Purification and structural characterization of a D-amino acid-containing conopeptide, conomarphin, from Conus marmoreus

Cone snails, a group of gastropod animals that inhabit tropical seas, are capable of producing a mixture of peptide neurotoxins, namely conotoxins, for defense and predation. Conotoxins are mainly disulfide-rich short peptides that act on different ion channels, neurotransmitter receptors, or transporters in the nervous system. They exhibit highly diverse compositions, structures, and biological functions. In this work, a novel Cys-free 15-residue conopeptide from Conus marmoreus was purified and designated as conomarphin. Conomarphin is unique because of its D-configuration Phe at the third residue from the C-terminus, which was identified using HPLC by comparing native conomarphin fragments and the corresponding synthetic peptides cleaved by different proteases. Surprisingly, the cDNA-encoded precursor of conomarphin was found to share the conserved signal peptide with other M-superfamily conotoxins, clearly indicating that conomarphin should belong to the M-superfamily, although conomarphin shares no homology with other six-Cys-containing M-superfamily conotoxins. Furthermore, NMR spectroscopy experiments established that conomarphin adopts a well-defined structure in solution, with a tight loop in the middle of the peptide and a short 3(10)-helix at the C-terminus. By contrast, no loop in L-Phe13-conomarphin was found, which suggests that D-Phe13 is essential for the structure of conomarphin. In conclusion, conomarphin may represent a new conotoxin family, whose biological activity remains to be identified.     

Characterization of novel M-superfamily conotoxins with new disulfide linkage

The M-superfamily with the typical Cys framework (-CC-C-C-CC-) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M-superfamily conotoxins can be provisionally categorized into four branches (M-1, M-2, M-3, M-4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176-8186]. Here we report the purification of seven M-superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M-1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M-superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M-1 and M-2 and only one to M-3. The disulfide analyses of two M-1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1-C5, C2-C4, C3-C6) which is different from those of the M-4 branch (C1-C4, C2-C5, C3-C6) and M-2 branch (C1-C6, C2-C4, C3-C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.     

Definition of the M-conotoxin superfamily: characterization of novel peptides from molluscivorous Conus venoms

Most of the >50,000 different pharmacologically active peptides in Conus venoms belong to a small number of gene superfamilies. In this work, the M-conotoxin superfamily is defined using both biochemical and molecular criteria. Novel excitatory peptides purified from the venoms of the molluscivorous species Conus textile and Conus marmoreus all have a characteristic pattern of Cys residues previously found in the mu-, kappaM-, and psi-conotoxins (CC-C-C-CC). The new peptides are smaller (12-19 amino acids) than the mu-, kappaM-, and psi-conotoxins (22-24 amino acids). One peptide, mr3a, was chemically synthesized in a biologically active form. Analysis of the disulfide bridges of a natural peptide tx3c from C. textile and synthetic peptide mr3a from C. marmoreus showed a novel pattern of disulfide connectivity, different from that previously established for the mu- and psi-conotoxins. Thus, these peptides belong to a new group of structurally and pharmacologically distinct conotoxins that are particularly prominent in the venoms of mollusc-hunting Conus species. Analysis of cDNA clones encoding the novel peptides as well as those encoding mu-, kappaM-, and psi-conotoxins revealed highly conserved amino acid residues in the precursor sequences; this conservation in both amino acid sequence and in the Cys pattern defines a gene superfamily, designated the M-conotoxin superfamily. The peptides characterized can be provisionally assigned to four distinct groups within the M-superfamily based on sequence similarity within and divergence between each group. A notable feature of the superfamily is that two distinct structural frameworks have been generated by changing the disulfide connectivity on an otherwise conserved Cys pattern.     

Isolation and characterization of a novel conus peptide with apparent antinociceptive activity

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.     

Isolation and cloning of a conotoxin with a novel cysteine pattern from Conus caracteristicus

A new conotoxin, ca16a, containing 8 cysteine residues was purified, sequenced, and cloned from a worm-hunting snail, Conus caracteristicus. This conotoxin is an extremely hydrophilic peptide comprising 34 residues, with 4 acidic and 4 basic residues. It is rich in polar Gly, Ser, and Thr residues and includes a hydroxylated Pro residue. The cysteine arrangement pattern of ca16a (-C-C-CC-C-CC-C-, designated as framework #16) is distinct from that of other known conotoxins. Furthermore, the signal peptide sequence of this conotoxin does not share any homology with those of other conotoxins. Leu residues account for almost 50% of its 20-residue signal peptide. The unique cysteine framework and signal peptide sequence of ca16a suggest that it belongs to a new conotoxin superfamily.     

