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Assembling an arsenal, the scorpion way.

Kozminsky-Atias A, Bar-Shalom A, Mishmar D, Zilberberg N - BMC Evol. Biol. (2008)

Bottom Line: Upon fixation, the mature toxin-coding domain was subjected to diversifying selection resulting in a significantly higher substitution rate that can be explained solely by diversifying selection.We interpret this as resulting from purifying selection acting on both the peptide and, as reported here for the first time, the DNA sequence, to create a toxin family-specific codon bias.We thus propose that scorpion toxin genes were shaped by selective forces acting at three levels, namely (1) diversifying the mature toxin, (2) conserving the leader peptide amino acid sequence and intriguingly, (3) conserving the leader DNA sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. adiko@bgu.ac.il

ABSTRACT

Background: For survival, scorpions depend on a wide array of short neurotoxic polypeptides. The venoms of scorpions from the most studied group, the Buthida, are a rich source of small, 23-78 amino acid-long peptides, well packed by either three or four disulfide bridges that affect ion channel function in excitable and non-excitable cells.

Results: In this work, by constructing a toxin transcripts data set from the venom gland of the scorpion Buthus occitanus israelis, we were able to follow the evolutionary path leading to mature toxin diversification and suggest a mechanism for leader peptide hyper-conservation. Toxins from each family were more closely related to one another than to toxins from other species, implying that fixation of duplicated genes followed speciation, suggesting early gene conversion events. Upon fixation, the mature toxin-coding domain was subjected to diversifying selection resulting in a significantly higher substitution rate that can be explained solely by diversifying selection. In contrast to the mature peptide, the leader peptide sequence was hyper-conserved and characterized by an atypical sub-neutral synonymous substitution rate. We interpret this as resulting from purifying selection acting on both the peptide and, as reported here for the first time, the DNA sequence, to create a toxin family-specific codon bias.

Conclusion: We thus propose that scorpion toxin genes were shaped by selective forces acting at three levels, namely (1) diversifying the mature toxin, (2) conserving the leader peptide amino acid sequence and intriguingly, (3) conserving the leader DNA sequences.

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B. occitanus israelis venom gland transcript distribution. (A) A phylogenetic tree analysis of putative toxins. Toxin families are highlighted according to their estimated blocking targets, i.e. Na+, K+, Cl- or Ca2+ channels, based on homology to known toxins [16]. Putative toxins with no known homologs were termed 'other toxins'. Only full-length distinct cDNA sequences were used for the protein data set construction. (B) Toxin families' transcript distribution. The number of distinct members of each family is presented as is their relative representation within the cDNA library. (C) Transcript expression levels. The number of clones obtained for each of the individual transcripts in the cDNA library is indicated. The estimated affiliation of each transcript is shown by shading of the bars.
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Figure 1: B. occitanus israelis venom gland transcript distribution. (A) A phylogenetic tree analysis of putative toxins. Toxin families are highlighted according to their estimated blocking targets, i.e. Na+, K+, Cl- or Ca2+ channels, based on homology to known toxins [16]. Putative toxins with no known homologs were termed 'other toxins'. Only full-length distinct cDNA sequences were used for the protein data set construction. (B) Toxin families' transcript distribution. The number of distinct members of each family is presented as is their relative representation within the cDNA library. (C) Transcript expression levels. The number of clones obtained for each of the individual transcripts in the cDNA library is indicated. The estimated affiliation of each transcript is shown by shading of the bars.

Mentions: To study the evolutionary mechanisms underlying the diversity of scorpion toxins, a database of all major transcripts expressed in the venom gland of the Israeli scorpion, B. occitanus israelis (Boi), was constructed. In addition, a venom-gland based cDNA library was established. To increase the chances of identifying rare toxin cDNA (i.e. frequency < 0.005), approximately 450 clones were isolated and sequenced. To identify putative toxin-encoding clones, translated cDNA sequences were first compared to those of known toxins. Next, all other open reading frames (ORFs) were inspected for the presence of a putative leader sequence or a known cysteine-based pattern. This sequencing effort showed 78% of the clones to have sequence attributes of toxins, leading these sequences to be termed putative scorpion toxin cDNAs. Of these, seventy two novel individual toxin-like transcripts were identified (GenBank accession numbers FJ360768–FJ360843). Evolutionary relationships between the toxins cDNAs were examined by the construction of a phylogenetic tree (Fig. 1A) based on alignments of both cDNA and predicted protein sequences. The Na+-channel toxin family and some of the K+-channel toxins could be clearly dividable into distinct sub-families on the basis of conserved elements present in the precursor sequences. Since the cDNA and protein trees presented highly similar topologies, only the protein tree is shown (Fig. 1A). As shown in figure 1B, over 50% of Boi toxin sequences are predicted to be Na+ channel modifiers and can be divided, by sequence similarity, into 4 sub-families (Fig. 1B). The predicted K+ channel blockers comprise 28% of the total transcripts and can be divided into numerous sub-types (Fig. 1B). This diverse toxin family is characterized by its star-shaped phylogenetic tree pattern (Fig. 1A). Four putative transcripts were predicted to belong to the chlorotoxin-like family. Eight additional ORF-containing putative transcripts, were identified as possessing toxin-like characteristics, i.e. a leader sequence and multiple cysteine residues, yet showed no similarity to known toxins. All Boi toxin precursors possessed the known two-domain structure, corresponding to the leader and mature peptide regions [5,15,16].


Assembling an arsenal, the scorpion way.

