Hormone-like peptides in the venoms of marine cone snails
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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