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Originally published In Press as doi:10.1074/jbc.M309654200 on December 30, 2003

J. Biol. Chem., Vol. 279, Issue 17, 17596-17606, April 23, 2004
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The A-superfamily of Conotoxins

STRUCTURAL AND FUNCTIONAL DIVERGENCE*

Ameurfina D. Santos{ddagger}§, J. Michael McIntosh¶||, David R. Hillyard**, Lourdes J. Cruz{ddagger}{ddagger}{ddagger}, and Baldomero M. Olivera{ddagger}§§

From the Departments of {ddagger}Biology, Psychiatry, and **Pathology, University of Utah, Salt Lake City, Utah 84112 and the §National Institute of Molecular Biology and Biotechnology and the {ddagger}{ddagger}Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines

Received for publication, September 2, 2003 , and in revised form, December 26, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The generation of functional novelty in proteins encoded by a gene superfamily is seldom well documented. In this report, we define the A-conotoxin superfamily, which is widely expressed in venoms of the predatory cone snails (Conus), and show how gene products that diverge considerably in structure and function have arisen within the same superfamily. A cDNA clone encoding {alpha}-conotoxin GI, the first conotoxin characterized, provided initial data that identified the A-superfamily. Conotoxin precursors in the A-superfamily were identified from six Conus species: most (11/16) encoded {alpha}-conotoxins, but some (5/16) belong to a family of excitatory peptides, the {kappa}A-conotoxins that target voltage-gated ion channels. {alpha}-Conotoxins are two-disulfide-bridged nicotinic antagonists, 13–19 amino acids in length; {kappa}A-conotoxins are larger (31–36 amino acids) with three disulfide bridges. Purification and biochemical characterization of one peptide, {kappa}A-conotoxin MIVA is reported; five of the other predicted conotoxins were previously venom-purified. A comparative analysis of conotoxins purified from venom, and their precursors reveal novel post-translational processing, as well as mutational events leading to polymorphism. Patterns of sequence divergence and Cys codon usage define the major superfamily branches and suggest how these separate branches arose.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The predatory cone snails use their biochemically and pharmacologically complex venoms to capture prey, defend against predators, and compete with other animals in their environment (1). The active components of these venoms are mostly small, disulfide-rich peptides, 10–30 amino acids in length ("conotoxins") (2). As a group, cone snails (genus Conus) are very successful (>500 species), arguably the largest single genus of marine invertebrates (3). During speciation, a remarkable hypermutation of Conus venom peptides occurs (4, 5). The entire spectrum of 50–200 peptides that comprises the conotoxin repertoire of a particular Conus species is distinct from the peptide complement of the venom of any other Conus species. Thus, it is estimated that a total of >50,000 different peptides can potentially be expressed in the venoms of living Conus.

A simplifying conceptual framework for addressing the formidable molecular complexity of the conotoxins is that the 50,000 peptides are encoded by relatively few gene superfamilies, which have greatly diversified within this genus. In general, each conotoxin superfamily has distinctive and conserved sequence features in the conotoxin precursors encoded by the gene superfamily. Several superfamilies have been characterized, including the O-, T-, P-, I-, and M-superfamilies that have between two and five disulfide cross-links (4, 6–9).1 The mature conotoxins in each conotoxin superfamily have a characteristic arrangement of cysteine residues, the oxidative folding of which results in a three-dimensional scaffold with a specific disulfide connectivity ("the Cys framework"). The conserved Cys superfamily framework largely determines the three-dimensional conformation of the polypeptide backbone of all peptides that belong to the superfamily.

The very first toxin purified and characterized from a Conus venom was {alpha}-conotoxin GI, a competitive nicotinic antagonist from the venom of the geography cone, Conus geographus (10). {alpha}-GI was shown to be a 13-amino acid peptide with two disulfide cross-links. The biochemical studies on {alpha}-GI established for the first time that the biologically active constituents of Conus venoms were small, multiply cross-linked peptides, generically referred to as conotoxins. Although several conotoxin superfamilies have been described, the gene superfamily to which {alpha}-conotoxin GI belongs has not been previously defined in the literature. We show that the A-conotoxin gene superfamily, to which {alpha}-conotoxin GI belongs, is found in fish-hunting, snail-hunting, and worm-hunting species of Conus and has undergone both structural and functional diversification.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation and Sequencing of cDNA Clones of {alpha}-Conotoxin GI—cDNA clones encoding {alpha}-conotoxin GI were isolated from a size-fractionated cDNA library previously constructed by J. Hunspurger from Conus geographus duct mRNA using pUC13 as cloning vector and Escherichia coli strain MC1061F'laclq as the host cell. Clones were identified by screening them with a mixed oligonucleotide probe corresponding to the first six amino acids of GI (5'-GCXGGRTTRCARCAYTC-3', where R = AorG, Y = CorT,and X = AGT or C). The probe was end-labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase (11) and hybridized to nitrocellulose filters for 40–60 h at 48 °C in 3 M tetramethylammonium chloride (TMAC),2 0.1 M sodium phosphate, 1 mM EDTA, 5x Denhardt's, 0.6% SDS, and 100 µg/ml salmon sperm DNA. After washing twice in 3 M TMAC/50 mM Tris/0.2% SDS and two times in 2x standard saline citrate/0.1% SDS, positive colonies were identified by autoradiography. Some clones gave the sequence of {alpha}-GIB, a homologous {alpha}-conotoxin. {alpha}-GIB clones were eliminated by subtractive screening using an additional probe corresponding to the five amino acids at the C terminus of {alpha}-GI. Positive clones were grown in LB/amp/kan broth at 37 °C, harvested, then lysed with alkali (12). Plasmid DNA was purified by equilibrium centrifugation in CsCl ethidium bromide gradient. Sequencing of inserts in both strands was done by the dideoxy method (13) using Sequenase version 2.0 enzyme from US Biochemicals. The sequence shown in Table I was obtained by walking through the cDNA with five primers in the forward direction and five primers in the reverse direction.


