An impressive biodiversity (>10,000 species) of marine snails (suborder Toxoglossa or superfamily Conoidea) have complex venoms, containing ca. 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.
venom peptides; accelerated evolution; Conidae; Turridae
A short (259 nucleotide) conserved intronic sequence (CIS) is surprisingly informative for delineating deep phylogenetic relationships in cone snails. Conus species previously have been assigned to clades based on the evidence from mitochondrial 12S and 16S rRNA gene sequences (1129 bp). Despite their length, these genes lack the phylogenetic information necessary to resolve the relationships among the clades. Here we show that the relationships can be inferred from just 46 sites in the very short CIS sequence (a portion of “intron 9” of the γ-glutamyl carboxylase gene). This is counterintuitive because in short sequences sampling error (noise) often drowns out phylogenetic signal. The intron 9 CIS is rich in synapomorphies that define the divergence patterns among eight clades of worm- and fish-hunting Conus, and it contains almost no homoplasy. Parsimony, maximum-likelihood and Bayesian analyses of the combined sequences (mt rRNA + CIS) confirm most of the relationships among 23 Conus sequences. This phylogeny implies that fish-hunting behavior evolved at least twice during the history of Conus -once among New World species and independently in the Indo-Pacific clades.
episodic evolution; conserved intron; Conus; evolution
The M-superfamily of conotoxins currently comprises three major groups of peptides (the μ-, κM-, and ψ-families) that share a key structural characteristic, the six-Cysteine motif CC-C-C-CC, but differ with respect to their molecular targets. The ψ-family consists of M-superfamily conotoxins that are nicotinic acetylcholine receptor (nAChR) antagonists. To date, only two ψ-conotoxins, PIIIE and PIIIF, are known, both of which were isolated from a single Conus species, C. purpurascens. In this paper, we report the discovery and initial characterization of a ψ-conotoxin from another Conus species, C. parius, which we designated as PrIIIE. Its amino acid sequence, inferred from a cloned cDNA, differed significantly from those of PIIIE and PIIIF. Its bioactivity was investigated by using the synthetic form of the peptide in mice and fish bioassays. At 2.5 nmole, the synthetic peptide induced flaccid paralysis in goldfish in ca. 4 min but did not induce any remarkable behavior in mice (after i.c. and i.p. injection of up to 10 nmole of peptide) and did not block action potential in directly-stimulated frog muscle preparations. Electrophysiological experiments carried out to measure inhibition of ion currents through mouse nAChR receptors expressed in oocytes revealed that PrIIIE (IC50 ∼ 250 nM) was significantly more potent than PIIIE (IC50 ∼ 7000 nM) and that PrIIIE showed higher ihhibition potency against the adult-type than the fetal type nAChR. In similar electrophysiological assays, PrIIIE showed no inhibitory effects against the mouse muscle subtype Na+ channel isoform Nav 1.4. The discovery of this ψ-conotoxin from a Conus species that belongs to the subgenus Phasmoconus, which is distinct from and larger than the clade in which C. purpurascens belongs, suggests that greater structural and functional diversity of ψ-conotoxins remains to be discovered from the members of this subgenus.
Cone snails, genus Conus, are predatory marine snails that use venom to capture their prey. This venom contains a diverse array of peptide toxins, known as conotoxins, which undergo a diverse set of posttranslational modifications. Amidating enzymes modify peptides and proteins containing a C-terminal glycine residue, resulting in loss of the glycine residue and amidation of the preceding residue. A significant fraction of peptides present in the venom of cone snails contain C-terminal amidated residues, which are important for optimizing biological activity. This study describes the characterization of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), present in the venom duct of cone snails, Conus bullatus and Conus geographus.
