Uncovering intense protein diversification in a cone snail venom gland using an integrative venomics approach.

Marine cone snail venoms are highly complex mixtures of peptides and proteins. They have been studied in-depth over the past 3 decades, but the modus operandi of the venomous apparatus still remains unclear. Using the fish-hunting Conus consors as a model, we present an integrative venomics approach, based on new proteomic results from the venom gland and data previously obtained from the transcriptome and the injectable venom. We describe here the complete peptide content of the dissected venom by the identification of numerous new peptides using nanospray tandem mass spectrometry in combination with transcriptomic data. Results reveal extensive mature peptide diversification mechanisms at work in the venom gland. In addition, by integrating data from three different venom stages, transcriptome, dissected, and injectable venoms, from a single species, we obtain a global overview of the venom processing that occurs from the venom gland tissue to the venom delivery step. In the light of the successive steps in this venom production system, we demonstrate that each venom compartment is highly specific in terms of peptide and protein content. Moreover, the integrated investigative approach discussed here could become an essential part of pharmaceutical development, as it provides new potential drug candidates and opens the door to numerous analogues generated by the very mechanisms used by nature to diversify its peptide and protein arsenal.

δ-Conotoxins synthesized using an acid-cleavable solubility tag approach reveal key structural determinants for NaV subtype selectivity.

Conotoxins are venom peptides from cone snails with multiple disulfide bridges that provide a rigid structural scaffold. Typically acting on ion channels implicated in neurotransmission, conotoxins are of interest both as tools for pharmacological studies and as potential new medicines. δ-Conotoxins act by inhibiting inactivation of voltage-gated sodium channels (Nav). Their pharmacology has not been extensively studied because their highly hydrophobic character makes them difficult targets for chemical synthesis. Here we adopted an acid-cleavable solubility tag strategy that facilitated synthesis, purification, and directed disulfide bridge formation. Using this approach we readily produced three native δ-conotoxins from Conus consors plus two rationally designed hybrid peptides. We observed striking differences in Nav subtype selectivity across this group of compounds, which differ in primary structure at only three positions: 12, 23, and 25. Our results provide new insights into the structure-activity relationships underlying the Nav subtype selectivity of δ-conotoxins. Use of the acid-cleavable solubility tag strategy should facilitate synthesis of other hydrophobic peptides with complex disulfide bridge patterns.

Conus consors snail venom proteomics proposes functions, pathways, and novel families involved in its venomic system.

For some decades, cone snail venoms have been providing peptides, generally termed conopeptides, that exhibit a large diversity of pharmacological properties. However, little attention has been devoted to the high molecular mass (HMM) proteins in venoms of mollusks. In order to shed more light on cone snail venom HMM components, the proteins of dissected and injected venom of a fish-hunting cone snail, Conus consors, were extensively assessed. HMM venom proteins were separated by two-dimensional polyacrylamide gel electrophoresis and analyzed by mass spectrometry (MS). The MS data were interpreted using UniProt database, EST libraries from C. consors venom duct and salivary gland, and their genomic information. Numerous protein families were discovered in the lumen of the venom duct and assigned a biological function, thus pointing to their potential role in venom production and maturation. Interestingly, the study also revealed original proteins defining new families of unknown function. Only two groups of HMM proteins passing the venom selection process, echotoxins and hyaluronidases, were clearly present in the injected venom. They are suggested to contribute to the envenomation process. This newly devised integrated HMM proteomic analysis is a big step toward identification of the protein arsenal used in a cone snail venom apparatus for venom production, maturation, and function.

Recruitment of glycosyl hydrolase proteins in a cone snail venomous arsenal: further insights into biomolecular features of Conus venoms.