An O-conotoxin from the vermivorous Conus spurius active on mice and mollusks

Here, we report the purification, amino acid sequence and a preliminary biological characterization of a peptide, sr7a, from the venom of Conus spurius, a vermivorous species collected in the Yucatan Channel, Mexico. The peptide consists of 32 amino acid residues (CLQFGSTCFLGDDDICCSGECFYSGGTFGICS&; &, amidated C-terminus) and contains six cysteines arranged in the pattern (C-C-CC-C-C) that characterizes the O-superfamily of conotoxins. This superfamily includes several pharmacological families (omega-, kappa-, muO-, delta- and gamma-conotoxins) that target Ca(2+), K(+), Na(+) and pacemaker voltage-gated ion channels. Compared with other O-conotoxins that were purified from venoms, this peptide displays sequence similarity with omega-SVIA (from Conus striatus), delta-TxVIA/B (from Conus textile), omega-CVID (from Conus catus) and kappa-PVIIA (from Conus purpurascens). At a dose of 250 pmol, peptide sr7a elicited hyperactivity when injected intracranially into mice and produced paralysis when injected into the pedal muscle of freshwater snails, Pomacea paludosa, but it had no apparent effect after intramuscular injection into the limpet Patella opea or the freshwater fish Lebistes reticulatus.     

Oxidative folding of conotoxins sharing an identical disulfide bridging framework

Conotoxins are short, disulfide-rich peptide neurotoxins produced in the venom of predatory marine cone snails. It is generally accepted that an estimated 100,000 unique conotoxins fall into only a handful of structural groups, based on their disulfide bridging frameworks. This unique molecular diversity poses a protein folding problem of relationships between hypervariability of amino acid sequences and mechanism(s) of oxidative folding. In this study, we present a comparative analysis of the folding properties of four conotoxins sharing an identical pattern of cysteine residues forming three disulfide bridges, but otherwise differing significantly in their primary amino acid sequence. Oxidative folding properties of M-superfamily conotoxins GIIIA, PIIIA, SmIIIA and RIIIK varied with respect to kinetics and thermodynamics. Based on rates for establishing the steady-state distribution of the folding species, two distinct folding mechanisms could be distinguished: first, rapid-collapse folding characterized by very fast, but low-yield accumulation of the correctly folded form; and second, slow-rearrangement folding resulting in higher accumulation of the properly folded form via the reshuffling of disulfide bonds within folding intermediates. Effects of changing the folding conditions indicated that the rapid-collapse and the slow-rearrangement mechanisms were mainly determined by either repulsive electrostatic or productive noncovalent interactions, respectively. The differences in folding kinetics for these two mechanisms were minimized in the presence of protein disulfide isomerase. Taken together, folding properties of conotoxins from the M-superfamily presented in this work and from the O-superfamily published previously suggest that conotoxin sequence diversity is also reflected in their folding properties, and that sequence information rather than a cysteine pattern determines the in vitro folding mechanisms of conotoxins.     

The three-dimensional solution structure of mini-M conotoxin BtIIIA reveals a disconnection between disulfide connectivity and peptide fold

