Jan Tytgat

Universitair Psychiatrisch Centrum KU Leuven, Cortenberg, Flemish, Belgium

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Publications (241)869.31 Total impact

  • Drug Testing and Analysis 04/2015; · 2.82 Impact Factor
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    ABSTRACT: In the last decades, Ts1 has not only been the subject of many studies, it has also been considered as a very useful tool to investigate NaV channels and to explore the exact role of NaV channels in channelopathies. Ts1 is believed to modulate the activation process of NaV upon interaction at the neurotoxin binding site 4. Our aim was to carry out an in depth functional characterization of Ts1 on a wide array of Nav channels, in order to investigate its mechanism of action and to verify if Ts1 can indeed be considered as a prototype site 4 selective toxin, valid for all the Nav isoforms we know currently. Ts1 has been subjected to an in-depth functional investigation on 9 NaV isoforms expressed in Xenopus laevis oocytes. Ts1 does not only interfere with the activation process but also modulates the inactivation in a bell-shaped voltage-dependent matter. Furthermore, Ts1 altered the ion selectivity through insect NaV. without influencing the tetrodotoxin selectivity of the channels. Finally, Ts1 was also found to inhibit the sodium current through the cardiac Nav1.5 isoform. On the basis of the totally unexpected plethora of Nav modulations as induced by Ts1, we demonstrate that caution is required in interpretation the in vivo experiments when using Ts1. The electrophysiological characterization of Ts1 indeed shows that the general accepted contours of NaV binding sites are much more obscure than believed and that interpretation of NaV pharmacology upon toxin binding is more complex than believed thus far. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Neuropharmacology 04/2015; DOI:10.1016/j.neuropharm.2015.03.027 · 4.82 Impact Factor
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    ABSTRACT: Scorpion venom consists of a complex mixture of molecules including biologically active compounds. Because of their high potency and selectivity, toxins have medical applicability. In the last decades, scorpion toxins have thus gained considerable interest among scientist in the fields of pharmacology, biophysics and neurobiology. Identification of scorpion venom peptides and toxins can be achieved based on transcriptome approaches. We constructed the first cDNA library and Expressed Sequence Tag (EST) study to explore the transcriptomic composition of the telson from the southern African scorpion Hottentotta conspersus, belonging to the family Buthidae. We obtained 21 new venom-related sequences (8 contigs and 16 singlets) from a total of 98 ESTs analyzed, including putative neurotoxins (chloride, potassium, sodium and calcium channel toxins), bradykinin-potentiating peptides and other venom peptides without established function. These novel toxin-related sequences might serve as basis for further research both of pharmaceutical and phylogenetic nature. Copyright © 2015. Published by Elsevier Ltd.
    Toxicon 03/2015; DOI:10.1016/j.toxicon.2015.03.015 · 2.58 Impact Factor
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    ABSTRACT: The lesser Asian scorpion Mesobuthus eupeus (Buthidae) is one of the most widely spread and dispersed species of the Mesobuthus genus and its venom is actively studied. Nevertheless, a considerable amount of active compounds is still under-investigated due to the high complexity of this venom. Here we report a comprehensive analysis of putative potassium channel toxins (KTxs) from the cDNA library of M. eupeus venom glands and compare the deduced KTx structures with peptides purified from the venom. For the transcriptome analysis we used conventional tools as well as a search for structural motifs characteristic of scorpion venom components in the form of regular expressions. We found 59 candidate KTxs distributed in 30 subfamilies and presenting the cysteine-stabilized α/β (CSα/β) and inhibitor cystine knot (ICK) types of fold. M. eupeus venom was then separated to individual components by multi-stage chromatography. A facile fluorescent system based on the expression of the KcsA-Kv1.1 hybrid channels in Escherichia coli and utilization of a labeled scorpion toxin was elaborated and applied to follow Kv1.1 pore-binding activity during venom separation. As a result, eight high-affinity Kv1.1 channel blockers were identified including five novel peptides, which extend the panel of potential pharmacologically important Kv1 ligands. Activity of the new peptides against rat Kv1.1 channel was confirmed (IC50 in the range of 1-750 nM) by the two-electrode voltage-clamp technique using a standard Xenopus oocyte system. Our integrated approach is of general utility and efficiency to mine natural venoms for KTxs. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 03/2015; DOI:10.1074/jbc.M115.637611 · 4.60 Impact Factor
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    ABSTRACT: Cyclic nucleotide-sensitive ion channels are molecular pores that open in response to cAMP or cGMP, which are universal second messengers. Binding of a cyclic nucleotide to the carboxyterminal cyclic nucleotide binding domain (CNBD) of these channels is thought to cause a conformational change that promotes channel opening. The C-linker domain, which connects the channel pore to this CNBD, plays an important role in coupling ligand binding to channel opening. Current structural insight into this mechanism mainly derives from X-ray crystal structures of the C-linker/CNBD from hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels. However, these structures reveal little to no conformational changes upon comparison of the ligand-bound and unbound form. In this study, we take advantage of a recently identified prokaryote ion channel, SthK, which has functional properties that strongly resemble cyclic nucleotide-gated (CNG) channels and is activated by cAMP, but not by cGMP. We determined X-ray crystal structures of the C-linker/CNBD of SthK in the presence of cAMP or cGMP. We observe that the structure in complex with cGMP, which is an antagonist, is similar to previously determined HCN channel structures. In contrast, the structure in complex with cAMP, which is an agonist, is in a more open conformation. We observe that the CNBD makes an outward swinging movement, which is accompanied by an opening of the C-linker. This conformation mirrors the open gate structures of the Kv1.2 channel or MthK channel, which suggests that the cAMP-bound C-linker/CNBD from SthK represents an activated conformation. These results provide a structural framework for better understanding cyclic nucleotide modulation of ion channels, including HCN and CNG channels.
