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Characterization of glutamate carboxypeptidase 2 orthologs in trematodes
BACKGROUND: Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in m...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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BioMed Central
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9768917/ https://www.ncbi.nlm.nih.gov/pubmed/36539882 http://dx.doi.org/10.1186/s13071-022-05556-5 |
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author | Jedlickova, Lucie Peterkova, Kristyna Boateng, Enoch Mensah Ulrychova, Lenka Vacek, Vojtech Kutil, Zsofia Jiang, Zhenze Novakova, Zora Snajdr, Ivan Kim, Juan O’Donoghue, Anthony J. Barinka, Cyril Dvorak, Jan |
author_facet | Jedlickova, Lucie Peterkova, Kristyna Boateng, Enoch Mensah Ulrychova, Lenka Vacek, Vojtech Kutil, Zsofia Jiang, Zhenze Novakova, Zora Snajdr, Ivan Kim, Juan O’Donoghue, Anthony J. Barinka, Cyril Dvorak, Jan |
author_sort | Jedlickova, Lucie |
collection | PubMed |
description | BACKGROUND: Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets. METHODS: Identified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species. RESULTS: Single genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice. CONCLUSIONS: Our study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13071-022-05556-5. |
format | Online Article Text |
id | pubmed-9768917 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-97689172022-12-22 Characterization of glutamate carboxypeptidase 2 orthologs in trematodes Jedlickova, Lucie Peterkova, Kristyna Boateng, Enoch Mensah Ulrychova, Lenka Vacek, Vojtech Kutil, Zsofia Jiang, Zhenze Novakova, Zora Snajdr, Ivan Kim, Juan O’Donoghue, Anthony J. Barinka, Cyril Dvorak, Jan Parasit Vectors Research BACKGROUND: Glutamate carboxypeptidase 2 (GCP2) belongs to the M28B metalloprotease subfamily encompassing a variety of zinc-dependent exopeptidases that can be found in many eukaryotes, including unicellular organisms. Limited information exists on the physiological functions of GCP2 orthologs in mammalian tissues outside of the brain and intestine, and such data are completely absent for non-mammalian species. Here, we investigate GCP2 orthologs found in trematodes, not only as putative instrumental molecules for defining their basal function(s) but also as drug targets. METHODS: Identified genes encoding M28B proteases Schistosoma mansoni and Fasciola hepatica genomes were analyzed and annotated. Homology modeling was used to create three-dimensional models of SmM28B and FhM28B proteins using published X-ray structures as the template. For S. mansoni, RT-qPCR was used to evaluate gene expression profiles, and, by RNAi, we exploited the possible impact of knockdown on the viability of worms. Enzymes from both parasite species were cloned for recombinant expression. Polyclonal antibodies raised against purified recombinant enzymes and RNA probes were used for localization studies in both parasite species. RESULTS: Single genes encoding M28B metalloproteases were identified in the genomes of S. mansoni and F. hepatica. Homology models revealed the conserved three-dimensional fold as well as the organization of the di-zinc active site. Putative peptidase activities of purified recombinant proteins were assayed using peptidic libraries, yet no specific substrate was identified, pointing towards the likely stringent substrate specificity of the enzymes. The orthologs were found to be localized in reproductive, digestive, nervous, and sensory organs as well as parenchymal cells. Knockdown of gene expression by RNAi silencing revealed that the genes studied were non-essential for trematode survival under laboratory conditions, reflecting similar findings for GCP2 KO mice. CONCLUSIONS: Our study offers the first insight to our knowledge into M28B protease orthologs found in trematodes. Conservation of their three-dimensional structure, as well as tissue expression pattern, suggests that trematode GCP2 orthologs may have functions similar to their mammalian counterparts and can thus serve as valuable models for future studies aimed at clarifying the physiological role(s) of GCP2 and related subfamily proteases. GRAPHICAL ABSTRACT: [Image: see text] SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s13071-022-05556-5. BioMed Central 2022-12-20 /pmc/articles/PMC9768917/ /pubmed/36539882 http://dx.doi.org/10.1186/s13071-022-05556-5 Text en © The Author(s) 2022 https://creativecommons.org/licenses/by/4.0/Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) . The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/ (https://creativecommons.org/publicdomain/zero/1.0/) ) applies to the data made available in this article, unless otherwise stated in a credit line to the data. |
spellingShingle | Research Jedlickova, Lucie Peterkova, Kristyna Boateng, Enoch Mensah Ulrychova, Lenka Vacek, Vojtech Kutil, Zsofia Jiang, Zhenze Novakova, Zora Snajdr, Ivan Kim, Juan O’Donoghue, Anthony J. Barinka, Cyril Dvorak, Jan Characterization of glutamate carboxypeptidase 2 orthologs in trematodes |
title | Characterization of glutamate carboxypeptidase 2 orthologs in trematodes |
title_full | Characterization of glutamate carboxypeptidase 2 orthologs in trematodes |
title_fullStr | Characterization of glutamate carboxypeptidase 2 orthologs in trematodes |
title_full_unstemmed | Characterization of glutamate carboxypeptidase 2 orthologs in trematodes |
title_short | Characterization of glutamate carboxypeptidase 2 orthologs in trematodes |
title_sort | characterization of glutamate carboxypeptidase 2 orthologs in trematodes |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9768917/ https://www.ncbi.nlm.nih.gov/pubmed/36539882 http://dx.doi.org/10.1186/s13071-022-05556-5 |
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