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Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata
Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investiga...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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MDPI
2015
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344632/ https://www.ncbi.nlm.nih.gov/pubmed/25654788 http://dx.doi.org/10.3390/toxins7020407 |
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author | Liew, Yvonne Jing Mei Soh, Wai Tuck Jiemy, William Febry Hwang, Jung Shan |
author_facet | Liew, Yvonne Jing Mei Soh, Wai Tuck Jiemy, William Febry Hwang, Jung Shan |
author_sort | Liew, Yvonne Jing Mei |
collection | PubMed |
description | Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1–4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding. |
format | Online Article Text |
id | pubmed-4344632 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2015 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-43446322015-03-18 Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata Liew, Yvonne Jing Mei Soh, Wai Tuck Jiemy, William Febry Hwang, Jung Shan Toxins (Basel) Article Actinoporins are small 18.5 kDa pore-forming toxins. A family of six actinoporin genes has been identified in the genome of Hydra magnipapillata, and HALT-1 (Hydra actinoporin-like toxin-1) has been shown to have haemolytic activity. In this study, we have used site-directed mutagenesis to investigate the role of amino acids in the pore-forming N-terminal region and the conserved aromatic cluster required for cell membrane binding. A total of 10 mutants of HALT-1 were constructed and tested for their haemolytic and cytolytic activity on human erythrocytes and HeLa cells, respectively. Insertion of 1–4 negatively charged residues in the N-terminal region of HALT-1 strongly reduced haemolytic and cytolytic activity, suggesting that the length or charge of the N-terminal region is critical for pore-forming activity. Moreover, substitution of amino acids in the conserved aromatic cluster reduced haemolytic and cytolytic activity by more than 80%, suggesting that these aromatic amino acids are important for attachment to the lipid membrane as shown for other actinoporins. The results suggest that HALT-1 and other actinoporins share similar mechanisms of pore formation and that it is critical for HALT-1 to maintain an amphipathic helix at the N-terminus and an aromatic amino acid-rich segment at the site of membrane binding. MDPI 2015-02-03 /pmc/articles/PMC4344632/ /pubmed/25654788 http://dx.doi.org/10.3390/toxins7020407 Text en © 2015 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Liew, Yvonne Jing Mei Soh, Wai Tuck Jiemy, William Febry Hwang, Jung Shan Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata |
title | Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata |
title_full | Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata |
title_fullStr | Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata |
title_full_unstemmed | Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata |
title_short | Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata |
title_sort | mutagenesis and functional analysis of the pore-forming toxin halt-1 from hydra magnipapillata |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4344632/ https://www.ncbi.nlm.nih.gov/pubmed/25654788 http://dx.doi.org/10.3390/toxins7020407 |
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