View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector FEBS Letters 580 (2006) 5979–5987 Molecular characterization of a possible progenitor sodium channel toxin from the Old World scorpion Mesobuthus martensii S. Zhu*, B. Gao Group of Animal Innate Immunity, State Key Laboratory of Integrated Management of Pest Insects & Rodents, Institute of Zoology, Chinese Academy of Sciences, No. 25 Beisihuan-Xi Road, Beijing 100080, PR China Received 5 September 2006; revised 26 September 2006; accepted 27 September 2006 Available online 12 October 2006 Edited by Maurice Montal ing diverse ecological niches, scorpions and other predatory Abstract Toxins affecting sodium channels widely exist in the venoms of scorpions throughout the world. These molecules com- organisms such as spiders, cone snails and snakes have devel- prise an evolutionarily related peptide family with three shared oped a large number of toxins commonly targeting sodium features including conserved three-dimensional structure and channels present in the excitable cells of preys and competitors gene organization, and similar function. Based on different phar- [2,6–8]. This may be a consequence of natural selection if we macological profiles and binding properties, scorpion sodium consider crucial roles of sodium channels in controlling the channel toxins are divided into a- and b-groups. However, their electrical activity of nerve and muscle systems [9]. Not surpris- evolutionary relationship is not yet established. Here, we report a ingly, modifying the pharmacological activities of these chan- gene isolated from the venom gland of scorpion Mesobuthus nels is capable of causing rapid immobilization of their preys. martensii which encodes a novel sodium channel toxin-like pep- Mechanically, these sodium channel toxins affect both per- tide of 64 amino acids, named Mesotoxin. The Mesotoxin gene meation and gating properties of the channels by targeting dis- is organized into three exons and two introns with the second in- tron location conserved across the family. This peptide is unusual tinct receptor sites and inducing conformational changes of the in that it has only three disulfides and a long cysteine-free tail channel protein [10]. Despite very different sequences and fold with loop size and structural characteristics close to b-toxins. types, some toxins from phylogentically distant venomous ani- Evolutionary analysis favors its basal position in the origin of mals convergently target the same receptor site. In this respect, scorpion sodium channel toxins as a progenitor. The discovery scorpion a-toxins, sea anemone toxins and spider toxins pro- of Mesotoxin will assist investigations into the key issue regard- vide good examples in which three unrelated structural types ing the origin and evolution of scorpion toxins. commonly target the site 3 of the channel [10]. Divergent evo- Ó 2006 Federation of European Biochemical Societies. Pub- lution stemming from gene duplication and speciation repre- lished by Elsevier B.V. All rights reserved. sents another common strategy in toxin evolution in which structural cores conserves but function changes [11,12]. Keywords: Ion channel; Disulfide bridge; Functional In the past few years, due to the combination of gene cloning evolution; Toxin origin; Peptide scaffold; Gene structure techniques and traditional biochemical methods, the family of scorpion toxins affecting sodium channels (NaScTx) has been enlarged to more than 200 members [13]. A typical NaScTx is composed of about 64 residues cross-linked by four disulfide bridges. On the basis of different pharmacological profiles and 1. Introduction binding properties, the NaScTxs are classified into two distinc- tive groups: a- and b-toxins [14]. The a-toxins prolong the Scorpions belong to venomous arachnids and exist at an inactivation of the channel whereas b-toxins affect activation intermediate level in food chains [1]. As generalist predators, of the channel. This disparate pharmacological property they feed on insects, spiders, and other small animals. In the roughly reflects the separation of Old World and New World mean time, these organisms themselves are prey to a variety scorpion species: the a-toxins affecting the channel inactivation of larger predators including both invertebrates and verte- extensively distribute in Old World scorpions, whereas the b- brates. Evolutionary emergence of lethally toxic peptides (tox- toxins affecting activation are mainly present in New World ins) in scorpion venoms provides highly efficient means for scorpions. capturing prey and protecting themselves [2–4]. This could Studies on the origin and evolution of NaScTxs have at- compensate their physical