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.
Highlights
Actinoporins are a group of potent α-pore forming toxins (α-PFTs) that were first identified in sea anemones [1]
Having discovered homologous actinoporins in Hydra, which belong to another class, Hydrozoa, it is of interest to determine the evolutionary constraint of HALT-1
In Glasser et al [12], HALT-1 has been proven to be the member of actinoporin family producing the most important findings, such as exerting haemolytic activity, targeting human cell membranes, and forming a larger pore size than Equinatoxin II (EqtII)
Summary
Actinoporins are a group of potent α-pore forming toxins (α-PFTs) that were first identified in sea anemones [1]. They are low molecular weight proteins with 18.5 kDa and are able to destroy cells containing sphingomyelin, a major component of plasma membrane lipids [2]. A cluster of exposed aromatic amino acids including a phosphocholine (POC) binding site has been shown to be functionally important for membrane binding. The aromatic amino acids provide initial contact between the protein and the cell membrane while the POC binding site recognizes the headgroup of sphingomyelin in the plasma membrane [6]
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