While cone snails have a very efficient mechanism to incapacitate their prey, they are not as mobile as other species, and therefore rely on high-efficiency hunting mechanisms. Consequently, the implications of a failed envenomation event are dramatically larger for snails than they are for other venomous animals that are more able to pursue their prey as it attempts to escape. Therefore it is likely that the evolutionary pressures on cone snails are stronger than those on other venomous animals. For instance, if an injection of venom takes two minutes to paralyze the prey, the cone snail would remain hungry, where a snake or scorpion would likely not. This is probably the explanation for the atypically high evolutionary rate of venom peptides in Conus.49
However, as discussed below, conotoxins offer a unique opportunity to study evolutionary and molecular mechanisms by which genetic information is translated into folding and activity of disulfide-rich polypeptides.
As illustrated in , the high rate of evolution of Conus venom toxins is in striking contrast with discrete conservation of various portions of a toxin precursor gene.49
From a mechanistic point of view, even more puzzling is the fact that the cysteine scaffold remains highly conserved at the level of Cys codon usage (TGC or TGT), while adjacent codons belong to hypervariable sequences of intercysteine loops.50–52
Despite this extreme conservation of the cysteine scaffold, this does not imply a conservation of pharmacological properties.10
For example, shows two common disulfide scaffolds found in conotoxins blocking sodium channels,53, 54
but one scaffold (i) also blocks calcium channels and potassium channels (ω- and κ-conotoxins, respectively), as well as delaying the inactivation of sodium channels (δ-conotoxins), whereas the other scaffold (ii) is also capable of functioning as a noncompetitive inhibitor of nicotinic acetylcholine receptors.55
Noteworthily, despite an evolutionary conservation of Cys positions and codons, both scaffolds provide examples of conotoxins containing functionally non-critical disulfide bridges.
Figure 6 Biochemical synthesis of cysteine rich conotoxins and disulfide scaffolds found among conotoxins. (a) The precursor prepropeptide with highlighted conserved and variable regions. (b) Two disulfide scaffolds found in conotoxins. (i) shows the disulfide (more ...)
However, despite the highly conserved disulfide scaffolds in Conus, there are several instances of evolutionary divergence of disulfide scaffolds in these marine snails. While it often is difficult to reliably determine which is the “ancestral” fold, the conservation of cysteine codons and pro-peptide region strongly suggest evolutionary linkage between the α-conotoxins (bearing two disulfide bridges) and the αA-conotoxins (bearing three disulfide bridges).50
Additionally, the examples of Kunitz domains in conotoxins indicate that the evolution of disulfide bridges does not bear an inherent directionality (gain/loss of bridges). The Kunitz domain is a very ancient fold, being present in both plants56
with bovine pancreatic trypsin inhibitor (BPTI) being the prototypical example. From this ancient fold/cysteine scaffold, cone snails have developed a class of conotoxins, referred to as conkunitzins. The conkunitzin family has sequences that show a deletion of a disulfide bridge (illustrated in ) that is highly conserved in non-Conus Kunitz domains,58
as well as in other conkunitzins that contain two Kunitz domains in tandem, with addition of a fourth disulfide bridge on the N-terminal Kunitz scaffold.59
One outstanding question that remains is that the mechanism by which the cysteine scaffold is so highly conserved remains unknown. The extent of conservation decreases from the signal peptide to the mature toxin region (). However, the placement of cysteine, and even the cysteine codons within the mature toxin region are much more highly conserved than the remainder of the toxin region. While it has been proposed that this is due to a polymerase that preferentially preserves cysteine codons,60
a DNA polymerase capable of recognizing frame shifts has yet to be discovered. Consequently, it is possible that the cysteine scaffold is preserved by another mechanism, likely relying on evolutionary pressures. A given disulfide bond may be either critical to oxidative folding or to biological activity (or both), or may even be expendable. Thus, the extent to which the sequence encodes folding information to direct the folding to the native disulfide connectivity is critical to the ability to add and/or remove disulfide bridges to an existing scaffold. Herein we explore the ability to delete a native disulfide bridge from ω-conotoxin GVIA, and consider the evolutionary pressures present to preserve a given disulfide scaffold.
The potential to dissect the roles of individual disulfide bridges in a short, disulfide-rich peptide allows for a much more thorough analysis of the evolutionary pressures present to maintain each bridge. In , it is evident that the first disulfide bridge is critical to the efficient folding of GVIA, while shows that the same disulfide has a minimal effect on the biological activity of GVIA. Other work on deleting the second and third disulfide bridges demonstrated significant loss of the activity.32
Using ω-conotoxin GVIA as a model for inhibitory cystine knot (ICK) peptides, it is evident that each of the disulfides serves a critical purpose in generating the final, biologically active compound. Consequently, the evolutionary pressure to efficiently produce active peptide has caused the retention of the disulfide bridges in almost all ICK peptides, with the only potential exception being U1-liotoxin-Lw1a, from the scorpion Liocheles waigiensis,61
which may also be a precursor which never acquired a third disulfide bridge.