Publications by authors named "Nicolas Gilles"

56 Publications

A snake toxin as a theranostic agent for the type 2 vasopressin receptor.

Theranostics 2020 18;10(25):11580-11594. Epub 2020 Sep 18.

Université Paris Saclay, CEA, INRAE, Département Médicaments et Technologies pour la Santé (DMTS), SIMoS, 91191 Gif-sur-Yvette, France.

MQ1, a snake toxin which targets with high nanomolar affinity and absolute selectivity for the type 2 vasopressin receptor (V2R), is a drug candidate for renal diseases and a molecular probe for imaging cells or organs expressing V2R. MQ1's pharmacological properties were characterized and applied to a rat model of hyponatremia. Its PK/PD parameters were determined as well as its therapeutic index. Fluorescently and radioactively labeled MQ1 were chemically synthesized and associated with moderate loss of affinity. MQ1's dynamic biodistribution was monitored by positron emission tomography. Confocal imaging was used to observe the labeling of three cancer cell lines. The inverse agonist property of MQ1 very efficiently prevented dDAVP-induced hyponatremia in rats with low nanomolar/kg doses and with a very large therapeutic index. PK (plasma MQ1 concentrations) and PD (diuresis) exhibited a parallel biphasic decrease. The dynamic biodistribution showed that MQ1 targets the kidneys and then exhibits a blood and kidney biphasic decrease. Whatever the approach used, we found a T1/2α between 0.9 and 3.8 h and a T1/2β between 25 and 46 h and demonstrated that the kidneys were able to retain MQ1. Finally, the presence of functional V2R expressed at the membrane of cancer cells was, for the first time, demonstrated with a specific fluorescent ligand. As the most selective V2 binder, MQ1 is a new promising drug for aquaresis-related diseases and a molecular probe to visualize and V2R expressed physiologically or under pathological conditions.
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http://dx.doi.org/10.7150/thno.47485DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7545998PMC
September 2020

A Venomics Approach Coupled to High-Throughput Toxin Production Strategies Identifies the First Venom-Derived Melanocortin Receptor Agonists.

J Med Chem 2020 08 16;63(15):8250-8264. Epub 2020 Jul 16.

Centre National de la Recherche Scientifique, Architecture et Fonction des Macromolécules Biologiques, Campus de Luminy, Marseille 13288 France.

Animal venoms are rich in hundreds of toxins with extraordinary biological activities. Their exploitation is difficult due to their complexity and the small quantities of venom available from most venomous species. We developed a Venomics approach combining transcriptomic and proteomic characterization of 191 species and identified 20,206 venom toxin sequences. Two complementary production strategies based on solid-phase synthesis and recombinant expression in generated a physical bank of 3597 toxins. Screened on hMC4R, this bank gave an incredible hit rate of 8%. Here, we focus on two novel toxins: N-TRTX-Preg1a, exhibiting an inhibitory cystine knot (ICK) motif, and N-BUTX-Ptr1a, a short scorpion-CSαβ structure. Neither N-TRTX-Preg1a nor N-BUTX-Ptr1a affects ion channels, the known targets of their toxin scaffolds, but binds to four melanocortin receptors with low micromolar affinities and activates the hMC1R/Gs pathway. Phylogenetically, these two toxins form new groups within their respective families and represent novel hMC1R agonists, structurally unrelated to the natural agonists.
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http://dx.doi.org/10.1021/acs.jmedchem.0c00485DOI Listing
August 2020

Can IM-MS Collision Cross Sections of Biomolecules Be Rationalized Using Collision Cross-Section Trends of Polydisperse Synthetic Homopolymers?

J Am Soc Mass Spectrom 2020 Apr 26;31(4):990-995. Epub 2020 Feb 26.

Mass Spectrometry Laboratory, University of Liège, MolSys Research unit, Quartier Agora, Allée du Six Aout 11, B-4000 Liège, Belgium.

In the past, we developed a method inferring physicochemical properties from ion mobility mass spectrometry (IM-MS) data from polydisperse synthetic homopolymers. We extend here the method to biomolecules that are generally monodisperse. Similarities in the IM-MS behavior were illustrated on proteins and peptides. This allows one to identify ionic species for which intramolecular interactions lead to specific structures.
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http://dx.doi.org/10.1021/jasms.9b00106DOI Listing
April 2020

Combination of Capillary Zone Electrophoresis-Mass Spectrometry, Ion Mobility-Mass Spectrometry, and Theoretical Calculations for Cysteine Connectivity Identification in Peptides Bearing Two Intramolecular Disulfide Bonds.

Anal Chem 2020 02 13;92(3):2425-2434. Epub 2020 Jan 13.

Mass Spectrometry Laboratory, MolSys Research Unit, Quartier Agora , University of Liège , Allée du Six Août 11 , B-4000 Liège , Belgium.

Disulfide bonds between cysteine residues are commonly involved in the stability of numerous peptides and proteins and are crucial for providing biological activities. In such peptides, the appropriate cysteine connectivity ensures the proper conformation allowing an efficient binding to their molecular targets. Disulfide bond connectivity characterization is still challenging and is a critical issue in the analysis of structured peptides/proteins targeting pharmaceutical or pharmacological utilizations. This study describes the development of new and fast gas-phase and in-solution electrophoretic methods coupled to mass spectrometry to characterize the cysteine connectivity of disulfide bonds. For this purpose, disulfide isomers of three peptides bearing two intramolecular disulfide bonds but different cysteine connectivity have been investigated. Capillary zone electrophoresis and ion mobility both coupled to mass spectrometry were used to perform the separation in both aqueous and gas phases, respectively. The separation efficiency of each technique has been critically evaluated and compared. Finally, theoretical calculations were performed to support and explain the experimental data based on the predicted physicochemical properties of the different peptides.
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http://dx.doi.org/10.1021/acs.analchem.9b03206DOI Listing
February 2020

Evaluation of the Spider ( genus) Phlotoxin 1 and Synthetic Variants as Antinociceptive Drug Candidates.

