Venom Of Conus Geographus Research Articles

Sensitive detection of α-conotoxin GI in human plasma using a solid-phase extraction column and LC-MS/MS

α-conotoxin GI, a short peptide toxin in the venom of Conus geographus, is composed of 13 amino acids and two disulfide bonds. It is the most toxic component of Conus geographus venom with estimated lethal doses of 0.029-0.038 mg/kg for humans. There is currently no reported analytical method for this toxin. In the present study, a sensitive detection method was developed to quantify GI in human plasma using a solid-phase extraction (SPE) column (polystyrene-divinyl benzene copolymer) combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the multiple reaction monitoring (MRM) mode. The plasma samples were treated with a protein precipitating solvent (methanol: acetonitrile = 50:50, v/v). GI in the solvent was efficiently extracted with an SPE column and was further separated by a Grace Alltima HP C18 (50 × 2.1 mm, 5 μm) column at a flow rate of 0.4 mL/min. Water (with 2% methanol) acetonitrile (with 0.1% acetic acid) was selected as the mobile phase combination used in a linear gradient system. α-Conotoxin GI was analyzed by an API 4000 triple quadrupole mass spectrometer. In the method validation, the linear calibration curve in the range of 2.0 to 300.0 ng/mL had correlation coefficients (r) above 0.996. The recovery was 57.6-66.8% for GI and the internal standard. The lower limit of quantification (LLOQ) was 2 ng/mL. The intra- and inter-batch precisions were below 6.31% and 8.61%, respectively, and the accuracies were all within acceptance. GI was stable in a bench-top autosampler through long-term storage and freeze/thaw cycles. Therefore, this method is specific, sensitive and reliable for quantitative analysis of α-conotoxin GI in human plasma.

Sensitive Detection of α-Conotoxin GI in Human Plasma Using a Solid-Phase Extraction Column and LC-MS/MS.

α-conotoxin GI, a short peptide toxin in the venom of Conus geographus, is composed of 13 amino acids and two disulfide bonds. It is the most toxic component of Conus geographus venom with estimated lethal doses of 0.029–0.038 mg/kg for humans. There is currently no reported analytical method for this toxin. In the present study, a sensitive detection method was developed to quantify GI in human plasma using a solid-phase extraction (SPE) column (polystyrene–divinyl benzene copolymer) combined with liquid chromatography/electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) in the multiple reaction monitoring (MRM) mode. The plasma samples were treated with a protein precipitating solvent (methanol: acetonitrile = 50:50, v/v). GI in the solvent was efficiently extracted with an SPE column and was further separated by a Grace Alltima HP C18 (50 × 2.1 mm, 5 μm) column at a flow rate of 0.4 mL/min. Water (with 2% methanol) acetonitrile (with 0.1% acetic acid) was selected as the mobile phase combination used in a linear gradient system. α-Conotoxin GI was analyzed by an API 4000 triple quadrupole mass spectrometer. In the method validation, the linear calibration curve in the range of 2.0 to 300.0 ng/mL had correlation coefficients (r) above 0.996. The recovery was 57.6–66.8% for GI and the internal standard. The lower limit of quantification (LLOQ) was 2 ng/mL. The intra- and inter-batch precisions were below 6.31% and 8.61%, respectively, and the accuracies were all within acceptance. GI was stable in a bench-top autosampler through long-term storage and freeze/thaw cycles. Therefore, this method is specific, sensitive and reliable for quantitative analysis of α-conotoxin GI in human plasma.

αS-conotoxin GVIIIB potently and selectively blocks α9α10 nicotinic acetylcholine receptors

