A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model
Abstract Connexins (Cxs) are fundamental in cell–cell communication, functioning as gap junction channels (GJCs) that facilitate solute exchange between adjacent cells and as hemichannels (HCs) that mediate solute exchange between the cytoplasm and the extracellular environment. Mutations in the GJB...
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BMC
2024-12-01
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| Series: | Cell Communication and Signaling |
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| Online Access: | https://doi.org/10.1186/s12964-024-01969-0 |
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| author | Abraham Tettey-Matey Viola Donati Chiara Cimmino Chiara Di Pietro Damiano Buratto Mariateresa Panarelli Alberto Reale Arianna Calistri Maria Vittoria Fornaini Ruhong Zhou Guang Yang Francesco Zonta Daniela Marazziti Fabio Mammano |
| author_facet | Abraham Tettey-Matey Viola Donati Chiara Cimmino Chiara Di Pietro Damiano Buratto Mariateresa Panarelli Alberto Reale Arianna Calistri Maria Vittoria Fornaini Ruhong Zhou Guang Yang Francesco Zonta Daniela Marazziti Fabio Mammano |
| author_sort | Abraham Tettey-Matey |
| collection | DOAJ |
| description | Abstract Connexins (Cxs) are fundamental in cell–cell communication, functioning as gap junction channels (GJCs) that facilitate solute exchange between adjacent cells and as hemichannels (HCs) that mediate solute exchange between the cytoplasm and the extracellular environment. Mutations in the GJB1 gene, which encodes Cx32, lead to X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare hereditary demyelinating disorder of the peripheral nervous system (PNS) without an effective cure or treatment. In Schwann cells, Cx32 HCs are thought to play a role in myelination by enhancing intracellular and intercellular Ca2+ signaling, which is crucial for proper PNS myelination. Single-point mutations (p.S85C, p.D178Y, p.F235C) generate pathological Cx32 HCs characterized by increased permeability (“leaky”) or excessive activity (“hyperactive”). We investigated the effects of abEC1.1-hIgG1, a fully human immunoglobulin G1 (hIgG1) monoclonal antibody, on wild-type (WT) and mutant Cx32D178Y HCs. Using HeLa DH cells conditionally co-expressing Cx and a genetically encoded Ca2+ biosensor (GCaMP6s), we demonstrated that mutant HCs facilitated 58% greater Ca2+ uptake in response to elevated extracellular Ca2+ concentrations ([Ca2+]ex) compared to WT HCs. abEC1.1-hIgG1 dose-dependently inhibited Ca2+ uptake, achieving a 50% inhibitory concentration (EC50) of ~ 10 nM for WT HCs and ~ 80 nM for mutant HCs. Additionally, the antibody suppressed DAPI uptake and ATP release. An atomistic computational model revealed that serine 56 (S56) of the antibody interacts with aspartate 178 (D178) of WT Cx32 HCs, contributing to binding affinity. Despite the p.D178Y mutation weakening this interaction, the antibody maintained binding to the mutant HC epitope at sub-micromolar concentrations. In conclusion, our study shows that abEC1.1-hIgG1 effectively inhibits both WT and mutant Cx32 HCs, highlighting its potential as a therapeutic approach for CMTX1. These findings expand the antibody’s applicability for treating diseases associated with Cx HCs and inform the rational design of next-generation antibodies with enhanced affinity and efficacy against mutant HCs. |
| format | Article |
| id | doaj-art-b229756db10d4abb859284512f10f9bb |
| institution | OA Journals |
| issn | 1478-811X |
| language | English |
| publishDate | 2024-12-01 |
| publisher | BMC |
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| series | Cell Communication and Signaling |
