Sequence diversity of O-superfamily conopetides from Conus marmoreus native to Hainan

The full-length cDNAs of six new O-superfamily conotoxins (CTX) were cloned and sequenced from Conus marmoreus native to Hainan in China South Sea using RT-PCR and 3′-RACE. Six novel conotoxin precursors encoded by these cDNAs consist of three typical regions of signal, pro-peptide and mature peptide. All the six toxin regions share a common O-superfamily cysteine pattern (C-C-CC-C-C, with three disulfide bridges). The predicted precursors are composed of 73–88 amino acids, and the predicted mature peptides consist of 26–34 amino acids. Phylogenetic analysis of new conotoxins from C. marmoreus from the present study and published homologue T-superfamily sequences from other Conus species was performed systematically. Patterns of sequence divergence for three regions of signal, pro-region and mature peptides, as well as Cys codon usage define the major O-superfamily branches and suggest how these separate branches arose. Percent identities of the 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 pro-region was also high with intermediate divergence between that observed in signal and toxin regions. Amino acid sequences and their mode of action (target) of previously identified conotoxins from molluscivorous C. marmoreus for the known conotoxins classes are discussed in detail. The data presented are new and should pave the way for chemical synthesis of these unique conotoxins for to allow determination of the molecular targets of these peptides, and also to provide clues for a better understanding of the phylogeny of these peptides.     

Conotoxin TVIIA, a novel peptide from the venom of Conus tulipa 1. Isolation, characterization and chemical synthesis.

A novel conotoxin belonging to the 'four-loop' structural class has been isolated from the venom of the piscivorous cone snail Conus tulipa. It was identified using a chemical-directed strategy based largely on mass spectrometric techniques. The new toxin, conotoxin TVIIA, consists of 30 amino-acid residues and contains three disulfide bonds. The amino-acid sequence was determined by Edman analysis as SCSGRDSRCOOVCCMGLMCSRGKCVSIYGE where O = 4-transL-hydroxyproline. Two under-hydroxylated analogues, [Pro10]TVIIA and [Pro10,11]TVIIA, were also identified in the venom of C. tulipa. The sequences of TVIIA and [Pro10]TVIIA were further verified by chemical synthesis and coelution studies with native material. Conotoxin TVIIA has a six cysteine/four-loop structural framework common to many peptides from Conus venoms including the omega-, delta- and kappa-conotoxins. However, TVIIA displays little sequence homology with these well-characterized pharmacological classes of peptides, but displays striking sequence homology with conotoxin GS, a peptide from Conus geographus that blocks skeletal muscle sodium channels. These new toxins and GS share several biochemical features and represent a distinct subgroup of the four-loop conotoxins.     

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.     

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 helical conotoxin from Conus imperialis has a novel cysteine framework and defines a new superfamily

Cone snail venoms are a rich source of peptides, many of which are potent and selective modulators of ion channels and receptors. Here we report the isolation and characterization of two novel conotoxins from the venom of Conus imperialis. These two toxins contain a novel cysteine framework, C-C-C-CC-C, which has not been found in other conotoxins described to date. We name it framework XXIII and designate the two toxins im23a and im23b; cDNAs of these toxins exhibit a novel signal peptide sequence, which defines a new K-superfamily. The disulfide connectivity of im23a has been mapped by chemical mapping of partially reduced intermediates and by NMR structure calculations, both of which establish a I-II, III-IV, V-VI pattern of disulfide bridges. This pattern was also confirmed by synthesis of im23a with orthogonal protection of individual cysteine residues. The solution structure of im23a reveals that im23a adopts a novel helical hairpin fold. A cluster of acidic residues on the surface of the molecule is able to bind calcium. The biological activity of the native and recombinant peptides was tested by injection into mice intracranially and intravenously to assess the effects on the central and peripheral nervous systems, respectively. Intracranial injection of im23a or im23b into mice induced excitatory symptoms; however, the biological target of these new toxins has yet to be identified.     

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.     

