Cloning, Sequencing, and Expression of Equinatoxin II

Anderluh, Gregor; Pungerčar, Jože; Štrukelj, Borut; Maček, Peter; Gubenšek, Franc

doi:10.1006/bbrc.1996.0391

Cited by 108 publications

(66 citation statements)

References 16 publications

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“…All other materials were from Sigma unless specified differently. recombinant wild-type EqtII and mutants EqtII I18C , EqtII A179C , EqtII V8C,I18C,K69C , and EqtII V8C,K69C,A179C were prepared as described (30). The expression vectors of the single and triple cysteine mutants were prepared by replacing corresponding EqtII residues with cysteine as described previously (23,25).…”

Section: Methodsmentioning

confidence: 99%

“…The expression vectors of the single and triple cysteine mutants were prepared by replacing corresponding EqtII residues with cysteine as described previously (23,25). All proteins were expressed in an E. coli BL21 (DE3) strain and purified from the bacterial cytoplasm as described elsewhere (30). Wild-type EqtII and mutants were purified to homogeneity as observed on SDS-polyacrylamide gels.…”

Section: Methodsmentioning

confidence: 99%

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Membrane Damage by an α-Helical Pore-forming Protein, Equinatoxin II, Proceeds through a Succession of Ordered Steps

Rojko

Kristan

Viero

et al. 2013

Journal of Biological Chemistry

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Background: Actinoporins are pore-forming toxins that damage cellular membranes by ␣-helices. Results: An engineered mutant of actinoporin equinatoxin II reveals sequential steps during pore formation. Conclusion: Pore formation is composed of a succession of ordered steps: fast membrane binding followed by the N-terminal region association with the membrane and oligomerization. Significance: Equinatoxin II pore formation does not require stable prepore intermediate as is often found in other poreforming toxins.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Membrane Damage by an α-Helical Pore-forming Protein, Equinatoxin II, Proceeds through a Succession of Ordered Steps

Rojko

Kristan

Viero

et al. 2013

Journal of Biological Chemistry

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“…All EqtII mutants (S114C, V8C/K69C, and tryptophan mutants W116F, W112F* (Trp-45 and Trp-149 were mutated to Phe in addition to Trp-112), and W112.116F) were prepared by site-directed mutagenesis as explained earlier (18,32,33). They were purified from the Escherichia coli cytoplasm exactly as described in (34). The fusion between the third domain of TolA (E. coli periplasmic protein) and EqtII was expressed in E .coli by the use of pTolT plasmid.…”

Section: Methodsmentioning

confidence: 99%

Two-step Membrane Binding by Equinatoxin II, a Pore-forming Toxin from the Sea Anemone, Involves an Exposed Aromatic Cluster and a Flexible Helix

