Actinoporins: From the Structure and Function to the Generation of Biotechnological and Therapeutic Tools

Actinoporins (APs) are a family of pore-forming toxins (PFTs) from sea anemones. These biomolecules exhibit the ability to exist as soluble monomers within an aqueous medium or as constitutively open oligomers in biological membranes. Through their conformational plasticity, actinoporins are considered good candidate molecules to be included for the rational design of molecular tools, such as immunotoxins directed against tumor cells and stochastic biosensors based on nanopores to analyze unique DNA or protein molecules. Additionally, the ability of these proteins to bind to sphingomyelin (SM) facilitates their use for the design of molecular probes to identify SM in the cells. The immunomodulatory activity of actinoporins in liposomal formulations for vaccine development has also been evaluated. In this review, we describe the potential of actinoporins for use in the development of molecular tools that could be used for possible medical and biotechnological applications

Transcriptome analysis of scorpion species belonging to the Vaejovis genus.

Scorpions belonging to the Buthidae family have traditionally drawn much of the biochemist's attention due to the strong toxicity of their venoms. Scorpions not toxic to mammals, however, also have complex venoms. They have been shown to be an important source of bioactive peptides, some of them identified as potential drug candidates for the treatment of several emerging diseases and conditions. It is therefore important to characterize the large diversity of components found in the non-Buthidae venoms. As a contribution to this goal, this manuscript reports the construction and characterization of cDNA libraries from four scorpion species belonging to the Vaejovis genus of the Vaejovidae family: Vaejovis mexicanus, V. intrepidus, V. subcristatus and V. punctatus. Some sequences coding for channel-acting toxins were found, as expected, but the main transcribed genes in the glands actively producing venom were those coding for non disulfide-bridged peptides. The ESTs coding for putative channel-acting toxins, corresponded to sodium channel β toxins, to members of the potassium channel-acting α or κ families, and to calcium channel-acting toxins of the calcin family. Transcripts for scorpine-like peptides of two different lengths were found, with some of the species coding for the two kinds. One sequence coding for La1-like peptides, of yet unknown function, was found for each species. Finally, the most abundant transcripts corresponded to peptides belonging to the long chain multifunctional NDBP-2 family and to the short antimicrobials of the NDBP-4 family. This apparent venom composition is in correspondence with the data obtained to date for other non-Buthidae species. Our study constitutes the first approach to the characterization of the venom gland transcriptome for scorpion species belonging to the Vaejovidae family

Relative distribution of the sequences found in the <i>V</i>. <i>intrepidus</i>, <i>V</i>. <i>mexicanus</i>, <i>V</i>. <i>subcristatus</i> and <i>V</i>. <i>punctatus</i> libraries with respect to the putative function of their encoded peptides.

<p>Relative distribution of the sequences found in the <i>V</i>. <i>intrepidus</i>, <i>V</i>. <i>mexicanus</i>, <i>V</i>. <i>subcristatus</i> and <i>V</i>. <i>punctatus</i> libraries with respect to the putative function of their encoded peptides.</p

Sequence alignment of the La1-like peptides found in the Vaejovis libraries.

<p>The precursor sequences of VmLa1lp1 from <i>V</i>. <i>mexicanus</i>, VsLa1lp1 from <i>V</i>. <i>subcristatus</i> and VpLa1lp1 from <i>V</i>. <i>punctatus</i> are aligned to the previously reported precursors of La1lp-15 (UniProt:AGA82761) from <i>U</i>. <i>yaschenkoi</i>, HsTx1 (UniProt:K7WMX6) from <i>H</i>. <i>spinifer</i>, VenPepPc (UniProt:H2CYP1) from <i>P</i>. <i>cavimanus</i>, and the mature La1 (UniProt:P0C5F3) from <i>L</i>. <i>australasiae</i>. The peptide length (aa) always refers to the confirmed (when the peptide has been isolated from the venom) or software-predicted mature peptides. The given percentages of identity (%I) correspond to the mature peptides only, and are relative to the first sequence. The predicted signal peptides are shown underlined, the propeptides in italics and bold.</p

Putative calcium channel toxins found in <i>Vaejovis</i>.