Cone snail venoms are considered an untapped reservoir of extremely diverse peptides, named conopeptides, displaying a wide array of pharmacological activities. We report here for the first time, the presence of high molecular weight compounds that participate in the envenomation cocktail used by these marine snails. Using a combination of proteomic and transcriptomic approaches, we identified glycosyl hydrolase proteins, of the hyaluronidase type (Hyal), from the dissected and injectable venoms ("injectable venom" stands for the venom variety obtained by milking of the snails. This is in contrast to the "dissected venom", which was obtained from dissected snails by extraction of the venom glands) of a fish-hunting cone snail, Conus consors (Pionoconus clade). The major Hyal isoform, Conohyal-Cn1, is expressed as a mixture of numerous glycosylated proteins in the 50 kDa molecular mass range, as observed in 2D gel and mass spectrometry analyses. Further proteomic analysis and venom duct mRNA sequencing allowed full sequence determination. Additionally, unambiguous segment location of at least three glycosylation sites could be determined, with glycans corresponding to multiple hexose (Hex) and N-acetylhexosamine (HexNAc) moieties. With respect to other known Hyals, Conohyal-Cn1 clearly belongs to the hydrolase-type of Hyals, with strictly conserved consensus catalytic donor and positioning residues. Potent biological activity of the native Conohyals could be confirmed in degrading hyaluronic acid. A similar Hyal sequence was also found in the venom duct transcriptome of C. adamsonii (Textilia clade), implying a possible widespread recruitment of this enzyme family in fish-hunting cone snail venoms. These results provide the first detailed Hyal sequence characterized from a cone snail venom, and to a larger extent in the Mollusca phylum, thus extending our knowledge on this protein family and its evolutionary selection in marine snail venoms.

Peak capacity optimisation for high resolution peptide profiling in complex mixtures by liquid chromatography coupled to time-of-flight mass spectrometry: application to the Conus consors cone snail venom.

The high resolution profiling of complex mixtures is indispensable for obtaining online structural information on the highest possible number of the analytes present. This is particularly relevant for natural extracts, as for the venom of the predatory marine snail Conus consors, which contains numerous bioactive peptides with molecular masses ranging between 1000 and 5000 Da. The goal of the present work was to maximise peak capacity of peptides separations by LC-MS while maintaining a reasonable analysis time. The best gradient performance using the C. consors venom as a real sample was obtained with a mobile phase flow rate as high as possible to maximise performance in the gradient mode, and gradient time comprised between 75 and 350 min when using a 150 mm column length. The present study also confirmed that an elevated temperature (up to 90 °C) improves performance under ultra-high pressure liquid chromatography (UHPLC) conditions. However, the thermal stability of the analytes had to be critically evaluated. For the profiling of C. consors, analyte degradation was not clearly observable at 90 °C with analysis times of approximately 100 min. Finally, the MS source was found to cause significant additional band broadening in the UHPLC mode (σ(ext)(2) was 10-24 times higher using TOF-MS vs. UV detection). Thus, if the MS contributes strongly to the peak capacity loss, classical 2.1mm I.D. columns can be replaced by 3.0mm I.D. to mitigate this problem. Based on these considerations, the optimal generic profiling conditions applied to the C. consors venom provided a peak capacity higher than 1100 for a gradient time of around 100 min, doubling the values reached by classical HPLC separation. UHPLC-QTOF-MS/MS experiments carried out in these conditions provided exploitable data that matched with peptides present in the C. consors venom. These optimal LC conditions are thus compatible with online peptide deconvolution and matching against transcriptomic data and, to some extent, de novo sequencing in such complex mixtures.

Large-scale discovery of conopeptides and conoproteins in the injectable venom of a fish-hunting cone snail using a combined proteomic and transcriptomic approach.

Predatory marine snails of the genus Conus use venom containing a complex mixture of bioactive peptides to subdue their prey. Here we report on a comprehensive analysis of the protein content of injectable venom from Conus consors, an indo-pacific fish-hunting cone snail. By matching MS/MS data against an extensive set of venom gland transcriptomic mRNA sequences, we identified 105 components out of ~400 molecular masses detected in the venom. Among them, we described new conotoxins belonging to the A, M- and O1-superfamilies as well as a novel superfamily of disulphide free conopeptides. A high proportion of the deduced sequences (36%) corresponded to propeptide regions of the A- and M-superfamilies, raising the question of their putative role in injectable venom. Enzymatic digestion of higher molecular mass components allowed the identification of new conkunitzins (~7 kDa) and two proteins in the 25 and 50 kDa molecular mass ranges respectively characterised as actinoporin-like and hyaluronidase-like protein. These results provide the most exhaustive and accurate proteomic overview of an injectable cone snail venom to date, and delineate the major protein families present in the delivered venom. This study demonstrates the feasibility of this analytical approach and paves the way for transcriptomics-assisted strategies in drug discovery.