Hong¹,

Gutiérrez-Aguirre²,

Barlič³

et al. 2002

Journal of Biological Chemistry

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195

235

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Equinatoxin II (EqtII) belongs to a unique family of 20-kDa pore-forming toxins from sea anemones. These toxins preferentially bind to membranes containing sphingomyelin and create cation-selective pores by oligomerization of 3-4 monomers. In this work we have studied the binding of EqtII to lipid membranes by the use of lipid monolayers and surface plasmon resonance (SPR). The binding is a two-step process, separately mediated by two regions of the molecule. An exposed aromatic cluster involving tryptophans 112 and 116 mediates the initial attachment that is prerequisite for the next step. Steric shielding of the aromatic cluster or mutation of Trp-112 and -116 to phenylalanine significantly reduces the toxin-lipid interaction. The second step is promoted by the N-terminal amphiphilic helix, which translocates into the lipid phase. The two steps were distinguished by the use of a double cysteine mutant having the N-terminal helix fixed to the protein core by a disulfide bond. The kinetics of membrane binding derived from the SPR experiments could be fitted to a two-stage binding model. Finally, by using membraneembedded quenchers, we showed that EqtII does not insert deeply in the membrane. The first step of the EqtII binding is reminiscent of the binding of the evolutionarily distant cholesterol-dependant cytolysins, which share a similar structural motif in the membrane attachment domain.Targeting and attachment of proteins to membranes is one of the key steps in many cellular processes (1-3). Protein-membrane interactions have been studied intensively in recent years with many different examples of proteins and membranes. These interactions can be promoted at the lipid-water interface by lipid anchors, electrostatic forces or surface-exposed aromatic and aliphatic residues (1, 2, 4). Compared with protein-protein interactions, details of protein-membrane interactions are poorly defined. Some of the best characterized examples are a phospholipase C pleckstrin homology domain specific for phosphatidylinositol trisphosphate (5) and small protein kinase-C-conserved (C2) domains specific for zwitterionic, particularly phosphatidylcholine membranes (6).Another group of proteins interacting with lipid membranes are pore-forming toxins (PFT) 1 (7-10), which bind to membranes before eliciting their toxic effects via the formation of transmembrane pores. The most studied PFT are bacterial since this group includes important virulence factors. Few examples of eukaryotic PFT have been well characterized, exceptions being the actinoporins, cytolysins found exclusively in sea anemones (10, 11). Members of this family have properties distinct from other PFT: they are composed of 175-179 amino acids, contain no cysteine residues, have pIϾ9.5, and show a preference for sphingomyelin (SM)-containing membranes. Actinoporins act on cellular and model lipid membranes by forming cation-selective pores with a hydrodynamic diameter of ϳ2 nm. The mechanism of pore formation involves at least two steps: binding of the water soluble m...

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“…Four major groups of these cytolysins are found in cnidarians (5,6). Among them, the 19-kDa pore-forming cytolysins are the most predominant (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). In previous studies, the sea anemone Heteractis magnifica was subjected to the analysis of different cytolysins including magnificalysins Hmg I, Hmg II and Hmg III (14)(15)(16)(17)(18).…”

Section: Introductionmentioning

confidence: 99%

“…In the laboratory, the live animal was induced through osmotic thermal stress to eject the epithelial mucus that contains the toxins (8). The cnidarian toxins are stored in the cnida, a capsule within specialized cells that contains a barbed, threadlike tube that delivers a paralyzing sting when propelled into attackers and prey.…”

Section: Preparation Of the Toxinmentioning

confidence: 99%

Characterization, purification and phylogenetic analysis of a cytolysin from the sea anemone Heteractis magnifica of the Indian Ocean

Karthikayalu¹,

Rama²,

Kirubagaran³

et al. 2010

J. Venom. Anim. Toxins incl. Trop. Dis

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ABSTRACT:It is well established that sea anemones comprise a rich source of cytolytic toxins. The present study reports the isolation and characterization of a cytolysin obtained from the sea anemone Heteractis magnifica collected in the Andaman Islands of the Indian Ocean. The crude extract was screened for hemolytic activity by a blood agar plate method and a 6-mm zone of clearance was observed after incubation. The hemolytic property of the crude extract, tested by the microtiter plate method, revealed positive results at concentrations as low as 120 ng/mL. Furthermore, it was favored by alkaline pH and was stable up to 60°C. On the other hand, the hemolytic effect was abolished by the addition of human serum. Purification steps involved ammonium sulfate precipitation and subsequent desalting by dialysis, followed by anion-and cation-exchange chromatographies. The purified fractions displayed the presence of a 19-kDa cytolysin when analyzed by SDS-PAGE. The conserved region of the cytolysin (with 303 bp) was amplified by RT-PCR and was sequenced. The sequence showed maximum homology (97%) with the already reported cytolysins from other sea anemone species.

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Cloning, Sequencing, and Expression of Equinatoxin II

Cited by 108 publications

References 16 publications

Membrane Damage by an α-Helical Pore-forming Protein, Equinatoxin II, Proceeds through a Succession of Ordered Steps

Membrane Damage by an α-Helical Pore-forming Protein, Equinatoxin II, Proceeds through a Succession of Ordered Steps

Two-step Membrane Binding by Equinatoxin II, a Pore-forming Toxin from the Sea Anemone, Involves an Exposed Aromatic Cluster and a Flexible Helix

Characterization, purification and phylogenetic analysis of a cytolysin from the sea anemone Heteractis magnifica of the Indian Ocean

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