Expression Cloning and Fusion Proteins as Tools to Study Receptor Structure

Barkas, T.; Mauron, Alex; Roth, Beat; Alliod, Christine; Tzartos, Socrates J.; Ballivet, Marc

doi:10.1007/978-3-642-71649-2_32

Cited by 10 publications

(16 citation statements)

References 8 publications

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“…The N-terminal extracellular domain of the ␣ chain (␣-(1-210)) contains both the binding sites for cholinergic ligands (4) and the MIR, the major target for autoantibodies in both MG and experimental models of MG (5-7). The major loop of the overlapping epitopes for several anti-MIR monoclonal antibodies (mAbs) has been localized between residues 67 and 76 of the ␣ subunit (8,9). Previous experiments have shown that the binding sites for both acetylcholine and ␣-BTX are located close to two adjacent cysteine residues at positions 192 and 193 of the ␣ subunit (10).…”

mentioning

confidence: 99%

Expression of Soluble Ligand- and Antibody-binding Extracellular Domain of Human Muscle Acetylcholine Receptor α Subunit in Yeast Pichia pastoris

Psaridi-Linardaki¹,

Mamalaki²,

Remoundos³

et al. 2002

Journal of Biological Chemistry

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The N-terminal extracellular domain (amino acids 1-210; h␣-(1-210)) of the ␣ subunit of the human muscle nicotinic acetylcholine receptor (AChR), bearing the binding sites for cholinergic ligands and the main immunogenic region, the major target for anti-AChR antibodies in patients with myasthenia gravis, was expressed in the yeast, Pichia pastoris. The recombinant protein was water-soluble and glycosylated, and fast protein liquid chromatography analysis showed it to be a monomer. h␣-(1-210) bound 125 I-␣-bungarotoxin with a high affinity (K d ‫؍‬ 5.1 ؎ 2.4 nM), and this binding was blocked by unlabeled d-tubocurarine and gallamine (K i ϳ7.5 mM). Interestingly, 125 I-␣-bungarotoxin binding was markedly impaired by in vitro deglycosylation of h␣-(1-210). Several monoclonal antibodies that show partial or strict conformation-dependent binding to the AChR were able to bind to h␣-(1-210), as did antibodies from a large proportion of myasthenic patients. These results suggest that the extracellular domain of the human AChR ␣ subunit expressed in P. pastoris has an apparently near native conformation. The correct folding of the recombinant protein, together with its relatively high expression yield, makes it suitable for structural studies on the nicotinic acetylcholine receptor and for use as an autoantigen in myasthenia gravis studies. The nicotinic acetylcholine receptor (AChR)1 at the neuromuscular junction is a member of the superfamily of ligandgated ion channels that also includes the glycine, ␥-aminobutiric acid A, and 5-HT 3 receptors (1). The AChR is a transmembrane glycoprotein (M r ϳ290000) consisting of five homologous subunits in the stoichiometry ␣ 2 ␤␥␦ (embryonic) or ␣ 2 ␤⑀␦ (adult). Each subunit consists of an N-terminal extracellular domain (ϳ210 residues) followed by three transmembrane domains, a large cytoplasmic loop, a fourth transmembrane domain, and a short, extracellular C-terminal tail (2, 3). The N-terminal extracellular domain of the ␣ chain (␣-(1-210)) contains both the binding sites for cholinergic ligands (4) and the MIR, the major target for autoantibodies in both MG and experimental models of MG (5-7). The major loop of the overlapping epitopes for several anti-MIR monoclonal antibodies (mAbs) has been localized between residues 67 and 76 of the ␣ subunit (8, 9). Previous experiments have shown that the binding sites for both acetylcholine and ␣-BTX are located close to two adjacent cysteine residues at positions 192 and 193 of the ␣ subunit (10). Moreover, other distinct regions on either the ␣ subunit (11, 12) or the adjacent ␥ or ␦ subunits (13) have been shown to contribute to ␣-BTX binding, whereas the role of glycosylation at residue ␣141 (14 -16) requires further study.These unique characteristics of the ␣ subunit have led to its being extensively studied in several laboratories. The expression of full-length Torpedo (15-17) or mouse (18) AChR ␣ subunits in heterologous protein expression systems has shown that the ␣ subunit, independently of other subunits, acquires a mature...