Although acetylcholine is widely utilized in vertebrate nervous systems, nicotinic acetylcholine receptors (nAChRs), including the α9α10 subtype, also are expressed in a wide variety of non-neuronal cells. These cell types include cochlear hair cells, adrenal chromaffin cells and immune cells. α9α10 nAChRs present in these cells may respectively play roles in protection from noise-induced hearing loss, response to stress and neuroprotection. Despite these critical functions, there are few available selective ligands to confirm mechanistic hypothesis regarding the role of α9α10 nAChRs. Conus, has been a rich source of ligands for receptors and ion channels. Here, we identified Conus geographus venom as a lead source for a novel α9α10 antagonist. The active component was isolated and the encoding gene cloned. The peptide signal sequence and cysteine arrangement had the signature of the σ-conotoxin superfamily. Previously isolated σ-conotoxin GVIIIA, also from Conus geographus, targets the 5-HT3 receptor. In contrast, αS-GVIIIB blocked the α9α10 nAChR with an IC50 of 9.8nM, yet was inactive at the 5-HT3 receptor. Pharmacological characterization of αS-GVIIIB shows that it is over 100-fold selective for the α9α10 nAChR compared to other nAChR subtypes. Thus, the S-superfamily represents a novel conotoxin scaffold for flexibly targeting a variety of receptor subtypes. Functional competition studies utilized distinct off-rate kinetics of conotoxins to identify the α10/α9 nAChR interface as the site of αS-GVIIIB binding; this adds to the importance of the (+) face of the α10 rather than the (+) face of the α9 nAChR subunit as critical to binding of α9α10-targeted conotoxins.

Evolution of separate predation- and defence-evoked venoms in carnivorous cone snails.

Venomous animals are thought to inject the same combination of toxins for both predation and defence, presumably exploiting conserved target pharmacology across prey and predators. Remarkably, cone snails can rapidly switch between distinct venoms in response to predatory or defensive stimuli. Here, we show that the defence-evoked venom of Conus geographus contains high levels of paralytic toxins that potently block neuromuscular receptors, consistent with its lethal effects on humans. In contrast, C. geographus predation-evoked venom contains prey-specific toxins mostly inactive at human targets. Predation- and defence-evoked venoms originate from the distal and proximal regions of the venom duct, respectively, explaining how different stimuli can generate two distinct venoms. A specialized defensive envenomation strategy is widely evolved across worm, mollusk and fish-hunting cone snails. We propose that defensive toxins, originally evolved in ancestral worm-hunting cone snails to protect against cephalopod and fish predation, have been repurposed in predatory venoms to facilitate diversification to fish and mollusk diets.

Physiology of invertebrate oxytocin and vasopressin neuropeptides

New findings• What is the topic of this review?This article describes the discovery and function of invertebrate oxytocin and vasopressin neuropeptides.• What advances does it highlight?The novel discovery of oxytocin-like peptides in arthropods is described. An up-to date overview is gven of the functional role (physiology and behaviour) of oxytocin and vasopressin signalling. The application of natural peptides for drug development is discussed.Neuropeptides and regulatory peptide hormones control many developmental, physiological and behavioural processes in animals, including humans. The nonapeptides oxytocin and arginine vasopressin are produced and released by the pituitary gland and have actions on many organs and tissues. Receptive cells possess particular receptors to which the peptides bind as ligands, leading to activation of G-protein-coupled receptors, hence cellular responses. In humans and other mammalian species, oxytocin and vasopressin mediate a range of peripheral and central physiological functions that are important for osmoregulation, reproduction, complex social behaviours, memory and learning. The origin of the oxytocin/vasopressin signalling system is thought to date back more than 600 million years. All vertebrate oxytocin- and vasopressin-like peptides have presumably evolved from the ancestral nonapeptide vasotocin by gene duplication and today are present in vertebrates, including mammals, birds, reptiles, amphibians and fish. Oxytocin- and vasopressin-like peptides have been identified in several invertebrate species, including molluscs, annelids, nematodes and arthropods. Members of this peptide family share high sequence similarity, and it is possible that they are functionally related across the entire animal kingdom. However, it is evident that not all animals express oxytocin/vasopressin neuropeptides and that there is little information available about the biology and physiology of this signalling system of invertebrates and, in particular, of insects, which represent more than half of all known living organisms. This report describes the discovery of novel oxytocin- and vasopressin-like peptides in arthropods and summarizes the status quo of the functional relevance of this neuropeptide signalling system in invertebrates, which will have beneficial implications for the design of selective and potent ligands to human oxytocin and vasopressin receptors.

Analysis of a cone snail's killer cocktail – The milked venom of Conus geographus

“Snails can kill” is a statement that receives much disbelief. Yet the venom from Conus geographus, as delivered by a disposable hypodermic-like needle, has indeed killed many unsuspecting human victims. Our understanding of their milked venom the essence of these fatalities, is in itself non-existent. Here, we present the molecular mass analysis of the milked venom of C. geographus, providing the first insight into the composition of its deadly cocktail.

