receptor (AcChR) antibodies in myasthenia gravis (MG) is modulated by specific Th (CD4+) lymphocytes that can recognize epitopes on the denatured AcChR alpha subunit. Thirty-two overlapping synthetic peptides corresponding to the complete sequence of human AcChR alpha subunit were used to investigate the anti-alpha subunit response of unselected lymphocytes and of CD8(+)-depleted, CD4(+)-enriched lymphocytes from the blood of nine MG patients and from four healthy controls. One subject was a newly diagnosed MG patient that was tested three times after the development of the disease. An anti-AcChR response of the CD4(+)-enriched cells was present that could be detected only after removal of the CD8+ population and that seems to be related to the clinical conditions of the patient. The high basal rate of the cell proliferation of the unselected unstimulated blood lymphocytes and the normal basal rate observed for the CD8(+)-depleted population suggested the presence of activated CD8+ cells. The study of surface markers of the T cells confirmed the existence of activated CD8+ and CD4+ cells in numbers correlated with the severity of the disease and the results of the in vitro response of the T cells. The anti-AcChR activity of the CD4+ cells in MG may be a useful marker of the activity of the disease and it seems to be influenced by activated CD8+ cells present in the patients' blood.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1990

Nonequivalence of alpha-bungarotoxin binding sites in the native nicotinic receptor molecule.

In the native, membrane-bound form of the nicotinic acetylcholine receptor (M-AcChR) the two sites for the cholinergic antagonist alpha-bungarotoxin (alpha-BGT) have different binding properties. One site has high affinity, and the M-AcChR/alpha-BGT complexes thus formed dissociate very slowly, similar to the complexes formed with detergent-solubilized AcChR (S-AcChR). The second site has much lower affinity (KD approximately 59 +/- 35 nM) and forms quickly reversible complexes. The nondenaturing detergent Triton X-100 is known to solubilize the AcChR in a form unable, upon binding of cholinergic ligands, to open the ion channel and to become desensitized. Solubilization of the AcChR in Triton X-100 affects the binding properties of this second site and converts it to a high-affinity, slowly reversible site. Prolonged incubation of M-AcChR at 4 degrees C converts the low-affinity site to a high-affinity site similar to those observed in the presence of Triton X-100. Although the two sites have similar properties when the AcChR is solubilized in Triton X-100, their nonequivalence can be demonstrated by the effect on alpha-BGT binding of concanavalin A, which strongly reduces the association rate of one site only. The Bmax of alpha-BGT to either Triton-solubilized AcChR or M-AcChR is not affected by the presence of concanavalin A. Occupancy of the high-affinity, slowly reversible site in M-AcChR inhibits the Triton X-100 induced conversion to irreversibility of the second site. At difference with alpha-BGT, the long alpha-neurotoxin from Naja naja siamensis venom (alpha-NTX) binds with high affinity and in a very slowly reversible fashion to two sites in the M-AcChR (Conti-Tronconi & Raftery, 1986). We confirm here that Triton-solubilized AcChR or M-AcChR binds in a very slowly reversible fashion the same amount of alpha-NTX.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Localization of sequence segments forming a kappa-bungarotoxin-binding site on the alpha 3 neuronal nicotinic receptor.

To identify the sequence segments of the alpha 3 subunit of the neuronal nicotinic acetylcholine receptor (N-nAChR) forming the binding site for the cholinergic antagonist kappa-bungarotoxin (kappa-BGT), overlapping peptides corresponding to the complete alpha 3 sequence were tested for their ability to bind 125I-labeled kappa-BGT. Two peptides located within the N-terminal extracellular domain specifically bound kappa-BGT in a solid phase assay, i.e. peptide N alpha(3)51-70 with a Kd approximately 300 nM and peptide N alpha(3)1-18 with slightly lower affinity (Kd approximately 500 nM). Preincubation of 125I-kappa-BGT with peptides N alpha(3)51-70 or N alpha(3)1-18 resulted in greater than 90% inhibition of kappa-125I-BGT binding to native N-nAChR expressed on the neuronal cell line PC12. Under the same conditions, two additional peptides, N alpha(3)180-199 and N alpha(3)183-201, were found to inhibit kappa-125I-BGT binding to PC12 by approximately 50%. These latter peptides represent sequences that are homologous to those shown previously to bind alpha-bungarotoxin. Peptide N alpha(3)51-70 (400 microM) also reduced by approximately 4-fold the observed rate of association of kappa-BGT to PC12 cells. The results of these experiments identify sequence segments of the alpha 3 subunit which are likely to interact with kappa-BGT and may indicate the relative contribution that these segments make in the formation of the high affinity kappa-BGT-binding site of this N-nAChR subtype.

Amino Acid Sequence↗
PubMed1990

Immunodominant regions for T helper-cell sensitization on the human nicotinic receptor alpha subunit in myasthenia gravis.

In myasthenia gravis an autoimmune response against the nicotinic acetylcholine receptor (AChR) occurs. The alpha subunit of the AChR contains both the epitope(s) that dominates the antibody response (main immunogenic region) and epitopes involved in T helper cell sensitization. In this study, overlapping synthetic peptides corresponding to the complete AChR alpha-subunit sequence were used to propagate polyclonal AChR-specific T helper cell lines from four myasthenic patients of different HLA types. Response of the T helper lines to the individual peptides was studied. Four immunodominant sequence segments were identified--i.e., residues 48-67, 101-120, 304-322, and 419-437. These regions did not include residues known to form the main immunogenic region or the cholinergic binding site, and they frequently contained sequence motifs that have been proposed to be related to T-epitope formation.

