José G. Gavilanes

Sticholysin II, a cytolysin from the sea anemone Stichodactyla helianthus, is a monomer-tetramer associating protein

Sticholysin II (Stn‐II) is a pore‐forming cytolysin. Stn‐II interacts with several supports for size exclusion chromatography, which results in an abnormal retardation precluding molecular mass calculations. Sedimentation equilibrium analysis has revealed that the protein is an associating system at neutral pH. The obtained data fit a monomer‐tetramer equilibrium with an association constant K c 4 of 109 M−3. The electrophoretic pattern of Stn‐II treated with different cross‐linking reagents, in a wide range of protein concentrations, corroborates the existence of tetrameric forms in solution. A planar configuration of the four monomers, C4 or D2 symmetry, is proposed from modelling of the cross‐linking data.

Kinetic study of the cytotoxic effect of alpha-sarcin, a ribosome inactivating protein from Aspergillus giganteus, on tumour cell lines: protein biosynthesis inhibition and cell binding.

Abstractα-Sarcin is a ribosome inactivating protein produced by the mouldAspergillus giganteus. The effect of this protein on eight different tumour cell lines has been studied in the absence of any agent affecting membrane permeability. The protein is cytotoxic for all the tumour cell lines considered. α-Sarcin modifies the cell proliferation pattern by inhibiting the protein biosynthesis of the cultured cells. No membrane damage produced by α-sarcin has been observed by measuring lactic dehydrogenase leakage. Alteration on the cell mitochondrial activity has not been detected upon treatment with α-sarcin. Differences on the extent of the protein binding to the cells have been observed by flow cytometric measurements. The kinetic analysis of the protein biosynthesis inhibition produced by α-sarcin reveals an α-sarcin concentration-dependent lag phase followed by a first order decrease of the protein synthesis rate. This parameter is dependent on the external α-sarcin concentration. A saturable component for the action of α-sarcin is also deduced from these experiments. Results are discussed in terms of the protein passage across the cell membrane as the potential rate-limiting step for the action of α-sarcin.

The behavior of sea anemone actinoporins at the water-membrane interface.

Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. They display high sequence identity and appear as multigene families. They show a singular behaviour at the water-membrane interface: In aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. These membranes contain sphingomyelin, display phase coexistence, or both. The water soluble structures of the actinoporins equinatoxin II (EqtII) and sticholysin II (StnII) are known in detail. The crystalline structure of a fragaceatoxin C (FraC) nonamer has been also determined. The three proteins fold as a β-sandwich motif flanked by two α-helices, one of them at the N-terminal end. Four regions seem to be especially important: A cluster of aromatic residues, a phosphocholine binding site, an array of basic amino acids, and the N-terminal α-helix. Initial binding of the soluble monomers to the membrane is accomplished by the cluster of aromatic amino acids, the array of basic residues, and the phosphocholine binding site. Then, the N-terminal α-helix detaches from the β-sandwich, extends, and lies parallel to the membrane. Simultaneously, oligomerization occurs. Finally, the extended N-terminal α-helix penetrates the membrane to build a toroidal pore. This model has been however recently challenged by the cryo-EM reconstruction of FraC bound to phospholipid vesicles. Actinoporins structural fold appears across all eukaryotic kingdoms in other functionally unrelated proteins. Many of these proteins neither bind to lipid membranes nor induce cell lysis. Finally, studies focusing on the therapeutic potential of actinoporins also abound.

Detergent-resistant membranes are platforms for actinoporin pore-forming activity on intact cells.

Sticholysin II is a pore‐forming toxin produced by the sea anemone Stichodactyla helianthus. We studied its cytolytic activity on COS‐7 cells. Fluorescence spectroscopy and flow cytometry revealed that the toxin permeabilizes cells to propidium cations in a dose‐dependent and time‐dependent manner. This permeabilization is impaired by preincubation of cells with cyclodextrin. Isolation of detergent‐resistant cellular membranes showed that sticholysin II colocalizes with caveolin‐1 in fractions corresponding to raft‐like domains. The interaction of sticholysin II with such domains is only lipid dependent as it also occurs in the absence of any other membrane‐associated protein. Toxin binding to raft‐like lipid vesicles inhibited cell permeabilization. The results suggest that sticholysin II promotes pore formation in COS‐7 cells through interaction with membrane domains which behave like cellular rafts.

Role of histidine‐50, glutamic acid‐96, and histidine‐137 in the ribonucleolytic mechanism of the ribotoxin α‐sarcin

α‐Sarcin is a ribotoxin secreted by the mold Aspergillus giganteus that degrades the ribosomal RNA by acting as a cyclizing ribonuclease. Three residues potentially involved in the mechanism of catalysis—histidine‐50, glutamic acid‐96, and histidine‐137—were changed to glutamine. Three dif‐ ferent single mutation variants (H50Q, E96Q, H137Q) as well as a double variant (H50/137Q) and a triple variant (H50/137Q/E96Q) were prepared and isolated to homogeneity. These variants were spectroscopically (circular dichroism, fluorescence emission, and proton nuclear magnetic resonance) characterized. According to these results, the three‐dimensional structure of these variants of α‐sarcin was preserved; only very minor local changes were detected. All the variants were inactive when assayed against either intact ribosomes or poly(A). The effect of pH on the ribonucleolytic activity of α‐sarcin was evaluated against the ApA dinucleotide. This assay revealed that only the H50Q variant still retained its ability to cleave a phosphodiester bond, but it did so to a lesser extent than did wild‐type α‐sarcin. The results obtained are interpreted in terms of His137 and Glu96 as essential residues for the catalytic activity of α‐sarcin (His137 as the general acid and Glu96 as the general base) and His50 stabilizing the transition state of the reaction catalyzed by α‐sarcin. Proteins 1999;37:474–484. ©1999 Wiley‐Liss, Inc.

