We have purified contulakin-G, a 16-amino acid Olinked glycopeptide (pGlu-Ser-Glu-Glu-Gly-Gly-SerAsn-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 -D-Galp-(133)-␣-D-GalpNAc-(13) attached to Thr 10 . The C-terminal sequence of contulakin-G shows a high degree of similarity to the neurotensin family of peptides. Synthetic peptide replicates of Gal(33) GalNAc(␣3)Thr 10 contulakin-G and its nonglycosylated analog were prepared using an Fmoc (9-fluorenylmethoxycarbonyl) protected solid phase synthesis strategy. The synthetic glycosylated contulakin-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 Thr 10 contulakin-G analog. The binding affinities of contulakin-G and the nonglycosylated Thr 10 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 Thr 10 contulakin-G was approximately an order of magnitude lower than that of neurotensin [1][2][3][4][5][6][7][8][9][10][11][12][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 Thr 10 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.
More than 100 species of venomous cone snails (genus Conus) are highly effective predators of fish. The vast majority of venom components identified and functionally characterized to date are neurotoxins specifically targeted to receptors, ion channels, and transporters in the nervous system of prey, predators, or competitors. Here we describe a venom component targeting energy metabolism, a radically different mechanism. Two fish-hunting cone snails, Conus geographus and Conus tulipa, have evolved specialized insulins that are expressed as major components of their venoms. These insulins are distinctive in having much greater similarity to fish insulins than to the molluscan hormone and are unique in that posttranslational modifications characteristic of conotoxins (hydroxyproline, γ-carboxyglutamate) are present. When injected into fish, the venom insulin elicits hypoglycemic shock, a condition characterized by dangerously low blood glucose. Our evidence suggests that insulin is specifically used as a weapon for prey capture by a subset of fish-hunting cone snails that use a net strategy to capture prey. Insulin appears to be a component of the nirvana cabal, a toxin combination in these venoms that is released into the water to disorient schools of small fish, making them easier to engulf with the snail's distended false mouth, which functions as a net. If an entire school of fish simultaneously experiences hypoglycemic shock, this should directly facilitate capture by the predatory snail.insulin shock | cone snails | conotoxins | nirvana cabal | venom
We purified and characterized a novel peptide from the venom of the fish-hunting cone snail Conus striatus that inhibits voltage-gated K+ channels. The peptide, kappaA-conotoxin SIVA, causes characteristic spastic paralytic symptoms when injected into fish, and in frog nerve-muscle preparations exposed to the toxin, repetitive action potentials are seen in response to a single stimulus applied to the motor nerve. Other electrophysiological tests on diverse preparations provide evidence that is consistent with the peptide blocking K+ channels. The peptide has three disulfide bonds; the locations of Cys residues indicate that the spastic peptide may be the first and defining member of a new family of Conus peptides, the kappaA-conotoxins, which are structurally related to, but pharmacologically distinct from, the alphaA-conotoxins. This 30 AA tricyclic toxin has several characteristics not previously observed in Conus peptides. In addition to the distinctive biological and physiological activity, a novel biochemical feature is the unusually long linear N-terminal tail (11 residues) which contains one O-glycosylated serine at position 7. This is the first evidence for O-glycosylation as a posttranslational modification in a biologically active Conus peptide.
Conkunitzin-S1 (Conk-S1) is a 60-residue neurotoxin from the venom of the cone snail Conus striatus that interacts with voltage-gated potassium channels. Conk-S1 shares sequence homology with Kunitz-type proteins but contains only two out of the three highly conserved cysteine bridges, which are typically found in these small, basic protein modules. In this study the three-dimensional structure of Conk-S1 has been solved by multidimensional NMR spectroscopy. The solution structure of recombinant Conk-S1 shows that a Kunitz fold is present, even though one of the highly conserved disulfide cross-links is missing. Introduction of a third, homologous disulfide bond into Conk-S1 results in a functional toxin with similar affinity for Shaker potassium channels. The affinity of Conk-S1 can be enhanced by a pore mutation within the Shaker channel pore indicating an interaction of Conk-S1 with the vestibule of potassium channels.Kunitz domain proteins, like the bovine pancreatic trypsin inhibitor (BPTI) 1 (1) or the dendrotoxins (2) are small, basic proteins that contain three highly conserved disulfide bonds. The three disulfide cross-links make these extracellular proteins extremely stable. Two different general functions are known for the different Kunitz proteins. One group, including BPTI, consists of potent protease inhibitors. The complex of BPTI and trypsin is exceptionally stable, with an association constant Ͼ10 MϪ1 (3). The dendrotoxins belong to another group of Kunitz peptides found in the venom of the black mamba, which block different potassium channels with a high degree of specificity and selectivity (4). In snake and scorpion venoms a diverse set of different potassium channel blockers have been characterized (2).Despite the great variety of toxins from the venoms of the predatory cone snails, relatively few have been identified so far that interact with potassium channels (5). Most conotoxins are small, peptidic toxins that typically contain 10 -30 amino acids and bind with high affinity and specificity to various ligand-or voltage-gated ion channels. One striking feature of these peptides is that they usually contain a diverse complement of posttranslational modifications, like C-terminal amidation, hydroxyprolines, or glycosylation of serine or threonine (6). Conotoxins can be broadly divided into two groups, the non-disulfide-rich peptides and the disulfide-rich conotoxins. The latter conotoxins are further separated into several families based on cysteine bridge pattern and biological activities (5).The potassium channel-targeted toxin conkunitzin-S1 (Conk-S1) from the venom of Conus striatus is the first member of a new family of polypeptides. Recently it has been shown that Conk-S1 blocks Shaker potassium channels with an IC 50 of less than 100 nM.2 Compared with most toxins from Conus venoms, Conk-S1 is unusually long (60 amino acids). The only post-translational modification of this peptide is the amidation of the C-terminal carboxylic acid. Conk-S1 shares 33% sequence identity with BPTI and 3...