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confidence: 99%

Expression of Soluble Ligand- and Antibody-binding Extracellular Domain of Human Muscle Acetylcholine Receptor α Subunit in Yeast Pichia pastoris

Psaridi-Linardaki¹,

Mamalaki²,

Remoundos³

et al. 2002

Journal of Biological Chemistry

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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).…”

Section: Discussionmentioning

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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“…The production and characteristics of the mAbs used have been described earlier (Tzartos and Lindstrom, 1980;Tzartos et al, 1981Tzartos et al, , 1983Ratnam et al, 1986;Barkas et al, 1987Barkas et al, , 1988)-All mAbs have been derived from rats immunized with AChR from various species. The preparations used were 50% ammonium sulfate precipitates from hybridoma supernatants, dialyzed against phosphate-buffered saline (NaC1/Pi; 8 mM NazHP04y mM KC& PH 7.4) containing 0.05% (maSS/Vol.)…”

Section: ~~~~~L Antibodiesmentioning

confidence: 99%

“…(-) in the sequences denote identical amino acids. Data on mAb binding to intact AChR were derived from Tzartos et al (1981,1983) for Torpedo and human, Sargent et al (1984) for Xenopus muscle, Swanson et al (1983) for chicken brain and Whitney and Lindstrom (1986) In another set of experiments, 32 continuously overlapping octapeptides were used to screen the novel 25-amino-acid segment of the human AChR a-subunit encoded by the exon P3A (Beeson et al, 1990). Screening of the 39-amino-acid region extending between a52 and a65 of the human AChR including the 25-amino-acid residue segment inserted between a58 and a59, by continuously overlapping octapeptides, did not reveal significant binding with any of the tested anti-(human MIR) mAbs 189,192,195 and 198, nor with the crossreacting mAb 6 (not shown).…”

Section: Localization Of Anti-mir Mab Binding To the Pentapeptide A67mentioning

confidence: 99%

“…Anti-MIR mAb binding has been gradually localized between amino acid residues 1-151 (Barkas et al, 1986), 46-120 (Ratnam et al, 1986), 37-85 (Barkas et al, 1987), 61-76 (Barkas et al, 1988) 67-78 (Wood et al, 1989 and finally 67-76 of the AChR ol-subunit . The conformation of the MIR decapeptide, either free (in dimethyl sulfoxide) or bound on the corresponding anti-MIR mAb, has been elucidated by the use of two-dimensional NMR experiments (Cung et al, 1989(Cung et al, , 1992.…”

mentioning

confidence: 99%

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High‐resolution epitope mapping and fine antigenic characterization of the main immunogenic region of the acetylcholine receptor

Papadouli

Sakarellos

Tzartos

1993

European Journal of Biochemistry

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The main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR) is an immunodominant area of the molecule, both in human and in experimental autoimmune myasthenia gravis. Anti-MII$ monoclonal antibody (mAb) binding has been earlier localized between amino acid residues 67-76 of the AChR a-subunit. A thorough study of the epitope(s) for anti-MIR mAbs, by the use of a large panel of overlapping synthetic peptides and multiple peptide analogues, is now presented and offers clues for potential therapeutic applications of the obtained data.Use of all possible overlapping hexapeptides within Torpedo and human 1x40-91 AChR and of selected peptides of various sizes, showed that the shortest peptide capable of significant antibody binding is the pentapeptide a67 -71. Systematic screening of peptide analogues, where each amino acid residue within a67-76 and a67-74 of both Torpedo and human AChRs was substituted by various amino acids, was performed. Asn68 and Asp71 were found to be indispensable for anti-MIR mAb binding, whereas Pro69 and Ala/Asp70 were less but still significantly important. mAb binding to a67-76 from various AChR species further supported the significance of these results. An additional series of selected peptide analogues was then constructed, aiming at the identification of analogues with high antigenic activity. Many analogues with either single substitutions of a76 or combinations of two substitutions at a73 and a76 were tested. Several of these analogues (mainly His76, Arg76, Va173Ala76, His73Ala76, Va173Arg76) exhibited dramatic mAb binding enhancement. Some anti-MIR mAbs that do not bind to a67 -76 bound significantly to certain analogues. Such analogues could find applications in studies of therapeutic models of myasthenia gravis.

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Expression Cloning and Fusion Proteins as Tools to Study Receptor Structure

Cited by 10 publications

References 8 publications

Expression of Soluble Ligand- and Antibody-binding Extracellular Domain of Human Muscle Acetylcholine Receptor α Subunit in Yeast Pichia pastoris

Expression of Soluble Ligand- and Antibody-binding Extracellular Domain of Human Muscle Acetylcholine Receptor α Subunit in Yeast Pichia pastoris

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.

High‐resolution epitope mapping and fine antigenic characterization of the main immunogenic region of the acetylcholine receptor

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