Publications by authors named "Julita S Imperial"

20 Publications

  • Page 1 of 1

Cannabinoid receptor agonists from Conus venoms alleviate pain-related behavior in rats.

Pharmacol Biochem Behav 2021 Mar 25;205:173182. Epub 2021 Mar 25.

University of Miami, Miller School of Medicine, Miami Project, 1095 NW 14(th) terrace, Miami, FL 33136, USA.

Cannabinoid (CB) receptor agonists show robust antinociceptive effects in various pain models. However, most of the clinically potent CB1 receptor-active drugs derived from cannabis are considered concerning due to psychotomimetic side effects. Selective CB receptor ligands that do not induce CNS side effects are of clinical interest. The venoms of marine snail Conus are a natural source of various potent analgesic peptides, some of which are already FDA approved. In this study we evaluated the ability of several Conus venom extracts to interact with CB1 receptor. HEK293 cells expressing CB1 receptors were treated with venom extracts and CB1 receptor internalization was analyzed by immunofluorescence. Results showed C. textile (C. Tex) and C. miles (C. Mil) samples as the most potent. These were serially subfractionated by HPLC for subsequent analysis by internalization assays and for analgesic potency evaluated in the formalin test and after peripheral nerve injury. Intrathecal injection of C. Tex and C. Mil subfractions reduced flinching/licking behavior during the second phase of formalin test and attenuated thermal and mechanical allodynia in nerve injury model. Treatment with proteolytic enzymes reduced CB1 internalization of subfractions, indicating the peptidergic nature of CB1 active component. Further HPLC purification revealed two potent antinociceptive subfractions within C. Tex with CB1 and possible CB2 activity, with mild to no side effects in the CB tetrad assessment. CB conopeptides can be isolated from these active Conus venom-derived samples and further developed as novel analgesic agents for the treatment of chronic pain using cell based or gene therapy approaches.
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http://dx.doi.org/10.1016/j.pbb.2021.173182DOI Listing
March 2021

Purification and Characterization of the Pink-Floyd Drillipeptide, a Bioactive Venom Peptide from (Gastropoda: Conoidea: Drilliidae).

Toxins (Basel) 2020 08 7;12(8). Epub 2020 Aug 7.

The Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines.

The cone snails (family Conidae) are the best known and most intensively studied venomous marine gastropods. However, of the total biodiversity of venomous marine mollusks (superfamily Conoidea, >20,000 species), cone snails comprise a minor fraction. The venoms of the family Drilliidae, a highly diversified family in Conoidea, have not previously been investigated. In this report, we provide the first biochemical characterization of a component in a Drilliidae venom and define a gene superfamily of venom peptides. A bioactive peptide, cdg14a, was purified from the venom of Fedosov and Puillandre, 2020. The peptide is small (23 amino acids), disulfide-rich (4 cysteine residues) and belongs to the J-like drillipeptide gene superfamily. Other members of this superfamily share a conserved signal sequence and the same arrangement of cysteine residues in their predicted mature peptide sequences. The cdg14a peptide was chemically synthesized in its bioactive form. It elicited scratching and hyperactivity, followed by a paw-thumping phenotype in mice. Using the Constellation Pharmacology platform, the cdg14a drillipeptide was shown to cause increased excitability in a majority of non-peptidergic nociceptors, but did not affect other subclasses of dorsal root ganglion (DRG) neurons. This suggests that the cdg14a drillipeptide may be blocking a specific molecular isoform of potassium channels. The potency and selectivity of this biochemically characterized drillipeptide suggest that the venoms of the Drilliidae are a rich source of novel and selective ligands for ion channels and other important signaling molecules in the nervous system.
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http://dx.doi.org/10.3390/toxins12080508DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7472735PMC
August 2020

Characterization of the First Conotoxin from , a Vermivorous Cone Snail from the Cabo Verde Archipelago.

Mar Drugs 2019 Jul 24;17(8). Epub 2019 Jul 24.

