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.

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.

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.

Self-homodimerization of an actinoporin by disulfide bridging reveals implications for their structure and pore formation.

The Trp111 to Cys mutant of sticholysin I, an actinoporin from Stichodactyla helianthus sea anemone, forms a homodimer via a disulfide bridge. The purified dimer is 193 times less hemolytic than the monomer. Ultracentrifugation, dynamic light scattering and size-exclusion chromatography demonstrate that monomers and dimers are the only independent oligomeric states encountered. Indeed, circular dichroism and fluorescence spectroscopies showed that Trp/Tyr residues participate in homodimerization and that the dimer is less thermostable than the monomer. A homodimer three-dimensional model was constructed and indicates that Trp147/Tyr137 are at the homodimer interface. Spectroscopy results validated the 3D-model and assigned 85° to the disulfide bridge dihedral angle responsible for dimerization. The homodimer model suggests that alterations in the membrane/carbohydrate-binding sites in one of the monomers, as result of dimerization, could explain the decrease in the homodimer ability to form pores.

Biophysical and biochemical strategies to understand membrane binding and pore formation by sticholysins, pore-forming proteins from a sea anemone.

Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that are able to bind and oligomerize in membranes, leading to cell swelling, impairment of ionic gradients and, eventually, to cell death. In this review we summarize the knowledge generated from the combination of biochemical and biophysical approaches to the study of sticholysins I and II (Sts, StI/II), two actinoporins largely characterized by the Center of Protein Studies at the University of Havana during the last 20 years. These approaches include strategies for understanding the toxin structure-function relationship, the protein-membrane association process leading to pore formation and the interaction of toxin with cells. The rational combination of experimental and theoretical tools have allowed unraveling, at least partially, of the complex mechanisms involved in toxin-membrane interaction and of the molecular pathways triggered upon this interaction. The study of actinoporins is important not only to gain an understanding of their biological roles in anemone venom but also to investigate basic molecular mechanisms of protein insertion into membranes, protein-lipid interactions and the modulation of protein conformation by lipid binding. A deeper knowledge of the basic molecular mechanisms involved in Sts-cell interaction, as described in this review, will support the current investigations conducted by our group which focus on the design of immunotoxins against tumor cells and antigen-releasing systems to cell cytosol as Sts-based vaccine platforms.

Gangliosides, Ab1 and Ab2 antibodies II. Light versus heavy chain: An idiotype-anti-idiotype case study.

The antibody heavy chain is generally more important than the light chain for the interaction with the antigen, although many reports demonstrate the influence of the light chain in the antibody binding properties. The heavy chains of anti-N-glycolyl-ganglioside P3 mAb and anti-idiotypic 1E10 mAb display complementary charged residues in their H-CDRs, particularly in H-CDR3. A basic residue in P3 mAb H-CDR1 was shown to be crucial for the interaction with the antigen and 1E10 mAb. The immunogenetic features of three other P3 mAb anti-idiotypic mAbs are now analyzed. One of them bears the same heavy chain as 1E10 mAb and a different light chain, but differs in its binding to P3 mAb mutants where H-CDR basic residues were replaced and in the binding to 1E10-specific phagotopes. Chimeric hybrid antibodies with P3 and 1E10 mAb heavy chains and unrelated light chains were obtained to further determine the importance of heavy chains in P3 and 1E10 mAb binding properties. One of the P3 heavy chain hybrid antibodies retained the specificity of P3 mAb with slight affinity differences. The heavy chains appear to play the main role in these mAb interactions, with the light chains modulating the affinity to their ligands.

Chimeric anti-N-glycolyl-ganglioside and its anti-idiotypic MAbs: immunodominance of their variable regions.

P3 monoclonal antibody (MAb) is a murine IgM that specifically recognizes N-glycolyl (NeuGc)-gangliosides and sulfatides. It also reacts with antigens expressed in human breast tumors and melanoma. In syngeneic model, P3 MAb is able to elicit a strong anti-idiotypic (Ab2) antibody response, even in the absence of adjuvants or carrier proteins. 1E10 MAb is an anti-idiotypic antibody specific for P3 MAb that has demonstrated anti-tumoral effects in syngeneic and allogeneic animals. Here we report the construction of the human IgG(1) chimeric P3 and 1E10 antibodies, and the evaluation of the maintenance of the main properties of the murine MAbs. Chimeric P3 antibody specifically reacted with GM3(NeuGc) and GM2(NeuGc) gangliosides, and not with their acetylated variants. Also, it strongly recognized the anti-idiotypic 1E10 MAb. Chimeric 1E10 antibody specifically reacted with P3 MAb. Upon immunization of Balb/c mice with both chimeric antibodies, we were able to demonstrate the immunodominance of their variable regions. The anti-idiotypic response induced by both antibodies was strong and in most of the mice was even significantly higher than the anti-isotypic response, despite the fact that 70% of the chimeric molecule is xenogenic with respect to the animal model.