    PLoS ONE 01/2015; 10(1):e0116369. DOI:10.1371/journal.pone.0116369 · 3.53 Impact Factor
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    ABSTRACT: 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.
    Journal of Biological Chemistry 12/2014; 289(51):35341-35350. DOI:10.1074/jbc.M114.610436 · 4.60 Impact Factor
  • Eva Cuypers, Elien Rosier, Jan Tytgat
    TIAFT 2014; 11/2014
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    ABSTRACT: We present a structural and functional study of a sodium channel activation inhibitor from crab spider venom. Hm-3 is an insecticidal peptide toxin consisting of 35 amino acid residues from the spider Heriaeus melloteei (Thomisidae). We produced Hm-3 recombinantly in Escherichia coli and determined its structure by NMR spectroscopy. Typical for spider toxins, Hm-3 was found to adopt the so-called ″inhibitor cystine knot″ (ICK) or ″knottin″ fold stabilized by three disulfide bonds. Its molecule is amphiphilic with a hydrophobic ridge on the surface enriched in aromatic residues and surrounded by positive charges. Correspondingly, Hm-3 binds to both neutral and negatively charged lipid vesicles. Electrophysiological studies showed that at a concentration of 1 μM Hm-3 effectively inhibited a number of mammalian and insect sodium channels. Importantly, Hm-3 shifted the dependence of channel activation to more positive voltages. Moreover, the inhibition was voltage-dependent, and strong depolarizing prepulses attenuated Hm-3 activity. The toxin is therefore concluded to represent the first sodium channel gating modifier from an araneomorph spider and feature a ″membrane-access″ mechanism of action. Its amino acid sequence and position of the hydrophobic cluster are notably different from other known gating modifiers from spider venom, all of which are described from mygalomorph species. We hypothesize parallel evolution of ICK toxins from Araneomorphae and Mygalomorphae suborders.
    Journal of Biological Chemistry 10/2014; 290(1). DOI:10.1074/jbc.M114.595678 · 4.60 Impact Factor
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    ABSTRACT: Cone snails (Conus sp.) are poisonous animals that can be found in all oceans where they developed a venomous strategy to prey or to defend. The venom of these species contains an undeniable source of unique and potent pharmacologically active compounds. Their peptide compounds, called conotoxins, are not only interesting for the development of new pharmaceutical ligands, but they are also useful for studying their broad spectrum of targets. One conotoxin family in particular, the α-conotoxins, acts on nicotinic acetylcholine receptors (nAChRs) which dysfunctions play important roles in pathologies such as epilepsy, myasthenic syndromes, schizophrenia, Parkinson's disease and Alzheimer's disease. Here we define a new subclass of the α-conotoxin family. We purified the venom of a yet unexplored cone snail species, i.e. Conus australis, and we isolated a 16-amino acid peptide named α-conotoxin AusIA. The peptide has the typical α-conotoxin CC-Xm-C-Xn-C framework, but both loops (m/n) contain 5 amino acids, which has never been described before. Using conventional electrophysiology we investigated the response of synthetically made globular (I-III, II-IV) and ribbon (I-IV, II-III) AusIA to different nicotinic acetylcholine receptors. The α7 nAChR was the only receptor found to be blocked with a similar potency by both peptide-configurations. This suggests that both α5/5 conotoxin isomers might be present in the venom gland of C. australis. NMR spectroscopy showed that no secondary structures define the peptides' three-dimensional topology. Moreover, the ribbon configuration, which is generally considered to be non-native, is more stable than the globular isoform. Accordingly, our findings show relevancy concerning the α-conotoxin classification which might be helpful in the design of novel therapeutic compounds.