Department of Biology, University of Utah, 257 S 1400 E, Salt Lake City, UT 84112, USA.

is a cone snail endemic to the west side of the island of Sal, in the Cabo Verde Archipelago off West Africa. We describe the isolation and characterization of the first bioactive peptide from the venom of this species. This 30AA venom peptide is named conotoxin AtVIA (δ-conotoxin-like). An excitatory activity was manifested by the peptide on a majority of mouse lumbar dorsal root ganglion neurons. An analog of AtVIA with conservative changes on three amino acid residues at the C-terminal region was synthesized and this analog produced an identical effect on the mouse neurons. AtVIA has homology with δ-conotoxins from other worm-hunters, which include conserved sequence elements that are shared with δ-conotoxins from fish-hunting . In contrast, there is no comparable sequence similarity with δ-conotoxins from the venoms of molluscivorous species. A rationale for the potential presence of δ-conotoxins, that are potent in vertebrate systems in two different lineages of worm-hunting cone snails, is discussed.
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http://dx.doi.org/10.3390/md17080432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6723684PMC
July 2019

Structure and Biological Activity of a Turripeptide from Unedogemmula bisaya Venom.

Biochemistry 2017 11 1;56(45):6051-6060. Epub 2017 Nov 1.

Marine Science Institute, University of the Philippines , P. Velasquez Street, Diliman, Quezon City 1101, Philippines.

The turripeptide ubi3a was isolated from the venom of the marine gastropod Unedogemmula bisaya, family Turridae, by bioassay-guided purification; both native and synthetic ubi3a elicited prolonged tremors when injected intracranially into mice. The sequence of the peptide, DCCOCOAGAVRCRFACC-NH (O = 4-hydroxyproline) follows the framework III pattern for cysteines (CC-C-C-CC) in the M-superfamily of conopeptides. The three-dimensional structure determined by NMR spectroscopy indicated a disulfide connectivity that is not found in conopeptides with the cysteine framework III: C-C C-C, C-C. The peptide inhibited the activity of the α9α10 nicotinic acetylcholine receptor with relatively low affinity (IC, 10.2 μM). Initial Constellation Pharmacology data revealed an excitatory activity of ubi3a on a specific subset of mouse dorsal root ganglion neurons.
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http://dx.doi.org/10.1021/acs.biochem.7b00485DOI Listing
November 2017

Glycine-rich conotoxins from the Virgiconus clade.

Toxicon 2016 Apr 4;113:11-7. Epub 2016 Feb 4.

Department of Biology, University of Utah, 257 South 1400 East, Salt Lake, UT 84112, USA.

Cone snails in the Virgiconus clade prey on marine worms. Here, we identify six related conotoxins in the O1-superfamily from three species in this clade, Conus virgo, Conus terebra and Conus kintoki. One of these peptides, vi6a, was directly purified from the venom of C. virgo by following its activity using calcium imaging of dissociated mouse dorsal root ganglion (DRG) neurons. The purified peptide was biochemically characterized, synthesized and tested for activity in mice. Hyperactivity was observed upon both intraperitoneal and intracranial injection of the peptide. The effect of the synthetic peptide on DRG neurons was identical to that of the native peptide. Using the vi6a sequence, five other homologs were identified. These peptides define a glycine-rich subgroup of the O1-superfamily from the Virgiconus clade, with biological activity in mice.
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http://dx.doi.org/10.1016/j.toxicon.2016.02.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5446245PMC
April 2016

Small Molecules in the Cone Snail Arsenal.

Org Lett 2015 Oct 30;17(20):4933-5. Epub 2015 Sep 30.

Interdisciplinary Centre of Marine and Environmental Research, CIIMAR/CIMAR, and Faculty of Sciences, University of Porto , 40150-123 Porto, Portugal.

Cone snails are renowned for producing peptide-based venom, containing conopeptides and conotoxins, to capture their prey. A novel small-molecule guanine derivative with unprecedented features, genuanine, was isolated from the venom of two cone snail species. Genuanine causes paralysis in mice, indicating that small molecules and not just polypeptides may contribute to the activity of cone snail venom.
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http://dx.doi.org/10.1021/acs.orglett.5b02389DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845746PMC
October 2015

Insights into the origins of fish hunting in venomous cone snails from studies of Conus tessulatus.

Proc Natl Acad Sci U S A 2015 Apr 6;112(16):5087-92. Epub 2015 Apr 6.

