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...

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Main Authors: Gaia Ziraldo, Damiano Buratto, Yuanyuan Kuang, Liang Xu, Andrea Carrer, Chiara Nardin, Francesco Chiani, Anna Maria Salvatore, Gaetano Paludetti, Richard A. Lerner, Guang Yang, Francesco Zonta, Fabio Mammano
Format: Article
Language:English
Published: Frontiers Media S.A. 2019-06-01
Series:Frontiers in Physiology
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Online Access:https://www.frontiersin.org/article/10.3389/fphys.2019.00392/full
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spelling doaj-b3ac6b95d41f4cd7a0dd04a751006fbd2020-11-24T21:30:38ZengFrontiers Media S.A.Frontiers in Physiology1664-042X2019-06-011010.3389/fphys.2019.00392447350A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel FunctionGaia Ziraldo0Gaia Ziraldo1Damiano Buratto2Yuanyuan Kuang3Liang Xu4Andrea Carrer5Andrea Carrer6Chiara Nardin7Chiara Nardin8Francesco Chiani9Anna Maria Salvatore10Gaetano Paludetti11Gaetano Paludetti12Richard A. Lerner13Guang Yang14Francesco Zonta15Fabio Mammano16Fabio Mammano17Fabio Mammano18CNR Institute of Cell Biology and Neurobiology, Monterotondo, ItalyInstitute of Otolaryngology, Università Cattolica del Sacro Cuore, Rome, ItalyShanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, ChinaShanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, ChinaShanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, ChinaCNR Institute of Cell Biology and Neurobiology, Monterotondo, ItalyDepartment of Physics and Astronomy “G. Galilei”, University of Padova, Padua, ItalyCNR Institute of Cell Biology and Neurobiology, Monterotondo, ItalyDepartment of Physics and Astronomy “G. Galilei”, University of Padova, Padua, ItalyCNR Institute of Cell Biology and Neurobiology, Monterotondo, ItalyCNR Institute of Cell Biology and Neurobiology, Monterotondo, ItalyInstitute of Otolaryngology, Università Cattolica del Sacro Cuore, Rome, ItalyFondazione Policlinico Universitario A. Gemelli IRCCS, Rome, ItalyShanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, ChinaShanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, ChinaShanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, ChinaCNR Institute of Cell Biology and Neurobiology, Monterotondo, ItalyShanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, Shanghai, ChinaDepartment of Physics and Astronomy “G. Galilei”, University of Padova, Padua, ItalyConnexin 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.https://www.frontiersin.org/article/10.3389/fphys.2019.00392/fullconnexin hemichannelsrare diseasesphage display librariestherapeutic monoclonal antibodiesmolecular dynamicspatch clamp
collection DOAJ
language English
format Article
sources DOAJ
author Gaia Ziraldo
Gaia Ziraldo
Damiano Buratto
Yuanyuan Kuang
Liang Xu
Andrea Carrer
Andrea Carrer
Chiara Nardin
Chiara Nardin
Francesco Chiani
Anna Maria Salvatore
Gaetano Paludetti
Gaetano Paludetti
Richard A. Lerner
Guang Yang
Francesco Zonta
Fabio Mammano
Fabio Mammano
Fabio Mammano
spellingShingle Gaia Ziraldo
Gaia Ziraldo
Damiano Buratto
Yuanyuan Kuang
Liang Xu
Andrea Carrer
Andrea Carrer
Chiara Nardin
Chiara Nardin
Francesco Chiani
Anna Maria Salvatore
Gaetano Paludetti
Gaetano Paludetti
Richard A. Lerner
Guang Yang
Francesco Zonta
Fabio Mammano
Fabio Mammano
Fabio Mammano
A Human-Derived Monoclonal Antibody Targeting Extracellular Connexin Domain Selectively Modulates Hemichannel Function
Frontiers in Physiology
connexin hemichannels
rare diseases
phage display libraries
therapeutic monoclonal antibodies
molecular dynamics
patch clamp
author_facet Gaia Ziraldo
Gaia Ziraldo
Damiano Buratto
Yuanyuan Kuang
Liang Xu
Andrea Carrer
Andrea Carrer
Chiara Nardin
Chiara Nardin
Francesco Chiani
Anna Maria Salvatore
Gaetano Paludetti
Gaetano Paludetti
Richard A. Lerner
Guang Yang
Francesco Zonta
Fabio Mammano
Fabio Mammano
Fabio Mammano
author_sort Gaia Ziraldo
title 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_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_sort human-derived monoclonal antibody targeting extracellular connexin domain selectively modulates hemichannel function
publisher Frontiers Media S.A.
series Frontiers in Physiology
issn 1664-042X
publishDate 2019-06-01
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.
topic connexin hemichannels
rare diseases
phage display libraries
therapeutic monoclonal antibodies
molecular dynamics
patch clamp
url https://www.frontiersin.org/article/10.3389/fphys.2019.00392/full
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