A vasopressin/oxytocin-related conopeptide with gamma-carboxyglutamate at position 8

Vasopressins and oxytocins are homologous, ubiquitous and multifunctional peptides present in animals. Conopressins are vasopressin/oxytocin-related peptides that have been found in the venom of cone snails, a genus of marine predatory molluscs that envenom their prey with a complex mixture of neuroactive peptides. In the present paper, we report the purification and characterization of a unique conopressin isolated from the venom of Conus villepinii, a vermivorous cone snail species from the western Atlantic Ocean. This novel peptide, designated gamma-conopressin-vil, has the sequence CLIQDCPgammaG* (gamma is gamma-carboxyglutamate and * is C-terminal amidation). The unique feature of this vasopressin/oxytocin-like peptide is that the eighth residue is gamma-carboxyglutamate instead of a neutral or basic residue; therefore it could not be directly classified into either the vasopressin or the oxytocin peptide families. Nano-NMR spectroscopy of the peptide isolated directly from the cone snails revealed that the native gamma-conopressin-vil undergoes structural changes in the presence of calcium. This suggests that the peptide binds calcium, and the calcium-binding process is mediated by the gamma-carboxyglutamate residue. However, the negatively charged residues in the sequence of gamma-conopressin-vil may mediate calcium binding by a novel mechanism not observed in other peptides of this family.     

Mollusc-specific toxins from the venom of Conus textile neovicarius.

Three peptide toxins exhibiting strong paralytic activity to molluscs, but with no paralytic effects on arthropods or vertebrates, were purified from the venom of the molluscivorous snail Conus textile neovicarius from the Red Sea. The amino acid sequences of these mollusc specific toxins are: TxIA, WCKQSGEMCNLLDQNCCDGYCI-VLVCT (identical to the so called 'King Kong peptide'); TxIB, WCKQSGEMCNVLDQNCCDGYCIVFVCT; TxIIA, WGGYSTYC gamma VDS gamma CCSDNCVRSYCT (gamma = gamma-carboxyglutamate). There is a similarity of the Cys framework of these toxins to that of the omega-conotoxins; however, their net negative charges, high content of hydrophobic residues and uneven number of Cys residues in TxIIA, are highly unusual for conotoxins. When assayed on isolated cultured Aplysia neurons, all three toxins induced membrane depolarization and spontaneous repetitive firing. The TxI toxins also induce a marked prolongation of the action potential duration, which is sodium dependent. These effects differ significantly from the blocking activities of piscivorous venom conotoxins. These mollusc specific conotoxins may therefore serve as new and selective probes for ion-channel functions in molluscan neuronal systems.     

Discovery of a novel class of conotoxin from Conus litteratus, lt14a, with a unique cysteine pattern

Conus litteratus is a worm-hunting cone snail with a highly sophisticated neuropharmacological defense strategy using small peptides in its venom. By analyzing different clones in the cDNA library of venom ducts from C. litteratus, we identified the peptide lt14a which displays a characteristic signal peptide sequence in its precursor and a unique arrangement of Cys residues (-C-C-C-C-) in its mature peptide region. RT-PCR analysis suggested that lt14a is abundantly expressed throughout the whole venom duct. An intensive analysis in sequence suggested that lt14a is similar to alpha-conotoxin qc1.1 cloned from Conus quercinus. We conducted the chemical synthesis of lt14a. The synthetic lt14a has a remarkable biological activity to suppress pain and inhibits the neuronal-type nicotinic acetylcholine receptors.     

