Scorpions from the primeval subgenus Archaeotityus produce putative homologs of Tityus serrulatus toxins active on voltage-gated sodium channels

It has been proposed that the subgenus Archaeotityus comprises the most ancient species group within the medically important scorpion genus Tityus. cDNA encoding sodium-channel active toxins from the type species of this subgenus, Tityus clathratus (central Venezuela), have been isolated and sequenced. Two cDNAs were retrieved that encoded 61 amino acid-long putative neurotoxins named Tcl1 and Tcl2. Sequence identity was highest (87%) when both were compared with β-toxin Ts1 from the Brazilian scorpion Tityus serrulatus and its homologs from T. bahiensis, T. stigmurus, and T. costatus. A Bayesian analysis indicated statistical support for the grouping of T. clathratus Tcl1 and Tcl2 with Brazilian gammalike β-toxins, reinforcing previous phylogenetic studies which suggested an evolutionary relationship between the subgenus Archaeotityus and scorpion species inhabiting southeast South America belonging to the subgenus Tityus.

Tityus is unquestionably the most complex genus of scorpions from a taxonomical standpoint (1)(2)(3)(4).It is annually responsible for numerous casualties in several endemic areas of Latin America and the Caribbean (1-7).With over 190 described species, there is toxinological and clinical evidence indicating diversity in venom action and/or composition across Tityus distribution range.For example, envenoming by T. obscurus (formerly T. cambridgei) in northeastern Brazil typically presents with central neurotoxicity as opposed to the mainly autonomic manifestations associated with envenoming by T. serrulatus in the southeast (8).In Venezuela, envenoming by T. zulianus (Andean range) often produces respiratory arrest and death by pulmonary edema, whereas T. discrepans (north-central range) sting causes mainly pancreatic and gastrointestinal disorders (6,9,10).Various degrees of toxicity have been associated with different Tityus venoms depending on the species, with medium lethal doses (in mice) ranging from 0.773 (T.stigmurus) to 12.136 mg/kg (T.obscurus) (11 ,12).Notably, the smallest Tityus species, now belonging to the subgenus Archaeotityus, are only mildly toxic to humans (e.g.T. uruguayensis, T. pussilus, and T. silvestris) (2,(13)(14)(15).Toxicity of Tityus venoms is mostly due to the action of peptides targeting voltage-gated sodium channels (Na v ), which have been classified as aand b-toxins depending on whether they alter the kinetics of Na v inactivation or activation respectively (16).
Medically important Tityus species belong to the subgenera Atreus and Tityus, proposed by Lourenço (2), together with Archaeotityus, Brazilotityus, and Caribetityus, in order to organize the morphological groups already described within the genus.Archaeotityus species (n = 24) comprise small (18-40 mm) and highly pigmented scorpions that are distributed throughout South America and the Caribbean and also Panamá and Costa Rica (2,3,17,18).According to Mello-Leitão (19) and Lourenço (20), Archaeotityus occupies a plesiomorphic position among Tityus morphological groups/ subgenera.Thus, its variegated pigmentation and the stronger distal tooth of the dorsal median carinae are considered primitive characters because they are only found in the juvenile stages of all remaining Tityus species (19).
While most toxins in the genus have been isolated and characterized from species in subgenera Atreus and Tityus, only scarce information is available on the venoms and toxins produced by Archaeotityus.Envenomation by Archaeotityus sp. is of poor medical relevance, probably due to the low amount of venom injected by scorpions in this subgenus (21).Primary structure determination of Archaeotityus toxins should throw light on their evolutionary relationship with other Tityus toxins that target ion channels considering the primitive status of this subgenus as suggested previously (19,20).
We undertook a molecular approach to recover amino acid sequences encoding Na v -active toxins from Tityus clathratus, the type species of Archaeotityus.This approach has identified protein sequences with molecular masses that correspond to bona fide toxins from other Tityus species confirmed by mass spectrometry (10).Adult scorpions (n = 12) were collected in Sanare, Lara State, western Venezuela (09°45´N, 69°20´W), and classified according to the taxonomic keys provided by González-Sponga (22).Total RNA was obtained from venom glands as described by Borges et al. (10).Animals were subjected to manual venom milking 48 hours before the dissection to increase production of toxin-encoding mRNAs (23) ´ and the degenerate oligonucleotide 5´-GTTTATYWGCTGCTTITTKC-3´.The latter primer was designed to anchor at the 3´-end of the DNA region coding for the leader peptide of Tityus long-chain toxins under the amplification conditions described by Borges et al. (10).