Conotoxins are bioactive peptides from the venoms of marine snails and have been divided into several superfamilies based on homologies in their precursor sequences. The M-superfamily conotoxins can be further divided into five branches based on the number of residues in the third loop of the peptide sequence. Recently two M-1 branch conotoxins (tx3a and mr3e) with a C1–C5, C2–C4, C3–C6 disulfide connectivity and one M-2 branch conotoxin (mr3a) with a C1–C6, C2–C4, C3–C5 disulfide connectivity were described. Here we report the disulfide connectivity, chemical synthesis and the three-dimensional NMR structure of the novel 14-residue conotoxin BtIIIA, extracted from the venom of Conus betulinus. It has the same disulfide connectivity as mr3a, which puts it in the M-2 branch conotoxins but has a distinctly different structure from other M-2 branch conotoxins. 105 NOE distance restraints and seven dihedral angle restraints were used for the structure calculations. The three-dimensional structure was determined with CYANA based on torsion angle dynamics and refinement in a water solvent box was carried out with CNS. Fifty structures were calculated and the 20 lowest energy structures superimposed with a RMSD of 0.49 ± 0.16 Å. Even though it has the M-2 branch disulfide connectivity, BtIIIA was found to have a ‘flying bird’ backbone motif depiction that is found in the M-1 branch conotoxin mr3e. This study shows that conotoxins with the same cysteine framework can have different disulfide connectivities and different peptide folds.     

A novel conotoxin from Conus delessertii with posttranslationally modified lysine residues

A major peptide, de13a from the crude venom of Conus delessertii collected in the Yucatan Channel, Mexico, was purified. The peptide had a high content of posttranslationally modified amino acids, including 6-bromotryptophan and a nonstandard amino acid that proved to be 5-hydroxylysine. This is the first report of 5-hydroxylysine residues in conotoxins. The sequence analysis, together with cDNA cloning and a mass determination (monoisotopic mass of 3486.76 Da), established that the mature toxin has the sequence DCOTSCOTTCANGWECCKGYOCVNKACSGCTH, where O is 4-hydroxyproline, W 6-bromotryptophan, and K 5-hydroxylysine, the asterisk represents the amidated C-terminus, and the calculated monoisotopic mass is 3487.09 Da. The eight Cys residues are arranged in a pattern (C-C-C-CC-C-C-C) not described previously in conotoxins. This arrangement, for which we propose the designation of framework #13 or XIII, differs from the ones (C-C-CC-CC-C-C and C-C-C-C-CC-C-C) present in other conotoxins which also contain eight Cys residues. This peptide thus defines a novel class of conotoxins, with a new posttranslational modification not previously found in other Conus peptide families.     

Amino acid sequence and biological activity of a gamma-conotoxin-like peptide from the worm-hunting snail Conus austini

A novel 31-residue toxin, named as7a, was isolated and characterized from the venom of Conus austini, a vermivorous cone snail collected in the western Gulf of Mexico. The complete amino acid sequence, TCKQKGEGCSLDVgammaCCSSSCKPGGPLFDFDC, was determined by automatic Edman sequencing after reduction and alkylation. The sequence shows six Cys residues arranged in the pattern that defines the O-superfamily of conotoxins, and the sequence motif -gammaCCS-, which has only been found in the gamma-conotoxin family. The molecular mass of the native peptide was determined by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, which confirmed the chemical analyses and suggested a free C-terminus. The purified peptide elicited toxic effects in the freshwater snail Pomacea paludosa after intramuscular injection, but it had no effect when injected intracerebrally into mice. The structural similarity of peptide as7a to other gamma-conotoxins suggests that modulation of pacemaker channels could be responsible for its biological activity.     

Alpha S-conotoxin RVIIIA: a structurally unique conotoxin that broadly targets nicotinic acetylcholine receptors

We report the purification and characterization of a new conotoxin from the venom of Conus radiatus. The peptide, alphaS-conotoxin RVIIIA (alphaS-RVIIIA), is biochemically unique with respect to its amino acid sequence, post-translational modification, and molecular targets. In comparison to other nicotinic antagonists from Conus venoms, alphaS-RVIIIA exhibits an unusually broad targeting specificity for nicotinic acetylcholine receptor (nAChR) subtypes, as assayed by electrophysiology. The toxin is paralytic to mice and fish, consistent with its nearly irreversible block of the neuromuscular nAChR. Similar to other antagonists of certain neuronal nAChRs, the toxin also elicits seizures in mice upon intracranial injection. The only previously characterized conotoxin from the S superfamily, sigma-conotoxin GVIIIA, is a specific competitive antagonist of the 5-HT3 receptor; thus, alphaS-RVIIIA defines a novel family of nicotinic antagonists within the S superfamily. All previously characterized competitive conotoxin nAChR antagonists have been members of the A superfamily of conotoxins. Our working hypothesis is that the particular group of fish-hunting Conus species that includes Conus radiatus uses the alphaS-conotoxin family to target the muscle nAChR and paralyze prey.     