Effects of synthetic omega-conotoxin GVIA (omega-CgTX GVIA) on the membrane calcium current of an identifiable giant neurone, d-RPLN, of an African giant snail (Achatina fulica Ferussac), measured under the voltage clamp condition

1. The sodium and the calcium inward currents (INa and ICa) of an identifiable giant neurone, d-RPLN (dorsal-right parietal large neurone), of an African giant snail (Achatina fulica Férussac), were measured separately under the voltage clamp condition. 2. The effects of synthetic omega-conotoxin GVIA (omega-CgTX GVIA) on the calcium current of the neuromembrane were examined. 3. omega-CgTX GVIA is a peptide venom originally isolated from a fish-hunting marine snail (Conus geographus L.); the peptide venom has been demonstrated to block markedly calcium channels in vertebrates. 4. In the case of the d-RPLN membrane, the ICa was much larger than INa. The command voltage (Vc) to get the ICa in maximum was about 0 mV; the maximum value of ICa in a representative experimental case, was measured as approximately 0.8 microA. 5. With respect to the ICa of a molluscan giant neurone, d-RPLN, synthetic omega-CgTX GVIA at a high concentration, 5 X 10(-5) M, showed almost no effect, in spite of reporting the peptide venom affecting the ICa in vertebrate preparations.     

The presence and distribution of crustacean cardioactive peptide in the central and peripheral nervous system of the stick insect, Baculum extradentatum

Crustacean cardioactive peptide (CCAP)-like immunoreactivity was localized and quantified in the central and peripheral nervous system of the Vietnamese stick insect, Baculum extradentatum, using immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). The brain, frontal ganglion, suboesophageal ganglion and ventral nerve cord displayed neurons and processes with CCAP-like immunoreactivity. The brain, in comparison to the other parts of the central nervous system, contained the greatest amount of CCAP (167 +/- 18 fmol), and showed CCAP-like staining in neurons, neuropil regions and the central complex. There were also CCAP-like varicosities and processes associated with the corpus cardiacum. The alimentary canal of B. extradentatum contained CCAP with the largest amount localized in the midgut (1110 +/- 274 fmol CCAP equivalents). The midgut contained numerous endocrine-like cells which stained positively for CCAP, whereas the foregut and hindgut revealed an extensive network of CCAP-like immunoreactive axons and varicosities. Based on physiological assays, the hindgut of the stick insect was found to be sensitive to CCAP, showing dose-dependent increases in contractions with threshold at 10(-10) M CCAP and maximal response at 5 x 10(-7) M CCAP. There were negligible quantities of CCAP in the oviducts and no CCAP-like immunoreactivity was associated with the oviducts. CCAP had no effect on spontaneous contractions of the oviducts. The presence of CCAP in the central nervous system, the stomatogastric nervous system, the corpus cardiacum and the alimentary canal, suggest broad ranging roles for CCAP in B. extradentatum.     

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

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 (M?rch, 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.     

Two new 4-Cys conotoxins (framework 14) of the vermivorous snail Conus austini from the Gulf of Mexico with activity in the central nervous system of mice

As part of continuing studies of the venom components present in Conus austini (syn.: Conus cancellatus), a vermivorous cone snail collected in the western Gulf of Mexico, Mexico, two major peptides, as14a and as14b, were purified and characterized. Their amino acid sequences were determined by automatic Edman sequencing after reduction and alkylation. Their molecular masses, established by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, confirmed the chemical analyses and indicated that as14a and as14b have free C-termini. Each peptide contains 4-Cys residues arranged in a pattern (C-C-C-C, framework 14). The primary structure of as14a is GGVGRCIYNCMNSGGGLNFIQCKTMCY (experimental monoisotopic mass 2883.92Da; calculated monoisotopic mass 2884.20Da), whereas that of as14b is RWDVDQCIYYCLNGVVGYSYTECQTMCT (experimental monoisotopic mass 3308.63Da; calculated monoisotopic mass 3308.34Da). Both purified peptides elicited scratching and grooming activity in mice, and as14b also caused body and rear limb extension and tail curling immediately upon injection. The high sequence similarity of peptide as14a with peptide vil14a from the vermivorous C. villepinii suggests that the former might block K+ channels.     

