Membrane Remodeling by the Lytic Fragment of SticholysinII: Implications for the Toroidal Pore Model.

Sticholysins are pore-forming toxins of biomedical interest and represent a prototype of proteins acting through the formation of protein-lipid or toroidal pores. Peptides spanning the N-terminus of sticholysins can mimic their permeabilizing activity and, together with the full-length toxins, have been used as a tool to understand the mechanism of pore formation in membranes. However, the lytic mechanism of these peptides and the lipid shape modulating their activity are not completely clear. In this article, we combine molecular dynamics simulations and experimental biophysical tools to dissect different aspects of the pore-forming mechanism of StII1-30, a peptide derived from the N-terminus of sticholysin II (StII). With this combined approach, membrane curvature induction and flip-flop movement of the lipids were identified as two important membrane remodeling steps mediated by StII1-30. Pore formation by this peptide was enhanced by the presence of the negatively curved lipid phosphatidylethanolamine in membranes. This lipid emerged not only as a facilitator of membrane interactions but also as a structural element of the StII1-30 pore that is recruited to the ring upon its assembly. Collectively, these, to our knowledge, new findings support a toroidal model for the architecture of the pore formed by StII1-30 and provide new molecular insight into the role of phosphatidylethanolamine as a membrane component that can easily integrate into the ring of toroidal pores, thus probably aiding in their stabilization. This study contributes to a better understanding of the molecular mechanism underlying the permeabilizing activity of StII1-30 and peptides or proteins acting via a toroidal pore mechanism and offers an informative framework for the optimization of the biomedical application of this and similar molecules.

Architecture of the pore forming toxin sticholysin I in membranes.

Sticholysin I (StI) is a toxin produced by the sea anemone Stichodactyla helianthus and belonging to the actinoporins family. Upon binding to sphingomyelin-containing membranes StI forms oligomeric pores, thereby leading to cell death. According to recent controversial experimental evidences, the pore architecture of actinoporins is a debated topic. Here, we investigated the StI topology in membranes by site-directed spin labeling and electron paramagnetic resonance spectroscopy. The results reveal that StI in membrane exhibits an oligomeric architecture with heterogeneous stoichiometry of predominantly eight or nine protomers, according to the available structural models. The StI topology resembles the conic pore structure reported for the actinoporin fragaceatoxin C. Our data show that StI coexists in two membrane-associated conformations, with the N-terminal segment either attached to the protein core or inserted in the membrane forming the pore. This finding suggests a 'pre-pore' to 'pore' transition determined by a conformational change that detaches the N-terminal segment.

Novel Adjuvant Based on the Pore-Forming Protein Sticholysin II Encapsulated into Liposomes Effectively Enhances the Antigen-Specific CTL-Mediated Immune Response.

Vaccine strategies to enhance CD8+ CTL responses remain a current challenge because they should overcome the plasmatic and endosomal membranes for favoring exogenous Ag access to the cytosol of APCs. As a way to avoid this hurdle, sticholysin (St) II, a pore-forming protein from the Caribbean Sea anemone Stichodactyla helianthus, was encapsulated with OVA into liposomes (Lp/OVA/StII) to assess their efficacy to induce a CTL response. OVA-specific CD8+ T cells transferred to mice immunized with Lp/OVA/StII experienced a greater expansion than when the recipients were injected with the vesicles without St, mostly exhibiting a memory phenotype. Consequently, Lp/OVA/StII induced a more potent effector function, as shown by CTLs, in vivo assays. Furthermore, treatment of E.G7-OVA tumor-bearing mice with Lp/OVA/StII significantly reduced tumor growth being more noticeable in the preventive assay. The contribution of CD4+ and CD8+ T cells to CTL and antitumor activity, respectively, was elucidated. Interestingly, the irreversibly inactive variant of the StI mutant StI W111C, encapsulated with OVA into Lp, elicited a similar OVA-specific CTL response to that observed with Lp/OVA/StII or vesicles encapsulating recombinant StI or the reversibly inactive StI W111C dimer. These findings suggest the relative independence between StII pore-forming activity and its immunomodulatory properties. In addition, StII-induced in vitro maturation of dendritic cells might be supporting these properties. These results are the first evidence, to our knowledge, that StII, a pore-forming protein from a marine eukaryotic organism, encapsulated into Lp functions as an adjuvant to induce a robust specific CTL response.

Damage of eukaryotic cells by the pore-forming toxin sticholysin II: Consequences of the potassium efflux.

