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Title: Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor

Abstract

A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.

Authors:
; ; ;
Publication Date:
Research Org.:
Mayo Clinic, Rochester, MN
OSTI Identifier:
6778136
Report Number(s):
CONF-8604222-
Journal ID: CODEN: FEPRA; TRN: 87-008954
Resource Type:
Conference
Resource Relation:
Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States); Journal Volume: 45:4; Conference: 70. annual meeting of the Federation of American Society for Experimental Biology, St. Louis, MO, USA, 13 Apr 1986
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; NERVOUS SYSTEM DISEASES; PATHOGENESIS; PEPTIDES; CHEMICAL PREPARATION; ACETYLCHOLINE; ANTIBODIES; IODINE 125; LYMPHOCYTES; RATS; RECEPTORS; TRACER TECHNIQUES; AMINES; AMMONIUM COMPOUNDS; ANIMAL CELLS; ANIMALS; AUTONOMIC NERVOUS SYSTEM AGENTS; BETA DECAY RADIOISOTOPES; BIOLOGICAL MATERIALS; BLOOD; BLOOD CELLS; BODY FLUIDS; CONNECTIVE TISSUE CELLS; DAYS LIVING RADIOISOTOPES; DISEASES; DRUGS; ELECTRON CAPTURE RADIOISOTOPES; ESTERS; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; LEUKOCYTES; MAMMALS; MATERIALS; MEMBRANE PROTEINS; NEUROREGULATORS; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; PARASYMPATHOMIMETICS; PROTEINS; QUATERNARY COMPOUNDS; RADIOISOTOPES; RODENTS; SOMATIC CELLS; SYNTHESIS; VERTEBRATES 550901* -- Pathology-- Tracer Techniques

Citation Formats

McCormick, D.J., Griesmann, G.E., Huang, Z., and Lennon, V.A. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor. United States: N. p., 1986. Web.
McCormick, D.J., Griesmann, G.E., Huang, Z., & Lennon, V.A. Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor. United States.
McCormick, D.J., Griesmann, G.E., Huang, Z., and Lennon, V.A. 1986. "Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor". United States. doi:.
@article{osti_6778136,
title = {Synthetic. cap alpha. subunit peptide 125-147 of human nicotinic acetylcholine receptor induces antibodies to native receptor},
author = {McCormick, D.J. and Griesmann, G.E. and Huang, Z. and Lennon, V.A.},
abstractNote = {A synthetic peptide corresponding to residues 125-147 of the Torpedo acetylcholine receptor (AChR) ..cap alpha.. subunit proved to be a major antigenic region of the AChR. Rats inoculated with 50 ..mu..g of peptide (T ..cap alpha.. 125-147) developed T cell immunity and antibodies to native AChR and signs of experimental autoimmune myasthenia gravis. They report the synthesis and preliminary testing of a disulfide-looped peptide comprising residues 125-147 of the human AChR ..cap alpha.. subunit. Peptide H ..cap alpha.. 125-147 differs from T ..cap alpha.. 125-147 at residues 139 (Glu for Gln) and 143 (Ser for Thr). In immunoprecipitation assays, antibodies to Torpedo AChR bound /sup 125/I-labelled H..cap alpha.. 125-147 antibody bound H..cap alpha.. 125-147, but monoclonal antibodies to an immunodominant region of native AChR bound neither H..cap alpha.. 125-147 nor T ..cap alpha.. 125-147. Rats immunized with H ..cap alpha.. 125-147 produced anti-mammalian muscle AChR antibodies that induced modulation of AChRs from cultured human myotubes. Thus, region 125-147 of the human AChR ..cap alpha.. subunit is extracellular in muscle, and is both antigenic and immunogenic. It remains to be determined whether or not autoantibodies to this region may in part cause the weakness or myasthenia gravis in man.},
doi = {},
journal = {Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)},
number = ,
volume = 45:4,
place = {United States},
year = 1986,
month = 3
}