Department of Biology, University of Utah, Salt Lake City, UT 84112;

Prey shifts in carnivorous predators are events that can initiate the accelerated generation of new biodiversity. However, it is seldom possible to reconstruct how the change in prey preference occurred. Here we describe an evolutionary "smoking gun" that illuminates the transition from worm hunting to fish hunting among marine cone snails, resulting in the adaptive radiation of fish-hunting lineages comprising ∼100 piscivorous Conus species. This smoking gun is δ-conotoxin TsVIA, a peptide from the venom of Conus tessulatus that delays inactivation of vertebrate voltage-gated sodium channels. C. tessulatus is a species in a worm-hunting clade, which is phylogenetically closely related to the fish-hunting cone snail specialists. The discovery of a δ-conotoxin that potently acts on vertebrate sodium channels in the venom of a worm-hunting cone snail suggests that a closely related ancestral toxin enabled the transition from worm hunting to fish hunting, as δ-conotoxins are highly conserved among fish hunters and critical to their mechanism of prey capture; this peptide, δ-conotoxin TsVIA, has striking sequence similarity to these δ-conotoxins from piscivorous cone snail venoms. Calcium-imaging studies on dissociated dorsal root ganglion (DRG) neurons revealed the peptide's putative molecular target (voltage-gated sodium channels) and mechanism of action (inhibition of channel inactivation). The results were confirmed by electrophysiology. This work demonstrates how elucidating the specific interactions between toxins and receptors from phylogenetically well-defined lineages can uncover molecular mechanisms that underlie significant evolutionary transitions.
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http://dx.doi.org/10.1073/pnas.1424435112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4413319PMC
April 2015

Specialized insulin is used for chemical warfare by fish-hunting cone snails.

Proc Natl Acad Sci U S A 2015 Feb 20;112(6):1743-8. Epub 2015 Jan 20.

Department of Biology, University of Utah, Salt Lake City, UT 84112;

More than 100 species of venomous cone snails (genus Conus) are highly effective predators of fish. The vast majority of venom components identified and functionally characterized to date are neurotoxins specifically targeted to receptors, ion channels, and transporters in the nervous system of prey, predators, or competitors. Here we describe a venom component targeting energy metabolism, a radically different mechanism. Two fish-hunting cone snails, Conus geographus and Conus tulipa, have evolved specialized insulins that are expressed as major components of their venoms. These insulins are distinctive in having much greater similarity to fish insulins than to the molluscan hormone and are unique in that posttranslational modifications characteristic of conotoxins (hydroxyproline, γ-carboxyglutamate) are present. When injected into fish, the venom insulin elicits hypoglycemic shock, a condition characterized by dangerously low blood glucose. Our evidence suggests that insulin is specifically used as a weapon for prey capture by a subset of fish-hunting cone snails that use a net strategy to capture prey. Insulin appears to be a component of the nirvana cabal, a toxin combination in these venoms that is released into the water to disorient schools of small fish, making them easier to engulf with the snail's distended false mouth, which functions as a net. If an entire school of fish simultaneously experiences hypoglycemic shock, this should directly facilitate capture by the predatory snail.
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http://dx.doi.org/10.1073/pnas.1423857112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330763PMC
February 2015

Discovery by proteogenomics and characterization of an RF-amide neuropeptide from cone snail venom.

J Proteomics 2015 Jan 15;114:38-47. Epub 2014 Nov 15.

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, 381 Royal Parade, Parkville 3052, Australia.

Unlabelled: In this study, a proteogenomic annotation strategy was used to identify a novel bioactive peptide from the venom of the predatory marine snail Conus victoriae. The peptide, conorfamide-Vc1 (CNF-Vc1), defines a new gene family. The encoded mature peptide was unusual for conotoxins in that it was cysteine-free and, despite low overall sequence similarity, contained two short motifs common to known neuropeptides/hormones. One of these was the C-terminal RF-amide motif, commonly observed in neuropeptides from a range of organisms, including humans. The mature venom peptide was synthesized and characterized structurally and functionally. The peptide was bioactive upon injection into mice, and calcium imaging of mouse dorsal root ganglion (DRG) cells revealed that the peptide elicits an increase in intracellular calcium levels in a subset of DRG neurons. Unusually for most Conus venom peptides, it also elicited an increase in intracellular calcium levels in a subset of non-neuronal cells.

