Scientific paper
The Evolution of aD-Conopeptides Targeting Neuronal Nicotinic Acetylcholine Receptors
Dietrich Mebs,1* Dusan Kordis,2 Yvonne Kendel1 and Silke Kauferstein1
1	Institute of Legal Medicine, University of Frankfurt, Kennedyallee 104, D-60596 Frankfurt,
Germany
2	Department of Molecular and Biomedical Sciences, Jozef Stefan Institute, Jamova Street 39,
SI-1000 Ljubljana, Slovenia.
* Corresponding author: E-mail: mebs@em.uni-frankfurt.de
Received: 12-05-2011 Dedicated to the memory of Professor Franc Gubensek
Abstract
Venoms of the marine cone snails (Conus spp.) consist of numerous proteins and peptides showing a wide variety of biological activities such as on ion-channels and receptors. Peptides acting on neuronal nicotinic acetylcholine receptors belong to several peptide superfamilies including the recently described aD-conopeptides which are homodimers of identical peptides with 47-49 amino acids. Among the venom glands of 27 Conus species analyzed by cDNA cloning, precursors of aD-conopeptides were identified in four species only: C. betulinus, C. capitaneus, C. mustelinus, and C. vexillum. Phylogenetic analysis of the relationships among the aD-conopeptides revealed that they belong to clades, which are characterized by an AVV- and EMM-motif in the signal peptide sequence.
Keywords: Conus, conopeptides, aD-conopeptide superfamily, neuronal nicotinic acetylcholine receptor
1. Introduction
The marine snails of the genus Conus (family: Co-nidae) use a powerful venom to subdue prey such as fish, worms or other snails, and to use it also for defense. Each venom represents a cocktail of numerous peptides acting on a variety of receptors and ion channels.1'2 Among them, a group of peptides are targeting nicotinic acetylc-holine receptors (nAChRs), which they effectively block causing almost immediate paralysis of the prey. According to their structural properties, they have been assigned to several superfamilies: A- (a- and aA-conopepti-des), M-(y- conopeptides), S- (aS- conopeptides), C-(aC- conopeptides), L- (lt-conopeptides) and D-superfa-mily (aD-conopeptides).1,3-7 Peptides of the A- and L-su-perfamily such as the small a-conopeptides consist of 12 to 15 amino acids in a chain cross-linked by two disulfide bridges. Depending on the number of amino acids between the third and fourth cysteine residue, they have
been classified into various subtypes which exhibit different activities to nAChRs, muscle as well as neuronal subtypes.7,8 The larger aA-conopeptides (30 amino acids and three disulfide bridges) inhibit specifically muscle n-AChRs like the aC- and y- conopeptides which act also as non-competitive antagonists.4,9-11 The aS- conopepti-des block muscle as well as various neuronal nAChRs subtypes.12
The recently described aD-conopeptides selectively inhibit a7 and 02 containing neuronal nAChRs only.5,6,13 They represent homodimers of identical peptides with 47-49 amino acid residues and show a characteristic arrangement of 10 cysteines. These novel conopeptides are the major constituents of venoms from Conus capitaneus, C. mustelinus and C. vexillum.5,6,13 In the present paper the screening of 27 Conus species for the occurrence of aD-conopeptides in their venom gland using cDNA cloning methods and the evolutionary implications are described.
2. Materials and Methods
2. 1. Materials
Specimens of Conus betulinus, C. capitaneus, C. cir-cumcisus, C. ebraeus, C. flavidus, C. generalis, C. geograp-hus, C. imperialis, C. litoglyphus, C. litteratus, C. marmo-reus, C. miles, C. mustulinus, C. planorbis, C. striatus, C. terebra, C. tesselatus, C. textile, C. vexillum, C. virgo, C. vulpinus were collected in the reefs of Olango Island, Cebu (Philippines), specimens of C. catus and C. distans of Taka-poto Island, Polynesia, and C. coelinae, C. eburneus, C. leopardus around Chesterfield Island, New Caledonia. The venom ducts of one or two specimens of each species were dissected, immediately placed in RNAlater® (Sigma-Al-drich, St. Louis, MO, USA) and stored at -20 °C.
