Identification of three extended antibody-binding segments in recombinant human muscle acetylcholine receptor α subunit extracellular domain 1-210

M. Sano, Daniel J Mc Cormick, S. Talib, G. E. Griesmann, Vanda A Lennon

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Abstract

The duplicated α subunits account for 40% of the total protein of the nicotinic acetylcholine receptor of muscle, and are implicated as targets for pathogenic autoantibodies in the neuromuscular disease myasthenia gravis (MG). This study reports some of the specificities of antibodies induced by a myasthenogenic recombinant protein (rHα1-210) corresponding to the proposed extracellular domain of the α subunit of human acetylcholine receptor, residues 1-210. Antisera produced by immunizing rats, rabbits, and mice were tested with a panel of overlapping synthetic peptides (each 16 amino acids) comprising residues 1-216 of the human α subunit. IgG antibodies produced in all three species bound only to peptides that were clustered in three segments: segment I (residues 9-24); segment II (57-96 in rats, 57-88 in rabbits, and 57-80 in mice); and segment III (137-184 in rats, 145-184 in rabbits and mice). Monoclonal antibodies were produced by 41 independent hybridomas derived from three rats immunized with rHα1-210; 12 reacted only with the recombinant or native protein, and 29 reacted additionally with peptides in segments II or III. Four mAbs bound to native human receptor; of these, three bound to peptides 57-72/65-80, 81-96, or 153-168, and one lacked peptide-binding activity. Lack of mAb reactivity with rat receptor precluded correlation of peptide reactivity with myasthenogenicity. Nevertheless, the data indicate that the human acetylcholine receptor's α subunit contains multiple sites in its extracellular domain that are potentially stimulatory for B cells.

Original languageEnglish (US)
Pages (from-to)983-989
Number of pages7
JournalInternational Immunology
Volume3
Issue number10
StatePublished - 1991
Externally publishedYes

Fingerprint

Cholinergic Receptors
Muscles
Peptides
Antibodies
Rabbits
Neuromuscular Diseases
Antibody Specificity
Myasthenia Gravis
Hybridomas
Nicotinic Receptors
Population Groups
Recombinant Proteins
Autoantibodies
Immune Sera
Proteins
B-Lymphocytes
Immunoglobulin G
Monoclonal Antibodies
Amino Acids

Keywords

  • myasthenia gravis
  • nicotinic receptor
  • recombinant autoantigen
  • synthetic peptides

ASJC Scopus subject areas

  • Immunology

Cite this

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title = "Identification of three extended antibody-binding segments in recombinant human muscle acetylcholine receptor α subunit extracellular domain 1-210",
abstract = "The duplicated α subunits account for 40{\%} of the total protein of the nicotinic acetylcholine receptor of muscle, and are implicated as targets for pathogenic autoantibodies in the neuromuscular disease myasthenia gravis (MG). This study reports some of the specificities of antibodies induced by a myasthenogenic recombinant protein (rHα1-210) corresponding to the proposed extracellular domain of the α subunit of human acetylcholine receptor, residues 1-210. Antisera produced by immunizing rats, rabbits, and mice were tested with a panel of overlapping synthetic peptides (each 16 amino acids) comprising residues 1-216 of the human α subunit. IgG antibodies produced in all three species bound only to peptides that were clustered in three segments: segment I (residues 9-24); segment II (57-96 in rats, 57-88 in rabbits, and 57-80 in mice); and segment III (137-184 in rats, 145-184 in rabbits and mice). Monoclonal antibodies were produced by 41 independent hybridomas derived from three rats immunized with rHα1-210; 12 reacted only with the recombinant or native protein, and 29 reacted additionally with peptides in segments II or III. Four mAbs bound to native human receptor; of these, three bound to peptides 57-72/65-80, 81-96, or 153-168, and one lacked peptide-binding activity. Lack of mAb reactivity with rat receptor precluded correlation of peptide reactivity with myasthenogenicity. Nevertheless, the data indicate that the human acetylcholine receptor's α subunit contains multiple sites in its extracellular domain that are potentially stimulatory for B cells.",
keywords = "myasthenia gravis, nicotinic receptor, recombinant autoantigen, synthetic peptides",
author = "M. Sano and {Mc Cormick}, {Daniel J} and S. Talib and Griesmann, {G. E.} and Lennon, {Vanda A}",
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T1 - Identification of three extended antibody-binding segments in recombinant human muscle acetylcholine receptor α subunit extracellular domain 1-210

AU - Sano, M.

AU - Mc Cormick, Daniel J

AU - Talib, S.

AU - Griesmann, G. E.

AU - Lennon, Vanda A

PY - 1991

Y1 - 1991

N2 - The duplicated α subunits account for 40% of the total protein of the nicotinic acetylcholine receptor of muscle, and are implicated as targets for pathogenic autoantibodies in the neuromuscular disease myasthenia gravis (MG). This study reports some of the specificities of antibodies induced by a myasthenogenic recombinant protein (rHα1-210) corresponding to the proposed extracellular domain of the α subunit of human acetylcholine receptor, residues 1-210. Antisera produced by immunizing rats, rabbits, and mice were tested with a panel of overlapping synthetic peptides (each 16 amino acids) comprising residues 1-216 of the human α subunit. IgG antibodies produced in all three species bound only to peptides that were clustered in three segments: segment I (residues 9-24); segment II (57-96 in rats, 57-88 in rabbits, and 57-80 in mice); and segment III (137-184 in rats, 145-184 in rabbits and mice). Monoclonal antibodies were produced by 41 independent hybridomas derived from three rats immunized with rHα1-210; 12 reacted only with the recombinant or native protein, and 29 reacted additionally with peptides in segments II or III. Four mAbs bound to native human receptor; of these, three bound to peptides 57-72/65-80, 81-96, or 153-168, and one lacked peptide-binding activity. Lack of mAb reactivity with rat receptor precluded correlation of peptide reactivity with myasthenogenicity. Nevertheless, the data indicate that the human acetylcholine receptor's α subunit contains multiple sites in its extracellular domain that are potentially stimulatory for B cells.

AB - The duplicated α subunits account for 40% of the total protein of the nicotinic acetylcholine receptor of muscle, and are implicated as targets for pathogenic autoantibodies in the neuromuscular disease myasthenia gravis (MG). This study reports some of the specificities of antibodies induced by a myasthenogenic recombinant protein (rHα1-210) corresponding to the proposed extracellular domain of the α subunit of human acetylcholine receptor, residues 1-210. Antisera produced by immunizing rats, rabbits, and mice were tested with a panel of overlapping synthetic peptides (each 16 amino acids) comprising residues 1-216 of the human α subunit. IgG antibodies produced in all three species bound only to peptides that were clustered in three segments: segment I (residues 9-24); segment II (57-96 in rats, 57-88 in rabbits, and 57-80 in mice); and segment III (137-184 in rats, 145-184 in rabbits and mice). Monoclonal antibodies were produced by 41 independent hybridomas derived from three rats immunized with rHα1-210; 12 reacted only with the recombinant or native protein, and 29 reacted additionally with peptides in segments II or III. Four mAbs bound to native human receptor; of these, three bound to peptides 57-72/65-80, 81-96, or 153-168, and one lacked peptide-binding activity. Lack of mAb reactivity with rat receptor precluded correlation of peptide reactivity with myasthenogenicity. Nevertheless, the data indicate that the human acetylcholine receptor's α subunit contains multiple sites in its extracellular domain that are potentially stimulatory for B cells.

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