Philippe Favreau

Genetic and Ecological Correlates of Intraspecific Variation in Pitviper Venom Composition Detected Using Matrix-Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) and Isoelectric Focusing

The ability to detect biochemical diversity in animal venoms has wide-ranging implications for a diverse array of scientific disciplines. Matrix-assisted laser desorption time-of-flight mass spectrometry (and, for comparative purposes, isoelectric focusing) were used to characterize venoms from a geographically diverse sample of Trimeresurus stejnegeri (n < 229) from Taiwan. Previously unrealised levels of heterogeneity were detected in venom phospholipase A2 isoforms (PLA2) and in whole venom profiles. Geographic variation in venom was primarily between Taiwan and two Pacific islets. Despite the common assumption that venom variation is a product of neutral molecular evolution, statistical testing failed to link venom variation with phylogenetic descent convincingly. Instead, pronounced differences in venom composition may be the product of natural selection for regional diets or of independent founder effects. More data are required on the functional differences between the isoforms to distinguish between these alternatives.

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.

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 animals 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 venoms 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.

Molecular phylogeny, classification and evolution of conopeptides.

Conopeptides are toxins expressed in the venom duct of cone snails (Conoidea, Conus). These are mostly well-structured peptides and mini-proteins with high potency and selectivity for a broad range of cellular targets. In view of these properties, they are widely used as pharmacological tools and many are candidates for innovative drugs. The conopeptides are primarily classified into superfamilies according to their peptide signal sequence, a classification that is thought to reflect the evolution of the multigenic system. However, this hypothesis has never been thoroughly tested. Here we present a phylogenetic analysis of 1,364 conopeptide signal sequences extracted from GenBank. The results validate the current conopeptide superfamily classification, but also reveal several important new features. The so-called “cysteine-poor” conopeptides are revealed to be closely related to “cysteine-rich” conopeptides; with some of them sharing very similar signal sequences, suggesting that a distinction based on cysteine content and configuration is not phylogenetically relevant and does not reflect the evolutionary history of conopeptides. A given cysteine pattern or pharmacological activity can be found across different superfamilies. Furthermore, a few conopeptides from GenBank do not cluster in any of the known superfamilies, and could represent yet-undefined superfamilies. A clear phylogenetically based classification should help to disentangle the diversity of conopeptides, and could also serve as a rationale to understand the evolution of the toxins in the numerous other species of conoideans and venomous animals at large.

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.

Engineering Potent and Selective Analogues of GpTx-1, a Tarantula Venom Peptide Antagonist of the NaV1.7 Sodium Channel

NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34-residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20× and 1000× over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp(29), Lys(31), and Phe(34) near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred 300-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000× selectivity against NaV1.4 and NaV1.5.

Marine snail venoms: use and trends in receptor and channel neuropharmacology

Venoms are rich mixtures of mainly peptides and proteins evolved by nature to catch and digest preys or for protection against predators. They represent extensive sources of potent and selective bioactive compounds that can lead to original active ingredients, for use as drugs, as pharmacological tools in research and for the biotechnology industry. Among the most fascinating venomous animals, marine snails offer a unique set of pharmacologically active components, targeting a wide diversity of receptors and ion channels. Recent advances still continue to demonstrate their huge neuropharmacological potential. In the quest for interesting pharmacological profiles, researchers face a vast number of venom components to investigate within time and technological constraints. A brief perspective on marine snail venoms complexity and features is given followed by the different discovery strategies and pharmacological approaches, exemplified with some recent developments. These advances will hopefully help further uncovering new pharmacologically important venom molecules.

High throughput screening of bradykinin-potentiating peptides in Bothrops moojeni snake venom using precursor ion mass spectrometry

Snake venoms are known to be an extensive source of bioactive peptides. Bradykinin-potentiating peptides (BPPs) are inhibitors of the angiotensin-converting enzyme that have already been identified in the venom of many snake, scorpion, spider and batrachian species. Their most characteristic structural features are an invariable N-terminal pyroglutamate residue (pGlu or Z) and two consecutive proline residues at the C-terminus. Fragmentation of BPPs by collision-induced dissociation during electrospray tandem mass spectrometry analysis (ESI-MS/MS) generates a predominant signal at m/z 213.1 corresponding to the y-ion of the terminal Pro-Pro fragment. In addition, signals at m/z 226.1 and 240.1 that correspond to the b ions of the N-terminus pGlu-Asn and pGlu-Lys, respectively, can often be observed. Based on these structural determinants, the present work describes an original methodology for the discovery of BPPs in natural extracts using liquid chromatography coupled to ESI-MS/MS operated in precursor ion-scan mode. The venom of the Bothrops moojeni snake was used as a model and the methodology was applied for subsequent structural analysis of the identified precursors by tandem mass spectrometry on quadrupole-time-of-flight (Q-TOF) and matrix-assisted laser-desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS) instruments. More than 40 peptides below 2500 Da could be detected, among them 20 were shown to belong to the BPP-like family including the related tripeptides pGlu-Lys-Trp and pGlu-Asn-Trp. A total of 15 new sequences have been identified using this approach.

A new conotoxin isolated from Conus consors venom acting selectively on axons and motor nerve terminals through a Na+‐dependent mechanism

A novel conotoxin was isolated and characterized from the venom of the fish‐hunting marine snail Conus consors. The peptide was identified by screening chromatography fractions of the crude venom that produced a marked contraction and extension of the caudal and dorsal fins in fish, and noticeable spontaneous contractions of isolated frog neuromuscular preparations. The peptide, named CcTX, had 30 amino acids and the following scaffold: X11CCX7CX2CXCX3C. At the frog neuromuscular junction, CcTx at nanomolar concentrations selectively increased nerve terminal excitability so that a single nerve stimulation triggered trains of repetitive or spontaneous synaptic potentials and action potentials. In contrast, CcTx had no noticeable effect on muscle excitability even at concentrations 100 × higher than those that affected motor nerve terminals, as revealed by direct muscle stimulation. In addition, CcTx increased miniature endplate potential (MEPP) frequency in a Ca2+‐free medium supplemented with ethylene glycol‐bis‐(β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid (EGTA). Blockade of voltage‐dependent sodium channels with tetrodotoxin (TTX) either prevented or suppressed the increase of MEPP frequency induced by the toxin. CcTx also produced a TTX‐sensitive depolarization of the nodal membrane in single myelinated axons giving rise, in some cases, to repetitive and/or spontaneous action potential discharges. In addition, CcTx increased the nodal volume of myelinated axons, as determined using confocal laser scanning microscopy. This increase was reversed by external hyperosmolar solutions and was prevented by pretreatment of axons with TTX. It is suggested that CcTx, by specifically activating neuronal voltage‐gated sodium channels at the resting membrane potential, produced Na+ entry into nerve terminals and axons without directly affecting skeletal muscle fibres. CcTx belongs to a novel family of conotoxins that targets neuronal voltage‐gated sodium channels.

A novel µ-conopeptide, CnIIIC, exerts potent and preferential inhibition of NaV1.2/1.4 channels and blocks neuronal nicotinic acetylcholine receptors.

The µ‐conopeptide family is defined by its ability to block voltage‐gated sodium channels (VGSCs), a property that can be used for the development of myorelaxants and analgesics. We characterized the pharmacology of a new µ‐conopeptide (µ‐CnIIIC) on a range of preparations and molecular targets to assess its potential as a myorelaxant.