PAM is known to carry out two functions, peptidyl α-hydroxylating monooxygenase (PHM) and peptidylamido-glycolate lyase (PAL). In some animals, such as Drosophila melanogaster, these two functions are present in separate polypeptides, working as individual enzymes. In other animals, such as mammals and in Aplysia californica, PAM activity resides in a single, bifunctional polypeptide. Using specific oligonucleotide primers and reverse transcription-polymerase chain reaction we have identified and cloned from the venom duct cDNA library, a cDNA with 49% homology to PAM from A. californica. We have determined that both the PHM and PAL activities are encoded in one mRNA polynucleotide in both C. bullatus and C. geographus. We have directly demonstrated enzymatic activity catalyzing the conversion of dansyl-YVG-COOH to dansyl-YV-NH2 in cloned cDNA expressed in Drosophila S2 cells.
Posttranslational modification; Conotoxins; Peptidylglycine α-amidating; monooxygenase
The venom peptides (i.e., conotoxins or conopeptides) that species in the genus Conus collectively produce are remarkably diverse, estimated to be around 50,000 to 140,000, but the pace of discovery and characterization of these peptides have been rather slow. To date, only a minor fraction have been identified and studied. However, the advent of next-generation DNA sequencing technologies has opened up opportunities for expediting the exploration of this diversity.
The whole transcriptome of a venom duct from the vermivorous marine snail C. pulicarius was sequenced using the 454 sequencing platform. Analysis of the data set resulted in the identification of over eighty unique putative conopeptide sequences, the highest number discovered so far from a Conus venom duct transcriptome. More importantly, majority of the sequences were potentially novel, many with unexpected structural features, hinting at the vastness of the diversity of Conus venom peptides that remains to be explored. The sequences represented at least 14 major superfamilies/types (disulfide- and non-disulfide-rich), indicating the structural and functional diversity of conotoxins in the venom of C. pulicarius. In addition, the contry-phans were surprisingly more diverse than what is currently known. Comparative analysis of the O-superfamily sequences also revealed insights into the complexity of the processes that drive the evolution and diversification of conotoxins.
Conotoxin; Conopeptide; Toxin; Transcriptome
There are over 10,000 species of venomous marine molluscs, the vast majority of these, which are generally referred to as “turrids”, are traditionally assigned to a single family, Turridae (Powell 1966). Here, we provide an initial molecular analysis of the type genus of the family, Turris Röding, 1798, thought to be among the most well characterized groups in the family. We show that the type genus is not monophyletic.
We analyzed specimens conventionally assigned to 9 different Turris species using molecular markers, combined with the shell morphology and radular anatomy whenever feasible. The results suggest that species assigned to the genus Turris, provisionally assigned to two different subgenera are not monophyletic. Five previously described species belong to the subgenus Turris (s.s.) Röding 1798: T. babylonia,(Linne, 1758), T. grandis, (J. E. Gray, 1834), T. dollyae, (Olivera, 1999), T. normandavidsoni (Olivera, 1999) and T. spectabilis (Reeve, 1843). With a change in species designation, T. assyria (formerlyT. babylonia1010) is added to a well-defined clade, which is in turn more closely related to Lophiotoma and Gemmula species than to the other five Turris species.
We show that these five species conventionally assigned to Turris do not belong in the same subgenus, and form a clade provisionally designated as Annulaturris Powell, 1966: T. annulata, (Reeve, 1843), T. undosa, (Lamarck, 1816), T. cristata, (Vera-Peláez, Vega-Luz, and Lozano-Francisco 2000) T. cryptorrhaphe (G. B. Sowerby, 1825) and T. nadaensis (Azuma, 1973). Implications of the molecular phylogenetic results and its correlation with radular morphology are discussed.
