Novel venom peptides from the cone snail Conus pulicarius discovered through next-generation sequencing of its venom duct transcriptome

Lluisma, Arturo O.; Milash, Brett; Moore, Barry; Olivera, Baldomero M.; Bandyopadhyay, Pradip K.

doi:10.1016/j.margen.2011.09.002

Cited by 59 publications

(71 citation statements)

References 29 publications

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“…However, using traditional protein-centric drug discovery approaches has been a tedious and time-consuming task that allows only superficial mining of the huge chemical diversity of natural products and that usually leads to the identification of only a few bioactive peptides per experiment (77). During the past decade, several studies focusing solely on cone snail venom duct (43,44,49,(78)(79)(80) or salivary gland (9,43,44,49,(78)(79)(80) transcriptomes, and later complemented by proteome profiling (46,47,50,59,81), have allowed the report of no more than only a hundred (47 on average) full-length precursor conotoxins each. The great majority of these studies used the ROCHE 454 next-generation sequencing platform because it produced low amounts of long reads that were possible to annotate by performing simple homology BLAST searches.…”

Section: Discussionmentioning

confidence: 99%

Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks

Lavergne

Harliwong

Jones

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Cone snails are predatory marine gastropods characterized by a sophisticated venom apparatus responsible for the biosynthesis and delivery of complex mixtures of cysteine-rich toxin peptides. These conotoxins fold into small highly structured frameworks, allowing them to potently and selectively interact with heterologous ion channels and receptors. Approximately 2,000 toxins from an estimated number of >70,000 bioactive peptides have been identified in the genus Conus to date. Here, we describe a high-resolution interrogation of the transcriptomes (available at www.ddbj.nig.ac.jp) and proteomes of the diverse compartments of the Conus episcopatus venom apparatus. Using biochemical and bioinformatic tools, we found the highest number of conopeptides yet discovered in a single Conus specimen, with 3,305 novel precursor toxin sequences classified into 9 known superfamilies (A, I1, I2, M, O1, O2, S, T, Z), and identified 16 new superfamilies showing unique signal peptide signatures. We were also able to depict the largest population of venom peptides containing the pharmacologically active C-C-CC-C-C inhibitor cystine knot and CC-C-C motifs (168 and 44 toxins, respectively), as well as 208 new conotoxins displaying odd numbers of cysteine residues derived from known conotoxin motifs. Importantly, six novel cysteine-rich frameworks were revealed which may have novel pharmacology. Finally, analyses of codon usage bias and RNA-editing processes of the conotoxin transcripts demonstrate a specific conservation of the cysteine skeleton at the nucleic acid level and provide new insights about the origin of sequence hypervariablity in mature toxin regions.

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Section: Discussionmentioning

confidence: 99%

Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks

Lavergne

Harliwong

Jones

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Interestingly, although A superfamily isoforms Pu1.1-Pu1.3 were previously discovered in the venom of C. pulicarius (40), they were not detected in the venom gland transcriptome (22). At the protein level, we were also unable to identify A-superfamily-related peptides when using NCBI cone-snail-related proteins as the searching database.…”

Section: Discussionmentioning

confidence: 74%

“…This high level of transcription for the superfamily also generated a high number of isoforms (77%). Interestingly, the O superfamily is also abundant (44%) in the transcriptome of another worm-hunting cone snail, C. pulicarius (22). The O superfamily has diverse pharmacology, with -, O, ␦, ␥-, and -conotoxins targeting voltage-gated calcium, sodium, and potassium channels (38).…”

Section: Discussionmentioning

confidence: 99%

Transcriptomic Messiness in the Venom Duct of Conus miles Contributes to Conotoxin Diversity

