Sticholysin II shows similar immunostimulatory properties to LLO stimulating dendritic cells and MHC-I restricted T cell responses of heterologous antigen.

Induction of CD8+ T cell responses against tumor cells and intracellular pathogens is an important goal of modern vaccinology. One approach of translational interest is the use of liposomes encapsulating pore-forming proteins (PFPs), such as Listeriolysin O (LLO), which has shown efficacy at priming strong and sustained CD8+ T cell responses. Recently, we have demonstrated that Sticholysin II (StII), a PFP from the sea anemone Stichodactyla helianthus, co-encapsulated into liposomes with ovalbumin (OVA) was able to stimulate, antigen presenting cells, antigen-specific CD8+ T cells and anti-tumor activity in mice. In the present study, we aimed to compare StII and LLO in terms of their abilities to stimulate dendritic cells and to induce major histocompatibility complex (MHC) class I restricted T cell responses against OVA. Interestingly, StII exhibited similar abilities to LLO in vitro of inducing dendritic cells maturation, as measured by increased expression of CD40, CD80, CD86 and MHC-class II molecules, and of stimulating OVA cross-presentation to a CD8+ T cell line. Remarkably, using an ex vivo Enzyme-Linked ImmunoSpot Assay (ELISPOT) to monitor gamma interferon (INF-γ) producing effector memory CD8+ T cells, liposomal formulations containing either StII or LLO induced comparable frequencies of OVA-specific INF-γ producing CD8+ T cells in mice that were sustained in time. However, StII-containing liposomes stimulated antigen-specific memory CD8+ T cells with a higher potential to secrete IFN-γ than liposomes encapsulating LLO. This StII immunostimulatory property further supports its use for the rational design of T cell vaccines against cancers and intracellular pathogens. In summary, this study indicates that StII has immunostimulatory properties similar to LLO, despite being evolutionarily distant PFPs.

The pore forming capacity of Sticholysin I in dipalmitoyl phosphatidyl vesicles is tuned by osmotic stress.

The osmotic condition modulates the properties of liposomes, particularly those related to their stability and response to external agents such as membrane-active proteins or peptides. In a previous work, we have demonstrated that an osmotic shock can increase, per se, water influx/efflux and the exit of the fluorophore calcein entrapped in the aqueous pool of dipalmitoylphosphatidylcholine (DPPC) and DPPC:sphingomyelin (SM) large unilamellar vesicles (LUVs), suggesting a loss of integrity of the liposome bilayer. In the present work, we have extended our study in order to assess how an osmotic imbalance prior to or synchronous with the addition of a recombinant variant of the pore-forming toxin sticholysin I (rSt I) modifies its pore forming capacity in DPPC and DPPC:SM (1:1) LUVs. Our results conclusively show the capacity of hypotonic gradients to improve the pore forming capacity of rSt I molecules, even in pure DPPC liposomes, rendering pore-formation less dependent on the presence of sphyngomyelin. In fact, non-active toxins in DPPC liposomes become active by a hypotonic imbalance in a similar way to those containing SM as a second component.

Response of unilamellar DPPC and DPPC:SM vesicles to hypo and hyper osmotic shocks: A comparison.

DPPC and DPPC:SM large unilamellar vesicles (LUVs), prepared by extrusion, readily respond to osmotic shocks (hypo- and hyper-osmotic) by water influx/efflux (evaluated by changes in turbidity) and by entrapped calcein liberation (measured by an increase in dye fluorescence intensity). On the other hand, small unilamellar vesicles (SUVs) prepared by sonication are almost osmotically insensitive. LUVs water transport, both in hypo- and hyper-osmotic conditions, takes place faster than calcein ejection towards the external solvent. Similarly, response to a hypotonic imbalance is faster than that associated to a hypertonic stress. This difference is particularly noticeable for the increase in calcein fluorescence intensity and can be related to the large reorganization of the bilayer needed to form pores and/or to adsorb the dye to the inner leaflet of the vesicle after water efflux. Conversely, addition of SM to the vesicles barely modify the rate of calcein permeation across the bilayer.

Cys mutants in functional regions of Sticholysin I clarify the participation of these residues in pore formation.

Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg5² in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu², Phe¹5) did not modify the toxin's permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation.

