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The cyclotides are distinguished from previously known small cyclic peptides biosynthesised by microorganisms in that the latter are not direct gene products but are assembled by peptide synthetases. Examples of the latter category include cyclosporin and a range of polyketides [44]. Such molecules are often characterised by the presence of modified amino acids or modified peptide linkages. In contrast, the cyclotides and other naturally occurring cyclic peptides are true gene products and as a consequence must first be expressed as a linear precursor with backbone cyclisation occurring as a post-translational modification. A great deal of interest has been shown in this cyclisation mechanism not only as a biological curio but also because such a mechanism would have an application in the cyclisation of peptide drugs, a process that is being explored to enhance the stability and resistance to proteolytic digestion of small peptides [11,45].

Figure 1.7: Organisation of the cyclotide precursor proteins as determined from cDNA transcripts Each precursor consists of an ER signal (coloured black), a Pro region, and one (OaK1), two (OaK2) or three (OaK4) copies of a mature cyclotide sequence with a preceding N-terminal repeat (ntr- single hashed segment). The OaK3 gene structure is a two-repeat motif similar to OaK2. Each of the precursors possesses a C-terminal tail that is predominately hydrophobic (double hashed region). The sequences of each ntr and tail region are shown boxed at the bottom of the Figure. A GLP (Oak1, 2 and 3) or SLP (Oak4) flanks each mature peptide sequence. In all peptides excluding Oak4 the GLP motif is present as the first three residues of the mature transcript (not shown) and as the first three residues of the C-terminal tail or ensuing ntr. In Oak4 the mature sequence includes the GLP (not shown) motif as the first three residues but contains a SLP motif in the C-terminal tail or ntr regions. This Gly/Ser substitution allowed the determination of the cleavage point for the cyclotide precursor (see Figure 1.8).

Several linear precursors of the cyclotides have now been identified in Oldenlandia affinis [46], from the Rubiaceae, and Viola odorata [47], from the Violaceae. As illustrated in Figure 1.7, these genes each encode multi-domain precursor proteins with one, two or three cyclotide domains. The general structure of a cyclotide precursor protein includes an endoplasmic reticulum (ER) signal, a relatively long and poorly conserved N-terminal segment, followed by one or more repeated units that include the mature cyclotide domain. Each one of these repeated domains contains a highly conserved N-terminal region, named the N-terminal repeated (ntr) fragment, preceding a mature cyclotide sequence. At the C-terminus the precursor has a small tail with a hydrophobic complexion. The function of the ntr is unknown but it has been shown to adopt an amphipatic helix in both Oldenlandia affinis and Viola odorata, and this helix that may assist in the correct folding of the precursor protein by stabilising the unusual hydrophobic surface of the cyclotides prior to processing [47]. Interestingly, although the ntr appears to be relatively well conserved within species there is little sequence conservation evident between species, suggesting that the conservation is at a structural rather than sequential level.

It would seem from the structure of these precursors that two cleavages are required to excise the mature peptide from the precursor sequence. In the originally discovered Oak clones, however, these processing points could not be determined as the mature sequence of the first three clones isolated were flanked by a GLP motif. As the mature cyclotides possessed only one GLP motif this meant that the processing site could occur at one of four positions (see Figure 1.8). This problem highlights the essential ``seamlessness'' of cyclisation as a post-translational modification. Cyclisation leaves no footprint in the mature peptide and there is no easy way to determine the processing points if there is ambiguity in the linear transcript or if only the mature peptide is available for examination.

Figure 1.8: Processing points of the cyclotide precursor Schematic of cyclotide biosynthesis showing the possible cleavage points of the precursor depending on the presence of the GLP or SLP motif. The first mature cyclotide sequence has four possible cleavage sites due to flanking GLP motifs situated at the beginning of the mature cyclotide sequence and in the subsequent ntr (single hash). In the second mature peptide sequence there is no ambiguity in the cleavage point due to the presence of the SLP motif in the tail (double hash). All five processing schemes produce a circular peptide with the same sequence. This can be seen in the sequence circle, which shows the mature cyclotide sequence, the disulfide connectivity and the position of the possible processing points in the mature peptide (asterisks). This highlights the ``seamlessness'' of the mature peptide as residues from either end of the mature sequence in the precursor are combined to form loop 6 of the mature peptide. X corresponds to either of the amino acids N or D.

The position of the processing points was solved by the discovery of Oak4 which had a Ser residue in place of Gly in the C-terminal GLP motif [46]. This clone contained the sequence of kalata B2 which does not contain a SLP sequence in the mature peptide. This indicated that proteolytic cleavage of the precursor must occur after the Lys at the N-termini and after the conserved Asn/Asp residue at the C termini. The consequent discovery of the three Voc clones, that contained no ambiguity, from Viola odorata confirmed the processing point and further reinforced the possible involvement of the conserved Asn/Asp in loop 6 in the cyclising reaction. The sequence of the Voc clones also differed in that N-terminal cleavage would need to occur after a Gly residue and not a Lys as in the Oak clones. If the same mechanism is used in the two plant families then the N-terminal cleavage reaction would not appear to be sequence specific.

As will become apparent as other cyclic proteins are examined further in the chapter, proteases have been implicated as possible mediators of cyclisation in a number of different species. Although there is no known enzyme that could cleave a peptide bond after both a Lys/Gly and an Asn, an interesting class of enzyme called the asparagine endopeptidases may have a role in the processing mechanism. The members of this class of peptidase, collectively known as the legumains, are reported to cleave various proteins in the secretory pathway by proteolytic cleavage on the C-terminal side of Asn residues [50,49,48]. Interestingly, an asparaginyl endopeptidase is also suspected of mediating a transpeptidation reaction in the processing of the concanavalin A precursor in Canavalia ensiformis (Jack bean) [51]. In this case the enzyme mediates the Asn-specific excision and transpeptidation of the precursor to produce an inversion of peptide fragments in the mature peptide. It is currently thought that plant and human legumains are derived from the GPI transamidases [52] and the transpeptidase activity of the legumains may be an echo of their evolutionary history.

Although the involvement of a legumain in cyclotide processing is a compelling theory it should be noted that, unlike the case in concanavalin A, cyclisation in the cyclotides also requires a second cleavage following the lysine residue. This could be provided by a decreased specificity of the putative processing legumain or by the involvement of another enzyme. Furthermore, the possibility of molecular chaperones and a wide variety of other enzymes abundant in the ER having some role in initial disulfide bond formation and folding of the precursor cannot be discounted.

Another theory that has been raised is the possibility of a self-excising intein-like processing mechanism [31]. Inteins are protein sequences that mediate the self-excision of protein `introns' from precursor proteins and they have been used to produce cyclic peptides in vivo and in vitro [54,45,53]. If this form of mechanism is applied to the cyclotides the mature domain would be homologous to the protein intron of a intein system, effectively reversing the nomenclature as the mature sequence is excised from the precursor. Inteins are identifiable via highly conserved protein motifs and no such motif has been found in the cyclotide precursors, but the possibility of a novel auto-catalytic mechanism should not be excluded.

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Next: Natural Activity of the Up: The Cyclotides Previous: Structural Characteristics of the
Jason Mulvenna