Segment α 182‐198 of <i>Torpedo californica</i> acetylcholine receptor contains a second toxin‐binding region and binds anti‐receptor antibodies

Mulac‐Jeričević, Biserka; Atassi, M. Zouhair

doi:10.1016/0014-5793(86)81225-x

Cited by 73 publications

(42 citation statements)

References 26 publications

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“…[6] and [3H]MPTA (this study) on the reduced AChR. It has also been reported that synthetic peptides corresponding to this region of the o~-chain can bind or-toxins [26][27][28], albeit with low affinities and in an agonist-insensitive manner. The present finding that the majority of the incorporated [3H]DDF was associated with the fragment ot 179-207 shows that residues located within this region are actually involved in the binding of cholinergic ligands in the native AChR.…”

Section: Discussionsupporting

confidence: 55%

A photoaffinity ligand of the acetylcholine‐binding site predominantly labels the region 179–207 of the α‐subunit on native acetylcholine receptor from Torpedo marmorata

et al. 1986

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Regions of the Torpedo marmorata acetylcholine receptor (AChR) α‐subunit involved in the binding of acetylcholine were probed with two different covalent ligands. The sulfhydryl‐directed affinity reagent 4‐(N‐maleimido)phenyltrimethylammonium iodide labeled a single α‐subunit cyanogen bromide fragment on the reduced AChR which was identified as α 179–207. The novel photoaffinity ligand p‐(N,N‐dimethylamino)‐benzenediazonium fluoroborate, on the other hand, labeled three distinct α‐chain cyanogen bromide fragments on the unmodified AChR in a carbamylcholine‐protectable manner. The major radiolabeled species was purified and identified by sequence analysis as α 179–207. The acetylcholine‐binding site on the native AChR may thus involve several distinct portions of the α‐chain, with the region α 179–207 making a major contribution to the site.

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Section: Discussionsupporting

confidence: 55%

A photoaffinity ligand of the acetylcholine‐binding site predominantly labels the region 179–207 of the α‐subunit on native acetylcholine receptor from Torpedo marmorata

et al. 1986

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“…The toxin-binding site(s) also resides in the a subunit of AcChoR (6). Recent studies from this laboratory using synthetic uniform-sized overlapping peptides encompassing the entire extracellular parts of the a chain of human AcChoR and Torpedo AcChoR enabled the localization of the full profile of the toxin-binding regions on the Torpedo (7)(8)(9) and human (10) receptors. Conversely, the binding sites for AcChoR on a-bungarotoxin (a-BTX) were mapped by synthetic peptides representing each of the a-BTX loops (11,12).…”

mentioning

confidence: 99%

Acetylcholine receptor-alpha-bungarotoxin interactions: determination of the region-to-region contacts by peptide-peptide interactions and molecular modeling of the receptor cavity.

Ruan

Spurlino

Quiocho

et al. 1990

Proc. Natl. Acad. Sci. U.S.A.

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In previous studies from this laboratory, the binding regions of a-neurotoxins on human and Torpedo acetylcholine (AcCho) receptors (AcChoRs) and the binding regions for the receptor on the toxin were characterized with synthetic peptides of the respective molecules. In the present work, peptides representing the active regions of one molecule are each allowed to bind to each of the active-region peptides of the other molecule. Thus, the interaction of three abungarotoxin (a-BTX) synthetic loop peptides with four synthetic peptides representing the toxin-binding regions on human AcChoR permitted the determination ofthe region-region interactions between a-BTX and the human receptor. Based on the known three-dimensional structure of the toxin, the active peptides of the receptor were then assembled to their appropriate toxin-contact regions by computer model building and energy minimization. This allowed the three-dimensional con- (10) receptors. Conversely, the binding sites for AcChoR on a-bungarotoxin (a-BTX) were mapped by synthetic peptides representing each of the a-BTX loops (11, 12). The present paper describes an approach for studying the details of protein-protein recognition. Each of the active peptides of one protein is allowed to interact with each of the active peptides of the other protein. Based on relative binding affinities of peptide-peptide interactions, the position of AcChoR peptides relative to the a-BTX molecule was assessed. The receptor peptides were docked onto the appropriate regions of a-BTX, whose three-dimensional (3-D) structure is known (13), by computer graphics and energy minimization, thus allowing a 3-D model to be constructed of the binding-site cavity for the toxin on human AcChoR. MATERIALS AND METHODSMaterials. Synthesis, purification, and characterization of the human AcChoR a-chain peptides have been reported (10). The a-neurotoxin binding regions on human AcChoR reside (10) in the peptides shown in Fig. 1. These peptides were used in the present work. The receptor-binding regions on a-BTX are present in the three loop peptides shown in Fig. 1 (12). The synthesis, purification, and characterization ofthe monomeric forms of the three cyclic peptides (Fig. 1) have been described (12). a-BTX and its synthetic peptides were labeled with iodine-125 by using the chloramine-T method (14). Radioiodinated materials were used immediately after labeling. The specific activities of the labeled peptides were: LIN, 3.1 x 103 cpm/pmol; L2, 2.4 x 103 cpm/pmol; and L3E, 2.1 x 103 cpm/pmol.Binding Studies. tTo whom reprint requests should be addressed at:

