Carlos Alvarez

Purification and characterization of two hemolysins from Stichodactyla helianthus

Two hemolysins, Sticholysin I (St I) and Sticholysin II (St II) were purified from the sea anemone Stichodactyla helianthus combining gel filtration and ion exchange chromatography. The amino acid composition of both cytolysins was determined revealing a high proportion of glycine, lysine, tyrosine and non-polar amino acids (alanine, leucine and valine). Cysteine was not found in either polypeptide. Molecular masses of St I and St II were 19401 and 19290 Da, respectively. N-terminal sequence analysis of St I and St II showed a high homology between them suggesting they are isoforms of the same cytolysin. Compared with other sea anemone cytolysins, St I and St II contain a 22 amino acid insertion fragment also present in Eq T II/Tn C and probably in CaT I and Hm T and absent in C III, the major hemolysin previously reported in this anemone.

Sizing the radius of the pore formed in erythrocytes and lipid vesicles by the toxin sticholysin I from the sea anemone Stichodactyla helianthus.

Abstract. The radius of the pore formed by sticholysin I and II (StI, StII) in erythrocytes and sticholysin I in lipid vesicles was investigated. The rate of colloid osmotic lysis of human erythrocytes, exposed to one of the toxins in the presence of sugars of different size, was measured. The relative permeability of each sugar was derived and the pore radius estimated with the Renkin equation. The radius was similar for sticholysin I and II and was independent of the reference sugar chosen and of the toxin concentration applied. It was also the same when erythrocytes were pretreated with different toxin doses in the presence of a polyethylene glycol (PEG) large enough to prevent lysis and thereafter transferred to solutions containing oligosaccharides of different size where they did lyse at different rates. The osmometric behavior of large unilamellar vesicles (LUV) was thereafter used to estimate the toxin lesion radius in a model system. LUV transferred to a hyperosmotic solution with a certain sugar immediately shrank and then re-swelled at a rate dependent on the bilayer permeability to water and sugar. When LUV were previously permeabilized with StI, only a fraction of them, namely those not carrying pores, continued to behave as osmometers. By increasing the size of the added sugar and approaching the pore radius, the fraction of osmometric LUV increased. Relative permeabilities were derived and used to estimate a channel radius around 1.2 nm, both for sugars and for PEGs. In conclusion the sticholysin pore has a constant size independent of toxin concentration and similar in natural and artificial membranes, suggesting it has a fixed predominant structure.

Sticholysins, two pore-forming toxins produced by the Caribbean Sea anemone Stichodactyla helianthus: Their interaction with membranes

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. As for the rest of the members of this family, Sts are cysteine-less proteins, with molecular weights around 20 kDa, high isoelectric points (>9.5), and a preference for sphingomyelin-containing membranes. A three-dimensional structure of StII, solved by X-ray crystallography, showed that it is composed of a hydrophobic beta-sandwich core flanked on the opposite sides by two alpha helices comprising residues 14-23 and 128-135. A variety of experimental results indicate that the first thirty N-terminal residues, which include one of the helices, are directly involved in pore formation. This region contains an amphipathic stretch, well conserved in all actinoporins, which is the only portion of the molecule that can change conformation without perturbing the general protein fold; in fact, binding to model membranes only produces a slight increase in the regular secondary structure content of Sts. Sts are produced in soluble form but they readily bind to different cell and model membrane systems such as lipidic monolayers, micelles, and lipid vesicles. Remarkably, both the binding and pore-formation steps are critically dependent on the physico-chemical nature of the membrane. In fact, a large population of toxin irreversibly binds with high affinity in membranes containing sphingomyelin whereas binding in membranes lacking this sphingolipid is relatively low and reversible. The joint presence of SM and cholesterol largely promotes binding and pore formation. Minor amounts of lipids favoring a non-lamellar organization also augment the efficiency of pore formation. The functional pore formed in cellular and model membranes has a diameter of approximately 2.0 nm and is presumably formed by the N-terminal alpha helices of four monomers tilted 31 degrees in relation to the bilayer normal. Experimental evidence supports the hypothesis that sticholysins, as well as equinatoxin II, another actinoporin, form a toroidal pore in membranes in which the polypeptide chains as well as the polar head groups of phospholipids are involved.

