Identification and characterization of a novel zebrafish (Danio rerio) pentraxin–carbonic anhydrase
Background Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15–21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only se...
| Main Authors: | , , , , , , , , , , , , , , , |
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PeerJ Inc.
2017-12-01
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| author | Maarit S. Patrikainen Martti E.E. Tolvanen Ashok Aspatwar Harlan R. Barker Csaba Ortutay Janne Jänis Mikko Laitaoja Vesa P. Hytönen Latifeh Azizi Prajwol Manandhar Edit Jáger Daniela Vullo Sampo Kukkurainen Mika Hilvo Claudiu T. Supuran Seppo Parkkila |
| author_facet | Maarit S. Patrikainen Martti E.E. Tolvanen Ashok Aspatwar Harlan R. Barker Csaba Ortutay Janne Jänis Mikko Laitaoja Vesa P. Hytönen Latifeh Azizi Prajwol Manandhar Edit Jáger Daniela Vullo Sampo Kukkurainen Mika Hilvo Claudiu T. Supuran Seppo Parkkila |
| author_sort | Maarit S. Patrikainen |
| collection | DOAJ |
| description | Background Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15–21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. Methods We isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI–PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI–PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. Results The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. Discussion These findings provide novel insights into the evolution, structure, and function of this unique CA form. |
| first_indexed | 2024-03-09T06:59:03Z |
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| spelling | doaj.art-54363a1d987547d0bf2f0282de05ff1b2023-12-03T09:56:05ZengPeerJ Inc.PeerJ2167-83592017-12-015e412810.7717/peerj.4128Identification and characterization of a novel zebrafish (Danio rerio) pentraxin–carbonic anhydraseMaarit S. Patrikainen0Martti E.E. Tolvanen1Ashok Aspatwar2Harlan R. Barker3Csaba Ortutay4Janne Jänis5Mikko Laitaoja6Vesa P. Hytönen7Latifeh Azizi8Prajwol Manandhar9Edit Jáger10Daniela Vullo11Sampo Kukkurainen12Mika Hilvo13Claudiu T. Supuran14Seppo Parkkila15Faculty of Medicine and Life Sciences, University of Tampere, Tampere, FinlandDepartment of Future Technologies, University of Turku, Turku, FinlandFaculty of Medicine and Life Sciences, University of Tampere, Tampere, FinlandFaculty of Medicine and Life Sciences, University of Tampere, Tampere, FinlandHiDucator Ltd., Kangasala, FinlandDepartment of Chemistry, University of Eastern Finland, Joensuu, FinlandDepartment of Chemistry, University of Eastern Finland, Joensuu, FinlandFaculty of Medicine and Life Sciences, University of Tampere, Tampere, FinlandFaculty of Medicine and Life Sciences, University of Tampere, Tampere, FinlandFaculty of Medicine and Life Sciences, University of Tampere, Tampere, FinlandDepartment of Epidemiology, Faculty of Health Sciences, Semmelweis University, Budapest, HungaryDipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di Firenze, Florence, ItalyFaculty of Medicine and Life Sciences, University of Tampere, Tampere, FinlandFaculty of Medicine and Life Sciences, University of Tampere, Tampere, FinlandDipartimento Neurofarba, Sezione di Scienze Farmaceutiche e Nutraceutiche, Università degli Studi di Firenze, Florence, ItalyFaculty of Medicine and Life Sciences, University of Tampere, Tampere, FinlandBackground Carbonic anhydrases (CAs) are ubiquitous, essential enzymes which catalyze the conversion of carbon dioxide and water to bicarbonate and H+ ions. Vertebrate genomes generally contain gene loci for 15–21 different CA isoforms, three of which are enzymatically inactive. CA VI is the only secretory protein of the enzymatically active isoforms. We discovered that non-mammalian CA VI contains a C-terminal pentraxin (PTX) domain, a novel combination for both CAs and PTXs. Methods We isolated and sequenced zebrafish (Danio rerio) CA VI cDNA, complete with the sequence coding for the PTX domain, and produced the recombinant CA VI–PTX protein. Enzymatic activity and kinetic parameters were measured with a stopped-flow instrument. Mass spectrometry, analytical gel filtration and dynamic light scattering were used for biophysical characterization. Sequence analyses and Bayesian phylogenetics were used in generating hypotheses of protein structure and CA VI gene evolution. A CA VI–PTX antiserum was produced, and the expression of CA VI protein was studied by immunohistochemistry. A knock-down zebrafish model was constructed, and larvae were observed up to five days post-fertilization (dpf). The expression of ca6 mRNA was quantitated by qRT-PCR in different developmental times in morphant and wild-type larvae and in different adult fish tissues. Finally, the swimming behavior of the morphant fish was compared to that of wild-type fish. Results The recombinant enzyme has a very high carbonate dehydratase activity. Sequencing confirms a 530-residue protein identical to one of the predicted proteins in the Ensembl database (ensembl.org). The protein is pentameric in solution, as studied by gel filtration and light scattering, presumably joined by the PTX domains. Mass spectrometry confirms the predicted signal peptide cleavage and disulfides, and N-glycosylation in two of the four observed glycosylation motifs. Molecular modeling of the pentamer is consistent with the modifications observed in mass spectrometry. Phylogenetics and sequence analyses provide a consistent hypothesis of the evolutionary history of domains associated with CA VI in mammals and non-mammals. Briefly, the evidence suggests that ancestral CA VI was a transmembrane protein, the exon coding for the cytoplasmic domain was replaced by one coding for PTX domain, and finally, in the therian lineage, the PTX-coding exon was lost. We knocked down CA VI expression in zebrafish embryos with antisense morpholino oligonucleotides, resulting in phenotype features of decreased buoyancy and swim bladder deflation in 4 dpf larvae. Discussion These findings provide novel insights into the evolution, structure, and function of this unique CA form.https://peerj.com/articles/4128.pdfPhylogenyProtein modelingPentraxinZebrafishCarbonic anhydrase VICarbonic anhydrase |
| spellingShingle | Maarit S. Patrikainen Martti E.E. Tolvanen Ashok Aspatwar Harlan R. Barker Csaba Ortutay Janne Jänis Mikko Laitaoja Vesa P. Hytönen Latifeh Azizi Prajwol Manandhar Edit Jáger Daniela Vullo Sampo Kukkurainen Mika Hilvo Claudiu T. Supuran Seppo Parkkila Identification and characterization of a novel zebrafish (Danio rerio) pentraxin–carbonic anhydrase PeerJ Phylogeny Protein modeling Pentraxin Zebrafish Carbonic anhydrase VI Carbonic anhydrase |
| title | Identification and characterization of a novel zebrafish (Danio rerio) pentraxin–carbonic anhydrase |
| title_full | Identification and characterization of a novel zebrafish (Danio rerio) pentraxin–carbonic anhydrase |
| title_fullStr | Identification and characterization of a novel zebrafish (Danio rerio) pentraxin–carbonic anhydrase |
| title_full_unstemmed | Identification and characterization of a novel zebrafish (Danio rerio) pentraxin–carbonic anhydrase |
| title_short | Identification and characterization of a novel zebrafish (Danio rerio) pentraxin–carbonic anhydrase |
| title_sort | identification and characterization of a novel zebrafish danio rerio pentraxin carbonic anhydrase |
| topic | Phylogeny Protein modeling Pentraxin Zebrafish Carbonic anhydrase VI Carbonic anhydrase |
| url | https://peerj.com/articles/4128.pdf |
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