Soluble expression, purification and functional identification of a disulfide-rich conotoxin derived from Conus litteratus

Conotoxins are a diverse array of small peptides mostly with multiple disulfide bridges. These peptides become an increasing significant source of neuro-pharmacological probes and drugs as a result of the high selectivity for ion channels and receptors. Usually, the analogue of natural conotoxins is produced by means of chemical synthesis. Here, we present a simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression. By fused with thioredoxin and His tag, a novel O-superfamily conotoxin lt7a was successfully expressed in Escherichia coli and purified, resulting in a high yield of recombinant lt7a about 6 mg/l. The purity of target protein is up to 95% as identified by HPLC results. Whole cell patch-clamp recording revealed that the new conotoxin blocked voltage-sensitive sodium channels in rat dorsal root ganglion neurons, indicating it might be a novel microO-conotoxin.     

Diversity of the O-superfamily conotoxins from Conus miles.

Conopeptides display prominent features of hypervariability and high selectivity of large gene families that mediate interactions between organisms. Remarkable sequence diversity of O-superfamily conotoxins was found in a worm-hunting cone snail Conus miles. Five novel cDNA sequences encoding O-superfamily precursor peptides were identified in C. miles native to Hainan by RT-PCR and 3'-RACE. They share the common cysteine pattern of the O-superfamily conotoxin (C-C-CC-C-C, with three disulfide bridges). The predicted peptides consist of 27-33 amino acids. We then performed a phylogenetic analysis of the new and published homologue sequences from C. miles and the other Conus species. Sequence divergence (%) and residue substitutions to view evolutionary relationships of the precursors' signal, propeptide, and mature toxin regions were analyzed. Percentage divergence of the amino acid sequences of the prepro region exhibited high conservation, whereas the sequences of the mature peptides ranged from almost identical with to highly divergent from inter- and intra-species. Despite the O-superfamily being a large and diverse group of peptides, widely distributed in the venom ducts of all major feeding types of Conus and discovered in several Conus species, it was for the first time that the newly found five O-superfamily peptides in this research came from the vermivorous C. miles. So far, conotoxins of the O-superfamily whose properties have been characterized are from piscivorous and molluscivorous Conus species, and their amino acid sequences and mode of action have been discussed in detail. The elucidated cDNAs of the five toxins are new and of importance and should attract the interest of researchers in the field, which would pave the way for a better understanding of the relationship of their structure and function.     

织锦芋螺ο家族芋螺毒素的序列分析

为了从织锦芋螺（Ｃｏｎｕｓｔｅｘｔｉｌｅ）中尽可能多地分离出ο家族的毒素序列和研究其应用价值,在克隆了织锦芋螺α芋螺毒素的基础上进行了织锦芋螺ο家族芋螺毒素基因的分离工作．从织锦芋螺毒管中提取ｍ ＲＮＡ,通过ＲＡＣＥ（ｒａｐｉｄ ａｍ ｐｌｉｆｉｃａｔｉｏｎ ｏｆｃＤＮＡ ｅｎｄｓ,ｃＤＮＡ 末端的快速扩增）－ＰＣＲ方法扩增获得ο家族芋螺毒素ｃＤＮＡ 片段,并进行克隆和序列分析．从织锦芋螺毒液中获得了６种新的芋螺毒素序列,且毒素序列的成熟肽部分均符合Ｃ－ Ｃ－ ＣＣ－ Ｃ－ Ｃ的保守半胱氨酸框架．这些是新的ο家族芋螺毒素序列,新序列的阐明为进一步研究其生物活性和应用打下了基础．     

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.     

Direct cDNA cloning of novel conotoxins of the T-superfamily from Conus textile

The T-superfamily is a large and diverse group of peptides, widely distributed in venom ducts of all major feeding types of Conus. These peptides are likely to be functionally diverse. A directed PCR-based approach using primers based on the conserved signal sequence was applied to investigate new conotoxins of the T-superfamily from Conus textile native to Hainan. Using RT-PCR and 3'-RACE, four novel cDNA sequences encoding precursor peptides were identified in C. textile. They share a common T-superfamily cysteine pattern (CC-CC, with two disulfide bridges). The predicted peptides are small (9-12 amino acids). TeAr193 composed of nine amino acid residues is one of the shortest T-superfamily conotoxins ever found. Patterns of sequence divergence and Cys codon usage define the major T-superfamily branches and suggest how these separate branches arose. The sequences of the signal regions exhibited highest conservation, whereas the sequences of the mature peptides were either almost identical or highly divergent; and conservation of the pro-region was intermediate between that observed in signal and toxin regions. The elucidated cDNAs of the four toxins will facilitate a better understanding of the relationship between structure and function.     