Pore-forming toxins (PFTs) form holes in membranes causing one of the most catastrophic damages to a target cell. Target organisms have evolved a regulated response against PFTs damage including cell membrane repair. This ability of cells strongly depends on the toxin concentration and the properties of the pores. It has been hypothesized that there is an inverse correlation between the size of the pores and the time required to repair the membrane, which has been for long a non-intuitive concept and far to be completely understood. Moreover, there is a lack of information about how cells react to the injury triggered by eukaryotic PFTs. Here, we investigated some molecular events related with eukaryotic cells response against the membrane damage caused by sticholysin II (StII), a eukaryotic PFT produced by a sea anemone. We evaluated the change in the cytoplasmic potassium, identified the main MAPK pathways activated after pore-formation by StII, and compared its effect with those from two well-studied bacterial PFTs: aerolysin and listeriolysin O (LLO). Strikingly, we found that membrane recovery upon StII damage takes place in a time scale similar to LLO in spite of the fact that they form pores by far different in size. Furthermore, our data support a common role of the potassium ion, as well as MAPKs in the mechanism that cells use to cope with these toxins injury.

Sticholysin I-membrane interaction: an interplay between the presence of sphingomyelin and membrane fluidity.

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer.

Role of B-1 cells in the immune response against an antigen encapsulated into phosphatidylcholine-containing liposomes.

B-1 lymphocytes comprise a unique subset of B cells that differ phenotypically, ontogenetically and functionally from conventional B-2 cells. A frequent specificity of the antibody repertoire of peritoneal B-1 cells is phosphatidylcholine. Liposomes containing phosphatidylcholine have been studied as adjuvants and their interaction with dendritic cells and macrophages has been demonstrated. However, the role of B-1 cells in the adjuvanticity of liposomes composed of phosphatidylcholine has not been explored. In the present work, we studied the contribution of B-1 cells to the humoral response against ovalbumin (OVA) encapsulated into dipalmitoylphosphatidylcholine (DPPC) and cholesterol-containing liposomes. BALB/X-linked immunodeficient (xid) mice, which are deficient in B-1 cells, showed quantitative and qualitative differences in the anti-OVA antibody response compared with wild-type animals after immunization with these liposomes. The OVA-specific immune response was significantly increased in the BALB/xid mice when reconstituted with B-1 cells from naive BALB/c mice. Our results indicate the internalization of DPPC-containing liposomes by these cells and their migration from the peritoneal cavity to the spleen. Phosphatidylcholine significantly contributed to the immunogenicity of liposomes, as DPPC-containing liposomes more effectively stimulated the anti-OVA response compared with vesicles composed of dipalmitoylphosphatidylglycerol. In conclusion, we present evidence for a cognate interaction between B-1 cells and phosphatidylcholine liposomes, modulating the immune response to encapsulated antigens. This provides a novel targeting approach to assess the role of B-1 cells in humoral immunity.

The Presence of Sterols Favors Sticholysin I-Membrane Association and Pore Formation Regardless of Their Ability to Form Laterally Segregated Domains.

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT. As for actinoporins, it has been proposed that the presence of cholesterol (Chol) and the coexistence of lipid phases increase binding to the target membrane and pore-forming ability. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, and the presence of lipid domains) on the activity of actinoporins or which regions of the membrane are the most favorable for protein insertion, oligomerization, and eventually pore formation. To gain insight into the role of membrane properties on the functional activity of St I, we studied its binding to monolayers and vesicles of phosphatidylcholine (PC), sphingomyelin (SM), and sterols inducing (ergosterol -Erg and cholesterol -Chol) or not (cholestenone - Cln) membrane phase segregation in liquid ordered (Lo) and liquid disordered (Ld) domains. This study revealed that St I binds and permeabilizes with higher efficiency sterol-containing membranes independently of their ability to form domains. We discuss the results in terms of the relevance of different membrane properties for the actinoporins mechanism of action, namely, molecular heterogeneity, specially potentiated in membranes with sterols inducers of phase separation (Chol or Erg) or Cln, a sterol noninducer of phase separation but with a high propensity to induce nonlamellar phase. The role of the Ld phase is pointed out as the most suitable platform for pore formation. In this regard, such regions in Chol-containing membranes seem to be the most favored due to its increased fluidity; this property promotes toxin insertion, diffusion, and oligomerization leading to pore formation.

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.

