Phenotypic selection and characterization of randomly produced non‐haemolytic mutants of the toxic sea anemone protein sticholysin II

Alegre-Cebollada, Jorge; Lacadena, Valle; Oñaderra, Mercedes; Mancheño, José M.; Gavilanes, José G.; Pozo, Álvaro Martı́nez del

doi:10.1016/j.febslet.2004.08.031

Cited by 34 publications

(34 citation statements)

References 16 publications

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“…The importance of tyrosyl side chains for toxin function was shown by Turk et al (66), where chemical modification of tyrosines in EqtII almost completely abolished hemolytic activity. In agreement with our model, in sticholysin II a mutation of the tyrosine equivalent to Tyr 113 (mutant Y111N) largely abolishes hemolytic activity (67). Furthermore, by introducing a 19 F label on EqtII tryptophans, it was shown that Trp 112 is important for SM recognition, as specific NMR chemical shifts were observed upon the addition of SM to PC micelles (31).…”

Section: Discussionsupporting

confidence: 81%

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Bakrač

Gutiérrez-Aguirre

Podlesek

et al. 2008

Journal of Biological Chemistry

165

169

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Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp 112 and Tyr 113 exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins. Sphingomyelin (SM)8 is an important eukaryotic membrane lipid, located for the most part in the outer leaflet of the plasma membrane in the form of specialized cholesterol-rich microdomains, so-called lipid rafts (1, 2). Many pathogens and toxic proteins employ lipid rafts to invade cells (3, 4), but currently little is known about the molecular details of the recognition mechanism of the lipid components present in the rafts. In the particular case of SM, the specific recognition occurs even though SM exposes the same phosphorylcholine headgroup as the other abundant lipid, phosphatidylcholine. SM-binding proteins are currently exploited as specific markers for cellular SM (5) and are used to identify other proteins involved in sphingolipid metabolism (6).Actinoporins are extremely potent cytolysins produced exclusively by sea anemones (7,8). They may be used to capture prey, in intraspecific aggression, or in preventing adhesion of other organisms (7, 9). The two most studied representatives are EqtII, isolated from the sea anemone Actinia equina, and sticholysin II (StII) from Stichodactyla helianthus. Actinoporins constitute a family of conserved proteins that cause hemolysis of red blood cells by colloid-osmotic lysis and exhibit cytolytic activity against various cell lines (7, 10 -12). Even the most distant members of the family share more than 60% sequence identity and the available threedimensional structures of EqtII and StII are nearly superimposable (13-15). The structure is composed of a tightly folded ␤-sandwich flanked b...

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Section: Discussionsupporting

confidence: 81%

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Bakrač

Gutiérrez-Aguirre

Podlesek

et al. 2008

Journal of Biological Chemistry

165

169

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“…Whereas R29Q and Y111N present a marked decrease in the association constant K, F106L showed an affinity constant only ~ 3 times lower than wild-type. Phe-106 is one of the aromatic residues presumably involved in the interaction with the lipid-water interface 16 . In fact, its relative binding and hemolytic activities are coincident ( Table 2), suggesting that the decrease in the hemolytic activity of this mutant may be explained only by a defective lipid binding.…”

Section: Discussionmentioning

confidence: 99%

“…probably establish cation-π interactions 16 . Therefore, Arg-29 seems to be a crucial residue in the mechanism of pore formation, since it may participate in both membrane recognition and conformational changes leading to pore formation.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, Arg-29 belongs to one cluster of basic amino acids that has been postulated as an important motif due to its situation between the N-terminus and the other lipidbinding regions 9 ; therefore, R29Q mutant has also been produced. Finally, has been shown to be necessary for pore formation of a His-tagged version of StnII 16 , though its exact role in the mechanism of pore formation is unknown. Thus, StnII K19E mutant has also been included in this study, together with the double mutant K19E/E23K.…”

Section: Mutants Production and Structural Characterizationmentioning

confidence: 99%

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Calorimetric Scrutiny of Lipid Binding by Sticholysin II Toxin Mutants

Alegre-Cebollada

Cunietti

Herrero-Galán

et al. 2008

Journal of Molecular Biology

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121

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The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid-protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporins family, have been produced and their hemolytic and lipidbinding properties compared to those of the wild-type protein. A thermodynamical approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpydriven process that occurs with a high affinity constant (1.7 x 10 8 M -1 ). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, even though it is necessary for correct pore 2 formation. Membrane binding is achieved through two separate mechanisms, i.e.recognition of the lipid-water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholine-binding site.Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation.3

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“…Regarding this study, the most important characteristic of EqtII is its remarkable SM specificity. EqtII recognizes SM during membrane binding via a phosphocholine (POC) binding site and an adjacent aromatic amino acid cluster (13,16,18). Because PC and SM possess the same POC headgroup and EqtII binds isolated SM analogues (16), the toxin therefore is sensitive to the characteristic SM chemistry below the headgroup rather than its headgroup or bulk phase behavior.…”

mentioning

confidence: 99%

A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin

Bakrač

Kladnik

Maček

et al. 2010

Journal of Biological Chemistry

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Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane. When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles.

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Phenotypic selection and characterization of randomly produced non‐haemolytic mutants of the toxic sea anemone protein sticholysin II

Cited by 34 publications

References 16 publications

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Calorimetric Scrutiny of Lipid Binding by Sticholysin II Toxin Mutants

A Toxin-based Probe Reveals Cytoplasmic Exposure of Golgi Sphingomyelin

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