PCR fragments were ligated to the vector pCR2.1-TOPO®(Invitrogen, USA) and transformed into competent Escherichia coli DH5a cells which were then plated onto Luria-Bertani/agar plates containing 5-bromo-4chloro-3-indolyl-beta-D-galactopyranoside (X-Gal, 40 µg/mL).Plasmids were isolated from recombinant colonies by the alkaline lysis method and sequenced using universal M13 primers in an automated ABI 3130XL DNA Sequencer (Applied Biosystems, USA) at the Nucleic Acid Sequencing Laboratory, Venezuelan Institute for Scientific Research (24).
Thirty seven clones were recovered from X-Galcontaining agar plates and subjected to DNA sequencing.Seven colonies encoded transcript Tcl1 and 30 encoded transcript Tcl2.Nucleotide sequences (312 and 314 bp respectively) are presented in Figure 1.GenBank accession numbers are HQ632799 (Tcl1) and HQ632800 (Tcl2).Both transcripts encode 71 amino acidlong proteins with the C-terminal-most 64 residues bearing high similarity to the mature peptide region of scorpion toxins targeting Na v channels based on comparisons using the BLAST server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and including the eight cysteines involved in formation of disulphide bridges.
The amino acid sequences of mature Tcl1 and Tcl2 are 79% identical to each other.Main differences occur at the C-terminus where Asp55, Arg56 and Arg60 (in Tcl1) are replaced by Ser55, Tyr56 and Thr60 in Tcl2.The deduced molecular masses of the processed 61-residuelong T. clathratus putative neurotoxins are (in Da) 6966 (for Tcl1) and 6913 (for Tcl2) assuming that the two C-terminal-most lysine residues of both proteins (Figure 1) are removed posttranslationally upon amidation via the amino group of Gly62, as is the case for other Tityus toxins (25).
It is clear that Tcl1 and Tcl2 belong to the b-toxin family, bearing the same secondary structure elements found by Polikarpov et al. (39) in Ts1, the most abundant T. serrulatus b-toxin (40).Since Tityus venoms also contain a-toxins (16), the fact that we did not retrieve cDNAs coding for putative T. clathratus a-toxins could be either due to their absence in T. clathratus venom or that their cDNAs were not amplified under our conditions due to variations in the leader-peptide nucleotide sequence of T. clathratus a-toxin genes, which may prevent the degenerate primer from anchoring at the selected site, identified as conserved amongst genes encoding other Tityus toxins (10).A thorough venom proteomic approach should help clarify whether this toxin group is produced by Archaeotityus species.
Both Tcl1 and Tcl2 share with Ts1 critical residues involved in the β-toxin pharmacophore (Glu26, Tyr22, Ile29) (41).Also, residue Gly24, which participates in antigenic recognition in Ts1, is conserved in both T. clathratus putative homologs (42).Gly28, also a residue of antigenic importance in Ts1, is not conserved in Tcl1 and Tcl2, but this position is also variable in other gamma-like toxins (42).Significantly, Gly24 and the pharmacophore residues lie within the a-helical region, which is highly conserved in Ts1, gamma-like toxins (Tb1, Tst1, Tcogmamma) and T. clathratus homologs (see alignment in Figure 2) (39,42).The discontinuous antigenic epitope encompassing the amino-( 1 KEGY 5 ) and carboxy-( 46 GLPXXVKV 53 ) terminal regions of T. serrulatus toxins is conserved in Tcl2, but the critical Gly46 is replaced in Tcl1 by Asp (43).At the gene level, both T. clathratus precursors exhibit 82% identity with respect to Ts1, with changes in Tcl1 and Tcl2 cDNAs mostly comprising thirdposition replacements in the region encoding the central domain (residues [27][28][29][30][31][32][33]. A Bayesian analysis was performed to investigate the phylogenetic relationships of T. clathratus Tcl1 and Tcl2 with Tityus toxins retrieved from GenBank. Figure 3 shows a consensus tree obtained after Bayesian reconstruction using the WAG model of protein evolution selected in ProtTest (44).Birtoxin, a three disulfide-bridge b-like toxin from the South African scorpion Parabuthus transvaalicus was chosen as outgroup since the probable ancestor of North and South American aand b-toxins was a three-disulfide bridge toxin related to birtoxin (27,45,46).