Calorimetric Scrutiny of Lipid Binding by Sticholysin II Toxin Mutants

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 actinoporin family, have been produced, and their hemolytic and lipid-binding properties were compared to those of the wild-type protein. A thermodynamic approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpy-driven process that occurs with a high affinity constant (1.7x10(8) M(-1)). Results suggest that conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation. Membrane binding is achieved through two separate mechanisms, that is, 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.

The cytotoxin α‐sarcin behaves as a cyclizing ribonuclease

The hydrolysis of adenylyl(3′→5′)adenosine (ApA) and guanylyl(3′→5′)adenosine (GpA) dinucleotides by the cytotoxic protein α‐sarcin has been studied. Quantitative analysis of the reaction has been performed through reverse‐phase chromatographic (HPLC) separation of the resulting products. The hydrolysis of the 3′‐5′ phosphodiester bond of these substrates yields the 2′‐3′ cyclic mononucleotide; this intermediate is converted into the corresponding 3′‐monophosphate derivative as the final product of the reaction. The values of the apparent Michaelis constant (K M), k cat and k cat/K M have also been calculated. The obtained results fit into a two‐step mechanism for the enzymatic activity of α‐sarcin and allow to consider this protein as a cyclizing RNase.

Characterization of a natural larger form of the antifungal protein (AFP) from Aspergillus giganteus

Two major proteins, alpha-sarcin and an antifungal polypeptide (AFP), are secreted by the mould Aspergillus giganteus MDH 18894 when it is cultured for 70-80 h. A third major protein is also found in the extracellular medium at 48-60 h, but it disappears as the culture proceeds. This protein has been isolated and characterized in terms of apparent molecular mass, electrophoretic and chromatographic behaviour, NH2-terminal primary structure, amino acid content, spectroscopical features, reactivity against anti-AFP antibodies, and antifungal activity. Based on the obtained results it would be an extracellular inactive precursor form of AFP, designated as the large form of AFP (lf-AFP). Its amino acid composition is identical to that of AFP but containing six extra residues. NH2-terminal sequence analysis of the first eight amino acid residues of this polypeptide revealed that the extra residues can be perfectly accommodated within the DNA-deduced sequence of the precursor form of AFP. Its alignment with precursor sequences of different proteins, secreted by a variety of Aspergillus spp., reveals the existence of a common tetrapeptide at the carboxy-terminal end of their leader peptides. This sequence would be Ile/Leu-Xaa-Yaa-Arg, being mostly Xaa and Yaa an acid residue (Asp/Glu) and alanine, respectively. The presence of lf-AFP as an extracellular protein would be in perfect agreement with the existence of this tetrapeptide motif, that can be involved in the protein secretion mechanisms of filamentous fungi.

Two-Dimensional Crystallization on Lipid Monolayers and Three-Dimensional Structure of Sticholysin II, a Cytolysin from the Sea Anemone Stichodactyla helianthus

Sticholysin II (Stn II), a potent cytolytic protein isolated from the sea anemone Stichodactyla helianthus, has been crystallized on lipid monolayers. With Fourier-based methods, a three-dimensional (3D) model of Stn II, up to a resolution of 15 A, has been determined. The two-sided plane group is p22(1)2, with dimensions a = 98 A, b = 196 A. The 3D model of Stn II displays a Y-shaped structure, slightly flattened, with a small curvature along its longest dimension (51 A). This protein, with a molecular mass of 19. 2 kDa, is one of the smallest structures reconstructed with this methodology. Two-dimensional (2D) crystals of Stn II on phosphatidylcholine monolayers present a unit cell with two tetrameric motifs, with the monomers in two different orientations: one with its longest dimension lying on the crystal plane and the other with this same axis leaning at an angle of approximately 60 degrees with the crystal plane.

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

A rapid screening method for haemolytic activity, using blood agar plates, has been developed to analyze randomly produced mutant variants of the pore‐forming protein sticholysin II (Stn II). Those exhibiting a reduced activity were selected and the DNA corresponding to each Stn II variant sequenced. Once the mutation produced was determined, protein variants were isolated and characterized in terms of structure (circular dichroism spectra and thermal stability) and haemolytic activity. Three single mutation protein variants, at residues K19, F106 and Y111, showed a significantly decreased haemolytic activity while their thermostability was identical to that of the wild‐type protein. Considering the obtained data and based on the three‐dimensional structure of the protein, the role of these residues on the mechanism of haemolysis has been analyzed.