Turris; Gemmula; Lophiotoma; radulae; molecular phylogeny; shell morphology; morphospecies
µ-SIIIA, a novel µ-conotoxin from Conus striatus, appeared to be a selective blocker of tetrodotoxin-sensitive sodium channels in frog preparations. It also exhibited potent analgesic activity in mice, although its selectivity profile against mammalian sodium channels remained unknown. We have determined the structure of µ-SIIIA in aqueous solution and characterized its backbone dynamics by NMR and its functional properties electrophysiologically. Consistent with the absence of hydroxyprolines, µ-SIIIA adopts a single conformation with all peptide bonds in the trans conformation. The C-terminal region contains a well-defined helix encompassing residues 11–16, while residues 3–5 in the N-terminal region form a helix-like turn resembling 310 helix. The Trp12 and His16 side chains are in close proximity, as in the related conotoxin µ-SmIIIA, but Asn2 is further away. Dynamics measurements show that the N-terminus and Ser9 have larger magnitude motions on the sub-ns timescale, while the C-terminus is more rigid. Cys4, Trp12 and Cys13 undergo significant conformational exchange on µs - ms timescales. µ-SIIIA is a potent, nearly irreversible blocker of NaV1.2, but also blocks NaV1.4 and NaV1.6 with submicromolar potency. The selectivity profile of µ-SIIIA, including poor activity against the cardiac sodium channel, NaV1.5, is similar to that of the closely related µ-KIIIA, suggesting that the C-terminal regions of both are critical for blocking neuronal NaV1.2. The structural and functional characterization described in this paper of an analgesic µ-conotoxin that targets neuronal subtypes of mammalian sodium channels provides a basis for the design of novel analogues with an improved selectivity profile.
Among the μ-conotoxins that block vertebrate voltage-gated sodium channels (VGSCs), some have been shown to be potent analgesics following systemic administration in mice. We have determined the solution structure of a new representative of this family, μ-BuIIIB, and established its disulfide connectivities by direct mass spectrometric collision induced dissociation fragmentation of the peptide with disulfides intact. The major oxidative folding product adopts a 1-4/2-5/3-6 pattern with the following disulfide bridges: Cys5-Cys17, Cys6-Cys23 and Cys13-Cys24. The solution structure reveals that the unique N-terminal extension in μ-BuIIIB, which is also present in μ-BuIIIA and μ-BuIIIC but absent in other μ-conotoxins, forms part of a short α-helix encompassing Glu3 to Asn8. This helix is packed against the rest of the toxin and stabilized by the Cys5-Cys17 and Cys6-Cys23 disulfide bonds. As such, the side chain of Val1 is located close to the aromatic rings of Trp16 and His20, which are located on the canonical helix that displays several residues found to be essential for VGSC blockade in related μ-conotoxins. Mutations of residues 2 and 3 in the N-terminal extension enhanced the potency of μ-BuIIIB for NaV1.3. One analog, [d-Ala2]BuIIIB, showed a 40-fold increase, making it the most potent peptide blocker of this channel characterized to date and thus a useful new tool with which to characterize this channel. Based on previous results for related μ-conotoxins, the dramatic effects of mutations at the N-terminus were unanticipated, and suggest that further gains in potency might be achieved by additional modifications of this region.
The μ-conotoxin μ-KIIIA, from Conus kinoshitai, blocks mammalian neuronal voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. We have determined its solution structure using NMR spectroscopy. Key residues identified previously as being important for activity against VGSCs (Lys7, Trp8, Arg10, Asp11, His12 and Arg14) all reside on an α-helix with the exception of Arg14. To further probe structure-activity relationships of this toxin against VGSC subtypes, we have characterised the analogue μ-KIIIA[C1A,C9A], in which the Cys residues involved in one of the three disulfides in μ-KIIIA were replaced with Ala. Its structure is quite similar to that of μ-KIIIA, indicating that the Cys1-Cys9 disulfide bond could be removed without any significant distortion of the α-helix bearing the key residues. Consistent with this, μ-KIIIA[C1A,C9A] retained activity against VGSCs, with its rank order of potency being essentially the same as that of μ-KIIIA, namely, NaV1.2 > NaV1.4 > NaV1.7 ≥ NaV1.1 > NaV1.3 > NaV1.5. Kinetics of block were obtained for NaV1.2, NaV1.4 and NaV1.7, and in each case both kon and koff values of μ-KIIIA[C1A,C9A] were larger than those of μ-KIIIA. Our results show that the key residues for VGSC binding lie mostly on an α-helix and that the first disulfide bond can be removed without significantly affecting the structure of this helix, although the modification accelerates the on- and off-rates of the peptide against all tested VGSC subtypes. These findings lay the groundwork for the design of minimized peptides and helical mimetics as novel analgesics.