Extracellular secretion of STa heat-stable enterotoxin by Escherichia coli after fusion to a heterologous leader peptide

The mature 19-amino acid STa heat-stable enterotoxin of E. coli has a preceding peptide of 53 amino acids which contains two domains called Pre (aa 1–19) and Pro (aa 20–53) sequences, proposed to be essential for extracellular toxin release by this host. The Pro sequence, however, has been proven not be indispensable for this process since Pro deletion mutants secrete STa. To find out if Pre and/or other unremoved natural STa flanking sequences are responsible for toxin secretion in those mutants we genetically fused mature STa directly to the leader peptide of the periplasmic E. coli heat-labile enterotoxin B-subunit (LTB). Expression of this gene fusion resulted in extracellular secretion of biologically active STa by E. coli independently of natural STa neighboring genetic sequences. Moreover, these results suggest that STa might be able to gain access to the extracellular milieu simply upon its entry into the E. coli periplasm once guided into this compartment by the LTB leader peptide. To test if extracellular secretion in this fashion might be extended to other disulfide bond-rich small peptides, the 13 amino acid conotoxin GI and a non-enterotoxic STa-related decapeptide were cloned. None of the two peptides was found in culture supernatants, in spite of high structural homology to the toxin. Failure to be secreted most likely leads to degradation as peptides were also not detected in bacterial sonicates. We hypothesize that cysteine-rich peptides must have an amino acid length and/or number of disulfide bridges closer to those in STa for them to follow this toxin secretory pathway in E. coli.     

Propeptide does not act as an intramolecular chaperone but facilitates protein disulfide isomerase-assisted folding of a conotoxin precursor

Conotoxins comprise a large and diverse group of peptide neurotoxins derived from Conus snail venoms; most contain multiple disulfide bonds. The conotoxin precursors consist of three distinct domains: the N-terminal signal sequence, an intervening propeptide region, and the C-terminal mature conotoxin. Formation of the native disulfide bonds during the oxidative folding of conotoxins is a prerequisite for their proper biological function, but in numerous in vitro folding experiments with mature conotoxins, a lack of specificity in formation of the native Cys-Cys connectivities is observed. The mechanisms that ensure that the native disulfide bonds are formed in venom ducts during biosynthesis remain unknown. To evaluate whether the propeptide could potentially function as an intramolecular chaperone, we studied the oxidative folding of a conotoxin precursor, pro-GI, belonging to the alpha-conotoxin family. Our results indicate that the propeptide sequence did not directly contribute to folding kinetics and thermodynamics. However, we found that the propeptide region of pro-GI played an important role when oxidative folding was catalyzed by protein disulfide isomerase (PDI). The PDI-assisted reaction was more efficient during the early folding in the context of the propeptide sequence (pro-GI), as compared to that of the mature conotoxin (alpha-GI). Taken together, our results suggest for the first time that the propeptide region may play a role in the PDI-catalyzed oxidative folding of conotoxin precursors.     

Pseudo amino acid composition and multi-class support vector machines approach for conotoxin superfamily classification

