Design, synthesis, and characterization of a 39 amino acid peptide mimic of the main immunogenic region of the Torpedo acetylcholine receptor

Vu B. Trinh, Alex J. Foster, Robert H Fairclough

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

•Designed a 39 amino acid peptide that mimics the AChR main immunogenic region with a conformation dependent structure.•Mimic is recognized by all six anti-MIR mAbs tested and not recognized by six agonist/antagonist site directed mAbs.•Mimic can remove all anti-MIR Abs from EAMG serum.•Mimic binds mAb 35 in a Western blot. We have designed a 39 amino acid peptide mimic of the conformation-dependent main immunogenic region (MIR) of the Torpedo acetylcholine receptor (TAChR) that joins three discontinuous segments of the Torpedo α-subunit, α(1-12), α(65-79), and α(110 - 115) with two GS linkers:. 123456789101112656667686970717273747576777879110 - 115SEHETRLVANLLGGGSLRWNPADYGGIKKIRGSLDYTGKThis 39MIR-mimic was expressed in E. coli as a fusion protein with an intein-chitin-binding domain (IChBD) to permit affinity collection on chitin beads. Six MIR-directed monoclonal antibodies (mAbs) bind to this complex and five agonist/antagonist site directed mAbs do not. The complex of MIR-directed mAb-132A with 39MIR has a Kd of (2.11±0.11)×10-10M, which is smaller than (7.13±1.20)×10-10M for the complex of mAb-132A with α(1-161) and about the same as 3.4×10-10M for that of mAb-132A with TAChR. Additionally, the 39MIR-IChBD adsorbs all MIR-directed antibodies (Abs) from an experimental autoimmune myasthenia gravis (EAMG) rat serum. Hence, the 39MIR-mimic has the potential to inactivate or remove pathogenic Torpedo MIR-directed Abs from EAMG sera and to direct a magic bullet to the memory B-cells that produce those pathogenic Abs. The hope is to use this as a guide to produce a mimic of the human MIR on the way to an antigen specific therapeutic agent to treat MG.

Original languageEnglish (US)
Pages (from-to)79-90
Number of pages12
JournalMolecular Immunology
Volume59
Issue number1
DOIs
StatePublished - May 2014

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Torpedo
Autoimmune Experimental Myasthenia Gravis
Cholinergic Receptors
Chitin
Monoclonal Antibodies
Inteins
Amino Acids
Peptides
Antibodies
Serum
Magic
B-Lymphocytes
Western Blotting
Escherichia coli
Antigens
tebufenozide
Proteins
Therapeutics

Keywords

  • Antibody mediated disease
  • Conformation-dependent epitope
  • Experimental autoimmune myasthenia gravis (EAMG)
  • Myasthenia gravis (MG)

ASJC Scopus subject areas

  • Molecular Biology
  • Immunology

Cite this

Design, synthesis, and characterization of a 39 amino acid peptide mimic of the main immunogenic region of the Torpedo acetylcholine receptor. / Trinh, Vu B.; Foster, Alex J.; Fairclough, Robert H.

In: Molecular Immunology, Vol. 59, No. 1, 05.2014, p. 79-90.

Research output: Contribution to journalArticle

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abstract = "•Designed a 39 amino acid peptide that mimics the AChR main immunogenic region with a conformation dependent structure.•Mimic is recognized by all six anti-MIR mAbs tested and not recognized by six agonist/antagonist site directed mAbs.•Mimic can remove all anti-MIR Abs from EAMG serum.•Mimic binds mAb 35 in a Western blot. We have designed a 39 amino acid peptide mimic of the conformation-dependent main immunogenic region (MIR) of the Torpedo acetylcholine receptor (TAChR) that joins three discontinuous segments of the Torpedo α-subunit, α(1-12), α(65-79), and α(110 - 115) with two GS linkers:. 123456789101112656667686970717273747576777879110 - 115SEHETRLVANLLGGGSLRWNPADYGGIKKIRGSLDYTGKThis 39MIR-mimic was expressed in E. coli as a fusion protein with an intein-chitin-binding domain (IChBD) to permit affinity collection on chitin beads. Six MIR-directed monoclonal antibodies (mAbs) bind to this complex and five agonist/antagonist site directed mAbs do not. The complex of MIR-directed mAb-132A with 39MIR has a Kd of (2.11±0.11)×10-10M, which is smaller than (7.13±1.20)×10-10M for the complex of mAb-132A with α(1-161) and about the same as 3.4×10-10M for that of mAb-132A with TAChR. Additionally, the 39MIR-IChBD adsorbs all MIR-directed antibodies (Abs) from an experimental autoimmune myasthenia gravis (EAMG) rat serum. Hence, the 39MIR-mimic has the potential to inactivate or remove pathogenic Torpedo MIR-directed Abs from EAMG sera and to direct a magic bullet to the memory B-cells that produce those pathogenic Abs. The hope is to use this as a guide to produce a mimic of the human MIR on the way to an antigen specific therapeutic agent to treat MG.",
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N2 - •Designed a 39 amino acid peptide that mimics the AChR main immunogenic region with a conformation dependent structure.•Mimic is recognized by all six anti-MIR mAbs tested and not recognized by six agonist/antagonist site directed mAbs.•Mimic can remove all anti-MIR Abs from EAMG serum.•Mimic binds mAb 35 in a Western blot. We have designed a 39 amino acid peptide mimic of the conformation-dependent main immunogenic region (MIR) of the Torpedo acetylcholine receptor (TAChR) that joins three discontinuous segments of the Torpedo α-subunit, α(1-12), α(65-79), and α(110 - 115) with two GS linkers:. 123456789101112656667686970717273747576777879110 - 115SEHETRLVANLLGGGSLRWNPADYGGIKKIRGSLDYTGKThis 39MIR-mimic was expressed in E. coli as a fusion protein with an intein-chitin-binding domain (IChBD) to permit affinity collection on chitin beads. Six MIR-directed monoclonal antibodies (mAbs) bind to this complex and five agonist/antagonist site directed mAbs do not. The complex of MIR-directed mAb-132A with 39MIR has a Kd of (2.11±0.11)×10-10M, which is smaller than (7.13±1.20)×10-10M for the complex of mAb-132A with α(1-161) and about the same as 3.4×10-10M for that of mAb-132A with TAChR. Additionally, the 39MIR-IChBD adsorbs all MIR-directed antibodies (Abs) from an experimental autoimmune myasthenia gravis (EAMG) rat serum. Hence, the 39MIR-mimic has the potential to inactivate or remove pathogenic Torpedo MIR-directed Abs from EAMG sera and to direct a magic bullet to the memory B-cells that produce those pathogenic Abs. The hope is to use this as a guide to produce a mimic of the human MIR on the way to an antigen specific therapeutic agent to treat MG.

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