Cyclotide Genetics

In contrast to the non-ribosomally produced, small cyclic peptides (5-12 aa) that are found in fungi and bacteria, the cyclotides are true gene products. Clones encoding cyclotides from O. affinis have been obtained using a PCR approach with a primer corresponding to a region of the cyclic protein in combination with oligo-dT (Jennings, et al., 2001). The amplified 400 bp fragment encoded the entire kalata B1 cyclotide together with a C-terminal extension of 4 amino acids. This PCR fragment was subsequently used to isolate cDNAs corresponding to four cyclotide genes from O. affinis. These cDNAs encode multi-domain precursor proteins with one, two or three cyclotide domains. The cyclotides corresponding to these mature domains, kalata B1, B2, B3, B6 and B7, have all been isolated from plant tissue, verifying the production and processing of the precursors.

Schematic of the gene structure of two cyclotide clones showing the precursor region as well as the N-terminal Repeat Fragment (NTRF).

The predicted precursors from Oak1-4 have a typical endoplasmic reticulum signal sequence and thus are likely to enter the secretory pathway where folding and disulfide bond formation occurs. Each precursor contains a relatively long N-terminal pro-domain that is not tightly conserved in sequence or length. This domain is followed by a relatively well-conserved 25 amino acid peptide that we have called the N-terminal repeated fragment (ntr). The ntr precedes each cyclotide domain in the single, double and triple cyclotide encoding precursors. We speculate that the ntr domain may assist in folding or processing of the cyclotide domain. The predicted precursors also have a C-terminal propeptide of 7 residues, but the function of this domain is unknown.

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