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.     

Three-dimensional structure of conotoxin tx3a: An m-1 branch peptide of the M-superfamily

The M-superfamily, one of eight major conotoxin superfamilies found in the venom of the cone snail, contains a Cys framework with disulfide-linked loops labeled 1, 2, and 3 (-CC (1) C (2) C (3) CC-). M-Superfamily conotoxins can be divided into the m-1, -2, -3, and -4 branches, based upon the number of residues located in the third Cys loop between the fourth and fifth Cys residues. Here we provide a three-dimensional solution structure for the m-1 conotoxin tx3a found in the venom of Conus textile. The 15-amino acid peptide, CCSWDVCDHPSCTCC, has disulfide bonds between Cys (1) and Cys (14), Cys (2) and Cys (12), and Cys (7) and Cys (15) typical of the C1-C5, C2-C4, and C3-C6 connectivity pattern seen in m-1 branch peptides. The tertiary structure of tx3a was determined by two-dimensional (1)H NMR in combination with the combined assignment and dynamics algorithm for nuclear magnetic resonance (NMR) applications CYANA program. Input for structure calculations consisted of 62 inter- and intraproton, five phi angle, and four hydrogen bond constraints. The root-mean-square deviation values for the 20 final structures are 0.32 +/- 0.07 and 0.84 +/- 0.11 A for the backbone and heavy atoms, respectively. Surprisingly, the structure of tx3a has a "triple-turn" motif seen in the m-2 branch conotoxin mr3a, which is absent in mr3e, the only other member of the m-1 branch of the M-superfamily whose structure is known. Interestingly, injection of tx3a into mice elicits an excitatory response similar to that of the m-2 branch peptide mr3a, even though the conotoxins have different disulfide connectivity patterns.     

Novel alpha-conotoxins identified by gene sequencing from cone snails native to Hainan, and their sequence diversity.

Conotoxins (CTX) from the venom of marine cone snails (genus Conus) represent large families of proteins, which show a similar precursor organization with surprisingly conserved signal sequence of the precursor peptides, but highly diverse pharmacological activities. By using the conserved sequences found within the genes that encode the alpha-conotoxin precursors, a technique based on RT-PCR was used to identify, respectively, two novel peptides (LiC22, LeD2) from the two worm-hunting Conus species Conus lividus, and Conus litteratus, and one novel peptide (TeA21) from the snail-hunting Conus species Conus textile, all native to Hainan in China. The three peptides share an alpha4/7 subfamily alpha-conotoxins common cysteine pattern (CCX(4)CX(7)C, two disulfide bonds), which are competitive antagonists of nicotinic acetylcholine receptor (nAChRs). The cDNA of LiC22N encodes a precursor of 40 residues, including a propeptide of 19 residues and a mature peptide of 21 residues. The cDNA of LeD2N encodes a precursor of 41 residues, including a propeptide of 21 residues and a mature peptide of 16 residues with three additional Gly residues. The cDNA of TeA21N encodes a precursor of 38 residues, including a propeptide of 20 residues and a mature peptide of 17 residues with an additional residue Gly. The additional residue Gly of LeD2N and TeA21N is a prerequisite for the amidation of the preceding C-terminal Cys. All three sequences are processed at the common signal site -X-Arg- immediately before the mature peptide sequences. The properties of the alpha4/7 conotoxins known so far were discussed in detail. Phylogenetic analysis of the new conotoxins in the present study and the published homologue of alpha4/7 conotoxins from the other Conus species were performed systematically. Patterns of sequence divergence for the three regions of signal, proregion, and mature peptides, both nucleotide acids and residue substitutions in DNA and peptide levels, as well as Cys codon usage were analyzed, which suggest how these separate branches originated. Percent identities of the DNA and amino acid sequences of the signal region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical to highly divergent between inter- and intra-species. Notably, the diversity of the proregion was also high, with an intermediate percentage of divergence between that observed in the signal and in the toxin regions. The data presented are new and are of importance, and should attract the interest of researchers in this field. The elucidated cDNAs of these toxins will facilitate a better understanding of the relationship of their structure and function, as well as the process of their evolutionary relationships.     