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“…Although the AChR a-subunit's identity as the immunodominant subunit of the receptor has been noted (21)(22)(23)(24), only recently by using synthetic peptides (5)(6)(7)(8)(9) have specific functional domains been identified. Such studies showed that residues around 192-193 of the AChR a-subunit from Torpedo californica include an a-BuTx binding site (5)(6)(7)(8)(9), although the same sequence from the HuAChR is not reactive with a-BuTx (6). Our studies, using affinity-purified autoantibodies to HuAChR a-subunit 157-170 from MG patient sera, show that this antibody significantly blocked the binding of a-BuTx to cultured rat muscle cells (myotubes).…”

Section: Discussionmentioning

confidence: 99%

“…tion with a-bungarotoxin (a-BuTx) (5)(6)(7)(8)(9). These interactions also map accessible sites on AChR that bind antibodies.…”

Section: Introductionmentioning

confidence: 99%

Molecular mimicry and myasthenia gravis. An autoantigenic site of the acetylcholine receptor alpha-subunit that has biologic activity and reacts immunochemically with herpes simplex virus.

Schwimmbeck¹,

Dyrberg²,

Drachman³

et al. 1989

J. Clin. Invest.

132

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The large majority of patients with the autoimmune disease myasthenia gravis characteristically have detectable antibodies against the acetykcholine receptor (AChR). We used synthetic peptides to identify antibodies in sera of myasthenia gravis patients reactive with the human acetycholine receptor (HuAChR) a-subunit, residues 160-167. Affinity purification of these antibodies, using the HuAChR a-subunit 157-170 peptide immobilized on thiopropyl-Sepharose, yielded IgG antibodies that bound to the native AChR and inhibited the binding of a-bungarotoxin to the receptor. The HuAChR a-subunit 160-167 peptide demonstrated specific immunological crossreactivity with a shared homologous domain on herpes simplex virus glycoprotein D, residues 286-293, by both binding and inhibition studies. Thus, HuAChR a-subunit, residues 160-167, elicits antibodies in myasthenic patients that binds to the native AChR protein and is capable of eliciting a biologic effect. Immunologic cross-reactivity of this "self" epitope with herpes simplex virus suggest that this virus may be associated with the initiation of some cases of myasthenia.

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Segment α 182‐198 of Torpedo californica acetylcholine receptor contains a second toxin‐binding region and binds anti‐receptor antibodies

Cited by 73 publications

References 26 publications

A photoaffinity ligand of the acetylcholine‐binding site predominantly labels the region 179–207 of the α‐subunit on native acetylcholine receptor from Torpedo marmorata

A photoaffinity ligand of the acetylcholine‐binding site predominantly labels the region 179–207 of the α‐subunit on native acetylcholine receptor from Torpedo marmorata

Acetylcholine receptor-alpha-bungarotoxin interactions: determination of the region-to-region contacts by peptide-peptide interactions and molecular modeling of the receptor cavity.

Molecular mimicry and myasthenia gravis. An autoantigenic site of the acetylcholine receptor alpha-subunit that has biologic activity and reacts immunochemically with herpes simplex virus.

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