99. The Venom of Conus geographus Revisited

99. The Venom of Conus geographus Revisited

Inhibition of Neuronal Nicotinic Acetylcholine Receptor Subtypes by α-Conotoxin GID and Analogues

alpha-Conotoxins are small disulfide-rich peptides from the venom of the Conus species that target the nicotinic acetylcholine receptor (nAChR). They are valuable pharmacological tools and also have potential therapeutic applications particularly for the treatment of chronic pain. alpha-Conotoxin GID is isolated from the venom of Conus geographus and has an unusual N-terminal tail sequence that has been shown to be important for binding to the alpha4beta2 subtype of the nAChR. To date, only four conotoxins that inhibit the alpha4beta2 subtype have been characterized, but they are of considerable interest as it is the most abundant nAChR subtype in the mammalian brain and has been implicated in a range of diseases. In this study, analysis of alanine-scan and truncation mutants of GID reveals that a conserved proline in alpha-conotoxins is important for activity at the alpha7, alpha3beta2, and alpha4beta2 subtypes. Although the proline residue was the most critical residue for activity at the alpha3beta2 subtype, Asp(3), Arg(12), and Asn(14) are also critical at the alpha7 subtype. Interestingly, very few of the mutations tested retained activity at the alpha4beta2 subtype indicating a tightly defined binding site. This lack of tolerance to sequence variation may explain the lack of selective ligands discovered for the alpha4beta2 subtype to date. Overall, our findings contribute to the understanding of the structure-activity relationships of alpha-conotoxins and may be beneficial for the ongoing attempts to exploit modulators of the neuronal nAChRs as therapeutic agents.

Conopressin-T from Conus tulipa Reveals an Antagonist Switch in Vasopressin-like Peptides

We report the discovery of conopressin-T, a novel bioactive peptide isolated from Conus tulipa venom. Conopressin-T belongs to the vasopressin-like peptide family and displays high sequence homology to the mammalian hormone oxytocin (OT) and to vasotocin, the endogenous vasopressin analogue found in teleost fish, the cone snail's prey. Conopressin-T was found to act as a selective antagonist at the human V 1a receptor. All peptides in this family contain two conserved amino acids within the exocyclic tripeptide (Pro7 and Gly9), which are replaced with Leu7 and Val9 in conopressin-T. Whereas conopressin-T binds only to OT and V 1a receptors, an L7P analogue had increased affinity for the V 1a receptor and weak V2 receptor binding. Surprisingly, replacing Gly9 with Val9 in OT and vasopressin revealed that this position can function as an agonist/antagonist switch at the V 1a receptor. NMR structures of both conopressin-T and L7P analogue revealed a marked difference in the orientation of the exocyclic tripeptide that may serve as templates for the design of novel ligands with enhanced affinity for the V 1a receptor.

Effect of Conantokin G on NMDA Receptor–Mediated Spontaneous EPSCs in Cultured Cortical Neurons

Conantokin G (Con G), derived from the venom of Conus geographus, is the most characterized natural peptide antagonist targeted to N-methyl-D-aspartate (NMDA) receptors. Although Con G is known to bind to the glutamate binding site on the NR2 subunit of the receptor, it is unclear whether it can allosterically modulate the function of the receptor through the glycine binding site on the NR1 subunit. This study was designed to evaluate the action of Con G on NMDA receptor-mediated spontaneous excitatory postsynaptic currents (sEPSCs) and its modulation by glycine in cultured cortical neurons (13-19 days in vitro) using the whole cell patch-clamp technique. Con G inhibited NMDA receptor-mediated sEPSCs in a concentration-dependent manner. Also, the potency of Con G decreased as a function of time in culture. The inhibition of EPSCs observed after application of Con G in the presence of high (10 microM) and nominal (no added) concentrations of glycine was not different at 13 days in vitro (DIV). Furthermore, similar results were obtained with experiments on Con G-induced inhibition of NMDA-evoked whole cell currents. These results indicate that glycine concentrations do not have a direct effect on Con G-induced inhibition of NMDA currents. In addition, age dependency in the action of Con G on cortical neurons in vitro suggests that this model system would be useful in examining the effects of different agonists/antagonists on native synaptic NMDA receptors.

