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

• 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 – 115SEHETRLVANLLGGGSLRWNPADYGGIKKIRGSLDYTGKFull-size tableTable optionsView in workspaceDownload as CSVThis 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) × 10−10 M, which is smaller than (7.13 ± 1.20) × 10−10 M for the complex of mAb-132A with α(1–161) and about the same as 3.4 × 10−10 M 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.