I-superfamily conotoxins: sequence and structure analysis

I-superfamily conotoxins have four-disulfide bonds with cysteine arrangement C-C-CC-CC-C-C, and they inhibit or modify ion channels of nerve cells. They have been characterized only recently and are relatively less well studied compared to other superfamily conotoxins. We have detected selective and sensitive sequence pattern for I-superfamily conotoxins. The availability of sequence pattern should be useful in protein family classification and functional annotation. We have built by homology modeling, a theoretical structural 3D model of ViTx from Conus virgo, a typical member of I-superfamily conotoxins. The modeling was based on the available 3D structure of Janus-atracotoxin-Hv1c of Janus-atracotoxin family whose members have been suggested as possible biopesticides. A study comparing the theoretically modeled structure of ViTx, with experimentally determined structures of other toxins, which share functional similarity with ViTx, reveals the crucial role of C-terminal region of ViTx in blocking therapeutically important voltage-gated potassium channels.     

Direct cDNA cloning of novel conopeptide precursors of the O-superfamily

Conotoxins from the venom of marine cone snails (genus Conus) represent large families of proteins exhibiting a similar precursor organization, but highly diverse pharmacological activities. A directed PCR-based approach using primers according to the conserved signal sequence was applied to investigate the diversity of conotoxins from the O-superfamily. Using 3' RACE, cDNA sequences encoding precursor peptides were identified in five Conus species (Conus capitaneus, Conus imperialis, Conusstriatus, Conus vexillum and Conus virgo). In all cases, the sequence of the signal region exhibited high conservancy, whereas the sequence of the mature peptides was either almost identical or highly divergent among the five species. These findings demonstrate that beside a common genetic pattern divergent evolution of toxins occurred in a highly mutating peptide family.     

A novel 13 residue acyclic peptide from the marine snail, Conus monile, targets potassium channels

A novel 13-residue peptide Mo1659 has been isolated from the venom of a vermivorous cone snail, Conus monile. HPLC fractions of the venom extract yielded an intense UV absorbing fraction with a mass of 1659Da. De novo sequencing using both matrix assisted laser desorption and ionization and electrospray MS/MS methods together with analysis of proteolytic fragments successfully yielded the amino acid sequence, FHGGSWYRFPWGY-NH(2). This was further confirmed by comparison with the chemically synthesized peptide and by conventional Edman sequencing. Mo1659 has an unusual sequence with a preponderance of aromatic residues and the absence of apolar, aliphatic residues like Ala, Val, Leu, and Ile. Mo1659 has no disulfide bridges distinguishing it from the conotoxins and bears no sequence similarity with any of the acyclic peptides isolated thus far from the venom of cone snails. Electrophysiological studies on the effect of Mo1659 on measured currents in dorsal root ganglion neurons suggest that the peptide targets non-inactivating voltage-dependent potassium channels.     

Solution structure of an M-1 conotoxin with a novel disulfide linkage

The M-superfamily of conotoxins has a typical Cys framework (-CC-C-C-CC-), and is one of the eight major superfamilies found in the venom of the cone snail. Depending on the number of residues located in the last Cys loop (between Cys4 and Cys5), the M-superfamily family can be divided into four branches, namely M-1, -2, -3 and -4. Recently, two M-1 branch conotoxins (mr3e and tx3a) have been reported to possess a new disulfide bond arrangement between Cys1 and Cys5, Cys2 and Cys4, and Cys3 and Cys6, which is different from those seen in the M-2 and M-4 branches. Here we report the 3D structure of mr3e determined by 2D (1)H NMR in aqueous solution. Twenty converged structures of this peptide were obtained on the basis of 190 distance constraints obtained from NOE connectivities, as well as six varphi dihedral angle, three hydrogen bond, and three disulfide bond constraints. The rmsd values about the averaged coordinates of the backbone atoms were 0.43 +/- 0.19 A. Although mr3e has the same Cys arrangement as M-2 and M-4 conotoxins, it adopts a distinctive backbone conformation with the overall molecule resembling a 'flying bird'. Thus, different disulfide linkages may be employed by conotoxins with the same Cys framework to result in a more diversified backbone scaffold.