Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin
Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxy...
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| Format: | Article |
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Frontiers Media S.A.
2018-09-01
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| Series: | Frontiers in Cell and Developmental Biology |
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| Online Access: | https://www.frontiersin.org/article/10.3389/fcell.2018.00117/full |
| _version_ | 1818881680803364864 |
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| author | Janna Blechman Savani Anbalagan Gary G. Matthews Gil Levkowitz |
| author_facet | Janna Blechman Savani Anbalagan Gary G. Matthews Gil Levkowitz |
| author_sort | Janna Blechman |
| collection | DOAJ |
| description | Presynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxytocin (OXT) neurons. This conclusion was based on immunoreactivity of a monoclonal antibody (mAb) clone L55/54, which was directed against the carboxy terminal tail of the CNGA2a. To study the role of CNGA2a in oxytocin neurons function, we generated zebrafish mutants of cnga2a, cnga2b and oxt genes using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing. We show that mAb L55/54 specifically recognizes CNGA2a protein when expressed in heterologous cell culture system. Surprisingly, anti-CNGA2a immunoreactivity was not eliminated following knockout of either cnga2a, cnga2b or both. However, knockout of oxt resulted in total loss of anti-CNGA2a mAb immunoreactivity despite the lack of sequence and structural similarities between OXT and CNGA2a proteins. Our results provide a noteworthy lesson of differences in antibody immunoreactivity, which could only be revealed using specific genetic tools. |
| first_indexed | 2024-12-19T15:05:43Z |
| format | Article |
| id | doaj.art-af1d4186b2dd42f48d97d62a45eb2e62 |
| institution | Directory Open Access Journal |
| issn | 2296-634X |
| language | English |
| last_indexed | 2024-12-19T15:05:43Z |
| publishDate | 2018-09-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Cell and Developmental Biology |
| spelling | doaj.art-af1d4186b2dd42f48d97d62a45eb2e622022-12-21T20:16:28ZengFrontiers Media S.A.Frontiers in Cell and Developmental Biology2296-634X2018-09-01610.3389/fcell.2018.00117405215Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward OxytocinJanna Blechman0Savani Anbalagan1Gary G. Matthews2Gil Levkowitz3Department of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, IsraelDepartment of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, IsraelDepartment of Neurobiology and Behavior, Stony Brook University, New York, NY, United StatesDepartment of Molecular Cell Biology, Weizmann Institute of Science, Rehovot, IsraelPresynaptic cGMP-gated ion (CNG) channels positively or negatively modulate neurotransmitter secretion as well as the strength of synaptic transmission. Zebrafish cGMP-gated ion channel, CNGA2a (a.k.a. CNGA5), was previously reported to be specifically enriched in synaptic terminals of zebrafish oxytocin (OXT) neurons. This conclusion was based on immunoreactivity of a monoclonal antibody (mAb) clone L55/54, which was directed against the carboxy terminal tail of the CNGA2a. To study the role of CNGA2a in oxytocin neurons function, we generated zebrafish mutants of cnga2a, cnga2b and oxt genes using clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome editing. We show that mAb L55/54 specifically recognizes CNGA2a protein when expressed in heterologous cell culture system. Surprisingly, anti-CNGA2a immunoreactivity was not eliminated following knockout of either cnga2a, cnga2b or both. However, knockout of oxt resulted in total loss of anti-CNGA2a mAb immunoreactivity despite the lack of sequence and structural similarities between OXT and CNGA2a proteins. Our results provide a noteworthy lesson of differences in antibody immunoreactivity, which could only be revealed using specific genetic tools.https://www.frontiersin.org/article/10.3389/fcell.2018.00117/fullneuropeptidecGMP-gated ion channelneurohypophysismonoclonal antibodyoxytocin |
| spellingShingle | Janna Blechman Savani Anbalagan Gary G. Matthews Gil Levkowitz Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin Frontiers in Cell and Developmental Biology neuropeptide cGMP-gated ion channel neurohypophysis monoclonal antibody oxytocin |
| title | Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin |
| title_full | Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin |
| title_fullStr | Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin |
| title_full_unstemmed | Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin |
| title_short | Genome Editing Reveals Idiosyncrasy of CNGA2 Ion Channel-Directed Antibody Immunoreactivity Toward Oxytocin |
| title_sort | genome editing reveals idiosyncrasy of cnga2 ion channel directed antibody immunoreactivity toward oxytocin |
| topic | neuropeptide cGMP-gated ion channel neurohypophysis monoclonal antibody oxytocin |
| url | https://www.frontiersin.org/article/10.3389/fcell.2018.00117/full |
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