    Toxicon 09/2014; DOI:10.1016/j.toxicon.2014.08.074 · 2.58 Impact Factor
  • Alternative Sampling Strategies in Toxicology and Therapeutic Drug Monitoring, Ghent, Belgium; 09/2014
  • Alternative Sampling Strategies in Toxicology and Therapeutic Drug Monitoring; 09/2014
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    ABSTRACT: Today, forensic hair analysis is considered to be a standard method for identifying chronic drug users since information about drug use stored and located in hair can cover several months to even years. When interpreting these results, one should be aware of all kind of pitfalls. External factors such as bleaching might influence the analytical result. Although the effect of hydrogen peroxide on cocaine in a solution was described before, it was never investigated whether the described reaction products (ecgonine methylester, benzoylecgonine, hydroxynorcocaine and dihydroxycocaine) are indeed found on contaminated or user hair. Since it is of great importance in forensic hair analysis to know whether cocaine and/or reaction products are detectable in hair after bleaching, matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was used to study the effect of hydrogen peroxide treatment on incorporated cocaine in hairs. Cocaine oxidation products were identified in a solution based on MS/MS spectra and spatial distribution of these products in hair was explored using MALDI TOF-MS. All images were accomplished by spraying α-Cyano-4-hydroxycinnamic acid (CHCA) as a MALDI-matrix. Images revealed a loss of detectability of cocaine and its reaction products in hairs already after a short bleaching period. Since all compounds of interest are found in the hydrogen peroxide and wash solution, these findings indicate that all evidence of cocaine use might be lost after a hair bleaching treatment. Therefore, forensic toxicologists should take into consideration whether hair samples were bleached before making any conclusions from hair analysis results.
    Forensic Science International 07/2014; 242C:103-110. DOI:10.1016/j.forsciint.2014.06.035 · 2.12 Impact Factor
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    ABSTRACT: Since it is an apocrine secretion, scorpion venom is a complex mixture that contains a variety of low-molecular-weight basic proteins (neurotoxins), mucus, salts, as well as a large number of other constituents. Diversity of scorpion venom peptides exists also at the transcript level. Two kinds of venom peptides are typically considered: the neurotoxins and the antimicrobial peptides. We constructed a cDNA library and carried an EST (Expressed Sequence Tag) approach to overview the different peptides in the transcriptome of the telson from Parabuthus stridulus. P. stridulus are psammophilous and highly venomous scorpions endemic to Namibia (Prendini 2004) with medical relevance because of important human envenomation occurrence. We obtained 111 ESTs, 20% of them corresponding to cellular process transcripts, 7% to hypothetical proteins and 17% were sequences without good matches, but the majority of ESTs, 56%, corresponds to transcripts encoding for different venom components, including voltage-gated sodium, potassium and calcium channel toxins, antimicrobial peptides and other venom and cell proteins. To the best of our knowledge this report contains the first transcriptome analysis of genes transcribed by the venomous gland of the scorpion species P. stridulus, belonging to the family of medically important Buthidae scorpions. One hundred and eleven ESTs were analyzed, showing an important number of genes that encode for products similar to known scorpion venom components. In total, 17 unique and novel sequences were indentified. The identification and characterization of these compounds will be a good source of novel pharmacological tools for studying ion channels and the understanding of the physiological effects of toxins in P.stridulus envenomations at a molecular level.
    Toxicon 06/2014; DOI:10.1016/j.toxicon.2014.03.001 · 2.58 Impact Factor
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    ABSTRACT: Marine snails of the genus Conus are a large family of predatory gastropods with an unparalleled molecular diversity of pharmacologically active compounds in their venom. Cone snail venom comprises of a rich and diverse cocktail of peptide toxins which act on a wide variety of ion channels such as voltage-gated sodium- (NaV), potassium- (KV), and calcium- (CaV) channels as well as nicotinic acetylcholine receptors (nAChRs) which are classified as ligand-gated ion channels. The mode of action of several conotoxins has been the subject of investigation, while for many others this remains unknown. This review aims to give an overview of the knowledge we have today on the molecular pharmacology of conotoxins specifically interacting with nAChRs along with the structure-function relationship data.
