Elsevier

Peptides

Volume 30, Issue 7, July 2009, Pages 1222-1227
Peptides

Remarkable inter- and intra-species complexity of conotoxins revealed by LC/MS

https://doi.org/10.1016/j.peptides.2009.03.019Get rights and content

Abstract

Cone snails have evolved an assortment of venom peptides as an evolutionary strategy for rapid prey immobilization and defence. Earlier studies estimated ∼100 conopeptides per species. In this study we optimized liquid chromatography and electrospray ionization mass spectrometry for the detection of conopeptides in crude venom to characterize conopeptides present in the venom of individual specimens of Conus textile, C. imperialis and C. marmoreus. Using this approach, we have expanded the predicted number of venom peptides 10-fold to an estimate of 1000–1900 conopeptides per species. Our investigation has also revealed a surprisingly high level of intra-species variation that distinguishes cone snails from other venomous species including spiders and scorpions. Given this inherent diversity and variability, more sensitive bioassays and sequencing techniques will be required to fully explore conotoxin bioactivity.

Introduction

The venoms of marine snails of the Conus genus are a rich source of bioactive peptides (conotoxins or conopeptides) that potently and selectively modulate a broad array of biological targets including G protein coupled receptors, ion channels and transporters [14]. Conus spp. are generally thought to contain up to a few hundred peptides, with <1% of these conopeptides presently pharmacologically characterized [14], [17]. Their relatively small size and structural diversity, enhanced by the presence of a large number of post-translational modifications, contribute to the value of conopeptides as both research tools and leads to new drugs.

Although there have been a number of studies on specific conopeptides, the true peptide diversity and the significance of intraspecific variation in cone snail venoms is poorly defined [20]. Both qualitative and quantitative differences in venom peptides have been reported for snails of the same species [10], [11], [20] that likely arise through multiple factors, including genetic heterogeneity, differential peptide expression and altered post-translational processing. This diversity contributes to the estimated 50–200 unique conopeptides present in each species [9], [11], [18], and challenges our ability to isolate and characterize the bioactives present using currently available methodologies.

Mass spectrometry (MS) is a central to the emerging field of venomics, the systematic analysis of venom components [6]. Cone snail venomics has the potential to reveal new classes of conopeptides or novel variants within known classes, potentially yielding peptides with novel target and target subtype specificity and/or peptides with improved physico-chemical properties. Towards this goal, we explored cone venom peptide diversity using optimized liquid chromatography/mass spectrometry (LC/MS). This approach allowed individual peptides to be identified and co-eluting peptides of different masses to be resolved. Comparing the venom peptides from individual cone snails has increased by at least 10-fold the number of conopeptides estimated to be present in Conus spp. venom, and revealed unprecedented intra- and inter-species variability in the venom peptides present.

Section snippets

Crude venom extraction

Venom was isolated from venom ducts of C. textile, C. marmoreus and C. imperialis collected in waters off the east Australian coast. Individual venom ducts of frozen specimens were dissected, finely cut and triturated with 2 mL chilled 30% acetonitrile (ACN) containing 5% formic acid (FA) and centrifuged for 5 min at 20 × g. The supernatant was removed and spun at 17,000 × g for 10 min to separate the fine insoluble granular material from the crude venom. For comparison, venom was also extracted from

Results

To study individual Conus venom peptide profiles, on-line LC/MS was performed on small amounts of crude venom with very minimal sample preparation. Fig. 1 shows examples of the LC/MS spectra of individual crude venoms from individual C. textile (Fig. 1A), C. marmoreus (Fig. 1B) and C. imperialis (Fig. 1C) demonstrating a surprising complexity of peptides present in each venom. C. textile venom was the most complex species examined, with one individual containing at least 703 different peptides (

Discussion

Conus venom peptides have evolved bioactive peptides acting at a diverse range of biological targets to allow the rapid paralysis and capture of their prey of fish, molluscs or worms. Using LC/MS, we have revealed an unprecedented level of conopeptide diversity, expanding the predicted numbers to well in excess of 1000 peptides per Conus species. We have also observed a high level of variation in the venom peptides expressed between individuals within a species. This high level of both inter-

Acknowledgement

This work was supported by an NHMRC Program Grant. RJL is an NHMRC Research Fellow.

References (20)

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