Discovery of an unusual biosynthetic origin for circular proteins in legumes

Abstract

Cyclotides are plant-derived proteins that have a unique cyclic cystine knot topology and are remarkably stable. Their natural function is host defense, but they have a diverse range of pharmaceutically important activities, including uterotonic activity and anti-HIV activity, and have also attracted recent interest as templates in drug design. Here we report an unusual biosynthetic origin of a precursor protein of a cyclotide from the butterfly pea, Clitoria ternatea, a representative member of the Fabaceae plant family. Unlike all previously reported cyclotides, the domain corresponding to the mature cyclotide from this Fabaceae plant is embedded within an albumin precursor protein. We confirmed the expression and correct processing of the cyclotide encoded by the Cter M precursor gene transcript following extraction from C. ternatea leaf and sequencing by tandem mass spectrometry. The sequence was verified by direct chemical synthesis and the peptide was found to adopt a classic knotted cyclotide fold as determined by NMR spectroscopy. Seven additional cyclotide sequences were also identified from C. ternatea leaf and flower, five of which were unique. Cter M displayed insecticidal activity against the cotton budworm Helicoverpa armigera and bound to phospholipid membranes, suggesting its activity is modulated by membrane disruption. The Fabaceae is the third largest family of flowering plants and many Fabaceous plants are of huge significance for human nutrition. Knowledge of Fabaceae cyclotide gene transcripts should enable the production of modified cyclotides in crop plants for a variety of agricultural or pharmaceutical applications, including plant-produced designer peptide drugs.cyclic peptides | structure | cystine knot | kalata B1

New Mode of Action for a Knottin Protein Bioinsecticide

“…PA1b and Cter M, the insecticidal part of the PA1 gene, appear to act via distinct mechanisms. In common with other cyclotides, Cter M binds membranes and forms pores (4). In contrast, our work shows that the PA1b peptide acts specifically via a membranous receptor.…”

“…Very few known molecules are really able to provide alternative solutions to chemical pesticides; plant insecticidal molecules either target digestive enzymes or are chitin-binding proteins (4). Thus, the inhibition of V-ATPase in the midgut of insects represents a new target for a bioinsecticide.…”

“…The structure of the PA1 gene is common among legumes for all PA1b toxins, and recently, this same structure was also discovered in Clitoria ternatea for the peptide PA1a, but here a cyclotide replaces PA1b ( Fig. 1) (4,5). The cyclotides are cyclic knottins that have been, together with knottins, studied increasingly because of their extensive agricultural and pharmaceutical applications.…”

Insights into Processing and Cyclization Events Associated with Biosynthesis of the Cyclic Peptide Kalata B1

“…The nature of the residue in the P1 position immediately upstream of the cyclotide domain is probably not important; it can be a Lys, as in the O. affinis cyclotides, or an Asn as occurs in other cyclotide subfamilies, as long as the Ntr is surfaceexposed and accessible to proteases and cleavage at this junction prior to the cleavage at the Ctr that results in cyclization. This is supported by the recent discovery of cyclotides in the Fabaceae family where the cyclotide domain is directly preceded by the ER signal peptide and the Ntr is absent (20). The cleavage of this signal peptide in the ER would free up the N terminus of the cyclotide, which presumably remains free until the cyclotide precursor reaches the compartment where cyclization occurs.…”

“…The importance of the P2Ј residue within the Ctr was exemplified with the truncation mutants that showed that Leu is essential for cyclization and this is not entirely surprising as a Leu in this position is completely conserved across all cyclic peptides found in all species except the Fabaceae, which contain an Ile (20). Cleavage and cyclization occurs 2 amino acids from this leucine.…”