Toxins (Basel) 2019 08 22;11(9). Epub 2019 Aug 22.

Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), CEA, Université Paris-Saclay, F-91191 Gif sur Yvette, France.

Over the two last decades, venom toxins have been explored as alternatives to opioids to treat chronic debilitating pain. At present, approximately 20 potential analgesic toxins, mainly from spider venoms, are known to inhibit with high affinity the Na1.7 subtype of voltage-gated sodium (Na) channels, the most promising genetically validated antinociceptive target identified so far. The present study aimed to consolidate the development of phlotoxin 1 (PhlTx1), a 34-amino acid and 3-disulfide bridge peptide of a genus spider, as an antinociceptive agent by improving its affinity and selectivity for the human (h) Na1.7 subtype. The synthetic homologue of PhlTx1 was generated and equilibrated between two conformers on reverse-phase liquid chromatography and exhibited potent analgesic effects in a mouse model of Na1.7-mediated pain. The effects of PhlTx1 and 8 successfully synthetized alanine-substituted variants were studied (by automated whole-cell patch-clamp electrophysiology) on cell lines stably overexpressing hNa subtypes, as well as two cardiac targets, the hCa1.2 and hK11.1 subtypes of voltage-gated calcium (Ca) and potassium (K) channels, respectively. PhlTx1 and D7A-PhlTx1 were shown to inhibit hNa1.1-1.3 and 1.5-1.7 subtypes at hundred nanomolar concentrations, while their affinities for hNa1.4 and 1.8, hCa1.2 and hK11.1 subtypes were over micromolar concentrations. Despite similar analgesic effects in the mouse model of Na1.7-mediated pain and selectivity profiles, the affinity of D7A-PhlTx1 for the Na1.7 subtype was at least five times higher than that of the wild-type peptide. Computational modelling was performed to deduce the 3D-structure of PhlTx1 and to suggest the amino acids involved in the efficiency of the molecule. In conclusion, the present structure-activity relationship study of PhlTx1 results in a low improved affinity of the molecule for the Na1.7 subtype, but without any marked change in the molecule selectivity against the other studied ion channel subtypes. Further experiments are therefore necessary before considering the development of PhlTx1 or synthetic variants as antinociceptive drug candidates.
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http://dx.doi.org/10.3390/toxins11090484DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6784069PMC
August 2019

High-Throughput Production of Oxidized Animal Toxins in Escherichia coli.

Methods Mol Biol 2019 ;2025:165-190

Architecture et Fonction des Macromolécules Biologiques (AFMB), Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS), Aix-Marseille Université, Marseille cedex 9, France.

High-throughput production (HTP) of synthetic genes is becoming an important tool to explore the biological function of the extensive genomic and meta-genomic information currently available from various sources. One such source is animal venom, which contains thousands of novel bioactive peptides with potential uses as novel therapeutics to treat a plethora of diseases as well as in environmentally benign bioinsecticide formulations. Here, we describe a HTP platform for recombinant bacterial production of oxidized disulfide-rich proteins and peptides from animal venoms. High-throughput, host-optimized, gene synthesis and subcloning, combined with robust HTP expression and purification protocols, generate a semiautomated pipeline for the accelerated production of proteins and peptides identified from genomic or transcriptomic libraries. The platform has been applied to the production of thousands of animal venom peptide toxins for the purposes of drug discovery, but has the power to be universally applicable for high-level production of various and diverse target proteins in soluble form. This chapter details the HTP protocol for gene synthesis and production, which supported high levels of peptide expression in the E. coli periplasm using a cleavable DsbC fusion. Finally, target proteins and peptides are purified using automated HTP methods, before undergoing quality control and screening.
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http://dx.doi.org/10.1007/978-1-4939-9624-7_7DOI Listing
March 2020

Disulfide Connectivity Analysis of Peptides Bearing Two Intramolecular Disulfide Bonds Using MALDI In-Source Decay.

J Am Soc Mass Spectrom 2018 Oct 9;29(10):1995-2002. Epub 2018 Jul 9.

Mass Spectrometry Laboratory, MolSys Research Unit, University of Liège, Quartier Agora, Allée du six Aout 11, B-4000, Liege, Belgium.

Disulfide connectivity in peptides bearing at least two intramolecular disulfide bonds is highly important for the structure and the biological activity of the peptides. In that context, analytical strategies allowing a characterization of the cysteine pairing are of prime interest for chemists, biochemists, and biologists. For that purpose, this study evaluates the potential of MALDI in-source decay (ISD) for characterizing cysteine pairs through the systematic analysis of identical peptides bearing two disulfide bonds, but not the same cysteine connectivity. Three different matrices have been tested in positive and/or in negative mode (1,5-DAN, 2-AB and 2-AA). As MALDI-ISD is known to partially reduce disulfide bonds, the data analysis of this study rests firstly on the deconvolution of the isotope pattern of the parent ions. Moreover, data analysis is also based on the formed fragment ions and their signal intensities. Results from MS/MS-experiments (MALDI-ISD-MS/MS) constitute the last reference for data interpretation. Owing to the combined use of different ISD-promoting matrices, cysteine connectivity identification could be performed on the considered peptides. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-018-2022-yDOI Listing
October 2018

In-Depth Glyco-Peptidomics Approach Reveals Unexpected Diversity of Glycosylated Peptides and Atypical Post-Translational Modifications in Dendroaspis angusticeps Snake Venom.

Int J Mol Sci 2017 Nov 18;18(11). Epub 2017 Nov 18.

