Purification and Characterization of the Pink-Floyd Drillipeptide, a Bioactive Venom Peptide from Clavus Davidgilmouri (Gastropoda: Conoidea: Drilliidae)
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toxins Article Purification and Characterization of the Pink-Floyd Drillipeptide, a Bioactive Venom Peptide from Clavus davidgilmouri (Gastropoda: Conoidea: Drilliidae) Victor M. Chua 1,2, Joanna Gajewiak 2, Maren Watkins 2, Samuel S. Espino 2, Iris Bea L. Ramiro 1,2,3 , Carla A. Omaga 1,2, Julita S. Imperial 2, Louie Paolo D. Carpio 1, Alexander Fedosov 4 , Helena Safavi-Hemami 2,3,5 , Lilibeth A. Salvador-Reyes 1 , Baldomero M. Olivera 2 and Gisela P. Concepcion 1,* 1 The Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines; [email protected] (V.M.C.); [email protected] (I.B.L.R.); [email protected] (C.A.O.); [email protected] (L.P.D.C.); [email protected] (L.A.S.-R.) 2 School of Biological Sciences, University of Utah, Salt Lake City, UT 84112, USA; [email protected] (J.G.); [email protected] (M.W.); [email protected] (S.S.E.); [email protected] (J.S.I.); [email protected] (H.S.-H.); [email protected] (B.M.O.) 3 Department of Biomedical Sciences, University of Copenhagen, 2200 Copenhagen N, Denmark 4 Severstov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninsky prospect 33, Moscow 119071, Russia; [email protected] 5 Department of Biochemistry, University of Utah, Salt Lake City, UT 84112, USA * Correspondence: [email protected] Received: 6 June 2020; Accepted: 4 August 2020; Published: 7 August 2020 Abstract: 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 Clavus davidgilmouri 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. Keywords: venom; Clavus; Drilliidae; drillipeptide Key Contribution: We report the discovery and chemical characterization of a peptide isolated from the drilliid gastropod, Clavus davidgilmouri. Toxins 2020, 12, 508; doi:10.3390/toxins12080508 www.mdpi.com/journal/toxins Toxins 2020, 12, 508 2 of 16 1. Introduction TheToxins successful 2020, 12, x FOR evolutionary PEER REVIEW radiation of the venomous marine snails in the superfamily2 Conoideaof 15 has generated1. Introduction a significant fraction of the biodiversity of living mollusks (>20,000 species) [1,2]. Traditionally, three groups of gastropods, the cone snails, the auger snails, and the turrids, were thought The successful evolutionary radiation of the venomous marine snails in the superfamily to comprise this superfamily. However, anatomical and molecular phylogenetic studies revealed Conoidea has generated a significant fraction of the biodiversity of living mollusks (>20,000 species) that while[1,2]. Traditionally, cone snails belongthree groups to a single of gastropods, monophyletic the cone lineage snails, (the the familyauger snails, Conidae), and the and turrids, the auger snailswere can similarlythought to be comprise grouped this into superfamily. a single clade However, (the family anatomical Terebridae), and molecular the conoidean phylogenetic snails that havestudies generally revealed been that referred while cone to as snails turrids belong (the to former a single family monophyletic Turridae, lineagesensu (the lato family) comprise Conidae), several phylogeneticallyand the auger unrelated snails can groups, similarly some be ofgrouped which into are morea single closely clade related (the family to the Terebridae), cone snails the than to the typeconoidean genus snails of the that Turridae, have generallyTurris [3 been–5]. referred to as turrids (the former family Turridae, sensu Inlato a) comprise proposed several reorganization phylogenetically of the unrelated superfamily groups, Conoidea some of which based are on more molecular closely datarelated with to the mostthe recent cone emendationssnails than to the [6 ],type the genus members of the ofTurridae, the old Turris family [3–5]. Turridae have been reclassified into 15 monophyleticIn a proposed families. reorganization One of the of most the superfamil diverse phylogeneticy Conoidea based lineages on molecular of former data “turrids” with the whose most recent emendations [6], the members of the old family Turridae have been reclassified into 15 distinctiveness has long since been acknowledged, is the family Drilliidae [1,6]. The venoms of this monophyletic families. One of the most diverse phylogenetic lineages of former “turrids” whose species-richdistinctiveness conoidean has long family since until been recentlyacknowledged, remained is the completely family Drilliidae uncharacterized, [1,6]. The venoms likely of this because mostspecies-rich drilliids are conoidean relatively family small, until uncommon, recently andrema predominantlyined completely collecteduncharacterized, offshore. likely Earlier because this year, resultsmost of thedrilliids transcriptomic are relatively analysis small, ofuncommon, two species and in predominantly the genus Clavus collectedwere offshore. published Earlier [7] featuringthis unparalleledyear, results diversity of the oftranscriptomic putative venom analysis components of two species in these in the gastropods. genus Clavus An were important published finding [7] of Lu etfeaturing al. [7] was unparalleled the high number diversity of of transcripts putative venom encoding components short cysteine-rich in these gastropods. mature An peptides, important sharing somefinding crucial structuralof Lu et al. features [7] was of the conotoxins high number [8]. Suchof transcripts structural encoding similarity short suggests cysteine-rich that drilliid mature venom peptidespeptides, are also sharing likely some to share crucial some structural pharmacological features of properties conotoxins of [8]. conotoxins. Such structural Taking similarity into account the broadsuggests interest that indrilliid conotoxins, venom discoveringpeptides are aalso principally likely to novelshare some source pharmacological of natural peptides properties with similarof conotoxins. Taking into account the broad interest in conotoxins, discovering a principally novel bioactivity would be of great importance. However, first, the predicted transcripts need to be validated source of natural peptides with similar bioactivity would be of great importance. However, first, the by thepredicted analysis transcripts of venom need proteins, to be and validated second, by the the proposed analysis bioactivityof venom proteins, has to be and demonstrated. second, the Inproposed this report, bioactivity we describehas to be demonstrated. the purification and characterization of cdg14a, isolated from the venom ofInClavus this report, davidgilmouri we describeFedosov the purification and Puillandre, and characterization 2020, the first bioactiveof cdg14a, peptide isolated offrom any the species in thevenom family of Drilliidae. Clavus davidgilmouri Following Fedosov Lu et al. and [7], Puillandre, we refer to2020, the venomthe first peptidesbioactive frompeptide species of any in this familyspecies as “drillipeptides” in the family Drilliidae. to distinguish Following these Lu fromet al. conopeptides[7], we refer to the and venom turripeptides. peptides from The speciesspecies that we havein this investigated, family as “drillipeptides”Clavus davidgilmouri to distinguish, measures these ~15–30 from mmconopeptides (Figure1 );and it is turripeptides. one of the species The in the broadlyspecies distributedthat we have Indo-Pacificinvestigated, ClavusClavus davidgilmouri canalicularis, complex,measures ~15–30 whose mm taxonomy (Figure 1); has it beenis one recently of the species in the broadly distributed Indo-Pacific Clavus canalicularis complex, whose taxonomy has revised [9] and for which transcriptomic data is available [7]. been recently revised [9] and for which transcriptomic data is available [7]. Figure 1. Shells of Clavus species. From left to right: Clavus davidgilmouri, Clavus canalicularis, Clavus Figure 1. Shells of Clavus species. From left to right: Clavus davidgilmouri, Clavus canalicularis, viduus, Clavus unizonalis, and Clavus suduirauti [9–11]. Clavus viduus, Clavus unizonalis, and Clavus suduirauti [9–11].