Humoral immune response against epidermal growth factor encapsulated in dehydration rehydration vesicles of different phospholipid composition.

We investigated the influence of dehydration-rehydration vesicles (DRV) phospholipid composition and the addition of other components on human recombinant epidermal growth factor (hrEGF) encapsulation efficiency and its release from liposomes. Encapsulation of EGF into DRV composed of phosphatidylcholine with different unsaturation levels was around 20-35%. The best result was obtained with dipalmitoyl phosphatidylcholine: cholesterol (DPPC:Ch) liposomes (35%) corresponding to the lowest hrEGF release during one month of storage. Even with this phospholipid composition, modification of the DRV procedure by including an extrusion step did not improve hrEGF encapsulation efficiency, rendering less stable particles. The inclusion of recombinant P64k from Neisseria meningitidis (rP64k), as such or conjugated to hrEGF, decreased the encapsulation efficiency of the latter protein into DRV or freeze and thaw multilamellar vesicles (FATMLV). The hrEGF release from liposomes could be related to the interaction between this polypeptide and the bilayer, as evidenced by increased carboxyfluorescein release from hrEGF-DRV; less susceptibility to fluorescence quenching by acrylamide in the presence of liposomes; and a measurable decrease of phospholipid phase transition Delta enthalpy (DeltaH). DRV comprising saturated phospholipids (DPPC:Ch or distearoyl phosphatidylcholine [DSPC]:Ch) and containing the conjugate EGF-P64k induced a more efficient immune response against hrEGF than unsaturated phospholipid and alum in terms of total IgG, IgG(2a), and IgG(2b) subclasses and the ability of antibody to inhibit the interaction of the EGF receptor with hrEGF.

Structural and functional characterization of a recombinant sticholysin I (rSt I) from the sea anemone Stichodactyla helianthus.

Sticholysins I and II (Sts I and II) are two potent cytolysins from the sea anemone Stichodactyla helianthus. These isoforms present 13 substitutions, with three non-conservative located at the N-terminus. St II is considerably more hemolytic than St I in human red blood cells, a result explained by the smaller number of negatively charged groups present at St II's N-terminus. In the present work, we have obtained a recombinant St I (rSt I), differing from the wild type in a single amino acid residue (E16Q). This pseudo-wild type is structurally similar to St I and shows a similar capacity to interact with and form pores in model membranes. This was assessed by the intrinsic fluorescence increase in the presence of liposomes, their adsorption to bilayers (measured by SPR), their concentration at the air-water interface, their interaction with lipid monolayers and their capacity to promote the release of carboxyfluorescein entrapped in liposomes. In spite of these similarities, rSt I presents a larger hemolytic activity in human red blood cells than St I, being intermediate in activity between Sts I and II. The results obtained in the present work emphasize that even the change of one single E by Q at the N-terminal segment may modify the toxin HA and show that this functional property is the most sensitive to subtle changes in the protein primary structure.

Role of endogenous channels in red blood cells response to their exposure to the pore forming toxin Sticholysin II.

Sticholysin II (St II) is a highly hemolytic cytolysin isolated from the sea anemone Stichodactyla heliantus. The toxin hemolytic action takes place through the formation of channels that provoke an electrolyte unbalance leading to osmotic shock. The lytic event must involve the exchange of electrolytes and the entrance of water, leading to red blood cell disruption. These processes can occur through St II pores and/or the endogenous red blood cells transporters. In order to evaluate the contribution of these channels to water, anion and cation transport, we have measured the hemolysis and K+ efflux rates in the presence of several specific inhibitors. The results obtained in the presence of Hg, an AQP1 blocker, indicate that water transport through these channels is not essential for the occurrence of the lytic process induced by St II. The data also support a partial role of K+ and anion transporters. In particular, they are compatible with a preferential K+ efflux though the K(+)/Cl- co-transport as a response to the promoted swelling. Furthermore, they suggest that chloride influx, a process that can regulate both K+ efflux and lysis, is partially mediated by the endogenous cell transporters, in particular, band-3 anion exchange system being relevant at early stages of the lytic process.

Construction of an immunotoxin with the pore forming protein StI and ior C5, a monoclonal antibody against a colon cancer cell line.