Chemical synthesis of analogs of the glycopeptide contulakin-G, an analgetically active conopeptide from Conus geographus

Cone snails are marine predators that use immobilizing venoms for catching prey. Chemical analysis of the venoms has revealed a variety of biologically active small and intermediate size peptides rich in post-translational modifications (modified amino acids, glycosylation). The glycopeptide contulakin-G (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-[β- d-Gal p-(1→3)-α- d-Gal pNAc-(1→]Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH) is a potent analgesic from Conus geographus venom. The in vivo activity of synthetic contulakin-G was previously found to be significantly higher compared to that of a peptide lacking the glycan. In order to further investigate the importance of the glycan, we have now synthesized analogs of contulakin-G where the glycan chain O-linked to threonine has been altered either to β- d-Gal p-(1→3)-β- d-Gal pNAc-, α- d-Gal p-(1→3)-α- d-Gal pNAc-, or β- d-Gal p-(1→6)-α- d-Gal pNAc-. The glycopeptides were assembled on a Wang resin using commercially available Fmoc amino acids and synthetically prepared Fmoc-protected threonine derivatives carrying O-acetyl protected sugar chains. The final products were thoroughly characterized by NMR and mass spectroscopy.

The effect of CGX-1007 and CI-1041, novel NMDA receptor antagonists, on kindling acquisition and expression

CGX-1007, a 17-amino acid polypeptide isolated from the venom of Conus geographus, is a novel NMDA receptor antagonist that is selective for the NR2B subunit. CI-1041 (PD 196860; Co 200461) is a novel, orally available NR2B selective antagonist. Both compounds possess anticonvulsant activity in a variety of well-established animal seizure models. The present study was designed to assess the effects of CGX-1007 and CI-1041 on the acquisition and expression of kindled seizures. In the corneal kindled rat, CGX-1007 [Epilepsia 36 (1998) 39] and CI-1041, administered p.o., 2 h prior to the kindling stimulation displayed time- and dose-dependent block of fully expressed corneal kindled seizures (ED 50=300 pmol and 2.5 mg/kg for CGX-1007 and CI-1041, respectively). In amygdala kindled rats, acute treatment with CGX-1007 blocked the secondarily generalized kindled seizure in a dose-dependent manner. Complete protection against the secondarily generalized seizure was only observed at a dose that produced behavioral impairment (4 nmol). Acute treatment with CI-1041 did not provide any notable protection against secondarily generalized seizures. Neither compound provided protection against the focal kindled seizure. Chronic i.c.v. infusion of CGX-1007 or chronic oral administration of CI-1041 did not delay the acquisition of amygdala kindling. The results from these studies suggest that NMDA receptors containing the NR2B subunit may contribute to the expression of fully kindled secondarily generalized seizures; however, they appear less important for the development of kindling. The differential results obtained with CGX-1007 and CI-1041 suggest that several classes of mechanistically distinct NR2B antagonists may exist and that CGX-1007 may be less specific as a NR2B receptor antagonist than initially reported.

The effect of CGX-1007 and CI-1041, novel NMDA receptor antagonists, on NMDA receptor-mediated EPSCs

The N-methyl- d-aspartate (NMDA) receptor-gated ion channel is comprised of at least one NR1 subunit and any of four NR2 subunits (NR2A–D). The NR2 subunit confers different pharmacological and kinetic properties to the receptor. CGX-1007 (Conantokin G), a 17-amino acid polypeptide isolated from the venom of Conus geographus, is a novel NMDA receptor antagonist that is thought to be selective for the NR2B subunit. CGX-1007 has been reported to have highly potent, broad-spectrum anticonvulsant activity in animal seizure models. CI-1041 is an investigational compound, which also possesses anticonvulsant activity and has been shown to be highly selective for NR2B containing NMDA receptors. Although both CI-1041 and CGX-1007 are reportedly NR2B specific antagonists, they differ in their ability to block amygdala-kindled seizures, suggesting that the mechanism of action of these compounds differs. The present study was designed to test the hypothesis that CI-1041 and CGX-1007 would differentially modulate the function of NMDA receptors at excitatory synapses. Using the whole cell patch clamp technique, CGX-1007 and CI-1041 were found to block CA1 pyramidal cell, NMDA receptor-mediated excitatory postsynaptic currents (N-EPSCs) in a concentration-dependent manner in hippocampal slices from P4–P6 animals. In contrast, only CGX-1007 decreased NMDA receptor-mediated EPSC peak amplitude in slices from adult animals. The CGX-1007 block of peak amplitude was accompanied by a similar concentration-dependent decrease in decay kinetics of NMDA receptor-mediated EPSCs. These results suggest that while CI-1041 may be selective for NMDA receptors containing the NR2B subunit, CGX-1007 appears to be less selective than previously reported.

