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Over recent years a large number of plants have been screened with the aim of discovering and identifying new peptides belonging to the cyclotide family [30,27,25,28,63,26,29,24]. To date cyclotides have been identified in every Violaceae plant screened as well as in a few examples of Rubiaceae species. Two related peptides also containing a CCK motif have been found in a plant from the Cucurbitaceae family [30,7] but they possess different loop spacings and their sequences are more homologous to acyclic squash trypsin inhibitors than they are to the cyclotides. The Rubiaceae and Violaceae are not closely related phylogenetically, with the branch point for the two lineages encompassing a great part of the core eudicots, the largest group within the flowering plants. The Violaceae contains approximately 800 species in 21 genera and belong to the Malpighiales (eurosids I, rosids), a large order which also includes species such as the passion fruit and mangrove trees. Conversely, the Rubiaceae constitutes the largest family of the Gentianales (euasterids I, asterids) and contains genera as diverse as the coffee tree and the cinchona. One evolutionary schema for the angiosperms is set out in Figure 1.9. If a common ancestral gene once existed it is possible that the cyclotides are distributed throughout core eudicots and the failure of screening programs to detect cyclotides in a more diverse range of plants presents an interesting evolutionary mystery.
A number of explanations are possible to explain this unusual distribution. One possibility is descent from an ancestral gene that has subsequently been lost in most lineages of plants, including the majority of the Rubiaceae. Differential gene loss is an active process in the evolution of angiosperm genomes [67,68] and, hence, it may not be surprising that the cyclotides are not more widely distributed. If this is the case, however, then it would be interesting to decipher why species of the Violaceae have found cyclotides more useful than have other species. Another possibility is that there has been lateral gene transfer from the Violaceae to the Rubiaceae, or, less likely, vice versa. Such gene transfer is relatively unknown, however several cases have now been documented in plants [69,70,71], and if lateral transfer has occurred it would be of immense interest. Finally, it is possible that shortcomings in the screening procedures have led to a bias toward the hydrophobic members of the family. Cyclotides are typically identified as late eluting peaks in RP-HPLC. This reliance on the unusual hydrophobicity of the cyclotides may be biasing screening programs toward hydrophobic members of the family. If less hydrophobic cyclotides exist, or are the norm, then current screening procedures would fail to identify these peptides.
Although the cyclotides are distinctive for containing both a cystine knot and a macrocyclic backbone they do share similarities with the knottins. Indeed, as mentioned, the squash trypsin inhibitors have two members possessing a cystine knot and a macrocyclic backbone. The relationship between the knottins and the cyclotides is uncertain, indeed the knottins encompass a wide variety of peptides from a wide variety of organisms and the evolutionary relationship between the members of the knottins is also uncertain. It has been proposed, however, that the knottins are derived from an elementary unit called the cystine stabilised -sheet (CSB) . This motif includes two of the three disulfides found in the cystine knot (II-V and III-VI) which stabilise a triple stranded -sheet. This motif has been found to be sufficient to stabilise the native structure of EETI [75,73,74], a squash trypsin inhibitor that contains a typical ICK in its native form. The CSB is common in other small disulfide rich folds and the possibility of an ancestral CSB containing protein linking the knottins, and cyclotides, to the other disulphide rich proteins, as illustrated in Figure 1.10, cannot be discounted, although it is also possible that the motif has evolved independently a number of times.
Interestingly, an evolutionary scheme has been proposed for the animal knottins that includes a precursor peptide exon shuffling event from a sea anemone to an ancestral non-secreting cystine knot and consequent divergent evolution between the cone snail and Arachnida cystine knot motifs . This scheme has been proposed based on the conservation of intronic insertion, gene organisation and three dimensional structure. All the single copy cystine knot precursors found in plants share the same precursor structure with a signal sequence, precursor region and mature peptide . However sequence identity between the non-cyclic ICK's and the CCK proteins is minimal and the structure of the cyclotide genes has not yet been elucidated so whether or not they possess introns is not known. Accordingly, a single evolutionary scheme that accounts for all cystine knot containing peptides in plants would need to account for the development of multi-copy transcripts, possible intronic insertions and a cyclising mechanism in a linear ancestral peptide.
Next: Summary Up: The Cyclotides Previous: Natural Activity of the Jason Mulvenna