Improved purification and enzymatic properties of a mixture of Sticholysin I and II: isotoxins with hemolytic and phospholipase A(2) activities from the sea anemone Stichodactyla helianthus.

Sticholysin I and Sticholysin II (StI and StII) are two potent hemolysins which form pores in natural and model membranes at nanomolar concentrations. These proteins were purified from the aqueous extract of the sea anemone Stichodactyla helianthus, Ellis 1768, by gel filtration and ionic exchange chromatography. This procedure rendered StI and StII with high purity (purification factors: 36 and 50, respectively) but a low yield of hemolytic activity, HA (<3%). Additionally, these toxins exhibited very low phospholipase activity (10(-3)U/mg of protein). In this work, a mixture StI-StII was obtained (yield >95%, with an increase in specific activity: 14 times) from the animal extract using an oxidized phospholipid-based affinity chromatographic matrix binding phospholipases. Cytolysin identification in the mixture was performed by immunoblotting and N-terminal sequence analyses. Phospholipase A2 (PLA2) activity of StI-StII was relatively high (1.85U/mg) and dependent of Ca(2+). The activity resulted optimum when was measured with the mostly unsaturated soybean phosphatidylcholine (PC), when compared to the less unsaturated egg PC or completely saturated dipalmitoyl PC, in the presence of 40mM Ca(2+) at pH 8.0. This Ca(2+) concentration did not exert any effect on binding of StI-StII with soybean PC monolayers. Then, PLA2 activity seems not be required to binding to membranes.

Unveiling Sticholysin II and plasmid DNA interaction: Implications for developing non-viral vectors.

Non-viral gene delivery systems offer significant potential for gene therapy due to their versatility, safety, and cost advantages over viral vectors. However, their effectiveness can be hindered by the challenge of efficiently releasing the genetic cargo from endosomes to prevent degradation in lysosomes. To overcome this obstacle, functional components can be incorporated into these systems. Sticholysin II (StII) is one of the pore-forming proteins derived from the sea anemone Stichodactyla helianthus, known for its high ability to permeabilize cellular and model membranes. In this study, we aimed to investigate the interaction between StII, and a model plasmid (pDNA) as an initial step towards designing an improved vector with enhanced endosomal escape capability. The electrophoretic mobility shift assay (EMSA) confirmed the formation of complexes between StII and pDNA. Computational predictions identified specific residues involved in the StII-DNA interaction interface, highlighting the importance of electrostatic interactions and hydrogen bonds in mediating the binding. Atomic force microscopy (AFM) of StII-pDNA complexes revealed the presence of nodular fiber and toroid shapes. These complexes were found to have a predominantly micrometer size, as confirmed by dynamic light scattering (DLS) measurements. Despite increase in the overall charge, the complexes formed at the evaluated nitrogen-to-phosphorus (N/P) ratios still maintained a negative charge. Moreover, StII retained its pore-forming capacity regardless of its binding to the complexes. These findings suggest that the potential ability of StII to permeabilize endosomal membranes could be largely maintained when combined with nucleic acid delivery systems. Additionally, the still remaining negative charge of the complexes would enable the association of another positively charged component to compact pDNA. However, to minimize non-specific cytotoxic effects, it is advisable to explore methods to regulate the protein's activity in response to the microenvironment.

Evaluation of specific humoral immune response and cross reactivity against Mycobacterium tuberculosis antigens induced in mice immunized with liposomes composed of total lipids extracted from Mycobacterium smegmatis.

The development of a new tuberculosis (TB) vaccine has become one of the main objectives of the scientific community. Protein antigens have been widely explored as subunit TB vaccines, however lipid antigens could be equally important to be used or included in such a vaccine. The aim of this study was to demonstrate the potential of a liposome formulation composed of an extract of lipids from Mycobacterium smegmatis (Ms) as a TB vaccine candidate. We evaluated the immunogenicity of this formulation as well as the cross reactive response against antigens from Mycobacterium tuberculosis (MTb) in BALB/c mice. We determined the anti-liposome IgG response in sera from TB patients and from healthy subjects who displayed a positive (PPD+) or negative (PPD-) tuberculin skin test. A significant increase in anti-liposome IgG (p<0.05) was detected in animals immunized with Bacille Calmette-Guérin (BCG) compared with all groups, and in the group immunized with liposomes from Ms (LMs) compared to animals immunized with either LMs adjuvanted with aluminium (LMs-A) or the negative control group (phosphate buffered saline, PBS) respectively. With respect to the cross reactive response against a cocktail of cell wall antigens (CWA) from MTb, significantly higher IgG levels were observed in animals immunized with BCG and LMs compared to negative controls and either, aluminium-adjuvanted liposomes (LMs-A) or montanide (LMs-M) (p<0.05). Furthermore, the anti-liposome IgG response was significantly superior in sera from pulmonary TB patients compared to PPD+ and PPD- healthy subjects (p<0.001) suggesting the expression of these antigens in vivo during active MTb infection. The results obtained provide some evidence for the potential use of liposomes containing total lipid extracts of Ms as a TB vaccine candidate.

