Global cyclotide adventure: a journey dedicated to the discovery of circular peptides from flowering plants

Abstract

Circular peptides and proteins of great number and diversity have been discovered in bacteria, plants, and animals. Cyclotides--disulfide-knotted and head-to-tail cyclized plant peptides that exhibit various bioactivities--are by far the largest group of circular proteins. Since their first discovery over three decades ago, there has been a lot of progress in the elucidation of structural characteristics and applications of cyclotides as novel peptide drug grafting frameworks, but there is a lack of information about their native occurrence in various plant families. The "global cyclotide adventure" was initiated as a plant collection and analysis project to advance our understanding of the origin and distribution of cyclotides in flowering plants. Here, I will provide a chronological overview of the preparation of this project, including background information on plant taxonomy and morphology, summarize, and comment on the recent progress about the discovery of cyclotide-producing plants and will give an outlook on the future of cyclotide analysis and further discoveries to be made.

Figure 1

Morphological features of Rubiaceae. Typical morphological Rubiaceae features, as shown with an example of the most famous family member, namely Coffea arabica L., which include (A) underside of leaf with domatia, (B) opposite leaf pair with axillary placed flowers, (C) dissected flower showing inside of corolla tube, anthers and style, (D) tip of stem showing stipules with secretion, (E) longitudinal section of ovary, (F) floral bud, and (G) bracts (Reproduced with permission from the National Botanical Garden Belgium; Artist: O. van de Kerckhove, 1995).

Figure 2

Illustration of cyclotide-containing Rubiaceae species. (A) Palicourea rigida Kunth is a small tree and a typical plant of the “Cerrado,” characterized by its orange inflorescences. (B) Palicourea coriacea (Cham.) K. Schum. is a shrub and another typical inhabitant of the Cerrado, characterized by its bright yellow inflorescences and very dry, but firm leaves. (C) Psychotria prunifolia (Kunth) Steyerm. is a small-sized tree and has typical five petal containing white flowers and small, pointed stipules (bi-fid). (D) Psychotria suterella Müll. Arg. is a tree with white, five-petalled flowers, typical whole, structured leaves, and blue fruits. (E) Psychotria brachyceras Müll. Arg. is a medium-sized tree. White five-petalled flowers, small, whole structured leaves, and pointed stipules are typical for this plant. (F) Geophila repens (L.) I.M. Johnst. is a ground growing herb with characteristic white, five-petalled flowers. (G) Chassalia discolour K. Schum. subsp. discolor is a medium-sized tree. Its inflorescences have a characteristic red/purple color. (H) Kadua centranthoides Hook. & Arn. growing on volcanic rock that results in the red color of the plant. Triangular stipules and actinomorphic flowers are typical Rubiaceae features. (I) Psychotria punctata Vatke with bright red fruits.

Figure 3

Cyclotide screening flowchart. Protein extracts of fresh or dried plant material were prepurified using C₁₈ chromatography. The extracts were analyzed by LC-MS before and after reduction and alkylation of cysteine residues. This chemical modification of the extracts and all peptides in the extract was used to characterize the number of cysteine residues per peptide. Cyclotides were identified by their typical hydrophobicity, their molecular weight, and the conserved number of cysteine residues. Finally, individual cyclotides were sequenced by reduction of the disulfide bonds and enzymatic cleavage of the stable peptide backbone to make them accessible for dissociation and sequencing via tandem mass spectrometry.

Figure 4

Presently known cyclotide-containing families within the flowering plants. The angiosperm phylogeny tree has been adapted and modified from the Angiosperm Phylogeny Group (Adapted and modified from Ref. 32, with permission from Academic Press). It shows the relationship of the different flowering plant classes, which are subdivided into subclasses and orders (some representative orders are shown and their number of estimated species is indicated). All cyclotide or cyclotide-like containing families are indicated in green. The family Rubiaceae belongs to the order of Gentianales (euasterids I), Violaceae to the order of Malphigiales (eurosids I), Cucurbitaceae to the order of Cucurbitales (eurosids I), and Poaceae, a monocotyledon plant, belongs to the order of Poales (commelinids). Other plant orders (e.g., Celestrales, Oxalidales, Fabales, Rosales, Lamiales, and Solanales) that are close to the respective positively-tested cyclotide containing orders are marked in yellow and could be potential candidate plant orders to continue the search for cyclotides. The number of estimated species in each order is indicated.