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  		TABLE I
cDNA sequence of the gene for {alpha}-conotoxin GI and the predicted translation product The signal sequence is shaded, and the toxin region is underlined. Nucleotides in the untranslated region are in small letters, and the poly(A) addition signal (aataaa) is shaded. In {alpha}-GIB, two amino acids (KG) corresponding to an insertion of six nucleotides (AAG GGA) are in the position marked by a dash (—) in the above sequence of {alpha}-GI.

 
Cloning and Sequencing of A-superfamily Peptides from Several Conus Species—The cDNAs were prepared from cDNA libraries of Conus striatus and C. magus, and the poly(A)+ mRNA obtained from the venom ducts of C. striatus, C. stercusmuscarum, C. bandanus, and C. caracteristicus; mRNA was from the venom ducts of several snails in each case. The cDNA libraries were prepared using either the protocol in Sambrook (11) or the Promega Magic Miniprep system. cDNAs were amplified using a pair of primers, one corresponding to the signal sequence and the other to the 3'-untranslated region near the open reading frame. Amplification with Vent polymerase was done for 30 cycles using an air thermocycler set as follows: denaturation at 94 °C, 0 s; annealing at 50 °C, 0 s; elongation at 72 °C, 15 s. Ten-microliter aliquots of PCR products were analyzed by electrophoresis in 1.5% agarose gel in Tris-acetate buffer with ethidium bromide, and the remaining products were purified by gel electrophoresis in a 1.5% low melting gel.

The plasmid vector used for cloning was a modified pDH52, which we named pDH78. pDH52 is basically pGEM 3Zf+ (Promega) with its polylinker region extended by cloning into the EcoRI and KpnI sites a synthetic oligomer encoding sites for EcoRI, Sacl, EcoRV, NotI, SphI, NsiI, StuI, BgIII, and KpnI. This was further modified by adding a 23-mer synthetic DNA fragment that has a KpnI site inserted into the XbaI and BglII sites to generate pDH78. Plasmid DNA digested with KpnI and the PCR-amplified products were treated with 2 units of T4 DNA polymerase per milligram of DNA in the presence of 0.5 mM dATP for the vector and 0.5 mM dTTP for the PCR product in a 10- to 20-µl solution of 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, 0.5 µg/µl bovine serum albumin. The mixture was incubated at 37 °C for 30 min, then heated for 10 min at 70–75 °C to stop the reaction. For ligation, a mixture of DNA polymerase-treated vector (0.1 µg) and PCR product (0.05 µg) was preincubated at 45 °C for 5 min before 5 nmol of ATP and 7–20 units of ligase were added, and the mixture was incubated at 16 °C for 16 h. Transformants were tested for the presence of the desired recombinant plasmids by digestion of the purified plasmid with XbaI and BglII, by PCR of the purified plasmid and/or by PCR of the colony after lysis. Solid phase sequencing (12) was done using magnetic beads from Dynal and the USB DNA sequencing kit.

Purification of {kappa}A-conotoxin MIVA—Freeze-dried Conus magus venom (500 mg) was extracted with 0.1% trifluoroacetic acid and filtered through Centriprep 30 microconcentrators as previously described by Cartier et al. (14). The extract was fractionated on a semi-preparative Vydac C18 column equipped with a guard module. All other chromatographic purifications involved an analytical Brownlee C8 column (4.6 mm x 22 cm, RP300 packing, 7-mm particle size, 300-Å pore size) with a guard cartridge that also had RP300 packing material. For all chromatograms, buffer A was 0.1% trifluoroacetic acid and buffer B was 60% acetonitrile in 0.1% trifluoroacetic acid. The activity of fractions was monitored by measuring synaptically evoked responses from cutaneous pectoris muscle of frog as previously described (15–17).

Pyridylethylation, Chymotrypsin Digestion, and Sequencing of MIVA—The peptide was pyridylethylated prior to sequencing. An aliquot of HPLC fraction containing 1 nmol of MIVA was concentrated down to 5 µl of MIVA and taken up in 50 µl of 0.25 M Tris/2 mM EDTA/6 M guanidine HCl, pH 7.7, mixed with 2 µl of 10% {beta}-mercaptoethanol, and incubated at room temperature for 30 min. Two microliters of 20% 4-vinylpyridine in ethanol was added, and the mixture was incubated for 15 min at room temperature in the dark. 25 µl of 2% heptafluorobutyric acid (HFBA) was added prior to HPLC on an analytical Brownlee C8 column (RP300, 220 x 4.6 mm), which was eluted with a gradient of acetonitrile in 0.05% HFBA.