Evaluation of the humoral immune response and cross reactivity against Mycobacterium tuberculosis of mice immunized with liposomes containing glycolipids of Mycobacterium smegmatis.

Mycobacterium smegmatis (Ms) is a nonpathogenic mycobacteria of rapid growth, which shares many characteristics with Mycobacterium tuberculosis (MTB), the major causative agent of tuberculosis. MTB has several cell wall glycolipids in common with Ms, which play an important role in the pathogenesis of tuberculosis and the induction of a protective immune response against MTB infection in some animal models. In this study, the humoral immune response and cross reactivity against MTB, of liposomes containing a mixture of cell wall glycolipids of Ms and commercial lipids was evaluated, in order to study its possible use as a component of a vaccine candidate against tuberculosis. Liposomes containing total lipids extracted from Ms, distearoyl phosphatidyl choline and cholesterol were prepared by the dehydration-rehydration technique. Balb/c mice were immunized with the liposomes obtained and the antibody response and cross reactivity against MTB were tested by ELISA. Total lipids extract from Ms showed the presence of several polar glycolipids in common with MTB, such as phosphatidylinositol mannosides. Liposomes that contained glycolipids of Ms were capable of inducing a specific IgG antibody response that allowed the recognition of surface antigens of MTB. The results of this study demonstrated the presence of immunogenic glycolipids in Ms, which could be included to enhance the protective effects of subunit vaccine formulations against tuberculosis.

Functional and topological studies with Trp-containing analogs of the peptide StII(1-30) derived from the N-terminus of the pore forming toxin sticholysin II: contribution to understand its orientation in membrane.

Sticholysin II (St II) is the most potent cytolysin produced by the sea anemone Stichodactyla helianthus, exerting hemolytic activity via pore formation in membranes. The toxin's N-terminus contains an amphipathic α-helix that is very likely involved in pore formation. We have previously demonstrated that the synthetic peptide StII(1-30) encompassing the 1-30 segment of St II forms pores of similar radius to that of the protein (around 1 nm), being a good model of toxin functionality. Here we have studied the functional and conformational properties of fluorescent analogs of StII(1-30) in lipid membranes. The analogs were obtained by replacing Leu residues at positions 2, 12, 17, and 24 with the intrinsically fluorescent amino acid Trp (StII(1-30L2W), StII(1-30L12W), StII(1-30L17W), or StII(1-30L24W), respectively). The exchange by Trp did not significantly modify the activity and conformation of the parent peptide. The blue-shift and intensity enhancement of fluorescence in the presence of membrane indicated that Trp at position 2 is more deeply buried in the hydrophobic region of the bilayer. These experiments, as well as assays with water-soluble or spin-labeled lipid-soluble fluorescence quenchers suggest an orientation of StII(1-30) with its N-terminus oriented towards the hydrophobic core of the bilayer while the rest of the peptide is more exposed to the aqueous environment, as hypothesized for sticholysins.

The Important Role of Membrane Fluidity on the Lytic Mechanism of the α-Pore-Forming Toxin Sticholysin I.

Actinoporins have emerged as archetypal α-pore-forming toxins (PFTs) that promote the formation of pores in membranes upon oligomerization and insertion of an α-helix pore-forming domain in the bilayer. These proteins have been used as active components of immunotoxins, therefore, understanding their lytic mechanism is crucial for developing this and other applications. However, the mechanism of how the biophysical properties of the membrane modulate the properties of pores generated by actinoporins remains unclear. Here we studied the effect of membrane fluidity on the permeabilizing activity of sticholysin I (St I), a toxin that belongs to the actinoporins family of α-PFTs. To modulate membrane fluidity we used vesicles made of an equimolar mixture of phosphatidylcholine (PC) and egg sphingomyelin (eggSM), in which PC contained fatty acids of different acyl chain lengths and degrees of unsaturation. Our detailed single-vesicle analysis revealed that when membrane fluidity is high, most of the vesicles are partially permeabilized in a graded manner. In contrast, more rigid membranes can be either completely permeabilized or not, indicating an all-or-none mechanism. Altogether, our results reveal that St I pores can be heterogeneous in size and stability, and that these properties depend on the fluid state of the lipid bilayer. We propose that membrane fluidity at different regions of cellular membranes is a key factor to modulate the activity of the actinoporins, which has implications for the design of different therapeutic strategies based on their lytic action.