High-resolution picture of a venom gland transcriptome: case study with the marine snail Conus consors.

Although cone snail venoms have been intensively investigated in the past few decades, little is known about the whole conopeptide and protein content in venom ducts, especially at the transcriptomic level. If most of the previous studies focusing on a limited number of sequences have contributed to a better understanding of conopeptide superfamilies, they did not give access to a complete panorama of a whole venom duct. Additionally, rare transcripts were usually not identified due to sampling effect. This work presents the data and analysis of a large number of sequences obtained from high throughput 454 sequencing technology using venom ducts of Conus consors, an Indo-Pacific living piscivorous cone snail. A total of 213,561 Expressed Sequence Tags (ESTs) with an average read length of 218 base pairs (bp) have been obtained. These reads were assembled into 65,536 contiguous DNA sequences (contigs) then into 5039 clusters. The data revealed 11 conopeptide superfamilies representing a total of 53 new isoforms (full length or nearly full-length sequences). Considerable isoform diversity and major differences in transcription level could be noted between superfamilies. A, O and M superfamilies are the most diverse. The A family isoforms account for more than 70% of the conopeptide cocktail (considering all ESTs before clustering step). In addition to traditional superfamilies and families, minor transcripts including both cysteine free and cysteine-rich peptides could be detected, some of them figuring new clades of conopeptides. Finally, several sets of transcripts corresponding to proteins commonly recruited in venom function could be identified for the first time in cone snail venom duct. This work provides one of the first large-scale EST project for a cone snail venom duct using next-generation sequencing, allowing a detailed overview of the venom duct transcripts. This leads to an expanded definition of the overall cone snail venom duct transcriptomic activity, which goes beyond the cysteine-rich conopeptides. For instance, this study enabled to detect proteins involved in common post-translational maturation and folding, and to reveal compounds classically involved in hemolysis and mechanical penetration of the venom into the prey. Further comparison with proteomic and genomic data will lead to a better understanding of conopeptides diversity and the underlying mechanisms involved in conopeptide evolution.

Dramatic intraspecimen variations within the injected venom of Conus consors: an unsuspected contribution to venom diversity.

With the advent of highly sensitive mass spectrometry techniques, the minute amount of various secretions produced by living animals can be studied to a level of details never attained before. In this study, we used LC-ESI-MS to analyse the injected venom of an indo-pacific piscivorous cone snail, Conus consors. While long-term follow up of several captive specimens have revealed a typical "venom fingerprint" for this species, dramatic variations were also observed. In the most extreme case, a single cone snail unexpectedly produced two very distinct venom profiles containing completely different sets of peptides with no overlap of detected masses. Surprisingly, there was no correlation between the peptides produced in the venom duct and those obtained after milking live cone snails, implying yet unknown mechanisms of selection and regulation. Our study defines the notion of intraspecimen variation and demonstrates how this phenomenon contributes to the overall venom diversity.

Comparative proteomic study of the venom of the piscivorous cone snail Conus consors.

In the context of an exhaustive study of the piscivorous cone snail Conus consors, we performed an in-depth analysis of the intact molecular masses that can be detected in the animal's venom, using MALDI and ESI mass spectrometry. We clearly demonstrated that, for the venom of this species at least, it is essential to use both techniques in order to obtain the broadest data set of molecular masses. Only 20% of the total number of molecules detected were found in both mass lists. The two data sets were also compared in terms of mass range and relative hydrophobicity of the components detected in each. With a view to an extensive analysis of this venom's proteome, we further performed a comparative study by ESI-MS between venom obtained after classical dissection of the venom duct versus venom obtained by milking live animals. Surprisingly, although many fewer components were found in the milked venom than in the dissected venom, approximately 50% of those found had not been seen in the dissected venom. Several questions raised by these observations are discussed. With regards to the current knowledge of the cone snail venom composition, our results emphasize the complementary nature of the mass spectrometry methods and of the two techniques used in venom collection.