Evolution of scorpion venom

The predominant pharmacologically active components in scorpion venoms are small polypeptide molecules, usually basic in nature. Scorpion venoms and their component elements have been studied for over 35 years; but lately the focus in these studies has shifted from their pharmacological and electrophysiological properties to their molecular structures. This came about due to the realization that many classes of peptides seem to bind to characteristic spots on their targets, which for the most part are ion channels. The peptide toxins also show considerable identity in their arrangement of cysteine residues within the polypeptide chain. These cysteine residues are terribly important because disulphide bonds are one of the major contributors to conformational stability in small peptides.

The first pharmacological studies on scorpion toxins concentrated on their effects on mammalian model systems, so mice were the first ones to draw the short end of the hypodermic syringe, for a while. Peptides were consequently classified into the alpha- and beta-types, where each type bound to its own special site on voltage-gated sodium channels. Eventually, however, it was discovered that some peptides worked against ion channels in insects, and now Sarcophaga argyrostoma blowfly larvae were adopted as the victim model system of choice. All anti-insect toxins induced paralysis, but one class of toxins was found to induce a contraction paralysis, while the other induced a depressant paralysis.

For a while it seemed that all peptides could be neatly docketed in this way, but continuing studies unearthed peptide toxins that were primarily anti-mammal but showed some anti-insect activity too. Subsequently peptides were found that showed comparable anti-insect and anti-mammal activities, one of which – showing anti-insect as well as alpha-type and beta-type anti-mammal activities – was first reported in the paper (Loret et al., 1991) referenced below. This sparked off a line of thought on how the types of peptides found in scorpions in different parts of the world could be used to put forward theories about where scorpions first appeared and how they diverged into the large number of species we see today.

The reason this cross-reactivity of toxins against insect and mammal receptors was such a big deal was this: Before this was discovered, toxins were found to be easily classifiable not only on the basis of their primary peptide sequences and pharmacological activities, but also based on geography. Alpha-type anti-mammal toxins came only from Old World scorpions and beta-type anti-mammal toxins came only from New World scorpions. All anti-insect toxins had been purified from Old World scorpions only. Deviations from this geographical structure brought to light interesting ideas about venom evolution in scorpions.

For example, the toxin mentioned above which had a high effect on mammals but a low effect on insects was a beta-type toxin from Centruroides (a New World species) which was toxic to insects, but 50 times weaker than Old World anti-insect toxins (these studies happened in the 80s). This could indicate that anti-insect activity is just starting to evolve in New World venoms, but has already become established in Old World ones, indicating that the Old World venoms appeared first in their evolutionary history.

The paper referenced below is a report of a toxin from Androctonus australis Hector (pictured below), called AaH IT4. The toxin was purified by successive steps of chromatography, on gel filtration, DEAE-Sephadex and C8 HPLC columns. Toxicities were tested on S. argyrostoma larvae and male C57/BL6 mice, and the ED₅₀ values (for larvae) and LD₅₀ values (for mice) were recorded. Binding assays using ¹²⁵I-iodinated toxins on synaptosomal preparations from cockroaches and rats; radioimmunoassay assays to check for cross-reactions with rabbit antisera against known anti-insect, alpha-type and beta-type toxins; and circular dichroism analyses for structural data were carried out. In addition, sequence analysis and sequence alignment against various other scorpion venoms were carried out.

The experimental data showed that AaH IT4 competed with all three types of toxins for target-binding, and that it cross-reacted with the antibody against a beta-type toxin, indicating some structural similarity with the beta-toxin class. The dendrogram generated from the sequence alignment showed that AaH IT4 is more closely related to beta-type toxins than to either alpha-type or anti-insect toxins (which supports the result of the RIA experiment), although the divergence between AaH IT4 and the beta-type toxin lineage took place a long time ago. This an important point because beta-type toxins come from New World scorpions while AaH IT4 was purified from an Old World toxin, so any sequence similarity suggests that some relationship may exist between the two.

Another point to be made about AaH IT4 is that the sequence analysis shows an absence of the amino acid proline. Proline had previously been found in every purified and studied peptide scorpion toxin, and was suspected to play a role in the stability of their conformations, since proline is more conformationally restricted as compared to the other amino acids. One explanation for the binding of AaH IT4 to three different kinds of binding sites is that the absence of proline allows a certain amount of backbone flexibility which may allow the peptide to switch between conformational states that preferentially bind to each of the target sites.

The authors themselves hypothesize that since the evolution of insect-specific toxins is clearly advantageous to scorpions (for whom insects form a major part of the diet), it is possible that AaH IT4 represents the closest approximation available right now to some kind of ancestral toxin sequence/structure, which later diverged into anti-mammal and anti-insect varieties.

I think it’s worth mentioning, in addition, that the isolated AaH IT4 corresponds to 0.06% of the total protein in the venom, so it’s possible that the production of this particular venom component may have undergone some down-regulation over the years, as a result of the development of other, more specialised toxins.

Post-1991, of course, the issue of toxin classification has become even more complicated, and the venoms being studied now include those with anti-arthropod specificity, and the set of recognised targets have grown to include voltage- and ligand-gated potassium- as well as calcium-channels instead of just the initial emphasis on voltage-gated sodium-channels. Peptides with microbial activity have also been reported.

References:

Loret, E., Martin-Eauclaire, M., Mansuelle, P., Sampieri, F., Granier, C., & Rochat, H. (1991). An anti-insect toxin purified from the scorpion Androctonus australis Hector also acts on the .alpha.- and .beta.-sites of the mammalian sodium channel: sequence and circular dichroism study Biochemistry, 30 (3), 633-640 DOI: 10.1021/bi00217a007