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  • The binding of the competitive antagonist {alpha}-bungarotoxin ({alpha}-Btx) and the noncompetitive inhibitor phencyclidine (PCP) to a synthetic peptide comprising residues 172-227 of the {alpha}-subunit of the Torpedo acetylcholine receptor has been characterized. {sup 125}I-{alpha}-Btx bound to the 172-227 peptide in a solid-phase assay and was competed by {alpha}-Btx d-tubocurarine and NaCl. In the presence of 0.02% sodium dodecyl sulfate, {sup 125}I-{alpha}-Btx bound to the 56-residue peptide with a K{sub D} of 3.5 nM, as determined by equilibrium saturation binding studies. Because {alpha}Btx binds to a peptide comprising residues 173-204 with the same affinity and does not bind to a peptidemore » comprising residues 205-227, the competitive antagonist and hence agonist binding site lies between residues 173 and 204. After photoaffinity labeling, ({sup 3}H)PCP was bound to the 172-227 peptide. ({sup 3}H)PCP binding was inhibited by chlorpromazine, tetracaine, and dibucaine. It is concluded that a high-affinity binding site for PCP is located between residues 205 and 227, which includes the first 18 residues of transmembrane segment M1, and that a low-affinity site is located in the competitive antagonist binding site between residues 173 and 204. These results show that a synthetic peptide comprising residues 172-227 of the {alpha} subunit contains three binding sites, one for {alpha}-Btx and two for PCP. Previous studies on the intact receptor indicate high-affinity PCP binding occurs in the receptor channel.« less
  • Using a human fetal brain genomic library and a P1 clone library from Genome Systems Inc, a cosmid and P1-clone walk has been initiated from the polymorphic markers D20S19, D20S20, D20S24, and D20S73. Standard methods were follwed for culture and preparation of the cosmids and P1 clones. Restriction mapping was performed by hybridization of T3 and T7 oligonucleotides to partial EcoRI/total NotI digests. A total of 43 cosmids and 4 P1 clones covering 420 kb in three contigs were isolated.
  • The human neuronal nicotinic acetylcholine receptor {alpha}4 subunit gene (CHRNA4) is located in the candidate region for three different phenotypes: benign familial neonatal convulsions, autosomal dominant nocturnal frontal lobe epilepsy, and low-voltage EEG. Recently, a missense mutation in transmembrane domain 2 of CHRNA4 was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy in one extended pedigree. We have determined the genomic organization of CHRNA4, which consists of six exons distributed over approximately 17 kb of genomic DNA. The nucleotide sequence obtained from the genomic regions adjacent to the exon boundaries enabled us to develop a set ofmore » primer pairs for PCR amplification of the complete coding region. The sequence analysis provides the basis for a comprehensive mutation screening of CHRNA4 in the above-mentioned phenotypes and possibly in other types of idopathic epilepsies. 29 refs., 3 figs., 1 tab.« less
  • The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) {alpha}subunit forms a binding site for {alpha}-bungarotoxin ({alpha}-BTX). Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR {alpha}1 subunits were tested for their ability to bind {sup 125}I-{alpha}-BTX, and differences in {alpha}-BTX affinity were determined by using solution (IC{sub 50}s) and solid-phase (K{sub d}s) assays. Panels of overlapping peptides corresponding to the complete {alpha}1 subunit of mouse and human were also tested for {alpha}-BTX binding, but other sequence segments forming the {alpha}-BTX site were not consistently detectable. The role of amore » putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased {alpha}-BTX binding, whereas oxidation of the peptides had little effect. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/{alpha}1-subunit interface a vicinal disulfide bound was not required for {alpha}-BTX binding.« less
  • Synthetic peptides corresponding to sequence segments of the nicotinic acetylcholine receptor (nAChR) {alpha} subunits have been used to identify regions that contribute to formation of the binding sites for cholinergic ligands. The authors have previously defined {alpha}-bungarotoxin ({alpha}-BTX) binding sequences between residues 180 and 199 of a putative rat neuronal nAChR {alpha} subunit, designated {alpha}5, and between residues 181 and 200 of the chick neuronal {alpha}7 and {alpha}8 subunits. These sequences are relatively divergent compared with the Torpedo and muscle nAChR {alpha}1 {alpha}-BTX binding sites, which indicates a serious limitation of predicting functional domains of proteins based on homology inmore » general. Given the highly divergent nature of the {alpha}5 sequence, they were interested in determining the critical amino acid residues for {alpha}-BTX binding. In the present study, the effects of single amino acid substitutions of Gly or Ala for each residue of the rat {alpha}(180-199) sequence were tested, using a competition assay, in which peptides compete for {sup 125}I-{alpha}-BTX binding with native Torpedo nAChR. These results indicate that a disulfide bridge between the vicinal cysteines at positions 191 and 192 of the {alpha}5 sequence is not an absolute requirement for {alpha}-BTX binding activity.« less