Sticholysin I (StI), a potent cytolysin isolated from the sea anemone Stichodactyla helianthus, was linked to the monoclonal antibody (mAb) ior C5. StI acts by forming hydrophilic pores in the membrane of the attacked cells leading to osmotic lysis. ior C5 is a murine IgG1, which recognizes the tumor associated antigen (TAA) ior C2. The cytolysin and the mAb were coupled by using the heterobifunctional cross-linking reagent sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). Two hybrid molecules composed by one ior C5 and one or two StI molecules were obtained (named conjugated I and II, respectively). The purified conjugates were evaluated by a binding affinity assay against an ior C2-positive colon cancer cell line (SW948). Both molecules were able to recognize the antigen (Ag) in the same way that unconjugated ior C5 does. The activity of both conjugates against human erythrocytes and SW948 cells was assessed. They lost most of their hemolytic activity but their residual activity was very similar. Nevertheless, when their cytotoxicity was studied on the SW948 cell line, only conjugate II killed efficiently the cells, indicating a specific mAb-Ag interaction. In this chimeric molecule the ratio between the cytotoxic and the hemolytic activity was larger than that of the free cytolysin. This fact indicates an increase of the specificity of the toxic effect toward the SW948 cell line and consequently an increase of the difference between its hemolytic and cytotoxic doses. The results herein support the feasibility of directing StI to the surface of cancer cells expressing ior C2 Ag via the mAb ior C5.

Comparison of pore-forming ability in membranes of a native and a recombinant variant of Sticholysin II from Stichodactyla helianthus.

Sticholysin II (St II) a potent cytolysin from the sea anemone Stichodactyla helianthus was obtained by recombinant procedures exhibiting six histidine residues in its N-terminus (St IIn6H). The functional comparison between St II and St IIn6H showed a lesser pore-forming ability for the recombinant than for the native in human or rat red blood cells (RBC) and in large unilamellar vesicles (LUV) of different phospholipid composition. However, binding of St IIn6H to small unilamellar vesicles (SUV) was higher with regard to St II. The explanation to the different permeabilizing capacity of both protein variants is not clear, but a different anchoring of St IIn6H to the lipid bilayer could delay the organization of the competent pore into membrane.

Effects of sodium dodecyl sulfate on the conformation and hemolytic activity of St I and St II, two isotoxins purified from Stichodactyla helianthus.

The effect of sodium dodecyl sulfate (SDS) upon the conformation and hemolytic activity of St I and St II strongly depends on its concentration. At relatively low surfactant concentrations (ca. 0.5-5mM range) the surfactant leads to the formation of aggregates, as suggested by the turbidity observed even at relatively low (micromolar range) protein concentrations. In this surfactant range, the proteins show an increase in intrinsic fluorescence intensity and reduced quenching by acrylamide, with an almost total loss of its hemolytic activity. At higher surfactant concentrations the protein adducts disaggregates. This produces a decrease in fluorescence intensity, increase in quenching efficiency by acrylamide, loss of the native tertiary conformation (as reported by the near UV-CD spectra), and increase in alpha-helix content (as evidenced by the far UV-CD spectra). However, and in spite of these substantial changes, the toxins partially recover their hemolytic activity. The reasons for this recovering of the activity at high surfactant concentrations is discussed.

Effect of a zwitterionic surfactant (HPS) on the conformation and hemolytic activity of St I and St II, two isotoxins purified from Stichodactyla helianthus.

N-hexadecyl-N-N'-dimethyl-3-ammonio-1-propane-sulfonate (BPS) is a zwitterionic surfactant that readily binds to sticholysins I and II, two sea toxins isolated from Stichodactyla helianthus. The binding constants, evaluated from changes in fluorescence intensities elicited by the surfactant, are approximately 0.5-0.7 microM(-1). The binding of the surfactant changes the conformation of the tertiary protein, without significant changes in its secondary structure, as reported from far-ultraviolet circular dichroism spectra. The changes elicited by HPS lead to loss of the native conformation (as reported from near-ultraviolet circular dichroism spectra) and to a shift of the intrinsic protein fluorescence toward longer wavelengths, an increase in fluorescence intensities and lifetimes, and a faster quenching by acrylamide. All these changes are indicative of a more expanded tertiary conformation. Despite this, the toxins fully retain their hemolytic activities, indicating that spectroscopic changes can be poor predictors of toxin activity.