Mass Spectrometry Laboratory, MolSys Research Unit, University of Liege, 4000 Liege, Belgium.

Animal venoms represent a valuable source of bioactive peptides that can be derived into useful pharmacological tools, or even innovative drugs. In this way, the venom of (DA), the Eastern Green Mamba, has been intensively studied during recent years. It mainly contains hundreds of large toxins from 6 to 9 kDa, each displaying several disulfide bridges. These toxins are the main target of venom-based studies due to their valuable activities obtained by selectively targeting membrane receptors, such as ion channels or G-protein coupled receptors. This study aims to demonstrate that the knowledge of venom composition is still limited and that animal venoms contain unexpected diversity and surprises. A previous study has shown that venom contains not only a cocktail of classical toxins, but also small glycosylated peptides. Following this work, a deep exploration of DA glycopeptidome by a dual nano liquid chromatography coupled to electrospray ionization mass spectrometry (nanoLC-ESI-MS) and Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analyses was initiated. This study reveals unsuspected structural diversity of compounds such as 221 glycopeptides, displaying different glycan structures. Sequence alignments underline structural similarities with natriuretic peptides already characterized in venoms. Finally, the presence of an -cysteinylation and hydroxylation of proline on four glycopeptides, never described to date in snake venoms, is also revealed by proteomics and affined by nuclear magnetic resonance (NMR) experiments.
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http://dx.doi.org/10.3390/ijms18112453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5713420PMC
November 2017

Green mamba peptide targets type-2 vasopressin receptor against polycystic kidney disease.

Proc Natl Acad Sci U S A 2017 07 19;114(27):7154-7159. Epub 2017 Jun 19.

Service d'Ingénierie Moléculaire des Protéines, Institut des Sciences du Vivant Frédéric Joliot, Commissariat à l'Energie Atomique, Université Paris-Saclay, F-91191 Gif sur Yvette, France;

Polycystic kidney diseases (PKDs) are genetic disorders that can cause renal failure and death in children and adults. Lowering cAMP in cystic tissues through the inhibition of the type-2 vasopressin receptor (V2R) constitutes a validated strategy to reduce disease progression. We identified a peptide from green mamba venom that exhibits nanomolar affinity for the V2R without any activity on 155 other G-protein-coupled receptors or on 15 ionic channels. Mambaquaretin-1 is a full antagonist of the V2R activation pathways studied: cAMP production, beta-arrestin interaction, and MAP kinase activity. This peptide adopts the Kunitz fold known to mostly act on potassium channels and serine proteases. Mambaquaretin-1 interacts selectively with the V2R through its first loop, in the same manner that aprotinin inhibits trypsin. Injected in mice, mambaquaretin-1 increases in a dose-dependent manner urine outflow with concomitant reduction of urine osmolality, indicating a purely aquaretic effect associated with the in vivo blockade of V2R. CD1-pcy/pcy mice, a juvenile model of PKD, daily treated with 13 [Formula: see text]g of mambaquaretin-1 for 99 d, developed less abundant (by 33%) and smaller (by 47%) cysts than control mice. Neither tachyphylaxis nor apparent toxicity has been noted. Mambaquaretin-1 represents a promising therapeutic agent against PKDs.
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http://dx.doi.org/10.1073/pnas.1620454114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5502595PMC
July 2017

Ancestral protein resurrection and engineering opportunities of the mamba aminergic toxins.

Sci Rep 2017 06 2;7(1):2701. Epub 2017 Jun 2.

CEA Institut des Sciences du Vivant Frédéric Joliot, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), Université Paris-Saclay, Gif-sur-Yvette, Paris, 91190, France.

Mamba venoms contain a multiplicity of three-finger fold aminergic toxins known to interact with various α-adrenergic, muscarinic and dopaminergic receptors with different pharmacological profiles. In order to generate novel functions on this structural scaffold and to avoid the daunting task of producing and screening an overwhelming number of variants generated by a classical protein engineering strategy, we accepted the challenge of resurrecting ancestral proteins, likely to have possessed functional properties. This innovative approach that exploits molecular evolution models to efficiently guide protein engineering, has allowed us to generate a small library of six ancestral toxin (AncTx) variants and associate their pharmacological profiles to key functional substitutions. Among these variants, we identified AncTx1 as the most α-adrenoceptor selective peptide known to date and AncTx5 as the most potent inhibitor of the three α2 adrenoceptor subtypes. Three positions in the ρ-Da1a evolutionary pathway, positions 28, 38 and 43 have been identified as key modulators of the affinities for the α and α adrenoceptor subtypes. Here, we present a first attempt at rational engineering of the aminergic toxins, revealing an epistasis phenomenon.
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http://dx.doi.org/10.1038/s41598-017-02953-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5457417PMC
June 2017

Discovery and characterization of EII a new α-conotoxin from Conus ermineus venom by nAChRs affinity capture monitored by MALDI-TOF/TOF mass spectrometry.

Toxicon 2017 May 24;130:1-10. Epub 2017 Feb 24.