The traditional taxonomy of ca. 700 cone snails assigns all species to a single genus, Conus Linnaeus, 1758. However an increasing body of evidence suggest that some belong to a genetically distinct clade alternatively referred to as the Conasprella (Thiele, 1929), Previously we showed that a short (259 bp) conserved intronic sequence (CIS) of the γ-glutamyl carboxylase gene (intron 9) is surprisingly informative for delineating deep phylogenetic relationships among other Conus snails (Kraus, et al. 2011). In this work, we once again use intron 9 (338 bp) to easily resolve problemaric relationships among the Conasprellans. Counterintuitively, we show that these relationships can be inferred from just 39 synapomorphic isites. The sequence is so well conserved that conflicting sites do not obscure the few informative sites that provide clear phylogenetic signal.
Unexpectedly we also found that intron 9 unambiguously distinguishes Conasprella species from the Conus species studied earlier. The respective alignments are so different from one another that the sequences from the two groups cannot be aligned and thus a phylogeny describing the genetic relationship between the formerly desginated congeners cannot be inferred from these data alone. This lack of homology between the intronic sequences belonging to each group clearly shows that they are separated by considerable evolutionary history.
nuclear genes; conserved intron; Conus; Conasprella; evolution
Using molecular phylogeny has accelerated the discovery of peptidic ligands targeted to ion channels and receptors. One clade of venomous cone snails, Asprella, appears to be significantly enriched in conantokins, antagonists of N-Methyl D-Asparate receptors (NMDARs). Here, we describe the characterization of two novel conantokins from Conus rolani, including conantokin conRl-B that has shown an unprecedented selectivity for blocking NMDARs that contain NR2B subunits. ConRl-B shares only some sequence similarity to the most studied NR2B-selective conantokin, conG. The divergence between conRl-B and conG in the second inter-Gla loop was used to design analogs for structure-activity studies; the presence of Pro10 was found to be key to the high potency of conRl-B for NR2B, whereas the ε-amino group of Lys8 contributed to discrimination in blocking NR2B- and NR2A-containing NMDARs. In contrast to previous findings from Tyr5 substitutions in other conantokins, conRl-B [L5Y] showed potencies on the four NR2 NMDA receptor subtypes that were similar to those of the native conRl-B. When delivered into the brain, conRl-B was active in suppressing seizures in the model of epilepsy in mice, consistent with NR2B-containing NMDA receptors being potential targets for antiepileptic drugs. Circular dichroism experiments confirmed that the helical conformation of conRl-B is stabilized by divalent metal ions. Given the clinical applications of NMDA antagonists, conRl-B provides a potentially important pharmacological tool for understanding the differential roles of NMDA receptor subtypes in the nervous system. This work shows the effectiveness of coupling molecular phylogeny, chemical synthesis and pharmacology for discovering new bioactive natural products.
Conus peptides; conantokin; NMDA antagonist; NR2B subunits; epilepsy; anticonvulsant
An accelerated rate of natural-product discovery is critical for the future of ion channel pharmacology. For the full potential of natural products to be realized, an interdisciplinary initiative is required that combines chemical ecology and ion channel physiology. A prime source of future drug leads targeted to ion channels is the vast assortment of compounds that mediate biotic interactions in the marine environment. Many animals have evolved a chemical strategy to change the behavior of their prey, predators or competitors, which appears to require a large set of ion-channel targeted compounds acting in concert. Some of these compounds (e.g. Ziconotide (Prialt)) have already found important biomedical applications. The elucidation of molecular mechanisms mediating biotic interactions should yield a rich stream of potent and selective natural products for the drug pipeline.