Crustacean cardioactive peptide is a modulator of oviduct contractions in Locusta migratoria

Crustacean cardioactive peptide (CCAP) stimulates the contractions of locust oviducts. CCAP increased the basal tonus and increased the frequency and amplitude of phasic contractions, as well as the amplitude of neurally-evoked oviduct contractions in a dose-dependent manner. Oviducts from Vth instar larvae and adult locusts aged 10 days or less, were more sensitive to CCAP than oviducts from adult locusts aged 12 days or more. This may be indicative of a differential expression of number or subtypes of CCAP receptors on the oviducts at different ages, and may be related to reproductive functions or to functions of CCAP on the oviducts during ecdysis. The oviducts appear more sensitive to CCAP when compared with previously published reports of CCAP actions on the hindgut. CCAP actions on the amplitude of neurally-evoked contractions of the oviducts are similar to those of proctolin, however, the oviducts are more sensitive to CCAP. No CCAP-like immunoreactive structures were discovered in the nerves innervating the oviducts, or on the oviducts themselves, confirming the previously published suggestion (Dircksen et al., 1991) that CCAP acts as a neurohormone at the oviducts. Cells showing CCAP-like immunoreactivity were discovered in the fat body associated with the oviducts and represent a potential source of CCAP, along with CCAP released from the transverse nerve and perivisceral organs.     

Reconstructing the Origins of the Somatostatin and Allatostatin-C Signaling Systems Using the Accelerated Evolution of Biodiverse Cone Snail Toxins

Somatostatin and its related peptides (SSRPs) form an important family of hormones with diverse physiological roles. The ubiquitous presence of SSRPs in vertebrates and several invertebrate deuterostomes suggests an ancient origin of the SSRP signaling system. However, the existence of SSRP genes outside of deuterostomes has not been established, and the evolutionary history of this signaling system remains poorly understood. Our recent discovery of SSRP-like toxins (consomatins) in venomous marine cone snails (Conus) suggested the presence of a related signaling system in mollusks and potentially other protostomes. Here, we identify the molluscan SSRP-like signaling gene that gave rise to the consomatin family. Following recruitment into venom, consomatin genes experienced strong positive selection and repeated gene duplications resulting in the formation of a hyperdiverse family of venom peptides. Intriguingly, the largest number of consomatins was found in worm-hunting species (>400 sequences), indicating a homologous system in annelids, another large protostome phylum. Consistent with this, comprehensive sequence mining enabled the identification of SSRP-like sequences (and their corresponding orphan receptor) in annelids and several other protostome phyla. These results established the existence of SSRP-like peptides in many major branches of bilaterians and challenge the prevailing hypothesis that deuterostome SSRPs and protostome allatostatin-C are orthologous peptide families. Finally, having a large set of predator–prey SSRP sequences available, we show that although the cone snail’s signaling SSRP-like genes are under purifying selection, the venom consomatin genes experience rapid directional selection to target receptors in a changing mix of prey.     

Intraspecies variability and conopeptide profiling of the injected venom of Conus ermineus

The venom of cone snails (ssp. Conus), a genus of predatory mollusks, is a vast source of bioactive peptides. Conus venom expression is complex, and venom composition can vary considerably depending upon the method of extraction and the species of cone snail in question. The injected venom from Conus ermineus, the only fish-hunting cone snail species that inhabits the Atlantic Ocean, was characterized using nanoNMR spectroscopy, MALDI-TOF mass spectrometry, RP-HPLC and nanoLC-ESI-MS. These methods allowed us to evaluate the variability of the venom within this species. Single specimens of C. ermineus show unchanged injected venom mass spectra and HPLC profiles over time. However, there was significant variability of the injected venom composition from specimen to specimen, in spite of their common biogeographic origin. Using nanoLC-ESI-MS, we determined that over 800 unique conopeptides are expressed by this reduced set of C. ermineus specimens. This number is considerably larger than previous estimates of the molecular repertoire available to cone snails to immobilize prey. These results support the idea of the existence of a complex regulatory mechanism to express specific venom peptides for injection into prey. These intraspecies differences can be a result of a combination of genetic and environmental factors. The differential expression of venom components represents a neurochemical paradigm that warrants further investigation.     

