Structural and functional characterisation of Tst2, a novel TRPV1 inhibitory peptide from the Australian sea anemone Telmatactis stephensoni.

Sea anemone venoms are complex mixtures of biologically active compounds, including disulfide-rich peptides, some of which have found applications as research tools, and others as therapeutic leads. Our recent transcriptomic and proteomic studies of the Australian sea anemone Telmatactis stephensoni identified a transcript for a peptide designated Tst2. Tst2 is a 38-residue peptide showing sequence similarity to peptide toxins known to interact with a range of ion channels (NaV, TRPV1, KV and CaV). Recombinant Tst2 (rTst2, which contains an additional Gly at the N-terminus) was produced by periplasmic expression in Escherichia coli, enabling the production of both unlabelled and uniformly 13C,15N-labelled peptide for functional assays and structural studies. The LC-MS profile of the recombinant Tst2 showed a pure peak with molecular mass 6 Da less than that of the reduced form of the peptide, indicating the successful formation of three disulfide bonds from its six cysteine residues. The solution structure of rTst2 was determined using multidimensional NMR spectroscopy and revealed that rTst2 adopts an inhibitor cystine knot (ICK) structure. rTst2 was screened using various functional assays, including patch-clamp electrophysiological and cytotoxicity assays. rTst2 was inactive against voltage-gated sodium channels (NaV) and the human voltage-gated proton (hHv1) channel. rTst2 also did not possess cytotoxic activity when assessed against Drosophila melanogaster flies. However, the recombinant peptide at 100 nM showed >50% inhibition of the transient receptor potential subfamily V member 1 (TRPV1) and slight (∼10%) inhibition of transient receptor potential subfamily A member 1 (TRPA1). Tst2 is thus a novel ICK inhibitor of the TRPV1 channel.

Genomic, functional and structural analyses elucidate evolutionary innovation within the sea anemone 8 toxin family.

BACKGROUND: The ShK toxin from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in Actiniarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T. stephensoni. RESULTS: We identified ten SA8-family genes in two clusters and six SA8-family genes in five clusters for T. stephensoni and A. tenebrosa, respectively. Nine SA8 T. stephensoni genes were found in a single cluster, and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While the functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. CONCLUSIONS: Our results provide the first demonstration that SA8 is a unique gene family in Actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T. stephensoni.

Alkyne-Bridged α-Conotoxin Vc1.1 Potently Reverses Mechanical Allodynia in Neuropathic Pain Models.

Several Conus-derived venom peptides are promising lead compounds for the management of neuropathic pain, with α-conotoxins being of particular interest. Modification of the interlocked disulfide framework of α-conotoxin Vc1.1 has been achieved using on-resin alkyne metathesis. Although introduction of a metabolically stable alkyne motif significantly disrupts backbone topography, the structural modification generates a potent and selective GABAB receptor agonist that inhibits Cav2.2 channels and exhibits dose-dependent reversal of mechanical allodynia in a behavioral rat model of neuropathic pain. The findings herein support the hypothesis that analgesia can be achieved via activation of GABABRs expressed in dorsal root ganglion (DRG) sensory neurons.

Structural and functional characterisation of a novel peptide from the Australian sea anemone Actinia tenebrosa.

Sea anemone venoms have long been recognised as a rich source of peptides with interesting pharmacological and structural properties. Our recent transcriptomic studies of the Australian sea anemone Actinia tenebrosa have identified a novel 13-residue peptide, U-AITx-Ate1. U-AITx-Ate1 contains a single disulfide bridge and bears no significant homology to previously reported amino acid sequences of peptides from sea anemones or other species. We have produced U-AITx-Ate1 using solid-phase peptide synthesis, followed by oxidative folding and purification of the folded peptide using reversed-phase high-performance liquid chromatography. The solution structure of U-AITx-Ate1 was determined based on two-dimensional nuclear magnetic resonance spectroscopic data. Diffusion-ordered NMR spectroscopy revealed that U-AITx-Ate1 was monomeric in solution. Perturbations in the 1D 1H NMR spectrum of U-AITx-Ate1 in the presence of dodecylphosphocholine micelles together with molecular dynamics simulations indicated an interaction of U-AITx-Ate1 with lipid membranes, although no binding was detected to 100% POPC and 80% POPC: 20% POPG lipid nanodiscs by isothermal titration calorimetry. Functional assays were performed to explore the biological activity profile of U-AITx-Ate1. U-AITx-Ate1 showed no activity in voltage-clamp electrophysiology assays and no change in behaviour and mortality rates in crustacea. Moderate cytotoxic activity was observed against two breast cancer cell lines.