2021 Fiscal Year Annual Research Report
Evaluation of an immunotherapy for chronic pain by expressing a single domain intrabody against the pain marker GCH1 in the rat dorsal root ganglion neurons
| Project/Area Number |
18K08853
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| Research Institution | Shiga University of Medical Science |
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Principal Investigator |
J・P Bellier 滋賀医科大学, 神経難病研究センター, 助教 (80346022)
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| Co-Investigator(Kenkyū-buntansha) |
守村 敏史 滋賀医科大学, 動物生命科学研究センター, 准教授 (20333338)
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| Project Period (FY) |
2018-04-01 – 2022-03-31
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| Keywords | GTP cyclohydrolase 1 / scfv / BH4 / conformational antibody / amyloid beta / 3D imaging |
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| Outline of Annual Research Achievements |
The metabolic pathway involved in the synthesis of the monoaminergic and nitrinergic neurotransmitters required the cofactor tetrahydrobiopterin (BH4). This project aimed at developing recombinant single-chain antibody (scFv) and express them as intrabody to inhibit the activity of the enzyme GTP cyclohydrolase 1 (GCH1), the rate-limiting enzyme responsible for the synthesis of BH4. Previously hybridomas synthesizing monoclonal antibody against GCH1 were cloned, then scFv were construct and expressed. Because of their tendency to aggregate and and efficiency of scFv for inhibiting GCH1 was reduced. In this context, we study the aggregation/dessagregation properties of molecules, using several techniques including atomic force microscopy (AFM), blue-native PAGE analysis, Photo-induced cross-linking of unmodified proteins (PICUP), and innovative 3D analysis of confocal microscopy. New construction were produced to modifying the aggregative properties of the scFv. The developped techniques were also used in collaborative studies focusing on the disassembly and toxicity of oligomeric amyloid-beta 42 peptides, and imaging of mitochondrial defect in the brain of patients with progressive supranuclear palsy.
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