Delayed pharmacological effects of proctolin and CCAP on heartbeat in pupae of the tobacco hornworm, Manduca sexta

Abstract. The effects in vivo of cardioactive peptides proctolin, CCAP and leucomyosuppressin (LMS) are investigated by means of noninvasive optocardiographic or thermographic techniques in postdiapause pupae of Manduca sexta. A constant pattern of heartbeat reversal in these pupae is manifested by regular alternations of the forward orientated (anterograde) and the backward orientated (retrograde) cardiac pulsations, with a periodicity of some 5–10 min. The heartbeat pattern is monitored continuously for several hours before and 24 h after injection of the investigated peptides. Injections of Ringer solution alone cause a slight, almost immediate increase of the rate of the pupal heartbeat (0–10%), which lasts only for 20–30 min. Injection of proctolin, CCAP or LMS does not show any immediate cardiostimulating effects (beyond those of Ringer) at concentrations up to 2 × 10⁻⁶ M (calculated from µg of the injected peptide and 70% pupal water content; 5–7 g pupal body mass). By contrast, injections of proctolin and CCAP in the range of 10^-9 − 10^-6 M concentrations cause delayed effects on the heartbeat, which are manifested only several hours after the injections. The delayed effects involve prolonged, or even continuous periods of unidirectional, more efficient and faster anterograde pulsations. Consequently, the flow of haemolymph through the head and thoracic parts of the pupal body increases. In the case of proctolin, the prolonged anterograde cardiac activity usually starts 5 h after the injections and the effect persists for 7–12 h. Using CCAP, the stimulation of anterograde activity starts 2.5–3 h after injections and lasts usually 7–8 h. Very small doses of peptides (10^-8 − 10^-9 M) do not change the latency period significantly, but they decrease the duration of the response. The frequency of the systolic contractions of the heart does not increase during the prolonged anterograde phase. Injections of LMS to produce a final concentration of 10⁻⁶ M in the pupa induce pathophysiological disturbances of heartbeat reversal and peristalsis. The effects start with a delay of some 1.5–2.5 h after the injections. By contrast to the effects of proctolin and CCAP, LMS does not produce delayed anterograde cardiac pulsations. These results show that the most intensively investigated cardiostimulating peptides in vitro, proctolin and CCAP, have no direct cardiostimulating activity under physiological conditions in vivo. It is concluded therefore that the delayed pharmacological effects of these peptides observed in the pupae of M. sexta, represent a secondary effect, resulting from stimulation of nonspecific, extracardiac myotropic or other physiological functions.     

Evidence for crustacean cardioactive peptide-like innervation of the gut in Locusta migratoria

Hindguts from female Vth instar larvae, young adults (1-2 days) and old adults (>10 days) are equally sensitive to the crustacean cardioactive peptide (CCAP), with changes in contraction occurring at a threshold concentration of 10(-9)M and maximal responses observed at concentrations ranging between 10(-7) and 5x10(-6)M. An immunohistochemical examination of the gut of Locusta migratoria with an antiserum raised against CCAP revealed an extensive network of CCAP-like immunoreactive processes on the hindgut and posterior midgut via the 11th sternal nerve arising from the terminal abdominal ganglion. Anterograde filling of the 11th sternal nerve with neurobiotin revealed extensive processes and terminals on the hindgut. Retrograde filling of the branch of the 11th sternal nerve which innervates the hindgut with neurobiotin revealed two bilaterally paired cells in the terminal abdominal ganglion which co-localized with CCAP-like immunoreactivity. Results suggest that a CCAP-like substance acts as a neurotransmitter/neuromodulator at the locust hindgut.     