Kozminsky-Atias A, Bar-Shalom A, Mishmar D, Zilberberg N - BMC Evol. Biol. (2008)

B. occitanus israelis venom gland transcript distribution. (A) A phylogenetic tree analysis of putative toxins. Toxin families are highlighted according to their estimated blocking targets, i.e. Na+, K+, Cl- or Ca2+ channels, based on homology to known toxins [16]. Putative toxins with no known homologs were termed 'other toxins'. Only full-length distinct cDNA sequences were used for the protein data set construction. (B) Toxin families' transcript distribution. The number of distinct members of each family is presented as is their relative representation within the cDNA library. (C) Transcript expression levels. The number of clones obtained for each of the individual transcripts in the cDNA library is indicated. The estimated affiliation of each transcript is shown by shading of the bars.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651877&req=5

Figure 1: B. occitanus israelis venom gland transcript distribution. (A) A phylogenetic tree analysis of putative toxins. Toxin families are highlighted according to their estimated blocking targets, i.e. Na+, K+, Cl- or Ca2+ channels, based on homology to known toxins [16]. Putative toxins with no known homologs were termed 'other toxins'. Only full-length distinct cDNA sequences were used for the protein data set construction. (B) Toxin families' transcript distribution. The number of distinct members of each family is presented as is their relative representation within the cDNA library. (C) Transcript expression levels. The number of clones obtained for each of the individual transcripts in the cDNA library is indicated. The estimated affiliation of each transcript is shown by shading of the bars.
Mentions: To study the evolutionary mechanisms underlying the diversity of scorpion toxins, a database of all major transcripts expressed in the venom gland of the Israeli scorpion, B. occitanus israelis (Boi), was constructed. In addition, a venom-gland based cDNA library was established. To increase the chances of identifying rare toxin cDNA (i.e. frequency < 0.005), approximately 450 clones were isolated and sequenced. To identify putative toxin-encoding clones, translated cDNA sequences were first compared to those of known toxins. Next, all other open reading frames (ORFs) were inspected for the presence of a putative leader sequence or a known cysteine-based pattern. This sequencing effort showed 78% of the clones to have sequence attributes of toxins, leading these sequences to be termed putative scorpion toxin cDNAs. Of these, seventy two novel individual toxin-like transcripts were identified (GenBank accession numbers FJ360768–FJ360843). Evolutionary relationships between the toxins cDNAs were examined by the construction of a phylogenetic tree (Fig. 1A) based on alignments of both cDNA and predicted protein sequences. The Na+-channel toxin family and some of the K+-channel toxins could be clearly dividable into distinct sub-families on the basis of conserved elements present in the precursor sequences. Since the cDNA and protein trees presented highly similar topologies, only the protein tree is shown (Fig. 1A). As shown in figure 1B, over 50% of Boi toxin sequences are predicted to be Na+ channel modifiers and can be divided, by sequence similarity, into 4 sub-families (Fig. 1B). The predicted K+ channel blockers comprise 28% of the total transcripts and can be divided into numerous sub-types (Fig. 1B). This diverse toxin family is characterized by its star-shaped phylogenetic tree pattern (Fig. 1A). Four putative transcripts were predicted to belong to the chlorotoxin-like family. Eight additional ORF-containing putative transcripts, were identified as possessing toxin-like characteristics, i.e. a leader sequence and multiple cysteine residues, yet showed no similarity to known toxins. All Boi toxin precursors possessed the known two-domain structure, corresponding to the leader and mature peptide regions [5,15,16].

Bottom Line: Upon fixation, the mature toxin-coding domain was subjected to diversifying selection resulting in a significantly higher substitution rate that can be explained solely by diversifying selection.We interpret this as resulting from purifying selection acting on both the peptide and, as reported here for the first time, the DNA sequence, to create a toxin family-specific codon bias.We thus propose that scorpion toxin genes were shaped by selective forces acting at three levels, namely (1) diversifying the mature toxin, (2) conserving the leader peptide amino acid sequence and intriguingly, (3) conserving the leader DNA sequences.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel. adiko@bgu.ac.il

ABSTRACT

Background: For survival, scorpions depend on a wide array of short neurotoxic polypeptides. The venoms of scorpions from the most studied group, the Buthida, are a rich source of small, 23-78 amino acid-long peptides, well packed by either three or four disulfide bridges that affect ion channel function in excitable and non-excitable cells.

Results: In this work, by constructing a toxin transcripts data set from the venom gland of the scorpion Buthus occitanus israelis, we were able to follow the evolutionary path leading to mature toxin diversification and suggest a mechanism for leader peptide hyper-conservation. Toxins from each family were more closely related to one another than to toxins from other species, implying that fixation of duplicated genes followed speciation, suggesting early gene conversion events. Upon fixation, the mature toxin-coding domain was subjected to diversifying selection resulting in a significantly higher substitution rate that can be explained solely by diversifying selection. In contrast to the mature peptide, the leader peptide sequence was hyper-conserved and characterized by an atypical sub-neutral synonymous substitution rate. We interpret this as resulting from purifying selection acting on both the peptide and, as reported here for the first time, the DNA sequence, to create a toxin family-specific codon bias.

Conclusion: We thus propose that scorpion toxin genes were shaped by selective forces acting at three levels, namely (1) diversifying the mature toxin, (2) conserving the leader peptide amino acid sequence and intriguingly, (3) conserving the leader DNA sequences.

Show MeSH
Related in: MedlinePlus