Laboratory of Mass Spectrometry- MolSys, Department of Chemistry, University of Liege, Liege, Belgium. Electronic address:

Animal toxins are peptides that often bind with remarkable affinity and selectivity to membrane receptors such as nicotinic acetylcholine receptors (nAChRs). The latter are, for example, targeted by α-conotoxins, a family of peptide toxins produced by venomous cone snails. nAChRs are implicated in numerous physiological processes explaining why the design of new pharmacological tools and the discovery of potential innovative drugs targeting these receptor channels appear so important. This work describes a methodology developed to discover new ligands of nAChRs from complex mixtures of peptides. The methodology was set up by the incubation of Torpedo marmorata electrocyte membranes rich in nAChRs with BSA tryptic digests (>100 peptides) doped by small amounts of known nAChRs ligands (α-conotoxins). Peptides that bind to the receptors were purified and analyzed by MALDI-TOF/TOF mass spectrometry which revealed an enrichment of α-conotoxins in membrane-containing fractions. This result exhibits the binding of α-conotoxins to nAChRs. Negative controls were performed to demonstrate the specificity of the binding. The usefulness and the power of the methodology were also investigated for a discovery issue. The workflow was then applied to the screening of Conus ermineus crude venom, aiming at characterizing new nAChRs ligands from this venom, which has not been extensively investigated to date. The methodology validated our experiments by allowing us to bind two α-conotoxins (α-EI and α-EIIA) which have already been described as nAChRs ligands. Moreover, a new conotoxin, never described to date, was also captured, identified and sequenced from this venom. Classical pharmacology tests by radioligand binding using a synthetic homologue of the toxin confirm the activity of the new peptide, called α-EII. The K value of this peptide for Torpedo nicotinic receptors was measured at 2.2 ± 0.7 nM.
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http://dx.doi.org/10.1016/j.toxicon.2017.02.023DOI Listing
May 2017

Diversity in sequences, post-translational modifications and expected pharmacological activities of toxins from four Conus species revealed by the combination of cutting-edge proteomics, transcriptomics and bioinformatics.

Toxicon 2017 May 20;130:116-125. Epub 2017 Feb 20.

Laboratory of Mass Spectrometry, MolSys, ULg, Liege, Belgium. Electronic address:

Venomous animals have developed a huge arsenal of reticulated peptides for defense and predation. Based on various scaffolds, they represent a colossal pharmacological diversity, making them top candidates for the development of innovative drugs. Instead of relying on the classical, low-throughput bioassay-guided approach to identify innovative bioactive peptides, this work exploits a recent paradigm to access to venom diversity. This strategy bypasses the classical approach by combining high-throughput transcriptomics, proteomics and bioinformatics cutting-edge technologies to generate reliable peptide sequences. The strategy employed to generate hundreds of reliable sequences from Conus venoms is deeply described. The study led to the discovery of (i) conotoxins that belong to known pharmacological families targeting various GPCRs or ion-gated channels, and (ii) new families of conotoxins, never described to date. It also focusses on the diversity of genes, sequences, folds, and PTM's provided by such species.
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http://dx.doi.org/10.1016/j.toxicon.2017.02.014DOI Listing
May 2017

High-throughput expression of animal venom toxins in Escherichia coli to generate a large library of oxidized disulphide-reticulated peptides for drug discovery.

Microb Cell Fact 2017 Jan 17;16(1). Epub 2017 Jan 17.

Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS) Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille, France.

Background: Animal venoms are complex molecular cocktails containing a wide range of biologically active disulphide-reticulated peptides that target, with high selectivity and efficacy, a variety of membrane receptors. Disulphide-reticulated peptides have evolved to display improved specificity, low immunogenicity and to show much higher resistance to degradation than linear peptides. These properties make venom peptides attractive candidates for drug development. However, recombinant expression of reticulated peptides containing disulphide bonds is challenging, especially when associated with the production of large libraries of bioactive molecules for drug screening. To date, as an alternative to artificial synthetic chemical libraries, no comprehensive recombinant libraries of natural venom peptides are accessible for high-throughput screening to identify novel therapeutics.

Results: In the accompanying paper an efficient system for the expression and purification of oxidized disulphide-reticulated venom peptides in Escherichia coli is described. Here we report the development of a high-throughput automated platform, that could be adapted to the production of other families, to generate the largest ever library of recombinant venom peptides. The peptides were produced in the periplasm of E. coli using redox-active DsbC as a fusion tag, thus allowing the efficient formation of correctly folded disulphide bridges. TEV protease was used to remove fusion tags and recover the animal venom peptides in the native state. Globally, within nine months, out of a total of 4992 synthetic genes encoding a representative diversity of venom peptides, a library containing 2736 recombinant disulphide-reticulated peptides was generated. The data revealed that the animal venom peptides produced in the bacterial host were natively folded and, thus, are putatively biologically active.

Conclusions: Overall this study reveals that high-throughput expression of animal venom peptides in E. coli can generate large libraries of recombinant disulphide-reticulated peptides of remarkable interest for drug discovery programs.
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http://dx.doi.org/10.1186/s12934-016-0617-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5242012PMC
January 2017

Gene design, fusion technology and TEV cleavage conditions influence the purification of oxidized disulphide-rich venom peptides in Escherichia coli.

Microb Cell Fact 2017 Jan 17;16(1). Epub 2017 Jan 17.

Unité Mixte de Recherche (UMR) 7257, Centre National de la Recherche Scientifique (CNRS)-Aix-Marseille Université, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille, France.

Background: Animal venoms are large, complex libraries of bioactive, disulphide-rich peptides. These peptides, and their novel biological activities, are of increasing pharmacological and therapeutic importance. However, recombinant expression of venom peptides in Escherichia coli remains difficult due to the significant number of cysteine residues requiring effective post-translational processing. There is also an urgent need to develop high-throughput recombinant protocols applicable to the production of reticulated peptides to enable efficient screening of their drug potential. Here, a comprehensive study was developed to investigate how synthetic gene design, choice of fusion tag, compartment of expression, tag removal conditions and protease recognition site affect levels of solubility of oxidized venom peptides produced in E. coli.

Results: The data revealed that expression of venom peptides imposes significant pressure on cysteine codon selection. DsbC was the best fusion tag for venom peptide expression, in particular when the fusion was directed to the bacterial periplasm. While the redox activity of DsbC was not essential to maximize expression of recombinant fusion proteins, redox activity did lead to higher levels of correctly folded target peptides. With the exception of proline, the canonical TEV protease recognition site tolerated all other residues at its C-terminus, confirming that no non-native residues, which might affect activity, need to be incorporated at the N-terminus of recombinant peptides for tag removal.