Biological Significance: Our findings illustrate the utility of proteogenomics for the discovery of novel, functionally relevant genes and their products. CNF-Vc1 should be useful for understanding the physiological role of RF-amide peptides in the molluscan and mammalian nervous systems.
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http://dx.doi.org/10.1016/j.jprot.2014.11.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4366139PMC
January 2015

A family of excitatory peptide toxins from venomous crassispirine snails: using Constellation Pharmacology to assess bioactivity.

Toxicon 2014 Oct 2;89:45-54. Epub 2014 Jul 2.

Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA.

The toxinology of the crassispirine snails, a major group of venomous marine gastropods within the superfamily Conoidea, is largely unknown. Here we define the first venom peptide superfamily, the P-like crassipeptides, and show that the organization of their gene sequences is similar to conotoxin precursors. We provide evidence that one peptide family within the P-like crassipeptide superfamily includes potassium-channel (K-channel) blockers, the κP-crassipeptides. Three of these peptides were chemically synthesized (cce9a, cce9b and iqi9a). Using conventional electrophysiology, cce9b was shown to be an antagonist of both a human Kv1.1 channel isoform (Shaker subfamily of voltage-gated K channels) and a Drosophila K-channel isoform. We assessed the bioactivity of these peptides in native mammalian dorsal root ganglion neurons in culture. We demonstrate that two of these crassipeptides, cce9a and cce9b, elicited an excitatory phenotype in a subset of small-diameter capsaicin-sensitive mouse DRG neurons that were also affected by κJ-conotoxin PlXIVA (pl14a), a blocker of Kv1.6 channels. Given the vast complexity of heteromeric K-channel isoforms, this study demonstrates that the crassispirine venoms are a potentially rich source for discovering novel peptides that can help to identify and characterize the diversity of K-channel subtypes expressed in native neurons and other cell types.
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http://dx.doi.org/10.1016/j.toxicon.2014.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4134995PMC
October 2014

Adaptive radiation of venomous marine snail lineages and the accelerated evolution of venom peptide genes.

Ann N Y Acad Sci 2012 Sep;1267:61-70

Biology Department, University of Utah, Salt Lake City, Utah, USA.

An impressive biodiversity (>10,000 species) of marine snails (suborder Toxoglossa or superfamily Conoidea) have complex venoms, each containing approximately 100 biologically active, disulfide-rich peptides. In the genus Conus, the most intensively investigated toxoglossan lineage (∼500 species), a small set of venom gene superfamilies undergo rapid sequence hyperdiversification within their mature toxin regions. Each major lineage of Toxoglossa has its own distinct set of venom gene superfamilies. Two recently identified venom gene superfamilies are expressed in the large Turridae clade, but not in Conus. Thus, as major venomous molluscan clades expand, a small set of lineage-specific venom gene superfamilies undergo accelerated evolution. The juxtaposition of extremely conserved signal sequences with hypervariable mature peptide regions is unprecedented and raises the possibility that in these gene superfamilies, the signal sequences are conserved as a result of an essential role they play in enabling rapid sequence evolution of the region of the gene that encodes the active toxin.
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http://dx.doi.org/10.1111/j.1749-6632.2012.06603.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3488454PMC
September 2012

Characterization of a venom peptide from a crassispirid gastropod.

Toxicon 2011 Dec 12;58(8):672-80. Epub 2011 Sep 12.

Marine Science Institute, University of the Philippines, Diliman, Quezon City.

The crassispirids are a large branch of venomous marine gastropods whose venoms have not been investigated previously. We demonstrate that crassispirids comprise a major group of toxoglossate snails in a clade distinct from all turrids whose venoms have been analyzed. The isolation and biochemical definition of the first venom component from any crassispirid is described. Crassipeptide cce9a from Crassispira cerithina (Anton, 1838) was purified from crude venom by following biological activity elicited in young mice, lethargy and a lack of responsiveness to external stimuli. Using Edman sequencing and mass spectrometry, the purified peptide was shown to be 29 amino acid residues long, with the sequence: GSCGLPCHENRRCGWACYCDDGICKPLRV. The sequence assignment was verified through the analysis of a cDNA clone encoding the peptide. The peptide was chemically synthesized and folded; the synthetic peptide was biologically active and coelution with the native venom peptide was demonstrated. When injected into mice of various ages, the peptide elicited a striking shift in behavioral phenotype between 14 and 16 days, from lethargy to hyperactivity.
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http://dx.doi.org/10.1016/j.toxicon.2011.09.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3223299PMC
December 2011

A novel Conus snail polypeptide causes excitotoxicity by blocking desensitization of AMPA receptors.