Jin

Dutertre

Kaas

et al. 2013

Molecular & Cellular Proteomics

113

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Marine cone snails have developed sophisticated chemical strategies to capture prey and defend themselves against predators. Among the vast array of bioactive molecules in their venom, peptide components called conotoxins or conopeptides dominate, with many binding with high affinity and selectivity to a broad range of cellular targets, including receptors and transporters of the nervous system. Whereas the conopeptide gene precursor organization has a conserved topology, the peptides in the venom duct are highly processed. Indeed, deep sequencing transcriptomics has uncovered on average fewer than 100 toxin gene precursors per species, whereas advanced proteomics has revealed >10-fold greater diversity at the peptide level. In the present study, second-generation sequencing technologies coupled to highly sensitive mass spectrometry methods were applied to rapidly uncover the conopeptide diversity in the venom of a worm-hunting species, Conus miles. A total of 662 putative conopeptide encoded sequences were retrieved from transcriptomic data, comprising 48 validated conotoxin sequences that clustered into 10 gene superfamilies, including 3 novel superfamilies and a novel cysteine framework (C-C-C-CCC-C-C) identified at both transcript and peptide levels. A surprisingly large number of conopeptide gene sequences were expressed at low levels, including a series of single amino acid variants, as well as sequences containing deletions and frame and stop codon shifts. Some of the toxin variants generate alternative cleavage sites, interrupted or elongated cysteine frameworks, and highly variable isoforms within families that could be identified at the peptide level. Together with the variable peptide processing identified previously, background genetic and phenotypic levels of biological messiness in venoms contribute to the hypervariability of venom peptides and their ability to evolve rapidly. Molecular & Cellular Proteomics 12:

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“…Table IV displays statistics on the gene superfamilies identified from 12 species of Conidae, including data from the recently reported venom duct transcriptomes of C. consors and C. pulicarius (17)(18)(19). Extensively studied mollusk-hunting species including C. marmoreus (this study), have a comparable distribution of transcripts across the different gene superfamilies, with gene superfamilies M, O1, and T dominating.…”

Section: Venomics Of Conus Marmoreusmentioning

confidence: 99%

“…However, recent reports showed the presence of Ͼ 1000 different peptides in a single venom using optimized liquid chromatography LC-MS approaches (15,16). Surprisingly, venom gland transcriptomes of several species have revealed a much more limited number of conopeptide genes (Ͻ 100) (17)(18)(19). This large discrepancy between the number of genes and the number of masses detected in the venom is currently not well understood.…”

mentioning

confidence: 99%

Deep Venomics Reveals the Mechanism for Expanded Peptide Diversity in Cone Snail Venom

Dutertre

Jin

Kaas

et al. 2013

Molecular & Cellular Proteomics

194

316

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Cone snails produce highly complex venom comprising mostly small biologically active peptides known as conotoxins or conopeptides. Early estimates that suggested 50 -200 venom peptides are produced per species have been recently increased at least 10-fold using advanced mass spectrometry. To uncover the mechanism(s) responsible for generating this impressive diversity, we used an integrated approach combining second-generation transcriptome sequencing with high sensitivity proteomics. From the venom gland transcriptome of Conus marmoreus, a total of 105 conopeptide precursor sequences from 13 gene superfamilies were identified. Over 60% of these precursors belonged to the three gene superfamilies O1, T, and M, consistent with their high levels of expression, which suggests these conotoxins play an important role in prey capture and/or defense. Seven gene superfamilies not previously identified in C. marmoreus, including five novel superfamilies, were also discovered. To confirm the expression of toxins identified at the transcript level, the injected venom of C. marmoreus was comprehensively analyzed by mass spectrometry, revealing 2710 and 3172 peptides using MALDI and ESI-MS, respectively, and 6254 peptides using an ESI-MS TripleTOF 5600 instrument. All conopeptides derived from transcriptomic sequences could be matched to masses obtained on the TripleTOF within 100 ppm accuracy, with 66 (63%) providing MS/MS coverage that unambiguously confirmed these matches. Comprehensive integration of transcriptomic and proteomic data revealed for the first time that the vast majority of the conopeptide diversity arises from a more limited set of genes through a process of variable peptide processing, which generates conopeptides with alternative cleavage sites, heterogeneous post-translational modifications, and highly variable N-and C-terminal truncations. Variable peptide processing is expected to contribute to the evolution of venoms, and explains how a limited set of ϳ 100 gene transcripts can generate thousands of conopeptides in a single species of cone snail. Molecular & Cellular

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Novel venom peptides from the cone snail Conus pulicarius discovered through next-generation sequencing of its venom duct transcriptome

Cited by 59 publications

References 29 publications

Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks

Optimized deep-targeted proteotranscriptomic profiling reveals unexplored Conus toxin diversity and novel cysteine frameworks

Transcriptomic Messiness in the Venom Duct of Conus miles Contributes to Conotoxin Diversity

Deep Venomics Reveals the Mechanism for Expanded Peptide Diversity in Cone Snail Venom

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