The multigene families of actinoporins (part II): Strategies for heterologous production in Escherichia coli

Valle, Aisel; Hervis, Yadira P.; Socas, Luis Benito Pérez; Canet, Liem; Faheem, Muhammad; Barbosa, J.A.R.G.; Lanio, María E.; Pazos, Fabiola

doi:10.1016/j.toxicon.2016.03.018

Cited by 6 publications

(5 citation statements)

References 109 publications

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“…Actinoporins have been intensively studied to understand their hemolytic mode of action and as models for protein-membrane interactions. The structures of three actinoporins have been elucidated, and the roles of many amino acid residues are known (recently reviewed by 60 – 62 ). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

The chemical armament of reef-building corals: inter- and intra-specific variation and the identification of an unusual actinoporin in Stylophora pistilata

Ben-Ari

Paz

Sher

2018

Sci Rep

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Corals, like other cnidarians, are venomous animals that rely on stinging cells (nematocytes) and their toxins to catch prey and defend themselves against predators. However, little is known about the chemical arsenal employed by stony corals, despite their ecological importance. Here, we show large differences in the density of nematocysts and whole-body hemolytic activity between different species of reef-building corals. In the branched coral Stylophora pistillata, the tips of the branches exhibited a greater hemolytic activity than the bases. Hemolytic activity and nematocyst density were significantly lower in Stylophora that were maintained for close to a year in captivity compared to corals collected from the wild. A cysteine-containing actinoporin was identified in Stylophora following partial purification and tandem mass spectrometry. This toxin, named Δ-Pocilopotoxin-Spi1 (Δ-PCTX-Spi1) is the first hemolytic toxin to be partially isolated and characterized in true reef-building corals. Loss of hemolytic activity during chromatography suggests that this actinoporin is only one of potentially several hemolytic molecules. These results suggest that the capacity to employ offensive and defensive chemicals by corals is a dynamic trait within and between coral species, and provide a first step towards identifying the molecular components of the coral chemical armament.

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Section: Resultsmentioning

confidence: 99%

The chemical armament of reef-building corals: inter- and intra-specific variation and the identification of an unusual actinoporin in Stylophora pistilata

Ben-Ari

Paz

Sher

2018

Sci Rep

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“…In our previous study, we observed significant cytolytic activity of recombinant HALT-4 despite the presence of additional residues from the pET28a vector [ 2 ]. However, it has been reported that the presence of fusion His-tag and vector residues at the N-terminal end led to reduced activity in either hemolysis or cytolysis for several actinoporins, including magnificalysin III from Heteractis magnifica and sticholysins I and II from Stichodactyla helianthus [ 12 ]. On the other hand, recombinant AvtI hemolytic toxin from Actineria illosa showed a 9.2-fold increase in its hemolytic activity when expressed in E. coli BL21 (DE3) as compared to the native AvtI [ 13 ].…”

Section: Resultsmentioning

confidence: 99%

Is Proteolytic Cleavage Essential for the Enhanced Activity of Hydra Pore-Forming Toxin, HALT-4?

Yap

Hwang

2023

Toxins

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Hydra actinoporin-like toxin 4 (HALT-4) differs from other actinoporins due to its N-terminal propart that contains approximately 103 additional residues. Within this region, we identified five dibasic residues and assumed that, when cleaved, they could potentially exhibit HALT-4′s cytolytic activity. We created five truncated versions of HALT-4 (tKK1, tKK2, tRK3, tKK4 and tKK5) to investigate the role of the N-terminal region and potential cleavage sites on the cytolytic activity of HALT-4. However, our results demonstrated that the propart-containing HALT-4 (proHALT-4), as well as the truncated versions tKK1 and tKK2, exhibited similar cytolytic activity against HeLa cells. In contrast, tRK3, tKK4 and tKK5 failed to kill HeLa cells, indicating that cleavage at the KK1 or KK2 sites did not enhance cytolytic activity but may instead facilitate the sorting of tKK1 and tKK2 to the regulated secretory pathway for eventual deposition in nematocysts. Moreover, RK3, KK4 and KK5 were unlikely to serve as proteolytic cleavage sites, as the amino acids between KK2 and RK3 are also crucial for pore formation.

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“…Most actinoporins do not contain Cys residues (Anderluh and Macek 2002;Valle et al 2015Valle et al , 2016, therefore, site-specific mutagenesis introducing a Cys residue has been a useful strategy to analyze the relevance of several amino acid positions to toxin function. Inclusion of a Cys residue in a specific position has allowed (1) the assessment of the structural and functional importance of the replaced residue; (2) the stabilization of conformational states by the non-native S-S bond (Hong et al 2002;Anderluh et al 2003;Kristan et al 2004;Penton et al 2011;Valle et al 2011;Hervis et al 2014); (3) the assessment of thiol-specific fluorescent labels (Malovrh et al 2003;Alegre-Cebollada et al 2007) or spin probe labels.…”

Section: Cys Mutants Of Stsmentioning

confidence: 99%

Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins, pore-forming proteins from a sea anemone

et al. 2017

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Actinoporins constitute a unique class of poreforming toxins found in sea anemones that are able to bind and oligomerize in membranes, leading to cell swelling, impairment of ionic gradients and, eventually, to cell death. In this review we summarize the knowledge generated from the combination of biochemical and biophysical approaches to the study of sticholysins I and II (Sts, StI/II), two actinoporins largely characterized by the Center of Protein Studies at the University of Havana during the last 20 years. These approaches include strategies for understanding the toxin structure-function relationship, the protein-membrane association process leading to pore formation and the interaction of toxin with cells. The rational combination of experimental and theoretical tools have allowed unraveling, at least partially, of the complex mechanisms involved in toxin-membrane interaction and of the molecular pathways triggered upon this interaction. The study of actinoporins is important not only to gain an understanding of their biological roles in anemone venom but also to investigate basic molecular mechanisms of protein insertion into membranes, protein-lipid interactions and the modulation of protein conformation by lipid binding. A deeper knowledge of the basic molecular mechanisms involved in Sts-cell interaction, as described in this review, will support the current investigations conducted by our group which focus on the design of immunotoxins against tumor cells and antigen-releasing systems to cell cytosol as Stsbased vaccine platforms.

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The multigene families of actinoporins (part II): Strategies for heterologous production in Escherichia coli

Cited by 6 publications

References 109 publications

The chemical armament of reef-building corals: inter- and intra-specific variation and the identification of an unusual actinoporin in Stylophora pistilata

The chemical armament of reef-building corals: inter- and intra-specific variation and the identification of an unusual actinoporin in Stylophora pistilata

Is Proteolytic Cleavage Essential for the Enhanced Activity of Hydra Pore-Forming Toxin, HALT-4?

Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins, pore-forming proteins from a sea anemone

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