Formation of angiotensin-(1–7) from angiotensin II by the venom of Conus geographus

The binding of [ 3H]angiotensin II to AT 1 receptors on Chinese Hamster Ovary cells expressing the human AT 1 receptor (CHO-AT 1 cells) is potently inhibited by venoms of the marine snails Conus geographus and C. betulinus. On the other hand, the binding of the nonpeptide AT 1 receptor-selective antagonist [ 3H]candesartan is not affected but competition binding curves of angiotensin II and the peptide antagonist [Sar 1,Ile 8]angiotensin II (sarile) are shifted to the right. These effects resulted from the breakdown of angiotensin II into smaller fragments that do not bind to the AT 1 receptor. In this context, angiotensin-(1–7) is the most prominent fragment and angiotensin-(1–4) and angiotensin-(1–5) are also formed but to a lesser extent. The molecular weight of the involved peptidases exceeds 50 kDa, as determined by gel chromatography and ultrafitration.

Synthesis and antibody recognition of mucin 1 (MUC1)-alpha-conotoxin chimera.

We synthesized and characterized new chimera peptides by inserting an epitope of the mucin 1 glycoprotein (MUC1) as a 'guest' sequence in the 'host' structure of alpha-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The Pro-Asp-Thr-Arg (PDTR) sequence of MUC1 selected for these studies is highly hydrophilic and adopts a beta-turn conformation. The alpha-conotoxin GI also contains a beta-turn in the 8-12 region, which is stabilized by two disulphide bridges in positions 2-7 and 3-13. Thus, the tetramer sequence of alpha-conotoxin, Arg9-His-Tyr-Ser12, has been replaced by PDTR, comprising the minimal epitope for MUC1 specific monoclonal antibodies (MAbs) HMFG1 (PDTR) and HMFG2 (DTR). Synthesis of the chimera peptide was carried out by Fmoc strategy on (4-(2',4'-dimethoxyphenyl-aminomethyl)phenoxy) (Rink) resin and either 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) or air oxidation was applied for the formation of the first Cys3-Cys13 or Cys2-Cys7 disulphide bridge, respectively. For the second disulphide bridge, three different oxidation procedures (iodine in acetic acid, 10% DMSO/1 M HCl or tallium trifluoroacetate (Tl(tfa)3) in TFA) were utilized. The HPLC purified peptides were characterized by electrospray mass spectrometry (ES-MS) and amino acid analysis. The CD spectra of the bicyclic MUC1-alpha-[Tyr1]-conotoxin chimera peptide showed partially ordered conformation with turn character. In antibody binding studies, the RIA data showed that both the linear and the bicyclic forms of MUC1-alpha-[Tyr1]-conotoxin chimera were recognized by MAb HMFG1 specific for PDTR sequence, while no binding was observed between MAb HMFG2 and various forms of the chimera. MAb HMFG1, using synthetic epitope conjugates or native MUC1 as target antigens, recognizes the PDTR motif more efficiently in the linear than in the bicyclic compound, but no reactivity was found with the monocyclic forms of MUC1-alpha-[Tyr1]-conotoxin chimera, underlining the importance of certain conformers stabilized by double cyclization.