Primary structure of two cytolysin isoforms from Stichodactyla helianthus differing in their hemolytic activity.

Sticholysin I (St-I) and sticholysin II (St-II) are cytolysins purified from the sea anemone Stichodactyla helianthus with a high degree of sequence identity (93%) but clearly differenced in their hemolytic activity. In order to go further into the structural determinants for the different behavior of St-I and St-II, we report here the complete amino acid sequences and the consensus secondary structure prediction of both proteins. The complete determination of St-II primary structure confirms the partial revision of cytolysin III amino acid sequence. All nonconservative changes between St-I and St-II are located at the N-terminal. According to our prediction these changes could be located at the same face of an alpha-helix during pore formation events and could account for the observed differences in hemolytic activity between St-I and St-II.

Properties of St I and St II, two isotoxins isolated from Stichodactyla helianthus: a comparison.

Sticholysins I and II are two highly hemolytic polypeptides purified from the Caribbean Sea anemone Stichodactyla helianthus. Their high sequence homology (93%) indicates that they correspond to isoforms of the same hemolysin. The spectroscopic measurements show a close similarity in the secondary structure content, conformation and stability of both toxins. Exposure of the toxins to high pHs (>11), a free radical source (AAPH), urea or temperature produce permanent changes in the toxin that lead to a significant loss of HA. It is significant to note that this loss of hemolytic activity occurs when other indicators, probably with the only exception of near-UV CD spectra, barely detect changes in the protein structure. This emphasizes the sensitivity of the protein function to changes in the macromolecule conformation. The most noticeable difference between both toxins is the considerably higher activity of St II, both measured in terms of erythrocyte internal K(+) exit or hemolysis; which is related to enthalpic factors. This difference is not due to an incomplete association of St I to the membrane. We consider then that the different pore forming capacity of both toxins in erythrocytes can be explained in terms of the difference in charge of the N-terminal fragment, than can considerably reduce the St I insertion rate in the membrane probably due to the negatively charged outer leaflet of the red blood cell, without a significant reduction of its capacity to bind to the cell membrane. This electrostatic effect, together with a slightly more relaxed structure in St II, could explain the higher pore forming capacity of St II in the red blood cell membrane.

Binding of sea anemone pore-forming toxins sticholysins I and II to interfaces--modulation of conformation and activity, and lipid-protein interaction.

Sticholysins I and II (St I and St II) are water-soluble toxins produced by the sea anemone Stichodactyla helianthus. St I and St II bind to biological and model membranes containing sphingomyelin (SM), forming oligomeric pores that lead to leakage of internal contents. Here we describe functional and structural studies of the toxins aiming at the understanding at a molecular level of their mechanism of binding, as well as their effects on membrane permeabilization. St I and St II caused potassium leakage from red blood cells and temperature-dependent hemolysis, the activation energy of the process being lower for the latter toxin. Protein intrinsic fluorescence measurements provided evidence for toxin binding to model membranes composed of 1:1 (mol:mol) egg phosphatidyl choline (ePC):SM. The fluorescence intensity increased and the maximum emission wavelength decreased as a result of binding. The changes were quantitatively different for both toxins. Circular dichroism spectra showed that both St I and St II exhibit a high content of beta-sheet structure and that binding to model membranes did not alter the toxins conformation to a large extent. Changing the lipid composition by adding 5 mol% of negatively charged phosphatidic acid (PA) or phosphatidyl glycerol (PG) had small, but detectable, effects on protein conformation. The influence of lipid composition on toxin-induced membrane permeabilization was assessed by means of fluorescence measurements of calcein leakage. The effect was larger for ePC:SM bilayers containing 5 mol% of negative curvature-inducing lipids. Electron paramagnetic resonance (EPR) spectra of intercalated fatty acid spin probes carrying the nitroxide moiety at different carbons (5, 7, 12, and 16) evidenced the occurrence of lipid-protein interaction. Upon addition of the toxins, two-component spectra were observed for the probe labeled at C-12. The broader component, corresponding to a population of strongly immobilized spin probes, was ascribed to boundary lipid. The contribution of this component to the total spectrum was larger for St II than for St I. Moreover, it was clearly detectable for the C-12-labeled probe, but it was absent when the label was at C-16, indicating a lack of lipid-protein interaction close to the lipid terminal methyl group. This effect could be either due to the fact that the toxins do not span the whole bilayer thickness or to the formation of a toroidal pore leading to the preferential interaction with acyl chain carbons closer to the phospholipids head groups.