limitation and benefit their survival tracted extensive attention in the recent years. A subfamily in a competitive environment [5]. Remarkably, despite having of NaScTx, represented by Birtoxin, is especially interesting different evolutionary histories and food sources, and occupy- in this aspect. All the members in this subfamily contain only three disulfides with a slightly smaller size (about 58 residues) *Corresponding author. Fax: +86 10 62579091. relative to the NaScTxs with four disulfides [15–18]. They E-mail address: [email protected] (S. Zhu). share 40–60% sequence identity to b-toxins but display more diverse pharmacological activity. Some act as modulators Abbreviations: NaScTx, scorpion toxin affecting sodium channels; TF, affecting sodium channel activation with a characteristic b-tox- transcription factor; TSS, transcriptional start site; WDB, wrapper disulfide bridge; ORF, open reading frame; UTR, untranslational in effect. Others serve as potassium channel blockers due to the region; 3D, three-dimensional development of a putative functional dyad in their a-helical 0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.09.071 5980 S. Zhu, B. Gao / FEBS Letters 580 (2006) 5979–5987 region. Previous phylogenetic analysis assigned the Birtoxin 2.3. DNA sequencing subfamily as the ancestor of all the NaScTxs [13,19]. Recently, Recombinant clones were analyzed by PCR using two vector prim- a new Birtoxin-like peptide, named BmKBT, was character- ers (SP6 and T7) and gel electrophoresis. Positive clones were se- quenced through the chain termination method using the primers ized from the Old World scorpion Mesobuthus martensii [20]. SP6 and T7. The nucleotide sequence of the Mesotoxin gene has been Genomic data indicates that BmKBT is in fact encoded by a deposited in the GenBank database (http://www.ncbi.nlm.nih.gov) transcript variant of the lipolysis activating peptide a-subunit under the accession number of DQ872676. gene, both differing in one base deletion only (Zhu and Gao, unpublished data). The a-subunit contains seven cysteines 2.4. Primer sequences forming three intramolecular disulfides and one intermolecular All primers used in this study are synthesized by SBS Genetech, Bei- jing and Takara, Dalian, and listed in Table 1. disulfide [21]. The latter links the a-subunit to the b-chain by the last cysteine. Such a deletion led to the production of 2.5. Searching TF binding sites BmKBT due to truncation and loss of the last cysteine. It thus Potential transcription factor (TF) binding sites in the upstream of appears that the Birtoxin subfamily is a mutated version of the transcriptional start site 2 (TSS2) of Mesotoxin gene was searched peptide with seven cysteines. Another study on the basis of a using the AliBaba2 program against the TRANSFAC database combination of sequence, structural and functional data (http://www.cs.uni-magdeburg.de/grabe/alibaba2). hypothesized a functionally unrelated but structurally con- 2.6. RNA secondary structure prediction served peptide – antifungal defensin as the ancestor of NaS- Mfold, which predicts RNA secondary structure by free energy min- cTxs. Some evolutionary events in terms of the origin were imization (http://www.bioinfo.rpi.edu/applications/mfold/), was used elucidated which included the adding of five-residue turn close to identify possible RNA hairpins in the non-coding regions of pre- to the N-terminus and the extension of C-terminus, and reor- transcripts (50 and 30 UTRs and introns). ganization of the fourth disulfide bridge [22]. Here, we report a full-length cDNA and its complete gene 2.7. Evolutionary analysis Multiple sequence alignment of scorpion sodium channel toxins and which encodes a unique peptide of 64 residues with only three related peptides was carried out using the CLUSTAL W program disulfide bridges. Sequence and structural analysis places it to (http://www2.ebi.ac.uk/clustalw/) and further refined by hand with the b-toxin group. Several characteristics together with a basal reference to the cysteine residue position. The aligned sequences were position in the evolutionary tree suggest that Mesotoxin might employed to make phylogenetic analysis using the neighbor-joining be a putative progenitor of the NaScTx family. method implemented in MGEA 3.1 (http://www.megasoftware.net). 2.8. Homology modeling Fold compatibility and template selection for comparative modeling 2. Materials and methods were performed through GenTHREADER [24]. The experimental structure of scorpion neurotoxin CsEv2a from Centruroides sculptura- 2.1. Preparation of total RNA and genomic DNA tus (PDB entry 1JZA) [25] was selected as a template for modeling the Scorpions (Mesobthus martensii)