Isolation and characterisation of conomap‐Vt, a <scp>d</scp>‐amino acid containing excitatory peptide from the venom of a vermivorous cone snail

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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 (Pro 7 and Gly 9 ), which are replaced with Leu 7 and Val 9 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 V 2 receptor binding. Surprisingly, replacing Gly 9 with Val 9 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.The vasopressin (AVP) 3 and oxytocin (OT) peptides were originally discovered and identified as neurohypophysial hormones in mammals (1). In humans, AVP acts via three vasopressin receptors (vascular V 1a R, pituitary V 1b R, and renal V 2 R), whereas OT acts via one OT receptor (OTR). All targets are members of the G protein-coupled receptor family (2). Peripherally, they regulate water balance, the control of blood pressure, and contraction of uterine smooth muscle and mammary myoepithelium (3). Centrally, these peptides affect levels of aggression, depression, and young parent bonding (4 -6). Endogenous analogues of OT and AVP have been reported in nonmammalian vertebrates, annelids, molluscs, and insects, suggesting an old lineage for these peptides (7). Surprisingly, two variants were also found in the venom of predatory cone snails. The original discovery of these two AVP analogues, named conopressins, was based on the characteristic "scratching" effect observed upon intracerebral injection into mice (8). Although the sequences of conopressins are similar to vasopressin itself, they have an additional positive charge in position 4, which is only found in two other endogenous vasopressin analogues, cephalotocin (Octopus vulgaris) and annetocin (Eisenia foetida). Conopressin-S was isolated from Conus striatus, whereas conopressin-G was first isolated from Conus geographus venom but later found in Conus imperialis venom as well as in tissue extracts of the nonvenomous snails Lymnea stagnalis and Aplysia californica and the leech Erpobdella octoculata (9 -11).Molluscs of the genus Conus produce bioactive peptides in a combinatorial fashion. As demonstrated for the snake toxins (12), most conotoxins or conopeptides are believed to be derived from an endogenous structural template (13). Because conopressin-G is widely distributed, it may represent the endogenous hormone in Gastropods and Annelid...

Section: Materials-t-butoxycarbonylmentioning

confidence: 99%

“…As demonstrated for the snake toxins (12), most conotoxins or conopeptides are believed to be derived from an endogenous structural template (13). Because conopressin-G is widely distributed, it may represent the endogenous hormone in Gastropods and Annelids.…”

mentioning

confidence: 99%

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

Dutertre

Croker²,

Daly

et al. 2008

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“…Conopeptides are typically expressed by exogenes with characteristic signal sequences followed by a pre-pro region and a hypervariable toxin region (8). Additionally, cone snail venom includes several families of endogenous-like peptides, such as the conopressins (vasopressin/oxytocin) (5, 9) contulakins (neurotensin) (4), conophysin (neurophysin) (10), conorfamides (RFamides) (11,12), conomaps (myoactive tetradecapeptide) (13), and conkunitzins (Kunitz-type proteins) (14). Here, we report the discovery and characterization of a new class of endogenous-like conopeptides in the venom duct of Conus vill-epinii that belongs to the superfamily of cardioactive peptides (CAPs).…”

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confidence: 99%

Functional Hypervariability and Gene Diversity of Cardioactive Neuropeptides

Möller

Melaun

Castillo

et al. 2010

Crustacean cardioactive peptide (CCAP) and related peptides are multifunctional regulatory neurohormones found in invertebrates. We isolated a CCAP-related peptide (conoCAP-a, for cone snail CardioActive Peptide) and cloned the cDNA of its precursor from venom of Conus villepinii. The precursor of conoCAP-a encodes for two additional CCAP-like peptides: conoCAP-b and conoCAP-c. This multi-peptide precursor organization is analogous to recently predicted molluscan CCAP-like preprohormones, and suggests a mechanism for the generation of biological diversification without gene amplification. While arthropod CCAP is a cardio-accelerator, we found that conoCAP-a decreases the heart frequency in Drosophila larvae, demonstrating that conoCAP-a and CCAP have opposite effects. Intravenous injection of conoCAP-a in rats caused decreased heart frequency and blood pressure in contrast to the injection of CCAP, which did not elicit any cardiac effect. Perfusion of rat ventricular cardiac myocytes with conoCAP-a decreased systolic calcium, indicating that conoCAP-a cardiac negative inotropic effects might be mediated via impairment of intracellular calcium trafficking. The contrasting cardiac effects of conoCAP-a and CCAP indicate that molluscan CCAP-like peptides have functions that differ from those of their arthropod counterparts. Molluscan CCAP-like peptides sequences, while homologous, differ between taxa and have unique sequences within a species. This relates to the functional hypervariability of these peptides as structure activity relationship studies demonstrate that single amino acids variations strongly affect cardiac activity. The discovery of conoCAPs in cone snail venom emphasizes the significance of their gene plasticity to have mutations as an adaptive evolution in terms of structure, cellular site of expression, and physiological functions.

“…In r11a, a 46-amino acid I 1 -conotoxin (41), has a phenylalanine at position 44, which undergoes epimerization from an L-Phe to a D-Phe, a transition linked to a dramatic gain in pharmacological activity (39). Similarly, isomerization of L-Phe to D-Phe also enhanced biological activity of conomap-Vt, a linear peptide from Conus vitulinus (40). The complete chemical synthesis of a toxin with only D-amino acids has been seldom reported.…”

Section: Discussionmentioning

confidence: 99%

“…The purity of D-MCa is illustrated by analytical C 18 reversed phase HPLC, and the folding/oxidation process is witnessed by the shift in elution time (Fig. 1B (20,40,60, 80, and 100°C). All CD spectra coincided well, indicating that the structure of D-MCa was remarkably stable with increasing temperatures (Fig.…”

Section: Chemical Synthesis Of D-mca and Enzymatic Stability-thementioning

confidence: 99%

d-Maurocalcine, a Pharmacologically Inert Efficient Cell-penetrating Peptide Analogue

Poillot

Dridi

Bichraoui

et al. 2010

Maurocalcine has been the first demonstrated animal toxin acting as a cell-penetrating peptide. Although it possesses competitive advantages, its use as a cell-penetrating peptide (CPP) requires that analogues be developed that lack its characteristic pharmacological activity on ryanodine-sensitive calcium channels without affecting its cell-penetrating and vector efficiencies. Here, we present the synthesis, three-dimensional 1 H NMR structure, and activity of D-maurocalcine. We demonstrate that it possesses all of the desired features for an excellent CPP: preserved structure, lack of pharmacological action, conserved vector properties, and absence of cell toxicity. This is the first report of a folded/oxidized animal toxin in its D-diastereomer conformation for use as a CPP. The protease resistance of this new peptide analogue, combined with its efficient cell penetration at concentrations devoid of cell toxicity, suggests that D-maurocalcine should be an excellent vector for in vivo applications.