Curr Biol 2009 Jun 28;19(11):900-8. Epub 2009 May 28.

Department of Biology, University of Utah, Salt Lake City, UT 84112-0840, USA.

Background: Ionotropic glutamate receptors (iGluRs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the central nervous system. Based on both molecular and pharmacological criteria, iGluRs have been divided into two major classes, the non-NMDA class, which includes both AMPA and kainate subtypes of receptors, and the NMDA class. One evolutionarily conserved feature of iGluRs is their desensitization in the continued presence of glutamate. Thus, when in a desensitized state, iGluRs can be bound to glutamate, yet the channel remains closed. However, the relevance of desensitization to nervous system function has remained enigmatic.

Results: Here, we report the identification and characterization of a novel polypeptide (con-ikot-ikot) from the venom of a predatory marine snail Conus striatus that specifically disrupts the desensitization of AMPA receptors (AMPARs). The stoichiometry of con-ikot-ikot appears reminiscent of the proposed subunit organization of AMPARs, i.e., a dimer of dimers, suggesting that it acts as a molecular four-legged clamp that holds the AMPAR channel open. Application of con-ikot-ikot to hippocampal slices caused a large and rapid increase in resting AMPAR-mediated current leading to neuronal death.

Conclusions: Our findings provide insight into the mechanisms that regulate receptor desensitization and demonstrate that in the arms race between prey and predators, evolution has selected for a toxin that blocks AMPAR desensitization, thus revealing the fundamental importance of desensitization for regulating neural function.
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http://dx.doi.org/10.1016/j.cub.2009.05.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2722447PMC
June 2009

Tyrosine-rich conopeptides affect voltage-gated K+ channels.

J Biol Chem 2008 Aug 27;283(34):23026-32. Epub 2008 May 27.

Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.

Two venom peptides, CPY-Pl1 (EU000528) and CPY-Fe1 (EU000529), characterized from the vermivorous marine snails Conus planorbis and Conus ferrugineus, define a new class of conopeptides, the conopeptide Y (CPY) family. The peptides have no disulfide cross-links and are 30 amino acids long; the high content of tyrosine is unprecedented for any native gene product. The CPY peptides were chemically synthesized and shown to be biologically active upon injection into both mice and Caenorhabditis elegans; activity on mammalian Kv1 channel isoforms was demonstrated using an oocyte heterologous expression system, and selectivity for Kv1.6 was found. NMR spectroscopy revealed that the peptides were unstructured in aqueous solution; however, a helical region including residues 12-18 for one peptide, CPY-Pl1, formed in trifluoroethanol buffer. Clones obtained from cDNA of both species encoded prepropeptide precursors that shared a unique signal sequence, indicating that these peptides are encoded by a novel gene family. This is the first report of tyrosine-rich bioactive peptides in Conus venom.
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http://dx.doi.org/10.1074/jbc.M800084200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2517004PMC
August 2008

I(1)-superfamily conotoxins and prediction of single D-amino acid occurrence.

Toxicon 2008 Feb 29;51(2):218-29. Epub 2007 Sep 29.

Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112, USA.

The considerable diversity of Conus peptides in the I(1)-superfamily provides a rare opportunity to define parameters important for the post-translational l- to d-isomerization of amino acids. This subtlest of post-translational modifications is not readily detectable by most techniques, and it would be a considerable advance if one could predict its potential occurrence purely from gene sequences. We previously described three I(1)-conotoxins, iota-RXIA (formerly designated r11a), r11b and r11c, each containing a d-amino acid at the third position from the C-terminus. In this work, we investigated two novel I(1)-superfamily members, r11d and ar11a, which we show have only l-amino acids. Based on these observations and an analysis of cDNA sequences of other group members, we suggest that there is a rule to predict d-amino acids in I(1)-superfamily peptides. Two factors are important: the residue to be modified should be three amino acids from the C-terminus of the precursor sequence, and it should be in a suitable sequence context. We apply the rule to other members of the I(1)-superfamily, to determine a priori which are probably modified.
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http://dx.doi.org/10.1016/j.toxicon.2007.09.006DOI Listing
February 2008

Venomous auger snail Hastula (Impages) hectica (Linnaeus, 1758): molecular phylogeny, foregut anatomy and comparative toxinology.

