Hormone-like peptides in the venoms of marine cone snails

https://doi.org/10.1016/j.ygcen.2015.07.012Get rights and content

Highlights

  • Several novel hormone-like toxins were identified in cone snail venom.

  • These included the peptides prohormone-4 and elevenin.

  • Hormone-like toxins are an integral part of the envenomation strategy of Conus.

Abstract

The venoms of cone snails (genus Conus) are remarkably complex, consisting of hundreds of typically short, disulfide-rich peptides termed conotoxins. These peptides have diverse pharmacological targets, with injection of venom eliciting a range of physiological responses, including sedation, paralysis and sensory overload. Most conotoxins target the prey’s nervous system but evidence of venom peptides targeting neuroendocrine processes is emerging. Examples include vasopressin, RFamide neuropeptides and recently also insulin. To investigate the diversity of hormone/neuropeptide-like molecules in the venoms of cone snails we systematically mined the venom gland transcriptomes of several cone snail species and examined secreted venom peptides in dissected and injected venom of the Australian cone snail Conus victoriae. Using this approach we identified several novel hormone/neuropeptide-like toxins, including peptides similar to the bee brain hormone prohormone-4, the mollusc ganglia neuropeptide elevenin, and thyrostimulin, a member of the glycoprotein hormone family, and confirmed the presence of insulin. We confirmed that at least two of these peptides are not only expressed in the venom gland but also form part of the injected venom cocktail, unambiguously demonstrating their role in envenomation. Our findings suggest that hormone/neuropeptide-like toxins are a diverse and integral part of the complex envenomation strategy of Conus. Exploration of this group of venom components offers an exciting new avenue for the discovery of novel pharmacological tools and drug candidates, complementary to conotoxins.

Introduction

Marine cone snails (genus Conus) are venomous. They use their venoms for prey capture, self-defense and possibly intraspecific competition (Dutertre et al., 2014, Olivera, 1999). Cone snail venoms are remarkably complex cocktails containing hundreds of small cysteine-rich peptide toxins (conotoxins) (Olivera et al., 1990, Robinson and Norton, 2014). Many conotoxins have unmatched potency and selectivity profiles for their respective targets, including specific subtypes of voltage- and ligand-gated ion channels, G protein-coupled receptors and neurotransmitter transporters. As such, conotoxins represent a rich source of valuable pharmacological tools and drug candidates.

Conotoxins are produced in a specialized venom gland, where they are translated as precursor peptides. As a general rule, conotoxin precursor peptides are comprised of an N-terminal signal sequence for targeting to the cellular secretory pathway, an intermediate pro-region that plays a role in vesicular transport (Conticello et al., 2003), post-translational modification (Bandyopadhyay et al., 1998) and folding (Buczek et al., 2004) and is presumably cleaved following secretion, and a single copy of the mature toxin peptide at the C-terminus. In many ways this process mirrors the production of endogenous hormones/neuropeptides.

While conotoxins are the major, and by far the most studied, constituent of Conus venom, several cases of hormone/neuropeptide-like components have been reported (Table 1). Examples include the conopressins (vasopressin analogues) from Conus geographus and Conus striatus (Cruz et al., 1987), contulakin-G (a neurotensin analogue) from C. geographus (Craig et al., 1999), RFamide neuropeptides from Conus spurius and Conus victoriae (Maillo et al., 2002, Robinson et al., 2015), conomap (a myoactive tetradecapeptide) from Conus vitulinus (Dutertre et al., 2006), conoCAPs (analogues of crustacean cardioactive peptide) from Conus villepinii (Möller et al., 2010) and neuropeptide-F/Y from Conus betulinus (Wu et al., 2010). Furthermore, we recently demonstrated that specialized insulins are an abundant and active component of some Conus venoms (Safavi-Hemami et al., 2015). When injected into fish, the venom insulin elicits hypoglycemic shock, thus facilitating capture of the physiologically impaired prey. It is thus becoming clear that Conus venoms are not limited to conotoxins targeting neuronal ion channels, but that other fascinating strategies are also being employed.

To systematically explore the full extent of hormone/neuropeptide-like venom components expressed, secreted and ultimately injected into the prey by cone snails, we used a combined transcriptomics/proteomics-based strategy (mass spectrometry (MS)-matching) on the extruded as well as the injected venom (venom collected from live snails during an envenomation event) of C. victoriae, a mollusc-hunting species endemic to the coast of north-western Australia. This combined approach led to the identification of several novel peptides that share high similarity with endogenous hormones/neuropeptides from other organisms. Furthermore, interrogation of venom gland transcriptomic data from additional cone snail species revealed that these peptides are widely distributed in the genus Conus.

Section snippets

Venom gland transcriptome

Specimens of C. victoriae were collected from Broome, Western Australia. Specimens were collected specifically for research use, under a commercial fishing license of the Western Australian Specimen Shell Managed Fishery (license number 2577). Ethics approval is not required, in Australia, for taking samples from Conus.

Preparation of the venom gland transcriptome of C. victoriae has been described previously (Robinson et al., 2014). Briefly, whole venom glands of live specimens were removed,

Results & Discussion

Matching of the C. victoriae venom gland transcriptome to MS/MS data of extruded venom revealed several novel peptides whose precursors did not belong to any conotoxin superfamilies (Robinson and Norton, 2014), but which belonged to hormone/neuropeptide families from other organisms. We were able to confirm the presence of two of these peptides in the injected venom of C. victoriae, unequivocally demonstrating their role as exogenous venom components. One of these peptides was related to

Conclusions

Our finding of hormone/neuropeptide-like components in the venom of C. victoriae, together with previous reports for this genus, clearly demonstrates that the Conus venom gland is not only highly specialized in the biosynthesis and secretion of conotoxins but also a rich source of hormone/neuropeptide-like peptides. Two of these peptides (elevenin and prohormone-4) were identified in the injected venom of C. victoriae, unambiguously demonstrating their role as exogenously targeted venom

Acknowledgments

We thank Dorothy Loo and Dr. Nicholas Williamson for technical assistance with mass spectrometry. The authors acknowledge financial support from a Discovery Grant (DP110101331) from the Australian Research Council (AWP), an NIH program project Grant (GM48677, B. Olivera Program Director) (PB) and NIH research project Grant (GM099939) (PKB and MY). RSN, AWP and ATP acknowledge fellowship support from the Australian National Health and Medical Research Council (NHMRC). ATP was supported by an

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