The pyridylethylated peptide, concentrated to ~5 µl, was mixed with 2.5 µl of acetonitrile before addition of the digestion buffer (0.25 M Tris/10 mM CaCl2, pH 7.8) and 2 µl of 0.05 µg/µl chymotrypsin (Roche Applied Science). The reaction mix was incubated overnight at room temperature, 25 µl of 2% HFBA was added before separation of the fragments by HPLC as described above for the pyridylethylated peptide. Sequencing of the alkylated peptide and the chymotrypsin fragments was performed on an Applied Biosystems model 477A protein sequencer at the Protein/DNA Core Facility at the University of Utah Cancer Center.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification and Analysis of a cDNA Clone Encoding {alpha}-Conotoxin GI—A cDNA sequence of the {alpha}-conotoxin GI prepropeptide matched the canonical organization originally established for conotoxin gene superfamilies by Hillyard and coworkers (4); the literature on conotoxin superfamilies was recently reviewed (1). The messenger RNA encoding {alpha}-conotoxin GI has the characteristic four-module organization, including a signal sequence, a "pro" region, the toxin-encoding region followed by a stop codon, and a 3'-untranslated region. For the {alpha}-conotoxin GI cDNA, the signal sequence is 21 amino acids, the pro region is 28 aa and the C-terminal toxin region comprises 15 amino acids that are predicted to be post-translationally processed to the 13-amino acid toxin. For {alpha}-conotoxin GI, the 3'-untranslated region is quite long: 823 nucleotides (Fig. 1). The generation of the mature toxin requires proteolytic cleavage of the N-terminal prepro region of the precursor, and standard processing at the C terminus from the sequence -CGR- to -C-NH2.



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  		FIG. 1.
Conus species that were analyzed for A-superfamily conotoxins. Shells of four fish-hunting species are shown: top row, left to right, C. striatus, C. geographus, and C. stercusmuscarum; bottom row, middle, C. magus. One mollusc-hunting species is shown in the bottom row, left, C. marmoreus, and one worm-hunting species is at the bottom row right, C. caracteristicus.

 
A closely related cDNA sequence was also found, which appears to be a polymorphism in the {alpha}-conotoxin GI gene. The nucleotide sequence is identical except for an insertion of six nucleotides, as shown in Table I, where the relevant {alpha}-GI sequence and the cDNA encoding the variant (designated here as {alpha}-conotoxin GIB) are shown. The insertion of the six nucleotides would lead to a mature toxin predicted to have two extra amino acids at the C terminus. Given the sequence identity between them, {alpha}-GI and {alpha}-GIB may be allelic variants.

Characterization of Related cDNA Clones from Another Fish-hunting Species, C. striatus—To compare related genes from another Conus species, we characterized cDNA clones from the venom duct of the striated cone, C. striatus, a fish-hunting species (see Fig. 1). This venom has been extensively characterized (18), and several {alpha}-conotoxins were previously purified from the venom of this species.

cDNA clones were analyzed from a C. striatus venom duct cDNA library as described under "Experimental Procedures." Five distinct sequences were obtained, which are compared with {alpha}-conotoxin GI in Table II. As expected, two of these sequences encode {alpha}-conotoxins previously characterized from C. striatus venom. These are {alpha}-conotoxin SI and {alpha}-conotoxin SII. The latter is an unusual peptide closely related in sequence to other {alpha}-conotoxins, but with an additional disulfide bond. A comparison of the amino acid sequences of the three predicted open reading frames (for {alpha}-conotoxins GI, SI, and SII) reveals that the signal sequence regions are identical. In general, sequence divergence increases from the N to C terminus of the open reading frame; in the signal sequence, there is at most a single amino acid that differs from the 21 aa consensus with greater divergence in the pro region, and considerable hypervariability at the C-terminal toxin-encoding region. There are clearly three groups of toxin sequences based on sequence similarity: the first four sequences ({alpha}-conotoxins GI, GIB, SI, and SII) show much greater sequence homology to each other than to the other two groups in Table II (S1.1 and SIVA/SIVB). The degree of sequence divergence is reflected in the biological properties of the peptides (see "Discussion").


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  		TABLE II
Comparison of conotoxin prepropeptide sequences Signal sequences are shaded, and the toxin regions are underlined. In A, the shaded sections of the cDNA sequences mark the 25-nucleotide alteration that explains the difference in length of {kappa}A-SIVA and {kappa}A-SIVB, and the 6-nucleotide change at the carboxyl terminus of SI to generate a longer conotoxin (SII). The sequence of {alpha}-conotoxin GI from C. geographus is given for comparison.

 
In addition to the {alpha}-conotoxin SI- and SII-encoding cDNA clones, the S1.1 clone from C. striatus encoded the precursor of an {alpha}-conotoxin-like peptide. Although the predicted mature peptide has the characteristic Cys pattern of the {alpha}-conotoxins (–CC–C–C–), it is very divergent in amino acid sequence from {alpha}-conotoxins GI, SI, and SII (which share considerable sequence homology). The predicted mature peptide has not been purified from venom.