Effect of pH on the conformation, interaction with membranes and hemolytic activity of sticholysin II, a pore forming cytolysin from the sea anemone Stichodactyla helianthus

Sticholysin II (St II) is a pore forming cytolysin obtained from the sea anemone Stichodactyla helianthus. Incubation of diluted St II solutions at different pHs (ranging from 2.0 to 12) slightly changes the secondary structure of the protein. These changes are particularly manifested at high pH. Similarly, the intrinsic fluorescence of the protein indicates a progressive opening of the protein structure when the pH increases from acidic (2.0) to basic (12). These modifications are only partially reversible and do not produce any significant increase in the small capacity of the protein to bind hydrophobic dyes (ANS or Prodan). Experiments carried out with model membranes show a reduced capacity of binding to egg phosphatidyl choline:sphingomyelin (1:1) liposomes both at low (2.3) and high (11.5) pH. Preincubation of the protein in the 2. 5-9.0 pH range does not modify its hemolytic activity, measured in human red blood cells at pH 7.4. On the other hand, preincubation at pH 11.5 drastically reduces the hemolytic activity of the toxin. This strong reduction takes place without measurable modification of the toxin ability to be adsorbed to the red blood cell surface. This indicates that preincubation at high pH irreversibly reduces the capacity of the toxin to form pores without a significant decrease in its binding capacity. The present results suggest that at pH > or = 10 St II experiences irreversible conformational changes that notably reduce its biological activity. This reduced biological activity is associated with a partial defolding of the protein, which seems to contradict what is expected in terms of a molten globule formalism.

Kinetics and mechanism of St I modification by peroxyl radicals.

St I is a toxin present in the Caribbean Sea anemone Stichodactyla helianthus which is highly hemolytic in the nanomolar concentration range. Exposure of the toxin to free radicals produced in the pyrolysis of 2,2′-azobis(2-amidinopropane) hydrochloride leads to a progressive loss of hemolytic activity. This loss of hemolytic activity is accompanied by extensive modification of tryptophan residues. On the average, three tryptophan residues are modified by each inactivated toxin. The loss of hemolytic activity of St I takes place without significant changes in the protein structure, as evidenced by the similarity of the fluorescence and CD spectra of native and modified proteins. Also, the native and modified ensembles present a similar resistance to their denaturation by guanidinium chloride. The hemolytic behavior and the performance of the toxin at the single-channel level when incorporated to black lipid membranes suggest that the modified ensemble can be considered as composed of inactive toxins and active toxins whose behavior is similar to that of the native proteins. These results, together with the lack of induction time in the activity loss, suggest that the fall of hemolytic activity takes place by an all-or-nothing inactivation mechanism in which the molecules become inactive when a critical amino acid residue is modified.

The role of ionic strength on the enhancement of the hemolytic activity of sticholysin I, a cytolysin from Stichodactyla helianthus.

Sticholysin I (St I) is a potent cytolytic polypeptide purified from the Caribbean sea anemone Stichodactyla helianthus. The hemolytic activity of sticholysin is potentiated by its preincubation at high ionic strengths. In the present work the mechanism of the potentiating action of the medium ionic strength on the toxin hemolytic capacity is investigated. It is suggested that preincubation with high saline concentration induces a transition of St I to a more relaxed conformation that facilitates the lytic process.

The sticholysin family of pore-forming toxins induces the mixing of lipids in membrane domains.

Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.