2. 2. Preparation of Total RNA, cDNA Cloning and Sequencing
Venom duct total RNA was extracted using the TRI-zol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. cDNA was prepared by reverse transcription of the RNA using the Omniscript RT Kit (QIAgen, Chatsworth, CA, USA). To determine the c-DNA sequence of the peptides the forward degenerated primer (5'-ACN CCN GGN TCN AAR TGG GGN-3') corresponding to the amino acid residues TPGSKWG, which was shown to be a part of the peptide sequence of VxXIIA by Loughnan et al.12, paired with an abridged universal amplification primer (Qa) devoid of the poly dT tail (5'-CCA GTG AGC AGA GTG ACG-3'), was first applied to obtain it's 3' partial sequence. The SMART first-strand cDNA was synthesized to serve as a template for 5'RACE with the SMART™ RACE cDNA Amplification Kit (Clontech, Mountain View, CA, USA). Based on the 3' partial sequence, the anti-sense primer (5'-TCT CCA ATG ATA GAC GCA GGT ACA-3') was designed corresponding to the peptide fragment CTCVYHWR, a part of the newly discovered peptide sequence, and paired with the 5' nested universal primer A (5'-AAG CAG TGG TAT CAA CGC AGA GT-3'). PCR amplification was performed by using a 3'-RACE-PCR protocol composed of an initial denaturation step of 95 °C for 10 min, 30 cycles of 95 °C for 1 min, 42 °C for 1 min, 72 °C for 1 min and a
final extension of 72 °C for 7 min and a 5'-RACE-PCR protocol composed of an initial denaturation step of 94 °C for 10 min, 30 cycles of 95 °C for 30 s, 64 °C 10 s and 72 °C for 2 min and a final extension of 72 °C for 7 min. All PCR products were analyzed on a 2.5% (w/v) agarose gel and ligated into the pGEM-T vector (Promega, Madison, WI, USA). Sequencing was performed on the ABI 3130 automated sequencer (Applied Biosystems, Foster City, CA, USA). Data analysis was achieved using the Sequencing Analysis 2.1.2 and Sequence Navigator 1.0.1 (Applied Biosystems, Foster City, CA, USA) software.
2. 3. Phylogenetic Analysis
The aD-conopeptide sequences were aligned using Clustal W2.13 Phylogenetic trees were constructed using the neighbour-joining (NJ)15 and the maximum likelihood (ML)16 methods. The reliability of the resulting topologies was evaluated by 1000 bootstrap replications. C. achati-nus S-superfamily conotoxin Ac8.1 (ACA63847) was used as outgroup. Phylogenetic analysis of aD-conopepti-de precursors or mature peptides was performed with the programs RAxML16, Treecon17 and MEGA 4.018.
3. Results
3. 1. Cloning of aD-conopeptide Precursor Sequences
aD-conopeptide precursor sequences were identified in venom glands of only four of the 27 Conus species
Figure 1. The cone snail species used in the present study (from left): Conus betulinus, C. mustelinus, C. capitaneus, C. vexillum.
Table 1. aD-conopeptide precursor amino acid sequences deduced from cDNA nucleotide , the mature peptide region is underlined, the AVV and EMM motif in the signal sequence are in bold. The prepro-region of Conus capitaneus was not completed.
C. mustelinus
MPKLAVVLLVLLILPLSYFDAAGGOVVOGDRRGNGLARYLORGDRDVRECOVNTPGSKWGKCCMTRMCGTMCCARSGCTCVYHWRRGHGCSCPG C. betulinus
MLKLEMMLVVLLILPLFYFDAAGGOVVOGDWRSDGLARYLORGDRDVRECNINTPGSSWGKCCLTRMCGPMCCARSGCACVYHWRRGHGCSCPG C. capitaneus
xxxxxxxxxxxxxxxxxxxxAAGGOVVOGDRRGNGLARYLORGDREVOECOVDTPGSSWGKCCMTRMCGTMCCSRSVCTCVYHWRRGHGCSCPG C. vexillum
MPKLEMMLLVLLIFPLSYFIAAGGOVVOVDRRGDGLAGYLORGDRDVODCOVSTPGSKWGRCCLNRVCGPMCCPASHCYCVYHRGRGHGCSC
investigated: Conus betulinus, capitaneus, mustelinus and vexillum (Figure 1). This adds another species, C. betulinus, to the six already known to contain these peptides (Table 1). The peptide from C. vexillum was found to be identical with Vx20.1 sequenced by Loughnan et al.,5 those from C. mustelinus and C. betulinus were new for these species. But in contrast to the results of Loughnan et al.5 screening of the cDNA from the venom gland of a C. miles specimen (from the Philippines) was negative for aD-conopeptide sequences.
The prepro-regions of the peptides consist of 44 (C. betulinus) and 45 (C. mustelinus and C. vexillum) amino acid residues and contain either the EMM- (C. betulinus) or AVV-motif (C. mustelinus, vexillum). The mature peptides have 47 (C. vexillum) and 49 (C. mustelinus, C. betulinus) amino acids including 10 cysteines, respectively (Table 1).