The bacterium Gordonia sp. 647W.R.1a.05 was cultivated from the venom duct of the cone snail, Conus circumcisus. The Gordonia sp. organic extract modulated the action potential of mouse dorsal root ganglion neurons. Assay-guided fractionation led to the identification of the new compound circumcin A (1) and 11 known analogs (2–12). Two of these compounds, kurasoin B (7) and soraphinol A (8), were active in a human norepinephrine transporter assay with Ki values of 2575 and 867 nM, respectively. No neuroactivity had previously been reported for compounds in this structural class. Gordonia species have been reproducibly isolated from four different cone snail species, indicating a consistent association between these organisms.
Natural product; symbiont; neuroassay
A multidisciplinary strategy for discovery of new Conus venom peptides combines molecular genetics and phylogenetics with peptide chemistry and neuropharmacology. Here we describe application of this approach to the conantokin family of conopeptides targeting NMDA receptors. A new conantokin from Conus rolani, ConRl-A, was identified using molecular phylogeny and subsequently synthesized and functionally characterized. ConRl-A is a 24-residue peptide containing three gamma-carboxyglutamic acid residues with a number of unique sequence features compared to conantokins previously characterized. The HPLC elution of ConRl-A suggested that this peptide exists as two distinct, slowly exchanging conformers. ConRl-A is predominantly helical (estimated helicity of 50%), both in the presence and absence of Ca++. The order of potency for blocking the four NMDA receptor subtypes by ConRl-A was NR2B>NR2D>NR2A>NR2C. This peptide has a greater discrimination between NR2B and NR2C then any other ligand reported so far. In summary, ConRl-A is a new member of the conantokin family that expands our understanding of structure/function of this group of peptidic ligands targeted to NMDA receptors. Thus, incorporating phylogeny in the discovery of novel ligands for the given family of ion channels or receptors is an efficient means of exploring the megadiverse group of peptides from genus Conus.
Conantokin; Molecular phylogeny; Conformational interconversion; Helical peptide; Electrophysiology
In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely-related μ-conotoxins. The two major isomers of synthetic μ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric CID fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of NaV1.2 (Kd 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on NMR data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the μ-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an α-helix spanning residues 7–12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as for μ-KIIIA, and both blocked NaV1.2 (Kd 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA/μ-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of μ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means to further expand the snail's repertoire of active peptides.
Disulfide bridges, which stabilize the native conformation of conotoxins impose a challenge in the synthesis of smaller analogs. In this work, we describe the synthesis of a minimized analog of the analgesic μ-conotoxin KIIIA that blocks two sodium channel subtypes, the neuronal NaV1.2 and skeletal muscle NaV1.4. Three disulfide-deficient analogs of KIIIA were initially synthesized in which the native disulfide bridge formed between either C1-C9, C2-C15 or C4-C16 was removed. Deletion of the first bridge only slightly affected the peptide’s bioactivity. To further minimize this analog, the N-terminal residue was removed and two non-essential Ser residues were replaced by a single 5-amino-3-oxapentanoic acid residue. The resulting “polytide” analog retained the ability to block sodium channels and to produce analgesia. Until now, the peptidomimetic approach applied to conotoxins has progressed only modestly at best; thus, the disulfide-deficient analogs containing backbone spacers provide an alternative advance toward the development of conopeptide-based therapeutics.
conopeptide; conotoxin; sodium channels; backbone spacers; disulfide bridges
This review presents recommended nomenclature for the biosynthesis of ribosomally synthesized and post-translationally modified peptides (RiPPs), a rapidly growing class of natural products. The current knowledge regarding the biosynthesis of the >20 distinct compound classes is also reviewed, and commonalities are discussed.