| spelling | doaj-art-b229756db10d4abb859284512f10f9bb2025-08-20T01:56:46ZengBMCCell Communication and Signaling1478-811X2024-12-0122111810.1186/s12964-024-01969-0A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro modelAbraham Tettey-Matey0Viola Donati1Chiara Cimmino2Chiara Di Pietro3Damiano Buratto4Mariateresa Panarelli5Alberto Reale6Arianna Calistri7Maria Vittoria Fornaini8Ruhong Zhou9Guang Yang10Francesco Zonta11Daniela Marazziti12Fabio Mammano13CNR Institute of Biochemistry and Cell BiologyCNR Institute of Biochemistry and Cell BiologyCNR Institute of Endocrinology and Experimental Oncology “G. Salvatore”CNR Institute of Biochemistry and Cell BiologyInstitute of Quantitative Biology, College of Life Science, Zhejiang UniversityDepartment of Molecular Medicine, University of PaduaDepartment of Molecular Medicine, University of PaduaDepartment of Molecular Medicine, University of PaduaDepartment of Molecular Medicine, University of PaduaInstitute of Quantitative Biology, College of Life Science, Zhejiang UniversityShanghai Institute for Advanced Immunochemical Studies, ShanghaiTech UniversityDepartment of Biosciences and Bioinformatics, School of Science, Xi’an Jiaotong-Liverpool UniversityCNR Institute of Biochemistry and Cell BiologyCNR Institute of Biochemistry and Cell BiologyAbstract Connexins (Cxs) are fundamental in cell–cell communication, functioning as gap junction channels (GJCs) that facilitate solute exchange between adjacent cells and as hemichannels (HCs) that mediate solute exchange between the cytoplasm and the extracellular environment. Mutations in the GJB1 gene, which encodes Cx32, lead to X-linked Charcot-Marie-Tooth type 1 (CMTX1), a rare hereditary demyelinating disorder of the peripheral nervous system (PNS) without an effective cure or treatment. In Schwann cells, Cx32 HCs are thought to play a role in myelination by enhancing intracellular and intercellular Ca2+ signaling, which is crucial for proper PNS myelination. Single-point mutations (p.S85C, p.D178Y, p.F235C) generate pathological Cx32 HCs characterized by increased permeability (“leaky”) or excessive activity (“hyperactive”). We investigated the effects of abEC1.1-hIgG1, a fully human immunoglobulin G1 (hIgG1) monoclonal antibody, on wild-type (WT) and mutant Cx32D178Y HCs. Using HeLa DH cells conditionally co-expressing Cx and a genetically encoded Ca2+ biosensor (GCaMP6s), we demonstrated that mutant HCs facilitated 58% greater Ca2+ uptake in response to elevated extracellular Ca2+ concentrations ([Ca2+]ex) compared to WT HCs. abEC1.1-hIgG1 dose-dependently inhibited Ca2+ uptake, achieving a 50% inhibitory concentration (EC50) of ~ 10 nM for WT HCs and ~ 80 nM for mutant HCs. Additionally, the antibody suppressed DAPI uptake and ATP release. An atomistic computational model revealed that serine 56 (S56) of the antibody interacts with aspartate 178 (D178) of WT Cx32 HCs, contributing to binding affinity. Despite the p.D178Y mutation weakening this interaction, the antibody maintained binding to the mutant HC epitope at sub-micromolar concentrations. In conclusion, our study shows that abEC1.1-hIgG1 effectively inhibits both WT and mutant Cx32 HCs, highlighting its potential as a therapeutic approach for CMTX1. These findings expand the antibody’s applicability for treating diseases associated with Cx HCs and inform the rational design of next-generation antibodies with enhanced affinity and efficacy against mutant HCs.https://doi.org/10.1186/s12964-024-01969-0Charcot–Marie–Tooth diseasesConnexonsCx32Ca2+ uptakeDye uptakeATP release |
| spellingShingle | Abraham Tettey-Matey Viola Donati Chiara Cimmino Chiara Di Pietro Damiano Buratto Mariateresa Panarelli Alberto Reale Arianna Calistri Maria Vittoria Fornaini Ruhong Zhou Guang Yang Francesco Zonta Daniela Marazziti Fabio Mammano A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model Cell Communication and Signaling Charcot–Marie–Tooth diseases Connexons Cx32 Ca2+ uptake Dye uptake ATP release |
| title | A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model |
| title_full | A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model |
| title_fullStr | A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model |
| title_full_unstemmed | A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model |
| title_short | A fully human IgG1 antibody targeting connexin 32 extracellular domain blocks CMTX1 hemichannel dysfunction in an in vitro model |
| title_sort | fully human igg1 antibody targeting connexin 32 extracellular domain blocks cmtx1 hemichannel dysfunction in an in vitro model |
| topic | Charcot–Marie–Tooth diseases Connexons Cx32 Ca2+ uptake Dye uptake ATP release |
| url | https://doi.org/10.1186/s12964-024-01969-0 |
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