Identification of six novel T-1 conotoxins from Conus pulicarius by molecular cloning

Cone snails are a group of ancient marine gastropods with highly sophisticated defense and prey strategies using conotoxins in their venom. Conotoxins are a diverse array of small peptides, mostly with multiple disulfide bridges. Using a 3' RACE approach, we identified six novel peptides from the venom ducts of a worm-hunting cone snail Conus pulicarius. These peptides are named Pu5.1-Pu5.6 as their primary structures show the typical pattern of T-1 conotoxin family, a large and diverse group of peptides widely distributed in venom ducts of all major feeding types of Conus. Except for the conserved signal peptide sequences in the precursors and unique arrangement of Cys residues (CC-CC) in mature domains, the six novel T-1 conotoxins show remarkable sequence diversity in their pro and mature regions and are, thus, likely to be functionally diversified. Here, we present a simple and fast strategy of gaining novel disulfide-rich conotoxins via molecular cloning and our detailed sequence analysis will pave the way for the future functional characterization of toxin-receptor interaction.     

lambda-conotoxins, a new family of conotoxins with unique disulfide pattern and protein folding. Isolation and characterization from the venom of Conus marmoreus

Conotoxins are multiple disulfide-bonded peptides isolated from marine cone snail venom. These toxins have been classified into several families based on their disulfide pattern and biological properties. Here, we report a new family of Conus peptides, which have a novel cysteine motif. Three peptides of this family (CMrVIA, CMrVIB, and CMrX) have been purified from Conus marmoreus venom, and their structures have been determined. Their amino acid sequences are VCCGYK-LCHOC (CMrVIA), NGVCCGYKLCHOC (CMrVIB), and GICCGVSFCYOC (CMrX), where O represents 4-trans-hydroxyproline. Two of these peptides (CMrVIA and CMrX) have been chemically synthesized. Using a selective protection and deprotection strategy during disulfide bond formation, peptides with both feasible cysteine-pairing combinations were generated. The disulfide pattern (C(1)-C(4), C(2)-C(3)) in native toxins was identified by their co-elution with the synthetic disulfide-isomeric peptides on reverse-phase high pressure liquid chromatography. Although cysteine residues were found in comparable positions with those of alpha-conotoxins, these toxins exhibited a distinctly different disulfide bonding pattern; we have named this new family "lambda -conotoxins." CMrVIA and CMrX induced different biological effects when injected intra-cerebroventricularly in mice; CMrVIA induces seizures, whereas CMrX induces flaccid paralysis. The synthetic peptide with lambda-conotoxin folding is about 1150-fold more potent in inducing seizures than the mispaired isomer with alpha-conotoxin folding. Thus it appears that the unique disulfide pattern, and hence the "ribbon" conformation, in lambda-conotoxins is important for their biological activity.     

The association of crustacean cardioactive peptide with the spermatheca of the African migratory locust, Locusta migratoria

Crustacean cardioactive peptide (CCAP)-like immunoreactivity was identified in neurons of the VIIIth abdominal ganglion and in axons in the nerves that project to the spermatheca of 3-4 week old adult female locusts. In addition, lightly stained CCAP-like immunoreactive processes were localized over the spermathecae. The amount of CCAP in the spermathecal tissue was quantified using an enzyme-linked immunosorbent assay (ELISA) performed on extracts of the whole spermatheca, and on its constituent parts, namely the sperm sac, coiled duct and straight duct. The spermatheca contains 920+/-273 fmol (mean+/-SE) of CCAP equivalents, with the majority localized in the coiled duct. There are age-related differences in the amount of CCAP present in the spermathecae with less content in spermathecae from 1 to 5 day old and greater content in spermathecae from 3 to 4 week old adults. There was also no difference in CCAP content of spermathecae in mated and virgin 3 to 4 week old adults. Reversed phase-high performance liquid chromatography (RP-HPLC) followed by ELISA further confirmed the presence of CCAP-like material in extracts of locust spermathecae. Physiological assays demonstrated that CCAP increased the basal tonus and frequency of spontaneous contractions of the spermatheca, with thresholds between 10(-10) and 10(-9)M and maxima at 10(-7)M CCAP. CCAP also increases the amplitude of neurally evoked contractions with a threshold less than 10(-11)M and a maximum of 10(-7)M CCAP. The present study suggests that CCAP acts as a neuromodulator/neurotransmitter at the spermathecal visceral tissue of female Locusta migratoria.