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A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function
Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a “leaky” character to the affected he...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Frontiers Media S.A.
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584803/ https://www.ncbi.nlm.nih.gov/pubmed/31263420 http://dx.doi.org/10.3389/fphys.2019.00392 |
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author | Ziraldo, Gaia Buratto, Damiano Kuang, Yuanyuan Xu, Liang Carrer, Andrea Nardin, Chiara Chiani, Francesco Salvatore, Anna Maria Paludetti, Gaetano Lerner, Richard A. Yang, Guang Zonta, Francesco Mammano, Fabio |
author_facet | Ziraldo, Gaia Buratto, Damiano Kuang, Yuanyuan Xu, Liang Carrer, Andrea Nardin, Chiara Chiani, Francesco Salvatore, Anna Maria Paludetti, Gaetano Lerner, Richard A. Yang, Guang Zonta, Francesco Mammano, Fabio |
author_sort | Ziraldo, Gaia |
collection | PubMed |
description | Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a “leaky” character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro. Altogether, these results indicate abEC1.1 is a promising tool for further translational studies. |
format | Online Article Text |
id | pubmed-6584803 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-65848032019-07-01 A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function Ziraldo, Gaia Buratto, Damiano Kuang, Yuanyuan Xu, Liang Carrer, Andrea Nardin, Chiara Chiani, Francesco Salvatore, Anna Maria Paludetti, Gaetano Lerner, Richard A. Yang, Guang Zonta, Francesco Mammano, Fabio Front Physiol Physiology Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a “leaky” character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro. Altogether, these results indicate abEC1.1 is a promising tool for further translational studies. Frontiers Media S.A. 2019-06-11 /pmc/articles/PMC6584803/ /pubmed/31263420 http://dx.doi.org/10.3389/fphys.2019.00392 Text en Copyright © 2019 Ziraldo, Buratto, Kuang, Xu, Carrer, Nardin, Chiani, Salvatore, Paludetti, Lerner, Yang, Zonta and Mammano. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Physiology Ziraldo, Gaia Buratto, Damiano Kuang, Yuanyuan Xu, Liang Carrer, Andrea Nardin, Chiara Chiani, Francesco Salvatore, Anna Maria Paludetti, Gaetano Lerner, Richard A. Yang, Guang Zonta, Francesco Mammano, Fabio A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function |
title | A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function |
title_full | A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function |
title_fullStr | A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function |
title_full_unstemmed | A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function |
title_short | A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function |
title_sort | human-derived monoclonal antibody targeting extracellular connexin domain selectively modulates hemichannel function |
topic | Physiology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6584803/ https://www.ncbi.nlm.nih.gov/pubmed/31263420 http://dx.doi.org/10.3389/fphys.2019.00392 |
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