Conclusions: This study reveals that E. coli is a convenient heterologous host for the expression of soluble and functional venom peptides. Using the optimal construct design, a large and diverse range of animal venom peptides were produced in the µM scale. These results open up new possibilities for the high-throughput production of recombinant disulphide-rich peptides in E. coli.
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http://dx.doi.org/10.1186/s12934-016-0618-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240416PMC
January 2017

Ion Mobility-Mass Spectrometry as a Tool for the Structural Characterization of Peptides Bearing Intramolecular Disulfide Bond(s).

J Am Soc Mass Spectrom 2016 10 3;27(10):1637-46. Epub 2016 Aug 3.

Laboratory of Mass Spectrometry, University of Liege, Quartier Agora, Allée du six Aout 11, B-4000, Liege, Belgium.

Disulfide bonds are post-translationnal modifications that can be crucial for the stability and the biological activities of natural peptides. Considering the importance of these disulfide bond-containing peptides, the development of new techniques in order to characterize these modifications is of great interest. For this purpose, collision cross cections (CCS) of a large data set of 118 peptides (displaying various sequences) bearing zero, one, two, or three disulfide bond(s) have been measured in this study at different charge states using ion mobility-mass spectrometry. From an experimental point of view, CCS differences (ΔCCS) between peptides bearing various numbers of disulfide bonds and peptides having no disulfide bonds have been calculated. The ΔCCS calculations have also been applied to peptides bearing two disulfide bonds but different cysteine connectivities (Cys1-Cys2/Cys3-Cys4; Cys1-Cys3/Cys2-Cys4; Cys1-Cys4/Cys2-Cys3). The effect of the replacement of a proton by a potassium adduct on a peptidic structure has also been investigated. Graphical Abstract ᅟ.
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http://dx.doi.org/10.1007/s13361-016-1443-8DOI Listing
October 2016

Mass-spectrometry-based method for screening of new peptide ligands for G-protein-coupled receptors.

Anal Bioanal Chem 2015 Jul 3;407(18):5299-307. Epub 2015 May 3.

Laboratory of Mass Spectrometry, Department of Chemistry, University of Liège, 4000, Liège, Belgium.

G-protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins. Although implicated in almost all physiological processes in the human body, most of them remain unexploited, mostly because of the lack of specific ligands. The objective of this work is to develop a new mass-spectrometry-based technique capable of identifying new peptide ligands for GPCRs. The strategy is based on the incubation of cellular membranes overexpressing GPCRs with a mixture of peptides that contains potential ligands. Peptide ligands bind to the receptors, whereas other peptides remain in the binding buffer. Bound peptides are eluted from membranes and directly detected, identified, and characterized by MALDI TOF-TOF. The results reveal the efficacy of the procedure for selecting a specific ligand of GPCRs in both simple and complex mixtures of peptides. This new approach may offer direct purification, identification, and characterization of the new ligand in a single workflow. The proposed method is labeling-free and, unlike radio-binding and other techniques, it does not require a previously known labeled ligand of the studied GPCR. All these properties greatly reduce the experimental constraints. Moreover, because it is not based on the principle of a competitive specific binding, this technique constitutes a new tool to discover new ligands not only for known GPCRs, but also for orphan GPCRs.
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http://dx.doi.org/10.1007/s00216-015-8692-4DOI Listing
July 2015

Combined use of ion mobility and collision-induced dissociation to investigate the opening of disulfide bridges by electron-transfer dissociation in peptides bearing two disulfide bonds.

Anal Chem 2015 7;87(10):5240-6. Epub 2015 May 7.

†Laboratory of Mass Spectrometry, Department of Chemistry, GIGA-R, University of Liege, Allée de la Chimie 3, B-4000 Liege, Belgium.

Disulfide bonds are post-translational modifications (PTMs) often found in peptides and proteins. They increase their stability toward enzymatic degradations and provide the structure and (consequently) the activity of such folded proteins. The characterization of disulfide patterns, i.e., the cysteine connectivity, is crucial to achieve a global picture of the active conformation of the protein of interest. Electron-transfer dissociation (ETD) constitutes a valuable tool to cleave the disulfide bonds in the gas phase, avoiding chemical reduction/alkylation in solution. To characterize the cysteine pairing, the present work proposes (i) to reduce by ETD one of the two disulfide bridges of model peptides, resulting in the opening of the cyclic structures, (ii) to separate the generated species by ion mobility, and (iii) to characterize the species using collision-induced dissociation (CID). Results of this strategy applied to several peptides show different behaviors depending on the connectivity. The loss of SH· radical species, observed for all the peptides, confirms the cleavage of the disulfides during the ETD process.
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http://dx.doi.org/10.1021/acs.analchem.5b00245DOI Listing
December 2015

Molecular exploration of the α(1A)-adrenoceptor orthosteric site: binding site definition for epinephrine, HEAT and prazosin.

FEBS Lett 2014 Dec 4;588(24):4613-9. Epub 2014 Nov 4.

CEA, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), F-91191 Gif sur Yvette, France. Electronic address:

Despite the physiological and pharmacological importance of the α1A-adrenoreceptor, the mode of interactions of classical agonists and radioactive ligands with this receptor is not yet clearly defined. Here, we used mutagenesis studies and binding experiments to evaluate the importance of 11 receptor sites for the binding of (125)I-HEAT, (3)H-prazosin and epinephrine. Only one residue (F312) commonly interacts with the three molecules, and, surprisingly, D106 interacts only with epinephrine in a moderate way. Our docking model shows that prazosin and HEAT are almost superimposed into the orthosteric pocket with their tetralone and quinazoline rings close to the phenyl ring of the agonist.
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http://dx.doi.org/10.1016/j.febslet.2014.10.033DOI Listing
December 2014

The European FP7 Venomics Project.