Conantokins are venom peptides from marine cone snails that are NMDA receptor antagonists. Here, we report the characterization of a 24 AA conantokin from Conus brettinghami (1), conantokin-Br (con-Br), the first conantokin that does not have the conserved glutamate residue at position 2. Molecular modeling studies suggest that con-Br has a helical structure between residues 2–13. In contrast to other characterized conantokins, con-Br has a high potency for NMDA receptors with NR2D subunits. To identify determinants for NR2D potency, we synthesized chimeras of con-Br and conantokin-R (con-R), the latter has a ~30-fold lower potency for the NR2D subtype. The characterization of two reciprocal chimeras (con-Br/R and con-R/Br), comprising the first 9–10 N-terminal AAs of each conantokin followed by the corresponding C-terminal AAs of the other conantokin demonstrates that determinants for NR2D selectivity are at the N-terminal region. Additional analogs comprising 1–3 amino acid substitutions from each peptide into the homologous region of the other led to the identification of a key determinant; a Tyr residue in position 5 increases potency for NR2D, while Val at this locus causes a decrease. The systematic definition of key determinants in the conantokin peptides for NMDA receptor subtype selectivity is an essential component in the development of conantokin peptides that are highly selective for each specific NMDA receptor subtype.
The present study details the purification, the amino acid sequence determination, and a preliminary characterization of the biological effects in mice of a new conotoxin from the venom of Conus cancellatus (jr. syn.: Conus austini), a worm-hunting cone snail collected in the western Gulf of Mexico (Mexico). The 23-amino acid peptide, called as24a, is characterized by the sequence pattern CX1CX2CX8CX1CCX5, which is, for conotoxins, a new arrangement of six cysteines (framework XXIV) that form three disulfide bridges. The primary structure (CKCPSCNFNDVTENCKCCIFRQP*; *, amidated C-terminus; calculated monoisotopic mass, 2,644.09 Da) was established by automated Edman degradation after reduction and alkylation, and MALDI-TOF and ESI mass spectrometry (monoisotopic mass, 2644.12/2644.08 Da). Upon intracranial injection in mice, the purified peptide provokes paralysis of the hind limbs and death with a dose of 240 pmoles (~0.635 ! g, ~24.9 ng/g). In addition, a post-translational variant of this peptide (as24b) was identified and determined to contain two hydroxyproline residues. These peptides may represent a novel conotoxin gene superfamily.
Conus cancellatus; Conus austini; vermivorous; conotoxin; six Cys; paralysis
In the oceans, toxic secondary metabolites often protect otherwise poorly defended, soft-bodied invertebrates such as shell-less mollusks from predation. The origins of these metabolites are largely unknown, but many of them are thought to be made by symbiotic bacteria. In contrast, mollusks with thick shells and toxic venoms are thought to lack these secondary metabolites due to reduced defensive needs. Here, we show that heavily defended cone snails also occasionally contain abundant secondary metabolites, γ-pyrones known as nocapyrones, and that these pyrones are synthesized by symbiotic bacteria. This study shows that symbiotic bacteria can produce metabolites isolated from gastropod mollusks. The symbiotic bacteria, Nocardiopsis alba CR167, are closely related to potentially widespread actinomycetes that we propose to be casual symbionts of invertebrates on land and in the sea. The natural roles of nocapyrones are not known, but they are active in neurological assays at low micromolar levels, revealing that mollusks with external shells are an overlooked source of secondary metabolite diversity.
The taxonomy of the genus Turris
Batsch, 1789, type genus of the family Turridae, widespread in shallow-water habitats of tropic Indo-Pacific, is revised. A total of 31 species of Turris, are here recognized as valid. New species described: Turris chaldaea, Turris clausifossata, Turris guidopoppei, Turris intercancellata, Turris kantori, T. kathiewayae. Homonym renamed: Turris bipartita nom. nov. for Pleurotoma variegata
Kiener, 1839 (non Philippi, 1836). New synonymies: Turris ankaramanyensis
Bozzetti, 2006 = Turris tanyspira
Kilburn, 1975; Turris imperfecti, T. nobilis, T. pulchra and T. tornatum
Röding, 1798, and Turris assyria
Olivera, Seronay & Fedosov, 2010 = T. babylonia; Turris dollyi
Olivera, 2000 = Pleurotoma crispa
Lamarck, 1816; Turris totiphyllis
Olivera, 2000 = Turris hidalgoi
Vera-Peláez, Vega-Luz & Lozano-Francisco, 2000; Turris kilburni
Vera-Peláez, Vega-Luz & Lozano-Francisco, 2000 = Turris pagasa
Olivera, 2000; Turris (Annulaturris) munizi
Vera-Peláez, Vega-Luz & Lozano-Francisco, 2000 = Gemmula lululimi
Olivera, 2000. Revised status: Turris intricata
Powell, 1964, Pleurotoma variegata Kiener, 1839 (non Philippi, 1836) and Pleurotoma yeddoensis Jousseaume, 1883, are regarded as full species (not subspecies of Turris crispa). Neotype designated: For Pleurotoma garnonsii
Reeve, 1843, to distinguish it from Turris garnonsii of recent authors, type locality emended to Zanzibar. New combination: Turris orthopleura
Kilburn, 1983, is transferred to genus Makiyamaia, family Clavatulidae.