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.

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.

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.

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.

Phase and surface properties of lipid bilayers containing neoglycolipids.

The physical properties conferred to DPPC bilayers by including neoglycolipids composed by two different trisaccharides: mannose-mannose-mannose (3M) and glucose-mannose-glucose (GMG) attached to a cholesterol (cho) and a distearylglycerol (diC18) lipid moiety by a spacer were evaluated by means of the measurement of the electrokinetic potential and interfacial fluorescent probes. The phase properties measured with diphenylhexatriene (DPH) were correlated with the surface properties measured with merocyanine 540, dansyl, and Laurdan probes. The results show that the surface properties of large unilamellar vesicles depend on the sugar exposure to the water phase and also on the hydrocarbon moiety by which it is anchored to the bilayer. The combination of the cholesterol moiety with the saccharide attenuates the cooperativity decrease induced by the cholesterol moiety without the sugar portion. The neoglycolipid GMG-diC18 promotes opposite effects affecting slightly the cooperativity at the hydrocarbon core of DPPC and displacing the phase transition temperature to higher values. The presence of neoglycolipid with diC18 introduces defects in the packing at the interface of the membrane in the gel state. It is concluded that a relatively low proportion of neoglycolipids affects significantly the interfacial properties of DPPC bilayers in large unilamellar vesicles in the absence of changes at the membrane bulk at 25 degrees C.

Modification of sticholysin II hemolytic activity by free radicals.

Sticholysin II is a highly hemolytic toxin present in the caribbean sea anemone Stichodactyla helianthus. Pre-incubation of St II with 2,2'-azobis(2-amidinopropane), a source of peroxyl radicals in air saturated solution, readily reduces its hemolytic activity. Analysis of the amino acids present in the protein after its modification shows that only tryptophan groups are significantly modified by the free radicals. According to this, the loss of hemolytic activity correlates with the loss of the protein intrinsic fluorescence. The results indicate that, at high toxin concentrations, nearly a tryptophan residue and 0.2 toxin molecules are inactivated by each radical introduced into the system. Association of St II to multilamellar liposomes (egg yolk phosphatidyl choline:sphingomyelin 1:1) increases the toxin intrinsic fluorescence, indicating a more hydrophobic average environment of the five tryptophan groups of the protein. In agreement with this, incorporation of St II to the liposomes reduces the rate of fluorescence loss during its modification by free radicals, particularly at long incubation times. These results are explained in terms of two populations of tryptophans that are quenched at different rates by acrylamide and whose rates of inactivation by free radicals are also different.

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.

Synthesis of a new neoglycolipid (AgH-1) and its effect upon the properties of dipalmitoylphosphatidylcholine: cholesterol liposomes.

A new neoglycolipid (AgH-1) bearing carbohydrate units that mimics the antigenic determinant of the O-blood group was synthesized and the effect of its incorporation in dipalmitoylphosphatidylcholine (DPPC): cholesterol liposomes was evaluated. The results obtained show that AgH-1 is readily incorporated into DPPC:cholesterol liposomes. The conditions leading to the optimal incorporation are the result of a compromise between incorporation efficiency and incorporation extent. The presence of AgH-1 produces liposomes of smaller size, with only small changes in the properties of the bilayer. However, the data obtained employing diphenylhexatriene and laurodan as fluorescence probes and merocyanine 540 as optical probe suggest that AgH-1 incorporation leads to a small rigidization of the liposomes at temperatures lower than ca. 42 degrees C.

Generation of a murine monoclonal antibody specific for N-glycolylneuraminic acid-containing gangliosides that also recognizes sulfated glycolipids.

The P3 murine monoclonal antibody (MAb) was generated by immunizing BALB/c mice with NeuGcGM3 included into liposomes. The specificity of this MAb was defined by an enzyme-linked immunosorbent assay and immunostaining on thin-layer chromatograms. P3 MAb binds to NeuGc-containing gangliosides and was shown also to react with sulfated glycolipids. A preliminary immunohistochemical study showed that the P3 MAb was able to recognize antigens expressed in human breast tumors.