Cys mutants as tools to study the oligomerization of the pore-forming toxin sticholysin I.

Sticholysin I (StI) is a water-soluble protein with the ability to bind membranes where it oligomerizes and forms pores leading to cell death. Understanding the assembly property of this protein may be valuable for designing potential biotechnological tools, such as stable or structurally defined nanopores. In order to get insights into the stabilization of StI oligomers by disulfide bonds, we designed and characterized single and double cysteine mutants at the oligomerization interface. The oligomer formation was induced in the presence of lipid membranes and visualized by SDS-PAGE. The contribution of the oligomeric structures to the membrane binding and pore-forming capacities of StI was assessed. Single and double cysteine introduction at the protein-protein oligomerization interface does not considerably affect the conformation and function of the monomeric protein. In the presence of membranes, a cysteine double mutation at positions 15 and 59 favored formation of different size oligomers stabilized by disulfide bonds. The results of this work highlight the relevance of these positions (15 and 59) to be considered for developing biosensors based on nanopores from StI.

Sticholysins, pore-forming proteins from a marine anemone can induce maturation of dendritic cells through a TLR4 dependent-pathway.

Sticholysins (Sts) I and II (StI and StII) are pore-forming proteins (PFPs), purified from the Caribbean Sea anemone Stichodactyla helianthus. StII encapsulated into liposomes induces a robust antigen-specific cytotoxic CD8+ T lymphocytes (CTL) response and in its free form the maturation of bone marrow-derived dendritic cells (BM-DCs). It is probable that the latter is partially supporting in part the immunomodulatory effect on the CTL response induced by StII-containing liposomes. In the present work, we demonstrate that the StII's ability of inducing maturation of BM-DCs is also shared by StI, an isoform of StII. Using heat-denatured Sts we observed a significant reduction in the up-regulation of maturation markers indicating that both PFP's ability to promote maturation of BM-DCs is dependent on their conformational characteristics. StII-mediated DC maturation was abrogated in BM-DCs from toll-like receptor (TLR) 4 and myeloid differentiation primary response gene 88 (MyD88)-knockout mice but not in cells from TLR2-knockout mice. Furthermore, the antigen-specific CTL response induced by StII-containing liposomes was reduced in TLR4-knockout mice. These results indicate that StII, and probably by extension StI, has the ability to induce maturation of DCs through a TLR4/MyD88-dependent pathway, and that this activation contributes to the CTL response generated by StII-containing liposomes.

Self-association and folding in membrane determine the mode of action of peptides from the lytic segment of sticholysins.

Sticholysin I and II (Sts: St I and St II) are proteins of biomedical interest that form pores upon the insertion of their N-terminus in the plasma membrane. Peptides spanning the N-terminal residues of StI (StI1-31) or StII (StII1-30) can mimic the permeabilizing ability of these toxins, emerging as candidates to rationalize their potential biomedical applications. These peptides have different activities that correlate with their hydrophobicity. However, it is not clear how this property contributes to peptide folding in solution or upon binding to membranes. Here we compared the conformational properties of these peptides and shorter versions lacking the hydrophobic segment 1-11 of StI (StI12-31) or 1-10 of StII (StII11-30). Folding of peptides was assessed in solution and in membrane mimetic systems and related with their ability to bind to membranes and to permeabilize lipid vesicles. Our results suggest that the differences in activity among peptides could be ascribed to their different folding propensity and different membrane binding properties. In solution, StII1-30 tends to acquire α-helical conformation coexisting with self-associated structures, while StI1-31 remains structureless. Both peptides fold as α-helix in membrane; but StII1-30 also self-associates in the lipid environment, a process that is favored by its higher affinity for membrane. We stress the contribution of the non-polar/polar balance of the 1-10 amino acid sequence of the peptides as a determining factor for different self-association capabilities. Such difference in hydrophobicity seems to determine the molecular path of peptides folding upon binding to membranes, with an impact in their permeabilizing activity. This study contributes to a better understanding of the molecular mechanisms underlying the permeabilizing activity of Sts N-terminal derived peptides, with connotation for the exploitation of these small molecules as alternative of the full-length toxins in clinical settings.