A cone snail venom peptide, μO §-conotoxin GVIIJ from Conus geographus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligandbinding site. μO §-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJ SSG ). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) Na V 1.2. A mutant channel of rNa V 1.2 lacking a cysteine near the pore loop of domain II (C910L), was >10 3 -fold less sensitive to GVIIJ SSG than was wild-type rNa V 1.2. In contrast, although rNa V 1.5 was >10 4 -fold less sensitive to GVIIJ SSG than Na V 1.2, an rNa V 1.5 mutant with a cysteine in the homologous location, rNa V 1.5[L869C], was >10 3 -fold more sensitive than wildtype rNa V 1.5. The susceptibility of rNa V 1.2 to GVIIJ SSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNa V β2 or rNa V β4, but not that of rNa V β1 or rNa V β3, protected rNa V 1.1 to -1.7 (excluding Na V 1.5) against block by GVIIJ SSG . Thus, GVIIJrelated peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which Na V β-and Na V α-subunits are present in native neurons.oltage-gated sodium channels (VGSCs) are responsible for the upstroke of action potentials in excitable tissues. Each VGSC is composed of a pore-and voltage sensor-bearing α-subunit and one or more auxiliary β-subunits. Mammals have nine α-subunit isoforms (Na V 1.1 to -1.9) and four β-subunit isoforms (Na V β1 to -β4) (1). An Na V 1 has about 2,000-aa residues arranged in four homologous domains, where each domain has six transmembrane spanning segments with an extracellular "pore" loop between segments 5 and 6 (1, 2); furthermore, each Na V 1 has about a dozen extracellular cysteine residues, all located in or near the pore loops. For the most part, not much is known about these cysteines (including whether they are disulfide bonded).Na V β-subunits can affect the function and cellular localization of Na V 1s (1, 3-5). Each Na V β-subunit has some 200-aa residues and consists of a single transmembrane segment with a large extracellular domain and a smaller intracellular domain (1). Na V β2-and Na V β4-subunits, unlike Na V β1-and Na V β3-subunits, are disulfide bonded to α-subunits (1, 6). A given neuron can have multiple isoforms of these subunits whose identities are challenging to appraise pharmacologically (7).Toxins that target VGSCs have been invaluable for probing the structure and function of these channels. Venoms are a rich source of such toxins. For example, in Conus snails, four families of neuroactive peptides have been characterized that target VGSCs:...
The major groups of Conus peptides previously characterized from fish-hunting cone snail venoms (the alpha-, mu-, and omega-conotoxins) all blocked neuromuscular transmission. A novel activity, the "lockjaw peptide", from the fish-hunting Conus purpurascens, caused a rigid (instead of flaccid) paralysis in fish and increased excitability at the neuromuscular junction (instead of a block). We report the purification, biological activity, biochemical and preliminary physiological characterization, and chemical synthesis of the lockjaw peptide and the sequence of a cDNA clone encoding its precursor. Taken together, the data lead us to conclude that the lockjaw peptide is a vertebrate-specific delta-conotoxin, which targets voltage-sensitive sodium channels. The sequence of the peptide, which we designate delta-conotoxin PVIA, is (O = 4-trans-hydroxyproline) EACYAOGTFCGIKOGLCCSEFCLPGVCFG-NH2. This is the first of a diverse spectrum of Conus peptides which are excitotoxins in vertebrate systems.
Summary Background Ionotropic glutamate receptors (iGluRs) are glutamate-gated ion channels that mediate excitatory neurotransmission in the central nervous system. Based on both molecular and pharmacological criteria, iGluRs have been divided into two major classes, the non-NMDA-class, which includes both AMPA and kainate subtypes of receptors, and the NMDA-class. One evolutionarily conserved feature of iGluRs is their desensitization in the continued presence of glutamate. Thus, when in a desensitized state, iGluRs can be bound to glutamate, yet the channel remains closed. However, the relevance of desensitization to nervous system function has remained enigmatic. Results Here, we report the identification and characterization of a novel polypeptide (con-ikot-ikot) from the venom of a predatory marine snail Conus striatus that specifically disrupts the desensitization of AMPA receptors (AMPARs). The stoichiometry of con-ikot-ikot appears reminiscent of the proposed subunit organization of AMPARs, i.e., a dimer of dimers, suggesting that it acts as a molecular four-legged clamp that holds the AMPAR channel open. Application of con-ikot-ikot to hippocampal slices caused a large and rapid increase in resting AMPAR-mediated current leading to neuronal death. Conclusions Our findings provide insight into the mechanisms that regulate receptor desensitization, and demonstrate that in the arms race between prey and predators, evolution has selected for a toxin that blocks AMPAR desensitization, thus revealing the fundamental importance of desensitization for regulating neural function.
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