Taxonomy; Turris; Indo-West Pacific; new species; new synonymies; new combinations
In cysteine-rich peptides, diselenides can be used as a proxy for disulfide bridges, since the energetic preference for diselenide bonding over mixed selenium-sulfur bonds simplifies folding. Herein we report that an intramolecular diselenide bond efficiently catalyzes the oxidative folding of selenopeptide analogs of conotoxins, and serves as a reagentless method to substantially accelerate formation of various native disulfide bridging patterns.
Diselenide; oxidative folding; kinetics; conotoxin; selenopeptide
Conotoxins comprise a large group of peptidic neurotoxins that employ diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of non-critical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of ω-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot (ICK) motif family and blocks N-type calcium channels. Removal of a non-critical Cys1–Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally non-critical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionary preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold.
disulfide bridges; conotoxins; structure-function; oxidative folding; calcium channels
Two new compounds, peptide-polyketide glycoside totopotensamide A (1) and its aglycone totopotensamide B (2), were isolated from Streptomyces sp. cultivated from the gastropod mollusk, Lienardia totopotens collected in the Philippines. The compounds contain a previously undescribed polyketide component, a novel 2,3-diaminobutyric acid-containing macrolactam, and a new amino acid, 4-chloro-5,7-dihydroxy-6-methylphenylglycine. The application of Marfey’s method to phenylglycine derivatives was explored using quantum mechanical calculations and NMR.
We investigated the functional expression of nicotinic acetylcholine receptors (nAChRs) in heterogeneous populations of dissociated rat and mouse lumbar dorsal root ganglion (DRG) neurons by calcium imaging. By this experimental approach, it is possible to investigate the functional expression of multiple receptor and ion-channel subtypes across more than 100 neuronal and glial cells simultaneously. Based on nAChR expression, DRG neurons could be divided into four subclasses: (1) neurons that express predominantly α3β4 and α6β4 nAChRs; (2) neurons that express predominantly α7 nAChRs; (3) neurons that express a combination of α3β4/α6β4 and α7 nAChRs; and (4) neurons that do not express nAChRs. In this comparative study, the same four neuronal subclasses were observed in mouse and rat DRG. However, the expression frequency differed between species: substantially more rat DRG neurons were in the first three subclasses than mouse DRG neurons, at all developmental time points tested in our study. Approximately 70–80% of rat DRG neurons expressed functional nAChRs, in contrast to only ~15–30% of mouse DRG neurons. Our study also demonstrated functional coupling between nAChRs, voltage-gated calcium channels, and mitochondrial Ca2+ transport in discrete subsets of DRG neurons. In contrast to the expression of nAChRs in DRG neurons, we demonstrated that a subset of non-neuronal DRG cells expressed muscarinic acetylcholine receptors and not nAChRs. The general approach to comparative cellular neurobiology outlined in this paper has the potential to better integrate molecular and systems neuroscience by uncovering the spectrum of neuronal subclasses present in a given cell population and the functionally integrated signaling components expressed in each subclass.
calcium imaging; DRG; nAChR; conotoxin; sensory neuron; neuronal subclass