Synthesis and antibody recognition of mucin 1 (MUC1)-α-conotoxin chimera

We synthesized and characterized new chimera peptides by inserting an epitope of the mucin 1 glycoprotein (MUC1) as a ‘guest’ sequence in the ‘host’ structure of α-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The Pro-Asp-Thr-Arg (PDTR) sequence of MUC1 selected for these studies is highly hydrophilic and adopts a β-turn conformation. The α-conotoxin GI also contains a β-turn in the 8–12 region, which is stabilized by two disulphide bridges in positions 2–7 and 3–13. Thus, the tetramer sequence of α-conotoxin, Arg9-His-Tyr-Ser12, has been replaced by PDTR, comprising the minimal epitope for MUC1 specific monoclonal antibodies (MAbs) HMFG1 (PDTR) and HMFG2 (DTR). Synthesis of the chimera peptide was carried out by Fmoc strategy on (4-(2′,4′-dimethoxyphenyl-aminomethyl)phenoxy) (Rink) resin and either 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB) or air oxidation was applied for the formation of the first Cys3–Cys13 or Cys2–Cys7 disulphide bridge, respectively. For the second disulphide bridge, three different oxidation procedures (iodine in acetic acid, 10% DMSO/1 M HCl or tallium trifluoroacetate (Tl(tfa)3) in TFA) were utilized. The HPLC purified peptides were characterized by electrospray mass spectrometry (ES-MS) and amino acid analysis. The CD spectra of the bicyclic MUC1-α-[Tyr1]-conotoxin chimera peptide showed partially ordered conformation with turn character. In antibody binding studies, the RIA data showed that both the linear and the bicyclic forms of MUC1-α-[Tyr1]-conotoxin chimera were recognized by MAb HMFG1 specific for PDTR sequence, while no binding was observed between MAb HMFG2 and various forms of the chimera. MAb HMFG1, using synthetic epitope conjugates or native MUC1 as target antigens, recognizes the PDTR motif more efficiently in the linear than in the bicyclic compound, but no reactivity was found with the monocyclic forms of MUC1-α-[Tyr1]-conotoxin chimera, underlining the importance of certain conformers stabilized by double cyclization. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.

Synthesis and immunological studies of alpha-conotoxin chimera containing an immunodominant epitope from the 268-284 region of HSV gD protein.

We have synthesized and characterized new chimeric peptides by inserting an epitope of the glycoprotein D (gD) of herpes simplex virus (HSV) serotype 1 as 'guest' sequence in the 'host' structure of alpha-conotoxin GI, a 13-residue peptide (ECCNPACGRHYSC) isolated from the venom of Conus geographus. The 276-284 region of HSV gD-1 selected for these studies is highly hydrophilic and adopts a beta-turn. The alpha-conotoxin GI also contains a beta-turn in the 8-12 region, stabilized by two disulfide bridges at positions 2-7 and 3-13. Thus, the tetramer sequence of alpha-conotoxin, 8Arg-His-Tyr-Ser12 has been replaced by Asp-Pro-Val-Gly (DPVG), identified previously as the epitope core. The syntheses were performed by Fmoc strategy on Rink resin and DTNB or air oxidation were applied for the formation of the first 3-13 disulfide bond in the presence of guanidinium hydrochloride. For the formation of the second disulfide Cys2-Cys7 three different oxidation procedures [iodine in 95% acetic acid, air oxidation in dimethyl sulfoxide/1 M HCl or Tl(tfa)3 in trifluoroacetic acid (TFE)] were compared. The high-performance liquid chromatography purified peptides were characterized by electrospray mass spectrometry and amino acid analysis. The bicyclic HSV-alpha-[Tyr1]-conotoxin chimeric peptide and native alpha-conotoxin GI showed similar circular dichroism spectra in phosphate-buffered saline (PBS) and in a PBS-TFE 1:1 (v/v) mixture, which might suggest that these compounds also share similar secondary structures. In immunologic studies the characteristics of the primary and of the memory immunoglobulin (Ig) M- and IgG-type antibody responses showed that the bicyclic HSV-alpha-[Tyr1]-conotoxin chimera is capable to induce strong antibody responses in C57/Bl/6 mice but was poorly immunogenic in CBA and BALB/c mice. Data obtained with the C57/Bl/6 serum indicate that the polyclonal antibodies recognize the DPVG motif presented in the bicyclic HSV-alpha-[Tyr1]-conotoxin and some reactivity was also found with the monocyclic but not with the linear form of the chimera. Results with two IgM type monoclonal antibodies from a bicyclic HSV-alpha-[Tyr1]-conotoxin immunized C57/Bl/6 mouse also point to the specific interaction with the DPVG sequence. Taken together these studies suggest, that the relative intensity of DPVG-specific responses was found to be dependent on the mouse strain and on the conformation of the chimeric molecules. We found that the IgM monoclonal antibodies are able to recognize the linear DPVG sequence, while the majority of IgG antibodies is directed to the same motif in a conformation stabilized by double cyclization.