A surprise from the analysis of the C. striatus cDNA was a class of clones that exhibited substantial sequence identity to those above but did not encode {alpha}-conotoxin precursors; instead, these encode two {kappa}A-conotoxins. One of the predicted mature conotoxins, {kappa}A-conotoxin SIVA, was previously characterized from venom (17). The two such sequences obtained originated from two different collection samples: the clones encoding {kappa}A-SIVA were obtained from a cDNA library made from snails collected in the Philippines. A second set of closely related clones, encoding a peptide designated as {kappa}A-SIVB was obtained from cDNA made from specimens of C. striatus collected in the Hawaiian Islands. As shown in Table II, the N-terminal sequences of these clones have considerable sequence identity to the clones that encode the {alpha}-conotoxins: the seven signal sequences ({alpha}-conotoxins GI, GIB, SI, and SII and {kappa}A-conotoxins SIVA, SIVB, and S1.1) were found to be virtually identical (see shaded amino acid sequences in Table IIA). However, in the propeptide region there is more divergence between the clones that encode {alpha}-conotoxins versus those that encode {kappa}A-conotoxins (see "Discussion"), and, in the encoded mature toxins (underlined sequences), there is essentially no homology detectable between the {alpha}-conotoxins and {kappa}A-conotoxins.

The two clones encoding {kappa}A-conotoxins are largely identical in the predicted amino acid sequence for the first 68 amino acids of the open reading frame (only 2 aa substitutions). However, the mature toxin region of {kappa}A-SIVA comprises 31 aa, whereas for {kappa}A-SIVB it is 42 aa. Thus, the SIVA precursor is a total of 69 aa versus the SIVB precursor, which is 80 aa. The C-terminal amino acid of SIVA is predicted to be a glycine followed by a stop codon. As shown in Table IIA, there is no stop codon at the homologous position in SIVB, resulting in an additional 11 amino acids at the C-terminal end. An examination of the nucleotide sequence in this region, shown in Table II, indicates that the conversion from SIVA to SIVB is not explained by a simple substitution, insertion, or deletion; instead, there is a substitution of a block of 25 nucleotides (shaded nucleotide sequences in Table IIA). At the 3'-end of the open reading frame of SIVB, the sequences become identical once again.

The discovery of the considerable extent of sequence identity between the {alpha}-conotoxin and {kappa}A-conotoxin precursors is unprecedented. The arrangement of Cys residues in the mature toxins are completely different for the two groups; furthermore, the {kappa}A-conotoxins are excitatory peptides targeted to voltage-gated ion channels and not nicotinic receptor antagonists, the physiological mechanism underlying the activity of {alpha}-conotoxins (19). Thus, although the two groups of peptides are genetically related (with considerable identity in signal sequences and the N-terminal side of their propeptide regions) and belong to the same gene superfamily, they have clearly diverged to yield two families of conotoxins that are both structurally and functionally distinct.

Analysis of cDNA Clones from Other Fish-hunting Conus Species—To explore which members of the A-conotoxin superfamily are highly expressed in other fish-hunting species, we investigated C. magus, the magician's cone, and Conus stercusmuscarum, the fly-speck cone (see Fig. 1). The results of the analysis are shown in Table IIB. From C. magus, one open reading frame encoding a precursor sequence for an {alpha}-conotoxin was identified; surprisingly, all of these encoded an {alpha}-conotoxin MII precursor. {alpha}-Conotoxin MII has been extensively characterized (14, 20) and shown not to interact with the nicotinic receptor at the neuromuscular junction. Instead, {alpha}-MII potently blocks two neuronal nicotinic receptor subtypes, {alpha}3{beta}2 and {alpha}6{beta}3{beta}2 (14, 21, 22).

The second A-superfamily sequence from C. magus encoded a peptide that had high sequence homology to {kappa}A-conotoxin SIVA, which we are designating {kappa}A-conotoxin MIVA. As will be described below, we have directly characterized {kappa}A-conotoxin MIVA from C. magus venom. The C. stercusmuscarum cDNA library did not yield any {alpha}-conotoxin precursors; all of the clones analyzed had open reading frames that encoded precursors of peptides homologous to {kappa}A-conotoxins SIVA and MIVA (designated {kappa}A-conotoxins SmIVA and SmIVB). These share considerable sequence identity and are likely to be allelic variants. The predicted open reading frames are shown in Table IIB.

Analysis of the A-superfamily in Non-piscivorous Conus Species—To compare the A-superfamily in Conus species that do not prey on fish to the A-superfamily conotoxins expressed in the fish-hunting Conus species investigated above, we identified A-superfamily members from cDNA libraries from a molluscivorous (snail-hunting) species, Conus bandanus, and from a vermivorous (worm-hunting) species, Conus caracteristicus (see Fig. 1). Using the nomenclature of Espiritu et al. (23), all of the fish-hunting species analyzed belong to either Clades I or II, whereas C. bandanus belongs to the mollusc-hunting Clade VI, and C. caracteristicus belongs to the worm-hunting Clade XIV. The analysis of cDNA clones yielded three different types of sequences for C. bandanus and two for C. caracteristicus (see Table IIC).

It is noteworthy that all five sequences elucidated from these species are predicted to encode {alpha}-conotoxins; however, none of the clones analyzed encoded a three-disulfide-cross-linked-containing Conus peptide (such as {kappa}A-conotoxin SIVA). Thus, in contrast to such species as C. striatus, C. magus, and C. stercusmuscarum, the non-piscivorous species we have analyzed are notable in encoding only {alpha}-conotoxin-like sequences.

