title: Mutagenesis and functional analysis of the pore-forming toxin HALT-1 from Hydra magnipapillata.
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.
authors: Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, Cheras, 56000 Kuala Lumpur, Malaysia. yvonneljm@gmail.com.
authors: Yvonne Jing Mei
authors: YJ
authors: Liew
authors: Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, Cheras, 56000 Kuala Lumpur, Malaysia. waitucksoh@outlook.com.
authors: Wai Tuck
authors: WT
authors: Soh
authors: Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, Cheras, 56000 Kuala Lumpur, Malaysia. willjie90@gmail.com.
authors: William Febry
authors: WF
authors: Jiemy
authors: Faculty of Applied Sciences, UCSI University, No. 1, Jalan Menara Gading, UCSI Heights, Cheras, 56000 Kuala Lumpur, Malaysia. jshanhwang@ucsiuniversity.edu.my.
authors: Jung Shan
authors: JS
authors: Hwang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000187:drug effects / Q000473:pathology; D006367:HeLa Cells; D006461:Hemolysis / Q000187:drug effects*; D006801:Humans; D006829:Hydra / Q000378:metabolism*; D008387:Marine Toxins / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D008969:Molecular Sequence Data; D005810:Multigene Family; D016297:Mutagenesis, Site-Directed; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics / Q000302:isolation & purification / Q000633:toxicity*; D011994:Recombinant Proteins
chemical_list: D008387:Marine Toxins; D052899:Pore Forming Cytotoxic Proteins; D011994:Recombinant Proteins
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