Contulakin-G, an O-Glycosylated Invertebrate Neurotensin

We have purified contulakin-G, a 16-amino acid O-linked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-Lys-Pro-Tyr-Ile-Leu-OH, pGlu is pyroglutamate) from Conus geographus venom. The major glycosylated form of contulakin-G was found to incorporate the disaccharide beta-D-Galp-(1-->3)-alpha-D-GalpNAc-(1-->) attached to Thr10. The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(beta-->3) GalNAc(alpha-->)Thr10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated con- tulakin-G, when administered intracerebroventricular into mice, was found to result in motor control-associated dysfunction observed for the native peptide. Contulakín-G was found to be active at 10-fold lower doses than the nonglycosylated Thr10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr10 contulakin-G for a number of neurotensin receptor types including the human neurotensin type 1 receptor (hNTR1), the rat neurotensin type 1 and type 2 receptors, and the mouse neurotensin type 3 receptor were determined. The binding affinity of the nonglycosylated Thr10 contulakin-G was approximately an order of magnitude lower than that of neurotensin1-13 for all the receptor types tested. In contrast, the glycosylated form of contulakin-G exhibited significantly weaker binding affinity for all of the receptors tested. However, both contulakin-G and nonglycosylated Thr10 contulakin-G were found to be potent agonists of rat neurotensin receptor type 1. Based on these results, we conclude that O-linked glycosylation appears to be a highly unusual strategy for increasing the efficacy of toxins directed against neurotransmitter receptors.

Refined solution structure of omega-conotoxin GVIA: implications for calcium channel binding.

The polypeptide omega-conotoxin GVIA (GVIA) is an N-type calcium channel blocker from the venom of Conus geographus, a fish-hunting cone shell. Here we describe a high-resolution solution structure of this member of the 'inhibitor cystine knot' protein family. The structure, based on NMR data acquired at 600 MHz, has mean pairwise RMS differences of 0.25 +/- 0.06 and 1.07 +/- 0.14 A over the backbone heavy atoms and all heavy atoms, respectively. The solvent-accessible side chains are better defined than in previously published structures and provide an improved basis for docking GVIA with models of the calcium channel. Moreover, some side chain interactions important in GVIA folding in vitro and in stabilizing the native structure are defined clearly in the refined structure. Two qualitatively different backbone conformations in the segment from Thr11 to Asn14 persisted in the restrained simulated annealing calculations until a small number of lower bound constraints was included to prevent close contacts from occurring that did not correspond with peaks in the NOESY spectrum. It is possible that GVIA is genuinely flexible at this segment, spending a finite time in the alternative conformation, and this may influence its interaction with the calcium channel.

Structure determination of the three disulfide bond isomers of α-conotoxin GI: a model for the role of disulfide bonds in structural stability

The three possible disulfide bonded isomers of α-conotoxin GI have been selectively synthesised and their structures determined by 1H NMR spectroscopy. α-Conotoxin GI derives from the venom of Conus geographus and is a useful neuropharmacological tool as it selectively binds to the nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel involved in nerve signal transmission. The peptide has the sequence ECCNPACGRHYSC-NH2, and the three disulfide bonded isomers are referred to as GI(2–7;3–13), GI(2–13;3–7) and GI(2–3;7–13). The NMR structure for the native isomer GI(2–7;3–13) is of excellent quality, with a backbone pairwise RMSD of 0.16 Å for a family of 35 structures, and comprises primarily a distorted 310helix between residues 5 to 11. The two non-native isomers exhibit multiple conformers in solution, with the major populated forms being different in structure both from each other and from the native form. Structure-activity relationships for the native GI(2–7;3–13) as well as the role of the disulfide bonds on folding and stability of the three isomers are examined. It is concluded that the disulfide bonds in α-conotoxin GI play a crucial part in determining both the structure and stability of the peptide. A trend for increased conformational heterogeneity was observed in the order of GI(2–7;3–13)<GI(2–13;3–7)<GI(2–3;7–13). It was found that the peptide bond joining Cys2 to Cys3 in GI(2–3;7–13) is predominantly trans, rather than cis as theoretically predicted. These structural data are used to interpret the varying nAChR binding of the non-native forms.A model for the binding of native GI(2–7;3–13) to the mammalian nAChR is proposed, with an α-subunit binding face made up of Cys2, Asn4, Pro5, Ala6 and Cys7 and a selectivity face, comprised of Arg9 and His10. These two faces orient the molecule between the α and δ subunits of the receptor. The structure of the CCNPAC sequence of the native GI(2–7;3–13) is compared to the structure of the identical sequence from the toxic domain of heat-stable enterotoxins, which forms part of the receptor binding region of the enterotoxins, but which has a different disulfide connectivity.