Conotoxins are disulfide rich small peptides that target a broad spectrum of ion-channels and neuronal receptors. They offer promising avenues in the treatment of chronic pain, epilepsy and cardiovascular diseases. Assignment of newly sequenced mature conotoxins into appropriate superfamilies using a computational approach could provide valuable preliminary information on the biological and pharmacological functions of the toxins. However, creation of protein sequence patterns for the reliable identification and classification of new conotoxin sequences may not be effective due to the hypervariability of mature toxins. With the aim of formulating an in silico approach for the classification of conotoxins into superfamilies, we have incorporated the concept of pseudo-amino acid composition to represent a peptide in a mathematical framework that includes the sequence-order effect along with conventional amino acid composition. The polarity index attribute, which encodes information such as residue surface buriability, polarity, and hydropathy, was used to store the sequence-order effect. Several methods like BLAST, ISort (Intimate Sorting) predictor, least Hamming distance algorithm, least Euclidean distance algorithm and multi-class support vector machines (SVMs), were explored for superfamily identification. The SVMs outperform other methods providing an overall accuracy of 88.1% for all correct predictions with generalized squared correlation of 0.75 using jackknife cross-validation test for A, M, O and T superfamilies and a negative set consisting of short cysteine rich sequences from different eukaryotes having diverse functions. The computed sensitivity and specificity for the superfamilies were found to be in the range of 84.0-94.1% and 80.0-95.5%, respectively, attesting to the efficacy of multi-class SVMs for the successful in silico classification of the conotoxins into their superfamilies.     

Factors governing selective formation of specific disulfides in synthetic variants of alpha-conotoxin

alpha-Conotoxin GI is a snail toxin protein consisting of 13 amino acids cross-linked by 2 intramolecular disulfide bridges. This toxin is an antagonist of acetylcholine receptors. The native sequence has been synthesized, along with nine additional variants in which non-cysteine residues are replaced by alanine or the cysteine positions are altered. Each reduced peptide has been oxidized by reaction with oxygen or glutathione both in a folding buffer and in 6 M guanidine hydrochloride. Purified products of oxidation have been characterized with respect to molecular weights and the positions of disulfides. The four cysteines in conotoxin can form two intramolecular disulfides in three different combinations. Relative yields of each of the three isomers have been determined, thereby permitting evaluation of the roles of non-cysteine residues and cysteine placements in the folding of conotoxin. Cysteine positions dominate factors directing formation of the nativelike isomer in a manner that may be predicted from equilibrium constants for loop formation in model peptides containing two cysteines. Alanine substitutions at several positions which are conserved in naturally occurring conotoxins affect the discrimination between the two most favored disulfide arrangements. Substitutions at three nonconserved positions have no structural effect on isomer yields. It therefore is possible to vary these latter three positions in a manner which might help to generate a functional binding surface which is complementary to receptors in the specific prey of a particular species of snail, without affecting the toxin's folding.     

Isolation and characterization of a T-superfamily conotoxin from Conus litteratus with targeting tetrodotoxin-sensitive sodium channels

A T-1-conotoxin, lt5d, was purified and characterized from the venom of vermivorous hunting cone snails Conus litteratus. The complete amino acid sequence of lt5d (DCCPAKLLCCNP) has been determined by Edman degradation. With two disulfide bonds, the calculated average mass is 1274.57 Da, which is confirmed by MALDI-TOF mass spectrometry (average mass 1274.8778). Under whole cell patch-clamp mode, lt5d inhibits tetrodotoxin-sensitive sodium currents on adult rat dorsal root ganglion neurons, but has no effects on tetrodotoxin-resistant sodium currents. The inhibition of TTX-sensitive sodium currents by lt5d was found to be concentration-dependent with the IC₅₀ value of 156.16 nM. Thus, this is the first T-superfamily conotoxin identified to block TTX-sensitive sodium channels.     

mr1e, a conotoxin from Conus marmoreus with a novel disulfide pattern

Conotoxins are well known for their highly variable structures and functions. Here we report the identification of a novel conotoxin named mr1e from Conus marmoreus . mr1e is composed of 11 amino acid residues cross-linked by two disulfide bonds (CCHSSWCKHLC). The spacing of intercysteine loops in mr1e is exactly the same as that in α4/3 conotoxins. However, the native mr1e peptide co-eluted on reverse-phase HPLC with the regioselectively synthesized ribbon disulfide linkage isomer (C¹-C⁴, C²-C³) but not the globular linkage isomer (C¹-C³, C²-C⁴). Although this peptide has the same disulfide connectivity as the χ-conotoxins, their sequences do not share significant homology. Thus, mr1e could be defined as a novel conotoxin family. By intracranial injection into mice, mr1e showed an excitatory effect. The characterization of mr1e certainly enriches our understanding of conotoxins, and also opens an avenue for further structural and functional investigation.