Conotoxin Diversity in Chelyconus ermineus (Born, 1778) and the Convergent Origin of Piscivory in the Atlantic and Indo-Pacific Cones

The transcriptome of the venom duct of the Atlantic piscivorous cone species Chelyconus ermineus (Born, 1778) was determined. The venom repertoire of this species includes at least 378 conotoxin precursors, which could be ascribed to 33 known and 22 new (unassigned) protein superfamilies, respectively.Most abundant superfamilies were T,W, O1, M, O2, and Z, accounting for 57% of all detected diversity. A total of three individuals were sequenced showing considerable intraspecific variation: each individual had many exclusive conotoxin precursors, and only 20% of all inferred mature peptides were common to all individuals. Three different regions (distal, medium, and proximal with respect to the venom bulb) of the venom duct were analyzed independently. Diversity (in terms of number of distinct members) of conotoxin precursor superfamilies increased toward the distal region whereas transcripts detected toward the proximal region showed higher expression levels. Only the superfamilies A and I3 showed statistically significant differential expression across regions of the venom duct. Sequences belonging to the alpha (motor cabal) and kappa (lightning-strike cabal) subfamilies of the superfamily A were mainly detected in the proximal region of the venom duct. The mature peptides of the alpha subfamily had the a4/4 cysteine spacing pattern, which has been shown to selectively target muscle nicotinic-acetylcholine receptors, ultimately producing paralysis. This function is performed by mature peptides having a a3/5 cysteine spacing pattern in piscivorous cone species from the Indo-Pacific region, thereby supporting a convergent evolution of piscivory in cones

Évolution de la suite logicielle FORMID pour la conception, l'exécution et le suivi de situations d'apprentissage à distance

This paper describes the work done in the internship of CNAM engineer cycle. This one was held in MeTAH team Grenoble Informatics Laboratory, around the FORMID suite. This suite consists of three modules which are dedicated to the design, implementation and monitoring of distance learning situations. It is described the choice of technological change, resulting in a migration of Java applets to a client-server architecture using the latest web technologies. In addition to the correction of anomalies related to security and application compatibility, it describes the implementation of FORMID industrialization and internationalization. Finally, the paper describes the modeling and implementation of a new tracking system with indicators of quality and the design module extension and its connection to an ontology of knowledge representation .Ce document retrace le travail effectué dans le cadre du stage de fin de cycle d’ingénieur CNAM. Ce stage s’est déroulé dans l’équipe MeTAH du Laboratoire Informatique de Grenoble, autour de la suite logicielle FORMID. Cette suite composée de trois modules est dédiée à la conception, l’exécution et le suivi de situations d’apprentissage à distance. Il y est décrit les choix de changements technologiques, entraînant une migration d’applets Java vers une architecture client-serveur utilisant les dernières technologies web. En plus de la correction des anomalies liées à la sécurité et à la compatibilité de l’application, il relate la mise en œuvre des moyens d’industrialisation et d’internationalisation de la suite FORMID. Pour finir, le document décrit la modélisation et l’implémentation d’un nouveau système de traçage avec des indicateurs de qualité et l’extension du module de conception ainsi que sa connexion à une ontologie de représentation des connaissances

Développement d'outils analytiques basés sur la Spectrométrie de Massepour la caractérisation de venins animaux

Animal venoms and toxins are now recognized as major sources of bioactive molecules that may be tomorrow’s new drug leads. Their complexity and their potential as drug sources have been demonstrated by application of modern analytical technologies, which have revealed venoms to be vast peptide combinatorial libraries. Structural as well as pharmacological diversity is immense, and mass spectrometry is now one of the major investigative tools for the structural investigation of venom components. The present work is dedicated to the development of new mass spectrometry-based methodologies for the study of animal venoms. The first methodology depicts an original approach to sequence snake toxins in a higher throughput way after an orthogonal separation of the crude venom. The second analytical development is devoted to decipher disulfide connectivity in cone snails’ toxins through a combination of partial reduction/oxidation followed by ion mobility separation of the semi-reduced/oxidized species and CID fragmentation. The last part of this work is focused on the capture of ligands of nAChRs in complex mixtures of peptides, especially in cone snail venoms. A new ligand has been discovered and fully characterized

Réarrangement de ponts disulfures dans les peptides partiellement réduit et alkylés révélé par spectrométrie de mobilité ionique et de masse.

