Structural and Functional Diversity of Peptide Toxins from Tarantula Haplopelma hainanum (Ornithoctonus hainana) Venom Revealed by Transcriptomic, Peptidomic, and Patch Clamp Approaches.

Spider venom is a complex mixture of bioactive peptides to subdue their prey. Early estimates suggested that over 400 venom peptides are produced per species. In order to investigate the mechanisms responsible for this impressive diversity, transcriptomics based on second generation high throughput sequencing was combined with peptidomic assays to characterize the venom of the tarantula Haplopelma hainanum. The genes expressed in the venom glands were identified, and the bioactivity of their protein products was analyzed using the patch clamp technique. A total of 1,136 potential toxin precursors were identified that clustered into 90 toxin groups, of which 72 were novel. The toxin peptides clustered into 20 cysteine scaffolds that included between 4 and 12 cysteines, and 14 of these groups were newly identified in this spider. Highly abundant toxin peptide transcripts were present and resulted from hypermutation and/or fragment insertion/deletion. In combination with variable post-translational modifications, this genetic variability explained how a limited set of genes can generate hundreds of toxin peptides in venom glands. Furthermore, the intraspecies venom variability illustrated the dynamic nature of spider venom and revealed how complex components work together to generate diverse bioactivities that facilitate adaptation to changing environments, types of prey, and milking regimes in captivity.

Screening for voltage-gated sodium channel interacting peptides.

The voltage-gated sodium channel (VGSC) interacting peptide is of special interest for both basic research and pharmaceutical purposes. In this study, we established a yeast-two-hybrid based strategy to detect the interaction(s) between neurotoxic peptide and the extracellular region of VGSC. Using a previously reported neurotoxin JZTX-III as a model molecule, we demonstrated that the interactions between JZTX-III and the extracellular regions of its target hNav1.5 are detectable and the detected interactions are directly related to its activity. We further applied this strategy to the screening of VGSC interacting peptides. Using the extracellular region of hNav1.5 as the bait, we identified a novel sodium channel inhibitor SSCM-1 from a random peptide library. This peptide selectively inhibits hNav1.5 currents in the whole-cell patch clamp assays. This strategy might be used for the large scale screening for target-specific interacting peptides of VGSCs or other ion channels.

[Inhibition of Jingzhaotoxin-V on Kv4.3 channel].

Kv4.3 channel is present in many mammalian tissues, predominantly in the heart and central nervous system. Its currents are transient, characterized by rapid activation and inactivation. In the hearts of most mammals, it is responsible for repolarization of the action potential of ventricular myocytes and is important in the regulation of the heart rate. Because of its central role in this important physiological process, Kv4.3 channel is a promising target for anti-arrhythmic drug development. Jingzhaotoxin-V (JZTX-V) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Whole-cell patch clamp recording showed that it partly blocked the transient outward potassium channels in dorsal root ganglion neurons of adult rats with an IC(50) value of 52.3 nmol/L. To investigate the effect of JZTX-V on Kv4.3 channel, JZTX-V was synthesized using the solid-phase chemical synthesis and separated by reverse phase high performance liquid chromatography (HPLC). The purity was tested by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MOLDI-TOF mass spectrometry). Two-electrode voltage-clamp technique was used to characterize the action of JZTX-V on Kv4.3 channels expressed in Xenopus laevis oocytes. As a result, JZTX-V displayed fast kinetics of inhibition and recovery from inactivation. Furthermore, it could inhibit Kv4.3 channel current in a time- and concentration-dependent manner with an IC(50) value of 425.1 nmol/L. The application of JZTX-V affected the activation and inactivation characteristics of Kv4.3 channel and caused a shift of the current-voltage relationship curve and the steady-state inactivation curve to depolarizing direction by approximately 29 mV and 10 mV, respectively. So we deduced that JZTX-V is a gating modifier toxin of Kv4.3 channel. Present findings should be helpful to develop JZTX-V into a molecular probe and drug candidate targeting to Kv4.3 channel in the myocardium.

Sequence-specific assignment of 1H-NMR resonance and determination of the secondary structure of Jingzhaotoxin-I.

Jingzhaotoxin-I (JZTX-I) purified from the venom of the spider Chilobrachys jingzhao is a novel neurotoxin preferentially inhibiting cardiac sodium channel inactivation by binding to receptor site 3. The structure of this toxin in aqueous solution was investigated using 2-D 1H-NMR techniques. The complete sequence-specific assignments of proton resonance in the 1H-NMR spectra of JZTX-I were obtained by analyzing a series of 2-D spectra, including DQF-COSY, TOCSY and NOESY spectra, in H2O and D2O. All the backbone protons except for Gln4 and more than 95% of the side-chain protons were identified by d alphaN, d alphadelta, d betaN and d NN connectivities in the NOESY spectrum. These studies provide a basis for the further determination of the solution conformation of JZTX-I. Furthermore, the secondary structure of JZTX-I was identified from NMR data. It consists mainly of a short triple-stranded antiparallel beta-sheet with Trp7-Cys9, Phe20-Lys23 and Leu28-Trp31. The characteristics of the secondary structure of JZTX-I are similar to those of huwentoxin-I (HWTX-I) and hainantoxin-IV (HNTX-IV), whose structures in solution have previously been reported.

Purification and characterization of raventoxin-I and raventoxin-III, two neurotoxic peptides from the venom of the spider Macrothele raveni.

The spider Macrothele raveni was recently identified as a new species of Genus Macrothele. The crude venom from M. raveni was found to be neurotoxic to mice and the LD(50) of the crude venom in mice was 2.852mg/kg. Two neurotoxic peptides, raventoxin-I and raventoxin-III, were isolated from the crude venom by ion-exchange and reverse phase high performance liquid chromatography. Raventoxin-I was the most abundant toxic component in the venom, while raventoxin-III was a lower abundant component. Both toxins can kill mice and block neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation, but have no effect on cockroaches. The LD(50) of raventoxin-I in mice is 0.772mg/kg. The complete amino acid sequences of raventoxin-I and raventoxin-III were determined and found to consist of 43 and 29 amino acid residues, respectively. It was determined by mass spectrometry that all Cys residues from raventoxin-I and raventoxin-III are involved in disulphide bonds. raventoxin-III showed no significant sequence homology with any presently known neurotoxins in the protein/DNA databases, while raventoxin-I has limited sequence identity with delta-AcTx-Hv1 and delta-AcTx-Ar1, which target both mammalian and insect sodium channels. Both raventoxin-I and raventoxin-III only work on vertebrates, but not on insects. Moreover, raventoxin-I could exert an effect of first exciting and then inhibiting the contraction of mouse diaphragm muscle caused by electrically stimulating the phrenic nerve, but raventoxin-III could not.