Amino Acid Sequence↗
PubMed1990

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

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 alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-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 alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-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 alpha 67-76 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 alpha 67-76 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 alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-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 alpha 331-351.

Animals↗
PubMed1989

The main immunogenic region of the nicotinic acetylcholine receptor. Identification of amino acid residues interacting with different antibodies.

In myasthenia gravis a highly conserved area of the nicotinic receptor (AcChR) dominates the autoantibody response (main immunogenic region, MIR), and it is formed by residues within the sequence segment 67-76 of the AcChR alpha-subunit. We have studied the binding of eight anti-MIR mAb to synthetic peptides containing the sequence segment 67-76 of the human alpha-subunit, and peptide analogues containing single residue substitutions of this sequence. We used also a peptide where both Asp70 and Asp71 were substituted by glycine residues. The binding of six anti-MIR mAb was strongly influenced by several substitutions. All these mAb required residues Asn68, and Pro69 for binding. Five of them required also Asp71 and Tyr72. Substitution of Asp70, which is an Ala residue in Torpedo AcChR, was irrelevant for the binding of an anti-Torpedo and an anti-Electrophorus mAb, and moderately reduced the binding of an anti-human mAb (no. 203). Substitution of Trp67 moderately reduced the binding of some of these mAbs. A mAb of this group (the antihuman mAb no. 198) bound in a manner only slightly influenced by ionic strength, whereas the binding of the other five mAb of this group was very sensitive to the ionic strength. Two anti-Electrophorus MIR mAb bound similarly to all peptide analogues in low ionic strength. At high ionic strength only the peptide analogue where Asp 70 was changed to a Gly residue bound significantly. This may indicate that the Electrophorus MIR has an uncharged residue at this position, as does Torpedo AcChR. Residues at position 73, 74, 75, and 76 were of little or no importance for the binding of all anti-MIR mAb. A free amino terminus was essential for the binding of most mAb. The results of competition experiments between different peptides and native AcChR for mAb binding were consistent with those obtained in direct binding experiments.

Acetylation↗
PubMed1989

Epitope mapping employing antibodies raised against short synthetic peptides: a study of the nicotinic acetylcholine receptor.

Antibodies were raised against eight synthetic peptides matching preselected portions of the amino acid sequence of nicotinic acetylcholine receptor (nAChR) from Torpedo marmorata. To increase the probability of obtaining antibodies specific for the exact sequence of the immunizing peptide, peptides of only five to seven amino acids in length were employed. Even under these limiting conditions some of the polyclonal rabbit immune sera showed cross-reactivity with other peptides and/or other sequence regions of the receptor. Further studies with polyclonal and monoclonal sera suggested that conformation and charge pattern rather than linear sequence are the essential determinants of antibody epitopes. Application of antibodies for topological studies therefore requires that the antibody specificity for a particular region of the antigen has been firmly established. Epitope mapping with the eight anti-peptide immune sera provides information on the accessibility to antibody of matching sequences within the receptor molecule. We find the sequence portions alpha 81-85, alpha 127-132, and alpha 190-195 to be freely accessible both at membrane-bound and at purified receptor. Binding of anti-alpha 387-392 serum does not prove accessibility of this region as the serum cross-reacts strongly with peptide fragments corresponding to the regions alpha 165-200 and beta 190-200 of nAChR from Torpedo californica. To permit binding of anti-alpha 137-142 immune serum, treatment of the receptor with endoglycosidase is required, showing that Asn-141 indeed is glycosylated in native nAChR. The homologous sequence of the other subunits differing only in one sequence position from alpha 137-142 is not accessible in native nAChR to antibody, indicating clear differences in folding of the receptor polypeptides. Sequence portions alpha 395-401 and alpha 161-166 must first be exposed by appropriate treatment to permit binding of respective serum. These results and previous epitope mapping studies by other laboratories are discussed with respect to the limited sequence specificity of antibodies.

Amino Acid Sequence↗
PubMed1988

Localization of the main immunogenic region of human muscle acetylcholine receptor to residues 67-76 of the alpha subunit.

The majority of antibodies to the acetylcholine receptor (AcChoR), both in the human disease myasthenia gravis and in its experimental models, are directed against an extracellular area of the AcChoR alpha subunit called the main immunogenic region (MIR). We have studied the binding of anti-AcChoR monoclonal antibodies (mAbs) to 26 synthetic peptides corresponding to the hydrophilic parts of the human AcChoR alpha subunit. The binding sites for eight anti-MIR mAbs and for eight anti-alpha-subunit, non-anti-MIR mAbs were localized. Anti-MIR mAbs bound to one peptide corresponding to residues 63-80 of the human alpha subunit. A second panel of peptides corresponding to the various parts of the alpha-subunit segment 63-80 was synthesized. Anti-MIR antibodies bound to a peptide that contained the alpha-subunit sequence 67-76. Thus, a main constituent loop of the MIR is localized between residues 67 and 76 of the alpha subunit.

Amino Acid Sequence↗