Animal venoms are mainly composed of peptide toxins, which are highly structured by many disulfide bridges. In these toxins, disulfides play different major roles such as increasing the toxins efficiency by lowering their immunogenicity or providing the adequate conformation to efficiently bind to the biological receptor. Peptide sequencing followed by determination of the cysteine pairings is still challenging and, therefore, an important step in structural analysis. This work was, in its beginning, focused on the development of ion mobility (IMS) based methodology used to assign disulfides. The strategy relies on the analysis of partially reduced/alkylated disulfide containing peptides. The resulting mixture is analyzed by ion mobility, followed by MS/MS acquisition on each mobility resolved species. Surprisingly, first investigations revealed, after partial reduction, a disulfide rearrangement phenomenon. Indeed, some of the cystein pairings were not those expected to be. These experiments were conducted on ¿-CnI and ¿-GI toxins purified from the venoms of Conus consors and Conus geographus marine snails, respectively. Each toxin contains four cysteines linked together with two disulfide bridges. Peptides were partially reduced by an excess of dithiothreitol and then alkylated by a large excess of iodoacetamide. The resulting mixture was purified on a microcolumn before being analyzed by nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Each toxin partially reduced/alkylated results, theoretically, in a mixture of fully oxidized (two disulfides oxidized), fully reduced (two disulfides reduced) and partially reduced forms (one of the two disulfides reduced). Thanks to the mass shift created by the alkylation, an isolation of the species which m/z ratio corresponds to one disulfide reduced and alkylated has been done in the quadrupole before the mobility separation. The arrival time distribution of triply charged ions reveals the presence of different species (4 in the case of ¿-GI and 2 for ¿-CnI), characterized by different relative cross sections in the gas-phase. As ion mobility resolved species give characteristic fragments upon fragmentation (after IMS), we were able to identify a scrambling of the disulfides (isomerization). In simple words, other disulfide bonds than expected ones were characterized. We suppose that the scrambling phenomenon occurs in solution,during the reduction step, since the alkylation cannot avoid rearrangement. The method is now being applied to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications

Assignement des ponts disulfures et caractérisation du repliement de toxines peptidiques par Spectrométrie de mobilité ionique couplée à la Spectrométrie de Masse

Main component of animal venoms is peptide toxins, which are highly structured by several disulfide bridges. Disulfide bridges fill different roles as increasing the toxins efficiency by lowering their immunogenicity or providing the adequate conformation to efficiently bind to the biological receptor. The sequencing and the determination of the cysteine pairing is still challenging and therefore an important step in structural analysis. In this work, we present a new strategy to sequence structured toxins and assign S-S bridges using ion mobility resolved MS/MS. The method relies on the analysis of partially reduced multiple-disulfide peptide. The mixture of the different forms is resolved by ion mobility, followed by MS/MS acquisition on each mobility separated species. The proof of concept has been successfully conducted on α-CnI, a toxin purified from the venom of Conus consors marine snail. The toxin’s sequence contains four cysteines linked together with two disulfide bridges. α-CnI was partially reduced by a small excess of tris(carboxyethyl)phosphine (10:1). The resulting mixture was purified before analysis by infusion nanoESI-Synapt-G2. Fragmentation was performed after the mobility cell, to obtain specific fragments of each species. Partial reduction of α-CnI results in a mixture of oxidized (the two disulfides are formed), reduced (the two disulfides have been reduced) and partially reduced forms (one of the two disulfides has been reduced). The arrival time distribution of triply charged ions reveals the presence of 4 different species, characterized by different relative cross sections in the gas-phase. Mass matching allows identifying the species: the first mobility (the most compact structure) was identified to be the oxidized folded toxin (M). The latest peak, corresponding to the larger cross-section, was identified as the fully reduced toxin (M+4Da). The second and the third mobility peaks were attributed to the two partially reduced forms in which only one disulfide bridge was reduced (M+2Da). The change in ion mobility depends on which S-S bridge is reduced. Ion mobility separated species give characteristic fragment ions upon fragmentation in the transfer cell (i.e. after ion mobility separator). Interestingly, fragment ions coming from partially reduced species, especially the C-S or S-S bond cleavages, clearly indicates that the disulfide linkage of α-CnI is (Cys1-Cys3) and (Cys2-Cys4) as expected from literature. The method is now being applied with success to more complex systems containing 3 or 4 disulfide bridges. The influence of the charge state on the mobility separation is systematically analyzed in terms of structural implications

Ion-Mobility mass spectrometry as a potential tool to assign disulfide bonds arrangements in peptides with multiple disulfide bridges.

Disulfide bridges play a major role in defining the structural properties of peptides and proteins. However, the determination of the cysteine pairing is still challenging. Peptide sequences are usually achieved using MS/MS spectra of the totally reduced unfolded species but the cysteine pairing information is lost. On the other hand, MS/MS experiments performed on native folded species show complex spectra composed of non-classical ions. MS/MS alone does not allow the cysteine pairing nor the full sequence of an unknown peptide to be determined. The major goal of this work is to set up a strategy for the full structural characterization of peptides including disulfide bridges annotation in the sequence. This strategy was developed by combining Ion Mobility Spectrometry (IMS)and Collision Induced Dissociation(CID). It is assumed that the opening of one S-S bridges in a peptide leads to a structural evolution which results in a modification of IMS drift time. In the presence of multiple S-S bridges, the shift in arrival time will depend on which disulfide(s) has (have) been reduced and on the shape adopted by the generated species. Due to specific fragmentations observed for each species, CID experiments performed after the mobility separation could provide not only information on peptide sequence, but also on the localization of the disulfide bridges. To achieve this goal, synthetic peptides containing two disulfides were studied. The openings of the bridges were carried out following different experimental conditions such as reduction, reduction/alkylation or oxidation. Due to disulfide scrambling highlighted with the reduction approaches, oxidation of S-S bonds into cysteic acids appeared to be the best strategy. Cysteines connectivity was then unambiguously determined for the two peptides, without any disulfide scrambling interference