None of the C. caracteristicus or C. bandanus peptides have been purified from venom. However, one of the C. bandanus peptides, encoded by clone Bn1.3, was chemically synthesized as described under "Experimental Procedures" and tested on six different cloned rat nicotinic acetylcholine receptor subtypes expressed in Xenopus oocytes by previously described methods (14) ({alpha}2{beta}2, {alpha}2{beta}4, {alpha}3{beta}4, {alpha}4{beta}2, {alpha}4{beta}4, and {alpha}6/{alpha}3{beta}2{beta}3). The peptide did not detectably affect any of the nicotinic receptor subtypes tested (IC50 > 5 µM for all subtypes). The lack of activity may be due to high specificity of the peptide for a molluscan target, although the possibility that a post-translational modification in the native peptide is required for activity is an alternative explanation (see next section).

Isolation and Characterization of {kappa}A-conotoxin MIVA—A highly potent excitatory peptide that elicits repetitive action potentials and causes the same spastic symptomatology as {kappa}A-conotoxin SIVA was purified from C. magus venom (Fig. 2). As shown in Table III, initial sequencing of the pyridylethylated peptide gave a 36-amino acid peptide with a faint Glu signal in position 3 and blanks in positions 7 and 9. The sequence is otherwise identical to that predicted from a cDNA clone from C. magus (underlined sequence in Table IIB), indicating that positions 7 and 9 are modified Thr residues.



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  		FIG. 2.
Purification of {kappa}A-Conotoxin MIVA. Samples were fractionated on C18 HPLC columns eluted with a gradient of acetonitrile in 0.1% trifluoroacetic acid indicated by the dashed line. A, fractionation of the venom extracts on semi-preparative C HPLC. B, fractions corresponding to the shaded area in A were pooled and applied on a C18 analytical column. C, fractions from seven HPLC runs corresponding to the peak marked by the arrow in B were pooled and chromatographed on the C18 analytical column. D, the peaks (marked by an arrow in C) from five runs were pooled and rerun on the C18 column. E, the peak marked by the arrow in D was cleaned by another run on the analytical column. Absorbance was monitored at 220 nm in A and at 280 nm in B–E. The active peaks (marked by arrow) eluted at 14.7–14.8 min in 30% acetonitrile-0.1% trifluoroacetic acid.

 


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  		TABLE III
Sequencing and mass spectrometry of {kappa}-A-conotoxin MIVA

 
Chymotrypsin digestion of the peptide gave three main fragments. Fragments 1 and 1a are essentially the same except for position 3, where a clear signal for Glu was found in fragment 1 and only a trace of Glu was found in fragment 1a, which is routinely observed when {gamma}-carboxyglutamate (Gla) is present. This is consistent with the mass spectrometry data that indicated a mass difference of 44 between fragments 1 and 1a. In fragment #2, there was an apparent cleavage after the Met in addition to the cleavage after the Tyr. Fragment #3 clearly ends with Hyp, suggesting that the GRR serves as an amidation signal. Amidation of this fragment was confirmed by mass spectrometry. The mass of the peptide obtained by liquid secondary ionization mass spectrometry (average MH+ = 5095.3) was 1053.6 Da higher than the mass predicted for the peptide containing Gla in position 3 and an amide group at the carboxyl end. This mass difference was localized to fragment 1 where the two threonines predicted from cDNA clones appear as blanks upon chemical sequencing. This is consistent with the two blank positions (7 and 9) being glycosylated Thr. The "missing mass" of 1053.6 presumably can be divided between two glycosylation sites on Thr7 and Thr9. The combination of nucleotide sequencing, amino acid sequencing, and mass spectrometry data confirms the sequence of {kappa}A-conotoxin MIVA as AO{gamma}LVVTATTNCCGYNOMTICOOCMCTYSCOOKRKO-NH2 ({gamma}, {gamma}-carboxyglutamate; T, modified threonine-putative O-glycosylation; O, hydroxyproline).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The small pharmacologically active peptides of Conus venoms have shown remarkable interspecific divergence. The ~50,000 different peptides found in Conus venoms can be grouped into Conus peptide families; in turn, several different Conus families may belong to a Conus peptide gene superfamily. Each Conus peptide gene superfamily has a number of shared diagnostic elements, the most striking of which is the highly conserved signal sequence. In contrast to families of conventional secreted gene products, in which the signal sequences at the N terminus are the least conserved elements, in conotoxin gene superfamilies the reverse is true: signal sequences are the most highly conserved region. Thus, the signal sequence is diagnostic of which superfamily a particular Conus peptide belongs to.

The A-conotoxin superfamily is defined here by 16 sequences from four species of fish-hunting Conus (C. geographus, C. striatus, C. magus, and C. stercusmuscarum) as well as from two non-fish-hunting species (C. bandanus, a snail-hunting Conus, and C. caracteristicus, a worm-hunting Conus).1 All species examined yielded cDNA clones that encode A-conotoxin superfamily precursors; these precursors have the diagnostic N-terminal signal sequence, an intervening pro region and a C-terminal mature toxin region. The exact 21-amino acid consensus sequence for the N-terminal signal sequence (MGMRMMFTVFLLVVLATTVVS) is found without any substitution whatsoever in a large majority of the different precursor sequences analyzed. This highly conserved signal sequence serves as a defining feature for members of the A-conotoxin gene superfamily. In striking contrast, there is a dramatic divergence of mature conotoxin sequences. To give a specific example, {alpha}-conotoxin MII and {kappa}A-conotoxin MIVA both from C. magus have identical signal sequences, but there is little, if any, homology in the mature toxin sequences (Table IV).