Future Med Chem 2014 Oct;6(15):1611-2

CEA. ibitecS/SIMOPRO/Toxins, Receptors & Channels Laboratory, 91191 Gif/Yvette, France.

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http://dx.doi.org/10.4155/fmc.14.85DOI Listing
October 2014

High-throughput production of two disulphide-bridge toxins.

Chem Commun (Camb) 2014 Aug;50(61):8408-11

CEA, DSV, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), CEA Saclay, Gif sur Yvette F-91191, France.

A quick and efficient production method compatible with high-throughput screening was developed using 36 toxins belonging to four different families of two disulphide-bridge toxins. Final toxins were characterized using HPLC co-elution, CD and pharmacological studies.
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http://dx.doi.org/10.1039/c4cc02679aDOI Listing
August 2014

Polypharmacology profiles and phylogenetic analysis of three-finger toxins from mamba venom: case of aminergic toxins.

Biochimie 2014 Aug 1;103:109-17. Epub 2014 May 1.

CEA, Institute of Biology and Technology (iBiTecS), Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), Gif-sur-Yvette 91191, France. Electronic address:

Composition of mamba's venom is quite atypical and characterized by the presence of a large diversity of three-finger fold toxins (3FTx) interacting with various enzymes, receptors and ion channels. In particular, 3FTx from mambas display the unique property to interact with class A GPCRs, sometimes with a high affinity and selectivity. A screening of five of these toxins (MT1, MT3, MT7, ρ-Da1a and ρ-Da1b) on 29 different subtypes of bioaminergic receptors, using competition binding experiments, highlights the diversity of their pharmacological profiles. These toxins may display either absolute selectivity for one receptor subtype or a polypharmacological property for various bioaminergic receptors. Nevertheless, adrenoceptor is the main receptor family targeted by these toxins. Furthermore, a new receptor target was identified for 3FTx and toxins in general, the ρ-Da1b interacting competitively with the human dopamine D3 receptor in the micromolar range. This result expands the diversity of GPCRs targeted by toxins and more generally highlights the multipotent interacting property of 3FTx. Phylogenic analyzes of these toxins show that muscarinic, adrenergic and dopaminergic toxins may be pooled in one family called aminergic toxins, this family coming probably from a specific radiation of ligands present in mamba venoms.
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http://dx.doi.org/10.1016/j.biochi.2014.04.009DOI Listing
August 2014

Peptidomic comparison and characterization of the major components of the venom of the giant ant Dinoponera quadriceps collected in four different areas of Brazil.

J Proteomics 2013 Dec 22;94:413-22. Epub 2013 Oct 22.

Laboratory of Mass Spectrometry, Department of Chemistry, University of Liège, Liège, Belgium.

Unlabelled: Despite the noxious effects inflicted by Dinoponera ant's envenomation, the information about the biological properties and composition of their venom is still very limited. Ants from the genus Dinoponera are believed to be the world's largest living ants with a body length of 3cm. Their occurrence is restricted to tropical areas of South America. In this work, we study the venom of the giant Dinoponera quadriceps ant collected in 4 different regions of Brazil. By using a combination of complementary mass spectrometric approaches, we aim at: (i) characterizing the venom composition of these ants; (ii) establishing a comparative analysis of the venom from four geographically different regions in Brazil. This approach demonstrates that ant venom is a copious source of new compounds. Several peptides were identified and selected for "de novo sequencing". Since most of the new peptides showed similarities with antimicrobial peptides (AMPs), antimicrobial assays were performed with the purpose of evaluating their activity. In regard to the comparative study of the four regions, we observed not only major differences in the venom compositions, but also that the venoms collected in closest areas are more similar than the ones collected in distant regions. These observations seem to highlight an adaption of the ant venoms to the local environment. Concerning the biological assays, the peptides called Dq-3162 and Da-3177 showed a wide-ranging antimicrobial activity. The characterization of new AMPs with a broad spectrum of activity and different scaffolds may aid scientists to design new therapeutic agents and understand the mechanisms of those peptides to interact with microbial membranes. The results obtained betoken the biotechnological potential of ant's venom.

Biological Significance: For the first time this manuscript describes an extensive proteomics characterization of the D. quadriceps venom. In addition this study reports the variation in venom composition of primitive ants from 4 geographically different areas of Brazil. The results reveal the presence of ~335 compounds for each venom/area and inter-colony variations were observed. 16 new peptides were characterized and 2 of them were synthesized and biologically assayed. These findings highlight the considerable and still unexplored diversity of ant's venom which could be used as valuable research tools in different areas of knowledge.
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http://dx.doi.org/10.1016/j.jprot.2013.10.017DOI Listing
December 2013

Orthosteric binding of ρ-Da1a, a natural peptide of snake venom interacting selectively with the α1A-adrenoceptor.

PLoS One 2013 25;8(7):e68841. Epub 2013 Jul 25.

Commissariat à l'Énergie Atomique Et Aux Énergies Alternatives, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines, Gif sur Yvette, France.