Cloning, purification and characterization of nigrelysin, a novel actinoporin from the sea anemone Anthopleura nigrescens.

Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that being secreted as soluble monomers are able to bind and permeabilize membranes leading to cell death. The interest in these proteins has risen due to their high cytotoxicity that can be properly used to design immunotoxins against tumor cells and antigen-releasing systems to cell cytosol. In this work we describe a novel actinoporin produced by Anthopleura nigrescens, an anemone found in the Central American Pacific Ocean. Here we report the amino acid sequence of an actinoporin as deduced from cDNA obtained from total body RNA. The synthetic DNA sequence encoding for one cytolysin variant was expressed in BL21 Star (DE3) Escherichia coli and the protein purified by chromatography on CM Sephadex C-25 with more than 97% homogeneity as verified by MS-MS and HPLC analyses. This actinoporin comprises 179 amino acid residues, consistent with its observed isotope-averaged molecular mass of 19 661 Da. The toxin lacks Cys and readily permeabilizes erythrocytes, as well as L1210 cells. CD spectroscopy revealed that its secondary structure is dominated by beta structure (58.5%) with 5.5% of α-helix, and 35% of random structure. Moreover, binding experiments to lipidic monolayers and to liposomes, as well as permeabilization studies in vesicles, revealed that the affinity of this toxin for sphingomyelin-containing membranes is quite similar to sticholysin II (StII). Comparison by spectroscopic techniques and modeling the three-dimensional structure of nigrelysin (Ng) showed a high homology with StII but several differences were also detectable. Taken together, these results reinforce the notion that Ng is a novel member of the actinoporin pore-forming toxin (PFT) family with a HA as high as that of StII, the most potent actinoporin so far described, but with peculiar structural characteristics contributing to expand the understanding of the structure-function relationship in this protein family.

The Vacuolar Pathway in Macrophages Plays a Major Role in Antigen Cross-Presentation Induced by the Pore-Forming Protein Sticholysin II Encapsulated Into Liposomes.

Cross-presentation is an important mechanism for the differentiation of effector cytotoxic T lymphocytes (CTL) from naïve CD8+ T-cells, a key response for the clearance of intracellular pathogens and tumors. The liposomal co-encapsulation of the pore-forming protein sticholysin II (StII) with ovalbumin (OVA) (Lp/OVA/StII) induces a powerful OVA-specific CTL activation and an anti-tumor response in vivo. However, the pathway through which the StII contained in this preparation is able to induce antigen cross-presentation and the type of professional antigen presenting cells (APCs) involved have not been elucidated. Here, the ability of mouse bone marrow-derived dendritic cells (BM-DCs) and macrophages (BM-MΦs) stimulated with Lp/OVA/StII to activate SIINFEKL-specific B3Z CD8+ T cells was evaluated in the presence of selected inhibitors. BM-MΦs, but not BM-DCs were able to induce SIINFEKL-specific B3Z CD8+ T cell activation upon stimulation with Lp/OVA/StII. The cross-presentation of OVA was markedly decreased by the lysosome protease inhibitors, leupeptin and cathepsin general inhibitor, while it was unaffected by the proteasome inhibitor epoxomicin. This process was also significantly reduced by phagocytosis and Golgi apparatus function inhibitors, cytochalasin D and brefeldin A, respectively. These results are consistent with the concept that BM-MΦs internalize these liposomes through a phagocytic mechanism resulting in the cross-presentation of the encapsulated OVA by the vacuolar pathway. The contribution of macrophages to the CTL response induced by Lp/OVA/StII in vivo was determined by depleting macrophages with clodronate-containing liposomes. CTL induction was almost completely abrogated in mice depleted of macrophages, demonstrating the relevance of these APCs in the antigen cross-presentation induced by this formulation.

Correlations between differences in amino-terminal sequences and different hemolytic activity of sticholysins.

Sticholysins I and II (St I and St II) are cytolysins produced by the sea anemone Stichodactyla helianthus. In spite of their 93% sequence homology, St II is more hemolytic against human erythrocytes than St I. In order to establish the possible causes of this difference, we studied the hemolytic activity of synthetic peptides containing sequences from the N-termini of both proteins. The results demonstrated that the differences in hemolytic activity of the toxins could be ascribed at least partly to differences in their N-termini.