<p>The peptide length (aa) always refers to the confirmed (when the peptide has been isolated from the venom) or software-predicted mature peptides. The given percentages of identity (%I) correspond to the mature peptides only, and refer to the first sequence. The predicted signal peptides are shown underlined, the propeptides in italics and bold. <b>A</b>) Sequence alignment of the three-disulfide-bridged putative calcinsVpCaTx1 from <i>V</i>. <i>punctatus</i> and ViCaTx1 from <i>V</i>. <i>intrepidus</i> with imperatoxin (UniProt:P59868) from <i>P</i>. <i>imperator</i>, maurocalcin (UniProt:P60254) from <i>S</i>. <i>maurus palmatus</i>, opicalcin-1 (UniProt:P60252) and opicalcin-2 (UniProt:P60253) from <i>Opistophthalmus carinatus</i>, UyCaTx20 (Calcium-channel toxin-like 20, UniProt:AGA82762) from <i>U</i>. <i>yaschenkoi</i> and hadrucalcin (UniProt:B8QG00) from <i>H</i>. <i>gertschi</i>. <b>B</b>) The precursors of the two-disulfide-bridged putative calcins VmCaTx1 and VmCaTx2 from <i>V</i>. <i>mexicanus</i> and VpCaTx2 from <i>V</i>. <i>punctatus</i>, are compared to previously reported sequences of their kind: LaIT1 (UniProt:P0C5F2) from <i>L</i>. <i>australasiae</i>, and U₁-LITX-Lw1a (UniProt:P0DJ08) from <i>L</i>. <i>waigiensis</i>.</p

Putative toxin sequences derived from the precursors found in the <i>Vaejovis</i> libraries.

<p>The peptide length (aa) always refers to the confirmed (when the peptide has been isolated from the venom) or software-predicted mature peptides. The identity (%I) is always relative to the first sequence of the alignment, and considers only the mature peptide regions. When present, the signal peptides are shown underlined and the propeptides are in italics and bold. The conserved cysteine arrangement typical of each family is highlighted. A) Sequence alignment of the putative sodium channel toxins. The precursor sequences of ViNaTx1 from <i>V</i>. <i>intrepidus</i>, VpNaTx1 and VpNaTx2 from <i>V</i>. <i>punctatus</i>, VsNaTx1 and VsNaTx2 from <i>V</i>. <i>subcristatus</i>, and VmNaTx1 from <i>V</i>. <i>mexicanus</i> are compared to the known sodium channel-specific ß-toxins Birtoxin (UniProt:P58752), Ikitoxin (UniProt:P0C1B8) and Altitoxin (UniProt:P0C1B5) from <i>Parabuthus transvaalicus</i>. B) Comparative alignments of the sequences found that belong to the two families of potassium channel-specific α-toxins. First, the precursor sequences of the six-cysteines α-toxins ViKTx1 form <i>V</i>. <i>intrepidus</i> and VpKTx4 from <i>V</i>. <i>punctatus</i> aligned to KTX-2 (Kaliotoxin-2, UniProt:P45696) from <i>Androctonus australis</i> and TdK1 (UniProt:P59925) from <i>Tityus discrepans</i> as references. Second, the precursor sequences coding for the toxins belonging to the family of the eight-cysteines α-toxins VmKTx1 from <i>V</i>. <i>mexicanus</i> and VpKTx3 from <i>V</i>. <i>punctatus</i>, aligned to Vm24 (UniProt:P0DJ31) from <i>V</i>. <i>mexicanus</i> and HgeTx1 (UniProt:P84864) from <i>Hadrurus gertschi</i> as references. C) The precursor sequence of VmKTx2 from <i>V</i>. <i>mexicanus</i> compared to the precursor sequence of HelaTx1 (UniProt:P0DJ41) from <i>Heterometrus laoticus</i> and κ-HfTx1 (UniProt:P82850) plus κ-HfTx2 (UniProt:P82851) from <i>Heterometrus fulvipes</i>, all belonging to the family of potassium channel-specific κ-toxins. D) The scorpine-like sequences ViScplp1–3 from <i>V</i>. <i>intrepidus</i>, VmScplp1–3 from <i>V</i>. <i>mexicanus</i>, VpScplp1 from <i>V</i>. <i>punctatus</i>, and the partial sequences of VsScplp1–2 from <i>V</i>. <i>subcristatus</i> are aligned. For comparison, the sequences of scorpine (UniProt:P56972) from <i>P</i>. <i>imperator</i>, HgeScplp1 (UniProt:Q0GY40) and HgeScplp2 (UniProt:P0C8W5) from <i>H</i>. <i>gertschi</i> are also included in the alignment.</p