    Marine Drugs 05/2014; 12(5):2970-3004. DOI:10.3390/md12052970 · 3.51 Impact Factor
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    ABSTRACT: We study the effect of all Tityus discrepans venom components on macrophage alterations. Only seven toxins called “Inflammatory Toxin” (InfTx1-7) induced cell changes. Incubation with InfTx1 through InfTx5 rose macrophage NO level at 2 h toxin exposure. Cells rose NO release by 4 h exposure with InfTx2 and InfTx5, the NO levels reached concentrations similar or higher than the induced by lipopolysaccharides (LPS) incubation. InfTx2, -6 and -7 increased cell TNF-α release. InfTx2 as LPS roses cell TNF-α secretion gradually in time. Macrophages were loaded with fluorescent dyes, exposed to all toxins and observed with a 3D wide field deconvolution setup. Cells exposed to whole venom or InfTx4 through InfTx7 developed pseudopodia, cytoplasm prolongations, blebs, and loss their rounded form. The molecular masses and N-terminal sequences of InfTx4 through InfTx7 were analyzed by MALDI-TOF mass spectrometry and Edman degradation. InfTx4-7 induced a remarkably rose of of intracellular Ca2+ levels ([Ca2+]i), measured as a rise of normalized cell green fluorescence intensity (FI) ×2.7, ×2.6, ×95 and ×2.9 the controls, respectively. InfTx6-7 action mechanisms were studied under different conditions. Results suggested that InfTx6 interact with a membrane sodium channel inducing cell depolarization with a consequently increase on intracellular [Na+], this would activate Na+/Ca2+ exchanger 3 (NCX) in the reverse mode and the phospholipase C inositol 1,4,5-trisphosphate (PLC-IP3) signaling pathway inducing [Ca2+]i overload. Inftx7 should activate the NCX in the reverse mode and/or should activate the Na+/H+ exchanger, increasing intracellular [Na+] which indirectly induce the activation of NCX3rv and the PLC-IP3 signaling pathway. All these mechanisms would cooperate with the [Ca2+]i overload. A rise of [Ca2+]i activates the synthesis and secretion of inflammatory molecules like TNF-α, which in turn, increases the gene transcription for inducible nitric oxide synthase, resulting on NO production.
    Toxicon 05/2014; DOI:10.1016/j.toxicon.2014.02.011 · 2.58 Impact Factor
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    ABSTRACT: Transcriptome approaches have revealed a diversity of venom compounds from a number of venomous species. Mesobuthus gibbosus scorpion showed a medical importance for the toxic effect of its sting. Previously, our group reported the first three transcripts that encode toxin genes in M. gibbosus. However, no additional toxin genes or venom components have been described for this species. Furthermore, only a very small number of reports on the genomic organization of toxin genes of scorpion species have been published. Up to this moment, no information on the gene characterization of M. gibbosus is available. This study provides the first insight into gene expression in venom glands from M. gibbosus scorpion. A cDNA library was generated from the venom glands and subsequently analyzed (301 clones). Sequences from 177 high-quality ESTs were grouped as 48 Mgib sequences, of those 48 sequences, 40 (29 "singletons" and 11 "contigs") correspond with one or more ESTs. We identified putative precursor sequences and were grouped them in different categories (39 unique transcripts, one with alternative reading frames), resulting in the identification of 12 new toxin-like and 5 antimicrobial precursors (transcripts). The analysis of the gene families revealed several new components categorized among various toxin families with effect on ion channels. Sequence analysis of a new KTx precursor provides evidence to validate a new KTx subfamily (alpha-KTx 27.x). A second part of this work involves the genomic organization of three Meg-chlorotoxin-like genes (ClTxs). Genomic DNA sequence reveals close similarities (presence of one same-phase intron) with the sole genomic organization of chlorotoxins ever reported (from M. martensii). Transcriptome analysis is a powerful strategy that provides complete information of the gene expression and molecular diversity of the venom glands (telson). In this work, we generated the first catalogue of the gene expression and genomic organization of toxins from M. gibbosus. Our result represents a relevant contribution to the knowledge of toxin transcripts and complementary information related with other cell function proteins and venom peptide transcripts. The genomic organization of the chlorotoxin genes may help to understand the diversity of this gene family.