ρ-Da1a is a three-finger fold toxin from green mamba venom that is highly selective for the α1A-adrenoceptor. This toxin has atypical pharmacological properties, including incomplete inhibition of (3)H-prazosin or (125)I-HEAT binding and insurmountable antagonist action. We aimed to clarify its mode of action at the α1A-adrenoceptor. The affinity (pKi 9.26) and selectivity of ρ-Da1a for the α1A-adrenoceptor were confirmed by comparing binding to human adrenoceptors expressed in eukaryotic cells. Equilibrium and kinetic binding experiments were used to demonstrate that ρ-Da1a, prazosin and HEAT compete at the α1A-adrenoceptor. ρ-Da1a did not affect the dissociation kinetics of (3)H-prazosin or (125)I-HEAT, and the IC50 of ρ-Da1a, determined by competition experiments, increased linearly with the concentration of radioligands used, while the residual binding by ρ-Da1a remained stable. The effect of ρ-Da1a on agonist-stimulated Ca(2+) release was insurmountable in the presence of phenethylamine- or imidazoline-type agonists. Ten mutations in the orthosteric binding pocket of the α1A-adrenoceptor were evaluated for alterations in ρ-Da1a affinity. The D106(3.32)A and the S188(5.42)A/S192(5.46)A receptor mutations reduced toxin affinity moderately (6 and 7.6 times, respectively), while the F86(2.64)A, F288(6.51)A and F312(7.39)A mutations diminished it dramatically by 18- to 93-fold. In addition, residue F86(2.64) was identified as a key interaction point for (125)I-HEAT, as the variant F86(2.64)A induced a 23-fold reduction in HEAT affinity. Unlike the M1 muscarinic acetylcholine receptor toxin MT7, ρ-Da1a interacts with the human α1A-adrenoceptor orthosteric pocket and shares receptor interaction points with antagonist (F86(2.64), F288(6.51) and F312(7.39)) and agonist (F288(6.51) and F312(7.39)) ligands. Its selectivity for the α1A-adrenoceptor may result, at least partly, from its interaction with the residue F86(2.64), which appears to be important also for HEAT binding.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0068841PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3723878PMC
March 2014

Crystallization of recombinant green mamba ρ-Da1a toxin during a lyophilization procedure and its structure determination.

Acta Crystallogr Sect F Struct Biol Cryst Commun 2013 Jun 29;69(Pt 6):704-9. Epub 2013 May 29.

CEA, DSV, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines, 91191 Gif-sur-Yvette, France.

ρ-Da1a toxin from eastern green mamba (Dendroaspis angusticeps) venom is a polypeptide of 65 amino acids with a strong affinity for the G-protein-coupled α(1A)-adrenoceptor. This neurotoxin has been crystallized from resolubilized lyophilized powder, but the best crystals grew spontaneously during lyophilization. The crystals belonged to the trigonal space group P3(1)21, with unit-cell parameters a = b = 37.37, c = 66.05 Å, and diffracted to 1.95 Å resolution. The structure solved by molecular replacement showed strong similarities to green mamba muscarinic toxins.
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http://dx.doi.org/10.1107/S1744309113011470DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3668600PMC
June 2013

New α-adrenergic property for synthetic MTβ and CM-3 three-finger fold toxins from black mamba.

Toxicon 2013 Dec 3;75:160-7. Epub 2013 May 3.

CEA, iBiTec-S, Service d'Ingénierie Moléculaire des Protéines (SIMOPRO), F-91191 Gif sur Yvette, France; UFR Sciences de la Vie, Université Pierre et Marie Curie (UPMC), 4 place Jussieu, Paris, France.

Despite their isolation more than fifteen years ago from the venom of the African mamba Dendroaspis polylepis, very few data are known on the functional activity of MTβ and CM-3 toxins. MTβ was initially classified as a muscarinic toxin interacting non-selectively and with low affinity with the five muscarinic receptor subtypes while no biological function was determined for CM-3. Recent results highlight the multifunctional activity of three-finger fold toxins for muscarinic and adrenergic receptors and reveal some discrepancies in the pharmacological profiles of their venom-purified and synthetic forms. Here, we report the pharmacological characterization of chemically-synthesized MTβ and CM-3 toxins on nine subtypes of muscarinic and adrenergic receptors and demonstrate their high potency for α-adrenoceptors and in particular a sub-nanomolar affinity for the α1A-subtype. Strikingly, no or very weak affinity were found for muscarinic receptors, highlighting that pharmacological characterizations of venom-purified peptides may be risky due to possible contaminations. The biological profile of these two homologous toxins looks like that one previously reported for the Dendroaspis angusticeps ρ-Da1a toxin. Nevertheless, MTβ and CM-3 interact more potently than ρ-Da1a with α1B- and α1D-AR subtypes. A computational analysis of the stability of the MTβ structure suggests that mutation S38I, could be involved in this gain in function.
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http://dx.doi.org/10.1016/j.toxicon.2013.04.017DOI Listing
December 2013

Identification and functional characterization of a novel α-conotoxin (EIIA) from Conus ermineus.

Anal Bioanal Chem 2013 Jun 14;405(15):5341-51. Epub 2013 Apr 14.

Laboratoire des Mécanismes Réactionnels, Département de Chimie, Ecole Polytechnique, CNRS UMR7651, 91128 Palaiseau Cedex, France.

Nicotinic acetylcholine receptors (nAChRs) are one of the most important families in the ligand-gated ion channel superfamily due to their involvement in primordial brain functions and in several neurodegenerative pathologies. The discovery of new ligands which can bind with high affinity and selectivity to nAChR subtypes is of prime interest in order to study these receptors and to potentially discover new drugs for treating various pathologies. Predatory cone snails of the genus Conus hunt their prey using venoms containing a large number of small, highly structured peptides called conotoxins. Conotoxins are classified in different structural families and target a large panel of receptors and ion channels. Interestingly, nAChRs represent the only subgroup for which Conus has developed seven distinct families of conotoxins. Conus venoms have thus received much attention as they could represent a potential source of selective ligands of nAChR subtypes. We describe the mass spectrometric-based approaches which led to the discovery of a novel α-conotoxin targeting muscular nAChR from the venom of Conus ermineus. The presence of several posttranslational modifications complicated the N-terminal sequencing. To discriminate between the different possible sequences, analogs with variable N-terminus were synthesized and fragmented by MS/MS. Understanding the fragmentation pathways in the low m/z range appeared crucial to determine the right sequence. The biological activity of this novel α-conotoxin (α-EIIA) that belongs to the unusual α4/4 subfamily was determined by binding experiments. The results revealed not only its selectivity for the muscular nAChR, but also a clear discrimination between the two binding sites described for this receptor.
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http://dx.doi.org/10.1007/s00216-013-6926-xDOI Listing
June 2013

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

Anal Chem 2013 May 10;85(9):4405-13. Epub 2013 Apr 10.