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  		TABLE IV
Comparison of nucleotide and amino acid sequences of signal sequence and mature toxin regions In A: Z, pyroglutamate; O, hydroxyproline; {gamma}, gamma-carboxyglutamate; underlined amino acids are glycosylated. All post-translational modifications are assumed to be homologous to isolated {kappa}A-conotoxins. Sequence differences are shaded in A.

 
Also shown in Table IV is a comparison of the nucleotide sequences of the signal sequence region, contrasted with the nucleotide sequences of mature toxin regions. It is noteworthy that not only are the amino acids of the signal sequences conserved, but codon usage at every locus is conserved. Because some of the sequences being compared are from pairs of Conus species believed to have diverged in the Miocene period or earlier (1, 23), the lack of even silent substitutions is notable.

The members of the O-conotoxin superfamily have a similarly highly conserved signal sequence; however, the O-superfamily signal sequences show no homology whatsoever to the A-superfamily signal sequences (4). Thus, each Conus peptide superfamily has its own characteristic signature signal sequence. A second diagnostic feature of members of a particular superfamily is the arrangement of cysteine residues, which in turn predicts the disulfide-bonding framework of the peptides belonging to that superfamily. Although most loci in the mature toxin region are hypervariable, the cysteine residues are conspicuously conserved; in general, at each Cys locus only one of the two codons is used, a feature previously noted for other conotoxins (24). Apparent exceptions are {alpha}-GI and {alpha}-SI; the first two Cys residues have an apparent reversal in codon usage from all of the other A-superfamily conotoxins in this study (TGT TGC in {alpha}-conotoxins GI and SI versus TGC TGT in all others; see Table IV). The possible origins of this codon reversal are discussed below. It should also be noted that cysteines 3, 4, and 5 in the {kappa}A conotoxins use TGC, whereas except for the first Cys residue, all other Cys residues are encoded by TGT in most other A-superfamily peptides.

Post-translational Processing—All conotoxins require processing from their precursors by proteolytic cleavage at a characteristic sequence (usually two basic amino acids, or alternatively –XR–) to yield the N terminus of the mature conotoxin. At the C-terminal end, sequences such as –XG, –XGR, –XGK, –XGRR, etc. are processed to –X-NH2 by a well characterized enzyme system (25, 26). The data presented here suggest that processing at both ends can be more complex for some members of the A-conotoxin superfamily. Some cases where one might not have predicted a priori from the cDNA clone what mature conotoxin sequence would be found in venom are shown in Table V. Thus, for {alpha}-conotoxin SI, there is unusual processing at the N-terminal end. For {alpha}-conotoxin SII, endoproteolytic cleavage after the arginine, followed by a carboxypeptidase removal of the C-terminal Arg residue presumably occurred.


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  		TABLE V
Some differences between natural toxin sequences and predicted cloned sequences Underlined amino acids are O-glycosylated; {gamma}, gamma-carboxyglutamate; O, hydroxyproline. {alpha}-Conotoxins SI and SII and {kappa}A-conotoxin SIVA were previously isolated by Zafaralla et al. (18), Ramilo et al. (32), and Craig et al. (17), respectively.

 
The most complex case of post-translational processing is observed in {kappa}A-conotoxin MIVA, as indicated in Table V. The proteolytic cleavage at the N terminus is conventional, but at the C terminus, initial cleavage after an arginine residue followed by the removal of an Arg by a carboxypeptidase and processing of the transiently generated Cys-Gly terminus to Cys-NH2 presumably takes place. In addition, in contrast to {alpha}-conotoxins in which only standard amino acids are found, {kappa}A-conotoxin MIVA has ten post-translationally modified amino acids. These include seven Pro residues that have been hydroxylated, a Glu residue at position 3 that has been {gamma}-carboxylated to Gla, and two modified residues of Thr at positions 7 and 9 that may be O-glycosylated.

In two peptides of this family directly purified from venom, {kappa}A-SIVA and a protein from Conus consors (27), a single Pro residue is not hydroxylated; this is immediately adjacent to a putative glycosylated Ser or Thr residue at position 7. This suggests a hierarchy in post-translational modifications. It is notable that in {kappa}A-SIVA and all homologous peptides ({kappa}A-conotoxin MIVA and the Conus consors peptide), position 7 is a serine or threonine residue that is always glycosylated, whereas Thr residues at positions 10 and 11 are not glycosylated (17, 27). {kappa}A-conotoxin MIVA is the first member of this family characterized that appears to have two glycosylated Thr residues.

The long N-terminal extension before the first Cys residue and the extensive suite of post-translational modifications present in {kappa}A-conotoxins further differentiate this branch of the A-superfamily from the {alpha}-conotoxin family. Thus, the A-conotoxin superfamily comprises at least two functionally differentiated (as well biochemically divergent) groups of peptides, the {alpha}-conotoxin family and the {kappa}A-conotoxin family. The former are targeted to nicotinic acetylcholine receptors, which belong to the ligand-gated channel superfamily, whereas the latter affect members of the voltage-gated ion channel superfamily.