    BMC Genomics 04/2014; 15(1):295. DOI:10.1186/1471-2164-15-295 · 4.04 Impact Factor
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    ABSTRACT: Several species of marine microalgae produce biotoxins that are responsible for massive fi sh kills and seafood-related poisonings in humans (e.g., paralytic shellfi sh poisoning and ciguatera poisoning). The symptoms are often neurological and/or gastrointestinal and linked to an altered cellular excitability. Voltage-gated ion channels are transmembrane proteins that regulate and control the cellular excitability in the entire body and therefore serve as primary molecular targets for marine guanidinium and cyclic polyether toxins (Fig. 1 in McNabb 2014). Here we focus on the molecularaspect of the mechanism of action of saxitoxins (and tetrodotoxins), brevetoxins, ciguatoxins and gambierol on voltage-gated sodium (NaV) and potassium (KV) channels.
    Toxins and Biologically Active Compounds from Microalgae, Volume 2: Biological Effects and Risk Management, Edited by Gian Paolo Rossini, 04/2014: chapter The mechanism of of action of microalgal toxins interacting with Nav and Kv channels.: pages 3-34; CRC Press 2014., ISBN: 978-1-4822-3146-5
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    ABSTRACT: Clathrodin is a marine alkaloid and believed to be a modulator of voltage-gated sodium (NaV) channels. Since there is an urgent need for small molecule NaV channel ligands as novel therapeutics, clathrodin could represent an interesting lead compound. Therefore, clathrodin was reinvestigated for its potency and NaV channel subtype selectivity. Clathrodin and its synthetic analogues were subjected to screening on a broad range of NaV channel isoforms, both in voltage clamp and patch clamp conditions. Even though clathrodin was not found to exert any activity, some analogues were capable of modulating the NaV channels, hereby validating the pyrrole-2-aminoimidazole alkaloid structure as a core structure for future small molecule-based NaV channel modulators.
    Marine Drugs 04/2014; 12(4):2132-43. DOI:10.3390/md12042132 · 3.51 Impact Factor
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    ABSTRACT: In this paper we present the spatial structure of the wheat antimicrobial peptide (AMP) Tk-AMP-X2 studied using NMR spectroscopy. This peptide is found to adopt a disulfide-stabilized α-helical hairpin fold and therefore belongs to the α-hairpinin family of plant defense peptides. Based on Tk-AMP-X2 structural similarity to cone snail and scorpion potassium channel blockers, a mutant molecule Tk-hefu and was engineered by incorporating the functionally important residues from κ-hefutoxin 1 onto Tk-AMP-X2 scaffold. The designed peptide contained the so-called essential dyad of amino acid residues significant for channel-blocking activity. Electrophysiological studies showed that while the parent peptide Tk-AMP-X2 did not present any activity against potassium channels, Tk-hefu blocked Kv1.3 channels with similar potency (IC50 ≈35 μM) to κ-hefutoxin 1 (IC50 ≈40 μM). We conclude that α-hairpinins are attractive in their simplicity structural templates, which may be used for functional engineering and drug design.
    Journal of Biological Chemistry 03/2014; 289(20). DOI:10.1074/jbc.M113.530477 · 4.60 Impact Factor

Publication Stats

5k Citations
869.31 Total Impact Points


  • 1996–2015
    • Universitair Psychiatrisch Centrum KU Leuven
      Cortenberg, Flemish, Belgium
  • 2014
    • The Catholic University of America
      Washington, Washington, D.C., United States
  • 1994–2013
    • University of Leuven
      • • Laboratory for Toxicology and Food Chemistry
      • • Faculty of Pharmaceutical Sciences
      Louvain, Flemish, Belgium
  • 2012
    • Chinese Academy of Sciences
      • Institute of Zoology
      Peping, Beijing, China
  • 2011
    • University of Wisconsin–Madison
      Madison, Wisconsin, United States
    • Nanyang Technological University
      Tumasik, Singapore
  • 2010
    • University of Chicago
      Chicago, Illinois, United States
  • 2006
    • Catholic University of Louvain
      Лувен-ла-Нев, Walloon, Belgium
  • 2005
    • Hunan University
      • College of Chemistry and Chemical Engineering
      Ch’ang-sha-shih, Hunan, China
  • 2004
    • Universität Ulm
      Ulm, Baden-Württemberg, Germany
    • Shanghai Jiao Tong University
      Shanghai, Shanghai Shi, China
  • 2003
    • Architecture et Fonction des Macromolécules Biologiques
      Marsiglia, Provence-Alpes-Côte d'Azur, France
    • University of Debrecen
      • Department of Biophysics and Cell Biology
      Debrecen, Hajdu-Bihar, Hungary
  • 1992
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 1990
    • Martin Luther University Halle-Wittenberg
      • Julius Bernstein Institute for Physiology
      Halle-on-the-Saale, Saxony-Anhalt, Germany