J Exp Zool B Mol Dev Evol 2007 Dec;308(6):744-56

Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.

The >10,000 living venomous marine snail species [superfamily Conoidea (Fleming, 1822)] include cone snails (Conus), the overwhelming focus of research. Hastula hectica (Linnaeus, 1758), a venomous snail in the family Terebridae (Mörch, 1852) was comprehensively investigated. The Terebridae comprise a major monophyletic group within Conoidea. H. hectica has a striking radular tooth to inject venom that looks like a perforated spear; in Conus, the tooth looks like a hypodermic needle. H. hectica venom contains a large complement of small disulfide-rich peptides, but with no apparent overlap with Conus in gene superfamilies expressed. Although Conus peptide toxins are densely post-translationally modified, no post-translationally modified amino acids were found in any Hastula venom peptide. The results suggest that different major lineages of venomous molluscs have strikingly divergent toxinological and venom-delivery strategies.
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http://dx.doi.org/10.1002/jez.b.21195DOI Listing
December 2007

Structure of conkunitzin-S1, a neurotoxin and Kunitz-fold disulfide variant from cone snail.

Acta Crystallogr D Biol Crystallogr 2006 Sep 19;62(Pt 9):980-90. Epub 2006 Aug 19.

Biology, University of Utah, 257 S 1400 E, Salt Lake City, Utah 84112-0840, USA.

Cone snails (Conus) are predatory marine mollusks that immobilize prey with venom containing 50-200 neurotoxic polypeptides. Most of these polypeptides are small disulfide-rich conotoxins that can be classified into families according to their respective ion-channel targets and patterns of cysteine-cysteine disulfides. Conkunitzin-S1, a potassium-channel pore-blocking toxin isolated from C. striatus venom, is a member of a newly defined conotoxin family with sequence homology to Kunitz-fold proteins such as alpha-dendrotoxin and bovine pancreatic trypsin inhibitor (BPTI). While conkunitzin-S1 and alpha-dendrotoxin are 42% identical in amino-acid sequence, conkunitzin-S1 has only four of the six cysteines normally found in Kunitz proteins. Here, the crystal structure of conkunitzin-S1 is reported. Conkunitzin-S1 adopts the canonical 3(10)-beta-beta-alpha Kunitz fold complete with additional distinguishing structural features including two completely buried water molecules. The crystal structure, although completely consistent with previously reported NMR distance restraints, provides a greater degree of precision for atomic coordinates, especially for S atoms and buried solvent molecules. The region normally cross-linked by cysteines II and IV in other Kunitz proteins retains a network of hydrogen bonds and van der Waals interactions comparable to those found in alpha-dendrotoxin and BPTI. In conkunitzin-S1, glycine occupies the sequence position normally reserved for cysteine II and the special steric properties of glycine allow additional van der Waals contacts with the glutamine residue substituting for cysteine IV. Evolution has thus defrayed the cost of losing a disulfide bond by augmenting and optimizing weaker yet nonetheless effective non-covalent interactions.
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http://dx.doi.org/10.1107/S0907444906021123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924234PMC
September 2006

A novel conotoxin inhibitor of Kv1.6 channel and nAChR subtypes defines a new superfamily of conotoxins.

Biochemistry 2006 Jul;45(27):8331-40

Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.

