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Toxins 2015, 7(2), 407-422; doi:10.3390/toxins7020407

Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata

Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, Cheras, 56000 Kuala Lumpur, Malaysia
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Author to whom correspondence should be addressed.
Academic Editor: John P. Berry
Received: 12 December 2014 / Accepted: 27 January 2015 / Published: 3 February 2015
(This article belongs to the Section Marine and Freshwater Toxins)
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Abstract

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. View Full-Text
Keywords: actinoporins; cytolysin; equinatoxin II; pore-forming protein; mutations; cnidarian actinoporins; cytolysin; equinatoxin II; pore-forming protein; mutations; cnidarian
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Liew, Y.J.M.; Soh, W.T.; Jiemy, W.F.; Hwang, J.S. Mutagenesis and Functional Analysis of the Pore-Forming Toxin HALT-1 from Hydra magnipapillata. Toxins 2015, 7, 407-422.

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