3. 2. Phylogenetic Analysis
Phylogenetic analysis of the aD-conopeptides revealed that they belong either to the AVV or EMM clades (Figure 2) as already reported by Loughnan et al.5 The position of the C. betulinus aD-conopeptide is in the AVV cla-de, although it contains the EMM motif in the signal pepti-de. However, the C. betulinus aD-conopeptide possesses sequence motifs in the mature peptide that are typical for members of the AVV clade and are not present in EMM cla-de sequences, i.e. LTRM, ARSG and the PG motif at the C-terminus. The distribution of the aD-conopeptides in Co-nus species is very limited. They are present in three vermi-vorous Conus clades: in Rhizoconus (Clade XII) - C. vexillum, C. mustelinus, C. miles, C. capitaneus, and C. rattus, in Clade IX (C. vitulinus) and in Clade X (C. betulinus). These Conus clades were proposed by Espiritu et al.19
Figure 2. Phylogenetic tree of the aD-conopeptides. The rooted neighbour-joining (NJ) tree shows the evolutionary relationship among the currently available aD-conopeptides. Sequences that were obtained in this (C. betulinus) and in previous study6 are marked with asterisks. The NJ tree represents the bootstrap consensus following 1000 replicates, nodes with confidence values greater than 50% are indicated. C. achatinus S-super-family conotoxin Ac8.1 (ACA63847) was used as outgroup. The following abbreviations have been used by Loughnan et al.5: Cp - Conus capitaneus, Ml - C. miles, Ms - C. mustelinus, Rt - C. rattus, Vt - C. vitulinus, Vx - C. vexillum.
4. Discussion
aD-conopeptides have been identified by screening cDNA from venom glands or by isolation from the venom of Conus capitaneus, miles, mustelinus, rattus, vitulinus, vexillum5'6'13 and C. betulinus (this paper). In several other species from the Philippines (Visaya Sea), Chesterfield Island (New Caledonia) and French Polynesia cDNA cloning did not indicate the presence of these peptides.
Loughnan et al.5 observed two disctinct groups among the aD-conopeptide precursors containing either an EMM or AVV signal peptide motif. The new peptide from C. betulinus exhibits the EMM motif. Variable signal peptide sequences defining superfamily subgroups are not uncommon among other conopeptides such as in O-,20'21 I-,22'23 M-24 and T-superfamilies.25-27 Although the C. be-tulinus aD-conopeptide contains the EMM motif in the signal peptide' it is placed in the AVV clade' because it possesses typical sequence motifs in the mature peptide' which are not present in EMM clade sequences.
Although the C. vitulinus and the other aD-cono-peptide possessing species diverged about 28 million years ago28 their conopeptides are more similar to the AVV clade representatives than to the EMM clade. The aD-conopeptides from the EMM clade possessing species (C. rattus and miles) show 34 to 47% amino acid divergence in their mature peptides. While the majority of sequence replacements are scattered throughout the whole mature peptide' they contain in the inter-cysteine loops 2 and 4 two sequence motifs that are typical for EMM cla-de, the LNR(V/M) and P(A/E)SH, and they are also lacking the PG motif at the C-terminus. These sequence motifs could be responsible for possible changes in specificity and targeting. However, it should be noted that for the EMM clade sequences no biological activity or physiological targets are known yet.
It has been demonstrated that aD-conopeptides from the venom of C. vexillum,5 C. capitaneus and C. mu-stelinus6 specifically block mammalian neuronal nAChRs of the a7, a3p2 and a4p2 subtypes in nanomolar concentrations. Since all cone snail species which have been tested positive for aD-conopeptides are vermivorous, it would be interesting to investigate the affinity of these peptides to similar receptors in the nervous system of marine worms (polychaetes). The overall dominance of these peptides and the low abundance or even lack of small a-conopeptides in the venoms of these Conus species6, 13 may suggest that aD-conopeptides are an adaptation to a specific type of prey, i.e. worms.
5. Acknowledgement
This work has been supported by a grant of the European Commission: CONCO, the cone snail genome project for health. Integrated Project ref. LSHB-CT-2007-
037592 (http://www.conco.eu). We thank the Government of New Caledonia and French Polynesia for granting permissions, the French Navy and IRD-Noumea for support as well as the members of the CONPOL 2007 and CON-FIELD 2008 expedition for collecting and preparing the samples.
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Povzetek
V strupih morskih polžev stožcev (Conus spp.) so našli številne biološko aktivne proteine in peptide, ki delujejo na ionske kanalčke in receptorje. Konopeptidi, ki delujejo na živčne nikotinske acetilholinske receptorje spadajo v različne ko-nopeptidne naddružine. Mednje spadajo tudi nedavno odkriti aD-konopeptidi, ki tvorijo homodimere z 4749 aminoki-slinskimi ostanki. Analiza prisotnosti aD-konopeptidov v strupnih žlezah 27 različnih vrst stožcev (rod Conus) s pomočjo RT-PCR in cDNA kloniranja je potrdila njihovo prisotnost le pri štirih vrstah (C. betulinus, C. capitaneus, C. mu-stelinus, C. vexillum). Filogenetska analiza vseh trenutno dostopnih aD-konopeptidov je pokazala, da spadajo v dve skupini, ki se razlikujeta po prisotnosti AVV- ali EMM-motiva v zaporedju signalnega peptida.