Sample Limited Characterization of a Novel Disulfide-Rich Venom Peptide Toxin from Terebrid Marine Snail Terebra variegata

Abstract

Disulfide-rich peptide toxins found in the secretions of venomous organisms such as snakes, spiders, scorpions, leeches, and marine snails are highly efficient and effective tools for novel therapeutic drug development. Venom peptide toxins have been used extensively to characterize ion channels in the nervous system and platelet aggregation in haemostatic systems. A significant hurdle in characterizing disulfide-rich peptide toxins from venomous animals is obtaining significant quantities needed for sequence and structural analyses. Presented here is a strategy for the structural characterization of venom peptide toxins from sample limited (4 ng) specimens via direct mass spectrometry sequencing, chemical synthesis and NMR structure elucidation. Using this integrated approach, venom peptide Tv1 from Terebra variegata was discovered. Tv1 displays a unique fold not witnessed in prior snail neuropeptides. The novel structural features found for Tv1 suggest that the terebrid pool of peptide toxins may target different neuronal agents with varying specificities compared to previously characterized snail neuropeptides.

Using Drosophila behavioral assays to characterize terebrid venom-peptide bioactivity

“…By contrast, Framework I conotoxins are in the A superfamily, which target either adrenergic receptors or nicotinic acetylcholine receptors (nAChR) 56 . An alignment of Tv1 and Tsu1.1 with known conotoxins confirms that, beyond their cysteine scaffold, and a conserved proline in Tsu1.1, Tv1 and Tsu1.1 are not homologous to members of their respective conotoxin superfamilies and are distinct from known conotoxins ( Figure 2C) 54 .…”

“…Previously, Tv1 injected into Nereis virens, a polychaete worm, has been shown to induce partial paralysis 54 . This effect was absent in Drosophila ( Figure 3A and C), suggesting that the Tv1 binds to targets which are not conserved between polychaetes and insect.…”

“…One of these candidates was a sequence with 21 amino acid residues (SAVEECCENPVCKHTSGCPTT), which we called Terebra subulata teretoxin 1.1 (Tsu1.1) ( Figure 2B). We compared Tsu1.1 with another peptide, Terebra variegata teretoxin 1 (Tv1) which was previously characerized in the Holford group 54 . Teretoxins Tv1 and Tsu1.1 have distinct cysteine frameworks-a feature that is used to group conotoxins into functional groups-which suggests they might have different molecular targets 31,55 .…”

Antitumor effects of Tv1 venom peptide in liver cancer

“…As teretoxins are present only in minute quantities in the venom, a greater quantity of the linear peptide Tv1 was chemically synthesized and purified by previously described methods and further oxidatively folded into biologically active form 32 (Figs. S11a & b).…”

“…Tv1, a 21 amino acid peptide with unique structural properties compared with known snail venom peptides. 32 Tv1 was chemically synthesized and assayed using both in vitro and in vivo systems. Our results suggest that Tv1 inhibits HCC selectively, and that its mechanism of action involves downstream manipulation of TRPC6 and/or TRPV6 channel activity, which were overexpressed in the HCC models used in this study.…”