Using assay-directed fractionation of the venom from the vermivorous cone snail Conus planorbis, we isolated a new conotoxin, designated pl14a, with potent activity at both nicotinic acetylcholine receptors and a voltage-gated potassium channel subtype. pl14a contains 25 amino acid residues with an amidated C-terminus, an elongated N-terminal tail (six residues), and two disulfide bonds (1-3, 2-4 connectivity) in a novel framework distinct from other conotoxins. The peptide was chemically synthesized, and its three-dimensional structure was demonstrated to be well-defined, with an alpha-helix and two 3(10)-helices present. Analysis of a cDNA clone encoding the prepropeptide precursor of pl14a revealed a novel signal sequence, indicating that pl14a belongs to a new gene superfamily, the J-conotoxin superfamily. Five additional peptides in the J-superfamily were identified. Intracranial injection of pl14a in mice elicited excitatory symptoms that included shaking, rapid circling, barrel rolling, and seizures. Using the oocyte heterologous expression system, pl14a was shown to inhibit both a K+ channel subtype (Kv1.6, IC50 = 1.59 microM) and neuronal (IC50 = 8.7 microM for alpha3beta4) and neuromuscular (IC50 = 0.54 microM for alpha1beta1 epsilondelta) subtypes of the nicotinic acetylcholine receptor (nAChR). Similarities in sequence and structure are apparent between the middle loop of pl14a and the second loop of a number of alpha-conotoxins. This is the first conotoxin shown to affect the activity of both voltage-gated and ligand-gated ion channels.
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http://dx.doi.org/10.1021/bi060263rDOI Listing
July 2006

Putative gamma-conotoxins in vermivorous cone snails: the case of Conus delessertii.

Peptides 2005 Jan;26(1):23-7

Laboratory of Marine Neuropharmacology, Institute of Neurobiology, Universidad Nacional Autónoma de México, Juriquilla, Qro. 76230, México.

Peptide de7a was purified from the venom of Conus delessertii, a vermivorous cone snail collected in the Yucatan Channel, Mexico. Its amino acid sequence was determined by automatic Edman degradation after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and several post-translationally modified residues. The determination of its molecular mass by means of laser desorption ionization time-of-flight mass spectrometry (average mass, 3170.0 Da) confirmed the chemical data and suggested amidation of the C-terminus. The primary structure (ACKOKNNLCAITgammaMAgammaCCSGFCLIYRCS*; O, hydroxyproline; gamma, gamma-carboxyglutamate; *, amidated C-terminus; calculated average mass, 3169.66 Da) of de7a contains a motif (gammaCCS) that has previously only been found in two other toxins, both from molluscivorous cone snails: TxVIIA from Conus textile and gamma-PnVIIA from Conus pennaceus. These toxins cause depolarization and increased firing of action potentials in molluscan neuronal systems, and toxin gamma-PnVIIA has been shown to act as an agonist of neuronal pacemaker cation currents. The similarities to toxins TxVIIA and gamma-PnVIIA suggest that peptide de7a might also affect voltage-gated nonspecific cation pacemaker channels.
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http://dx.doi.org/10.1016/j.peptides.2004.10.012DOI Listing
January 2005

The augertoxins: biochemical characterization of venom components from the toxoglossate gastropod Terebra subulata.

Toxicon 2003 Sep;42(4):391-8

Department of Biology, University of Utah, 257 South 1400 East, Salt Lake City, UT 84112-0840, USA.

We describe the purification and biochemical characterization of three components from the venom of the toxoglossate gastropod Terebra subulata. The three polypeptide venom components, augertoxins s6a, s7a and s11a, are 40-41AA in length with 3-4 disulfide linkages. The arrangement of Cys residues is reminiscent of certain conopeptide superfamilies, but molecular cloning failed to show the highly conserved sequence features diagnostic of the conopeptide gene superfamily with a similar arrangement of Cys residues. One of the purified peptides, s7a, elicited an uncoordinated twisting syndrome when injected into the nematode Caenorhabditis elegans, but had no effect on mice. T. subulata belongs to the family Terebridae, one of four major groups of toxoglossate gastropods in the superfamily Conacea. The results reveal that some features of the augertoxins and conotoxins are generally similar, such as the organization of prepropeptide precursors and their proteolytic processing into mature toxins; however, Terebra may have evolved generally larger venom components that are less highly post-translationally modified. The results suggest that Conus peptide gene superfamilies probably do not extend to the Terebridae, suggesting that distinctive venom gene superfamilies may be expressed in each major division of Conacean gastropods.
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http://dx.doi.org/10.1016/s0041-0101(03)00169-7DOI Listing
September 2003