There is reasonable (Bayesian posterior probability, BPP = 0.61) support for a monophyletic clade comprising toxins structurally and/or functionally related to the b-group which consists of two subclades.The first subclade (BPP = 0.98) contains T. serrulatus Ts2 and Ts2-like toxins.The second subclade (BPP = 0.87) comprises Ts1 and gamma-like toxins from T. bahiensis (Tb1), T. costatus (Tcogamma) and T. stigmurus (Tst1) with T. clathratus Tcl2 as a sister sequence.A subclade (BPP = 0.85) internal to the gammalike group includes toxins from Venezuela (Tz1, Td1-Td12, Na7), Colombia (Tpa2) and the Brazilian Amazon/French Guiana region (Tc49b, Tc48b, Tc49a) with Tcl1 as a distantly related (p-uncorrected distance: 40%) sister sequence.Both Tcl1 and Tcl2 are equally distant to the subclade comprising Ts1 and related proteins (p-uncorrected distance: 20%), in agreement with their sequence homology to gamma-like toxins.The topology of the tree in the remaining branches cannot be assessed with certainty given the lower node support (BPP < 0.50) for the groupings, with the exception of a-toxins from T. serrulatus (Ts3, Ts5), T. bahiensis (Tb3), T. stigmurus (Tst3), and T. discrepans (Na8) which form a monophyletic group (BPP = 1.0) lying outside the b-toxin clade as reported before (45,46).The fact that the T. clathratus genome encodes Na v toxins with a very close structural fingerprint to Ts1 and its homologs indicates a toxinological affinity not anticipated in previous studies, especially considering that T. clathratus inhabits an area about 4000 km north of its south eastern Brazilian congeners and that it is morphologically unrelated to them.Homologs of T. serrulatus Ts1 had only been found so far in other Brazilian Tityus such as T. bahiensis, T. stigmurus and T. costatus, species belonging to the subgenus Tityus (32,35).Significantly, T. discrepans and T. zulianus, two species sympatric with T. clathratus in Venezuela, do not produce such homologs but Na v toxins with divergent N-and C-termini with respect to Ts1 (10,33).In fact, in a recent phylogenetic analysis of Tityus scorpion Na v toxins, Guerrero-Vargas et al. (47) revealed a strong toxinological divergence among T. pachyurus, T. obscurus, T. discrepans and T. zulianus (in the subgenus Atreus) from the northern part of the Amazon Basin, and T. serrulatus, T. bahiensis, T. stigmurus, T. costatus and T. fasciolatus (in the subgenus Tityus), which live south of the Amazon.
Thus far T. clathratus is the only species from the north of the Amazon Basin producing putative toxin homologs of southern Brazilian Tityus, which provides further support for the evolutionary relationship between subgenera Archaeotityus (which has a trans-Amazonian distribution) and Tityus and its separation from the subgenus Atreus as suggested previously by Borges et al. (4).The latter authors, in their molecular phylogenetic analysis of 21 Tityus species using two mitochondrial DNA markers (cytochrome oxidase subunit 1 and ribosomal 16S rRNA), found that the Archaeotityus type species, T. clathratus, groups with the Brazilian T. serrulatus into a single clade which strongly diverges (53-57% nucleotide divergence) with respect to its other congeners in Venezuela, Trinidad and Panama in the subgenus Atreus.
No hypothesis for relationships amongst Tityus subgenera had been put forward prior to the present findings or those of Borges et al. (4).Only Lourenço (20) has suggested that the primitive Archaeotityus is closely related to Caribetityus, a subgenus endemic to the Caribbean island of Hispaniola (currently Dominican Republic and Haiti) and that the Cuban genera Alayotityus and Tityopsis, together with Caribetityus and Archaeotityus, are the possible proto-elements of continental Tityus, all sharing a common ancestor in South America.
Probably our results implicate that such common ancestor, evolutionary and toxinologically shared by subgenera Archaeotityus and Tityus, was distributed in South America before formation of the Amazon Basin and therefore prior to cladogenesis of the Atreus group, mainly restricted to the northern part of the subcontinent and whose species evolved different toxin repertoires (47).It is clear that the molecular analysis of more Archaeotityus species is needed to evaluate whether they produce Ts1 homologs as T. clathratus, and also to determine their phylogenetic affinity with species in the subgenus Tityus.
All in all, this research constitutes an initial point to study the evolution of Tityus venoms, the most speciose of all scorpion genera, comparing the putative toxins from an ancient species group with toxins from other species within the genus that have been well characterized.

Figure 1 .
Figure 1.Nucleotide sequences of Tityus clathratus clones Tcl1 and Tcl2 and translated amino acid sequences.The predicted protein sequence is shown below the nucleotide sequence and is numbered starting from the putative N-terminal residue, Lys.The signal peptide is underlined in the amino acid sequences; potential polyadenylation sites (AAUAAA and AAUUAA) are underlined in the nucleotide sequences(26).

Figure 3 .
Figure 3. Bayesian maximum likelihood consensus phylogram depicting phylogenetic relationships of Tityus Na v -active neurotoxins available from GenBank and T. clathratus Tcl1 and Tcl2 precursors isolated in this work.The sequence data set corresponds to the alignment presented in Figure 1.P. transvaalicus birtoxin was assigned as outgroup.Phylogenetic analysis was performed using the WAG model of protein evolution.Two Monte Carlo Markov chain were run 2,000,000 generations (burnin = 200,000).Clade support was inferred by bootstrapping.Bayesian posterior probability (BPP) was calculated for branching points.Only nodes supported by BPP > 0.5 are shown.