Polymorphism of Conotoxin Genes—The results of this study provide insight into conotoxin polymorphism at several levels. The majority of the sequences encoding the 16 different conotoxins are likely either different A-superfamily genes, or homologous genes from different Conus species. However, there are quite a few cases in which different sequences may be allelic variants, including {alpha}-conotoxins GI/GIB, {kappa}A-conotoxins SIVA/SIVB, {kappa}A-conotoxins SmIVA/SmIVB, {alpha}-conotoxins SI/SII, and Bn1.1/1.2. For several of these possible allelic pairs (such as {kappa}A-conotoxins SmIVA/SmIVB), there are a few amino acid substitutions when the two mature sequences are compared; these mostly arise from single non-synonymous nucleotide substitutions. For the {alpha}-conotoxin GI/GIB pair, there are two additional C-terminal amino acids in {alpha}-GIB compared with {alpha}-GI, but the two sequences are otherwise identical; this could have occurred either by the insertion of six nucleotides to the {alpha}-GI gene, or the deletion of six nucleotides from the {alpha}-GIB gene (a "block" insertion or deletion).

The most drastic difference in mature toxin sequences is between {kappa}A-conotoxins SIVA and SIVB; in this case, an additional 11 amino acids are present in SIVB compared with SIVA. As is demonstrated in Table II above, a block of 25 nucleotides in one gene has been substituted by a non-homologous block in the other gene (a "block substitution"). In regions 3' or 5' to the substituted blocks, the nucleotide sequences are identical in SIVA and SIVB. It is noteworthy, however, that {kappa}A-conotoxin SIVB was only found in C. striatus specimens collected from the Hawaiian Islands; these have a characteristically different shell morphology from Philippine specimens assigned to this species. It is interesting that, although a difference in Hawaiian and Philippine specimens was observed with respect to the {kappa}A-conotoxins expressed, no such differences were found for the {alpha}-conotoxins: identical {alpha}-conotoxin SI and SII cDNA clones were sequenced from both populations of C. striatus, and no sequence differences were observed between the two populations for these {alpha}-conotoxins.

Branches of the A-superfamily—The most unexpected result from this work was the discovery that peptides belonging to the A-superfamily do not have a single arrangement of cysteine residues in the mature conotoxin region, a general feature of other conotoxin superfamilies (1, 28). Thus, the O-superfamily and the M-superfamily have the Cys patterns XCXXCXCCX- CXXCX and XCCXCXXCXCCXX, respectively. {alpha}-Conotoxin GI and all other {alpha}-conotoxins have the cysteine residues arranged in the primary sequence as follows: XCCXXXCXXXCX (the Class I/II Cys pattern, referring to the formal nomenclature for conotoxins). In all known natural peptides with this arrangement, the disulfide connectivity is Cys1-Cys3, Cys2-Cys4, which we refer to as the A-1 scaffold (1). Our results establish that a second group of peptides, with a different Cys pattern, XCCXX- CXXCXCXXCXX (a Class IV Cys pattern), also belongs to the A-superfamily; this is typified by {kappa}A-conotoxin SIVA. Given these differences in Cys patterns, {alpha}-conotoxins and the {kappa}A-conotoxins would be expected to have quite different scaffolds, and therefore divergent polypeptide backbone structures. Because the latter peptides are excitotoxins and not nicotinic antagonists, the two groups are therefore both functionally and structurally divergent.

However, at a genetic level, there is also a clear-cut division between two branches of the {alpha}-conotoxin family, the {alpha}4/7 subfamily (consensus sequence XCC(X4)C(X7)CXX) and the {alpha}3/5 subfamily (consensus sequence XCC(X3)C(X5)CXX). It is noteworthy that, although C. magus and C. striatus belong to the same group of fish-hunting cone snails, the {alpha}-conotoxin MII precursor is not closely related at the sequence level of the prepro region to {alpha}-conotoxins SI and SII from C. striatus (nor to any of the {kappa}A-conotoxins); rather, in the first N-terminal 39 amino acids, {alpha}-conotoxin MII exhibits much less sequence divergence from two {alpha}-conotoxins predicted from C. bandanus and C. caracteristicus cDNA clones (see Bn1.1 and Ca 1.2 in Tables IV and VI). If the first 18 aa of the propeptide region of these precursor sequences are compared, at least three coherent groups can be defined: the {alpha}3/5 conotoxin subfamily, the {alpha}4/7 conotoxin subfamily, and the {kappa}A-conotoxin family. All peptides that belong within the same group do not differ in sequence in the interval analyzed in Table VI by more than three amino acids; in contrast, if peptides across different groups are compared, they diverge by no less than 5 out of 18 amino acids in the region indicated in the table. Three peptides (Bn1.3, S1.1, and Ca1.1) do not belong to any of the groups above and are divergent from each other and may define additional subfamilies within the {alpha}-conotoxins.


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  		TABLE VI
Non-identity in propeptide region (aa 22-39) The numbers indicate the number of non-identical amino acids for a pair of conotoxins. The horizontal and vertical lines between numbers delineate non-identities within a group and between groups of conotoxins.

