Isolation and Characterization of a Novel ConusPeptide with Apparent Antinociceptive Activity

Abstract

Cone snails are tropical marine mollusks that envenomate prey with a complex mixture of neuropharmacologically active compounds. We report the discovery and biochemical characterization of a structurally unique peptide isolated from the venom of Conus marmoreus. The new peptide, mr10a, potently increased withdrawal latency in a hot plate assay (a test of analgesia) at intrathecal doses that do not produce motor impairment as measured by rotarod test. The sequence of mr10a is NGVCCGYKLCHOC, where O is 4-trans-hydroxyproline. This sequence is highly divergent from all other known conotoxins. Analysis of a cDNA clone encoding the toxin, however, indicates that it is a member of the recently described T-superfamily. Total chemical synthesis of the three possible disulfide arrangements of mr10a was achieved, and elution studies indicate that the native form has a disulfide connectivity of Cys1-Cys4 and Cys2-Cys3. This disulfide linkage is unprecedented among conotoxins and defines a new family of Conus peptides.Conus is a genus of predatory marine gastropods that envenomate their prey. Prey capture is accomplished through a sophisticated arsenal of peptides that target specific ion channel and receptor subtypes. Each Conus venom appears to contain a unique set of 50 -200 peptides. The structure and function of only a small minority of these peptides have been determined to date. For peptides where function has been determined, three classes of targets have been elucidated: voltage-gated ion channels, ligand-gated ion channels, and G-protein-linked receptors.Conus peptides that target voltage-gated ion channels include those that delay the inactivation of sodium channels as well as blockers specific for sodium channels, calcium channels, and potassium channels. Peptides that target ligand-gated ion channels include antagonists of N-methyl-D-aspartate and serotonin receptors as well as competitive and non-competitive nicotinic receptor antagonists. Peptides that act on G protein receptors include neurotensin and vasopressin receptor agonists. The unprecedented targeting selectivity of conotoxins derives from specific disulfide bond frameworks combined with hypervariable amino acids within disulfide loops (see Ref. 1 for review). Due to the high potency and exquisite selectivity of the conopeptides, several are in various stages of clinical development for treatment of human disorders (2).In this report we describe the isolation of a new peptide from the venom of the marble cone, Conus marmoreus. C. marmoreus is found in the Indo-Pacific, from India to the Marshall Islands and Fiji. It preys upon various gastropods including other cone snails (3). We previously reported the isolation and characterization of a peptide from this venom that potently inhibits voltage-gated sodium channels (4). In this report, we describe the isolation of a novel peptide that appears antinociceptive and likely represents a defining member of a new family of Conus peptides. EXPERIMENTAL PROCEDURES Materials and HPLC 1 ConditionsThe venom of C...

Inhibition of the Norepinephrine Transporter by the Venom Peptide χ-MrIA

“…Given its lower potency in the rat brain, its only partial inhibitory effect and the modest degree of assumed specific (i.e. nisoxetine-or desipramine-sensitive) binding in the assay, it is perhaps not surprising that McIntosh et al (9) did not detect any effect of -MrIA (10 M) in their NET binding assay using conditions somewhat similar to those used here. A possible reason for the only partial inhibition of the specific Norepinephrine transport by the NET has been shown to be dependent on Na ϩ , reflecting the co-transport of Na ϩ with the substrate (46).…”

“…8). Intrathecal injection of -MrIA has been found to be analgesic in hot plate and neuropathic pain models (9,10). The inhibition of [ 3 H]norepinephrine uptake by the NET caused by -MrIA was found to be non-competitive, reducing the maximum rate of transport and not affecting the affinity of the transporter for substrate (7).…”

A New Level of Conotoxin Diversity, a Non-native Disulfide Bond Connectivity in α-Conotoxin AuIB Reduces Structural Definition but Increases Biological Activity

“…However there is a precedent for native ribbon isomers in other small conotoxins. The ribbon isomer appears to be selected as the native form in the recently discovered ␣-conotoxin MrIA (25)(26)(27). The structural data reported here for AuIB suggest that perhaps the ribbon isomer is unlikely to be in the venom due to the fact that it is substantially more flexible and hence perhaps less stable in vivo than the globular form.…”

“…Such studies are given further impetus by the fact that new families of conotoxins with alternative disulfide connectivities are now being found. For example a new family of conotoxins that displays the ribbon disulfide connectivity as their native form has been isolated recently (25)(26)(27). These molecules, referred to as or -conotoxins, are of similar size but exhibit different biological activity, primary sequences, and cysteine spacings to the ␣-conotoxins (Table I).…”

Sequencing of T-superfamily conotoxins from Conus virgo: Pyroglutamic acid identification and disulfide arrangement by MALDI mass spectrometry

“…T-1 conotoxins are characterized by a CCxxxxxCC motif with 1-3, 2-4 disulfide°connectivity°[2,°5,°14°-24],°while°the°cysteine framework in T-2 conotoxins is CCxxxxCxxC, with 1-4,°2-3°disulfide°connectivity° [25][26][27].°Table°1°shows the sequence alignment (based on the CC pair) of Conus peptides belonging to the T-1-superfamily, isolated from different species. The methodologies followed for the determination of sequences are also indicated.…”