Laboratory of Mass Spectrometry, GIGA-R, Department of Chemistry, University of Liege, Liege, Belgium.

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 tandem mass spectrometry (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 nonclassical ions. MS/MS alone does not allow either the cysteine pairing or 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 bridge 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. Cysteine connectivity was then unambiguously determined for the two peptides, without any disulfide scrambling interference.
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http://dx.doi.org/10.1021/ac303686wDOI Listing
May 2013

Secretion and maturation of conotoxins in the venom ducts of Conus textile.

Toxicon 2012 Dec 29;60(8):1370-9. Epub 2012 Sep 29.

Laboratory of Mass Spectrometry, Department of Chemistry, University of Liege, Liege, Belgium.

The 700 or more species of cone snail attack prey by employing complex venom which can vary considerably both within species and from one species to another. Cone snail venom is remarkable for the high proportion of conotoxins with varied post-translational modifications (PTMs) and for the production of more diverse toxin scaffolds than any other known venomous animal. The venom gland, which is several times longer than its shell, is also unique in being tubular. These unusual characteristics both raise questions, and provide the opportunity for research, concerning the secretion and maturation of conotoxins along the venom duct, a process which is currently not fully understood. This research uses the two mass spectrometric techniques of isotope Coded Affinity Tagging (ICAT) and label-free quantification to study each of five portions of the venom duct of Conus textile snails from New Caledonia. Fifteen conotoxins, several with different post-translational modifications (PTMs) were identified and quantified. One hundred and forty three non-identified conotoxins were also quantified. Distinctive patterns emerged, with the largest group of conotoxins increasing, then peaking in the central-proximal part, before decreasing; whilst the second largest group peaked in the distal region, generally displaying nothing in the first parts. Conotoxins from different superfamilies were commonly found to have similar distributions. A new conotoxin, PCCSKLHDNSCCGL*, was sequenced. A comparison is made with other studies to see how the process varies in cone snails from different regions.
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http://dx.doi.org/10.1016/j.toxicon.2012.09.013DOI Listing
December 2012

Engineering of three-finger fold toxins creates ligands with original pharmacological profiles for muscarinic and adrenergic receptors.

PLoS One 2012 14;7(6):e39166. Epub 2012 Jun 14.

CEA, iBiTecS, Service d'Ingénierie Moléculaire des Protéines, Laboratoire de Toxinologie Moléculaire et Biotechnologies, Gif-sur-Yvette, France.

Protein engineering approaches are often a combination of rational design and directed evolution using display technologies. Here, we test "loop grafting," a rational design method, on three-finger fold proteins. These small reticulated proteins have exceptional affinity and specificity for their diverse molecular targets, display protease-resistance, and are highly stable and poorly immunogenic. The wealth of structural knowledge makes them good candidates for protein engineering of new functionality. Our goal is to enhance the efficacy of these mini-proteins by modifying their pharmacological properties in order to extend their use in imaging, diagnostics and therapeutic applications. Using the interaction of three-finger fold toxins with muscarinic and adrenergic receptors as a model, chimeric toxins have been engineered by substituting loops on toxin MT7 by those from toxin MT1. The pharmacological impact of these grafts was examined using binding experiments on muscarinic receptors M1 and M4 and on the α(1A)-adrenoceptor. Some of the designed chimeric proteins have impressive gain of function on certain receptor subtypes achieving an original selectivity profile with high affinity for muscarinic receptor M1 and α(1A)-adrenoceptor. Structure-function analysis supported by crystallographic data for MT1 and two chimeras permits a molecular based interpretation of these gains and details the merits of this protein engineering technique. The results obtained shed light on how loop permutation can be used to design new three-finger proteins with original pharmacological profiles.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039166PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3375269PMC
December 2012

An unusual family of glycosylated peptides isolated from Dendroaspis angusticeps venom and characterized by combination of collision induced and electron transfer dissociation.

J Am Soc Mass Spectrom 2011 Nov 29;22(11):1891-7. Epub 2011 Jul 29.

Laboratoire de spectrométrie de masse, Département de Chimie-GIGA-R, Université de Liège, Liège 4000, Belgium.

This study describes the structural characterization of a totally new family of peptides from the venom of the snake green mamba (Dendroaspis angusticeps). Interestingly, these peptides differ in several points from other already known mamba toxins. First of all, they exhibit very small molecular masses, ranging from 1.3 to 2.4 kDa. The molecular mass of classical mamba toxins is in the range of 7 to 25 kDa. Second, the new peptides do not contain disulfide bonds, a post-translational modification commonly encountered in animal toxins. The third difference is the very high proportion of proline residues in the sequence accounting for about one-third of the sequence. Finally, these new peptides reveal a carbohydrate moiety, indicating a glycosylation in the sequence. The last two features have made the structural characterization of the new peptides by mass spectrometry a real analytical challenge. Peptides were characterized by a combined use of MALDI- TOF/TOF and nanoESI-IT-ETD experiments to determine not only the peptide sequence but also the composition and the position of the carbohydrate moiety. Anyway, such small glycosylated and proline-rich toxins are totally different from any other known snake peptide and form, as a consequence, a new family of peptides.
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http://dx.doi.org/10.1007/s13361-011-0210-0DOI Listing
November 2011