 
Because the {alpha}4/7 subfamily is widely distributed in fish-hunting, worm-hunting, and mollusc-hunting species, it has been suggested that this may be the ancestral Cys pattern of the {alpha}-conotoxin family (1). The {alpha}3/5 subfamily is much more narrowly distributed; it is only found in two clades of fish-hunting Conus and is regarded as a specialization that may have arisen later in the evolutionary history of the genus. If the {alpha}3/5 subfamily evolved from an {alpha}4/7 ancestor, the arrangement of codons shown in Table IV for {alpha}-conotoxin SII suggests how the conversion from 4 aa in the first loop (of {alpha}4/7 conotoxins) to 3 aa in the first loop (of {alpha}3/5 conotoxins) may have occurred. If an {alpha}-SII-like peptide was generated from an {alpha}4/7 ancestor by mutation to a Cys codon immediately after the first two Cys codons in an {alpha}4/7-like peptide, this would decrease the loop size from four to three (presumably this would be accompanied by the addition of another cysteine residue, such as is found in {alpha}-conotoxin SII). Subsequent loss of the first cysteine codon (TGC in {alpha}-conotoxin SII and all {alpha}4/7 subfamily members) would then result in the codon pattern actually observed in {alpha}-conotoxins GI and SI (thus, the proposed transitions would be: TGC TGT NNN (the {alpha}4/7 pattern) -> TGC TGT TGC (the SII pattern) -> NNN TGT TCG (the {alpha}3/5 pattern, where NNN is a codon that does not specify Cys). The result would be a decrease in the size of the first loop of non-Cys aa from four to three, concomitant with an apparent reversal in the order of the Cys codons. This scheme is shown in Fig. 3. Thus, the codon reversal can be rationalized by the first Cys residue in {alpha}3/5 conotoxins being derived from the second Cys residue of the {alpha}4/7 conotoxins, both of which use the TGT codon.



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  		FIG. 3.
Proposed mechanism for the evolution of {alpha}3/5- and {kappa}A-conotoxins from a presumptive ancestral {alpha}4/7 conotoxin. A, origin of {alpha}3/5 conotoxins. In Pathway I, the first step (Ia) involves mutation in the codon of the first amino acid in loop one of {alpha}4/7 to Cys (using a TGC codon). This is followed by two separate events (Ib), mutation in the first Cys residue to a non-Cys residue and the deletion of six nucleotides from loop 2 (dotted box). B, origin of {kappa}A-conotoxins. Pathway II consists of two steps: IIa, replacement of a block of nucleotides in the {alpha}4/7 ancestor (which includes the TGT codon for the third Cys residues) with another block of nucleotides containing the TGC codons for three Cys residues (solid boxes) to give the Cys pattern found in the {kappa}A-conotoxins, and IIb, insertion of a 12-nucleotide block (dotted boxes) in the first intercysteine loop.

 
In contrast, the {kappa}A-conotoxins have the conserved TGC TGT codons for the first two Cys residues as is generally found in the {alpha}-conotoxin family. However, Cys3, Cys4, and Cys5 all are encoded by TGC codons in all {kappa}A-conotoxins analyzed; no TGC codons are used for Cys residues except for the first two in any {alpha}-conotoxins. One explanation consistent with this codon usage is a "block substitution" mechanism, similar to that described above to explain the divergence between {kappa}A-conotoxins SIVA and SIVB. An ancestral {kappa}A-conotoxin could have been generated if a region containing Cys 3 of an {alpha}4/7 conotoxin ancestor were replaced by a block containing three Cys codons, all TGC (see Fig. 3 for a specific example of how this may have occurred).

In this study, we have defined the A-superfamily and delineated several major branches, one of which (the {kappa}A-conotoxins) is extremely divergent structurally and functionally. Moreover, the analysis of polymorphisms has uncovered genetic events (such as block substitution) that suggest how superfamily branches may have originated. Molecular mechanisms that underlie events such as block substitutions that drive toxin divergence are not known in detail at this time. It is known that A-superfamily genes have a large intron in the middle of the pro region3; it was previously suggested that the large introns found in conotoxin superfamily genes may play a role in the hypermutation observed in these peptides as speciation occurs (29). However, a detailed comparison of the introns from A-superfamily genes from different Conus species has not yet been made, and therefore this hypothesis has not been rigorously tested. Alternatively, block substitution events may arise from alternative splicing or retroviral-like template switching. However, the analysis presented here provides insight into potential transitional steps that occurred as structural and functional novelty evolved within a single conotoxin gene superfamily.


   FOOTNOTES
 
* This work was supported in part by Program Project GM-48677 from the National Institutes of Health. A preliminary account of some of this work was presented in an unpublished thesis (30) as well as in the patent literature (Olivera et al. (12/31/96), Process and primers for identifying nucleic acids encoding A-lineage conotoxin peptides, U.S. Patent 5,589,340 (33)). Two of the sequences in the unpublished thesis were independently discovered by Bai-Song et al. (31). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Also supported by National Institutes of Health Grant MH-53631. Back

§§ To whom correspondence should be addressed: Dept. of Biology, University of Utah, 257 S. 1400 E., Salt Lake City, UT 84112. Tel.: 801-581-8370; Fax: 801-585-5010; E-mail: olivera{at}biology.utah.edu.

1 G. P. Corpuz, R. B. Jacobsen, E. C. Jimenez, M. Watkins, C. Walker, C. Colledge, J. E. Garrett, D. McDougal, W. Li, W. R. Gray, D. R. Hillyard, J. Rivier, J. M. McIntosh, L. J. Cruz, and B. M. Olivera, submitted for publication. Back

2 The abbreviations used are: TMAC, tetramethylammonium chloride; {alpha}-, {alpha}-conotoxin; HFBA, heptafluorobutyric acid; HPLC, high-performance liquid chromatography; aa, amino acid(s). Back

3 R. Schoenfeld, B. Olivera, and D. Hillyard, unpublished results. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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