Cyclotides: From Structure to Function

Abstract

Early studies of cyclotides [7][8][9][13][14][15] were based on discovery and characterization efforts at the peptide level. The general approach for these studies involved either bioassay-guided or mass-guided fractionation of plant extracts, followed by peptide isolation and purification, amino acid analysis, Edman sequencing and chemical, NMR 9 or mass spectrometry-based 10 approaches to deduce disulfide connectivity. The first step in many of the early discovery programs was a pre-screening procedure to determine if a given plant tissue might contain cyclotides (Figure 3). Various protocols 13,15,57,69,116,123 have been reported for quickly screening if a plant might be cyclotide-positive, with the simplest based on extraction (using dichloromethane/methanol, aqueous acetonitrile, or aqueous buffer) and purification on a C18 reverse phase column followed by a main-screen. The latter typically involves (A) examination of a reversed phase HPLC-MS profile for late-eluting peaks that are (B) in the mass range 2800-4000 Da and (C) contain six Cys residues as judged by a mass shift of 348 Da after reduction and S-alkylation, as shown in Figure 3. 116 The rationale for the target HPLC elution range was that all of the cyclotides discovered in early studies were somewhat hydrophobic and eluted relatively late on HPLC. Similarly, many of the initial cyclotides were in the mass range 2500-4000 Da and it was assumed that new cyclotides would fall within a similar range. Finally, by definition, a cyclotide must contain six Cys residues so the mass shift test in step C of the screen provided a simple way of testing for that criterion. An example of the 348 Da mass shift after reduction and alkylation is illustrated for the identification of the cyclotide Cter A from Clitoria ternatea (butterfly pea) seeds in Figure 3. Chassalia chartacea chassatides 235 Chassalia curviflora chacur c Chassalia discolor CD-1 c Chassalia parvifolia circulins 7,22,406 Hedyotis biflora hedyotides 207,213,280,350 H. centranthoides hcf-1 c H. terminalis htf-1 c Oldenlandia affinis kalata peptides 1-5,9 Palicourea condensata palicoureins 25 P. rigida parigidin-br1 236,329,345 P. tetragona paltet 1 c Psychotria brachiata psybra 1 c Psychotria brachyceras Psyleio and psybry peptides c Psychotria leiocarpa Psyleio peptides 338 Psychotria leptothyrsa psyle peptides 157,158 Psychotria longipes cyclopsychotride A 8 Psychotria poeppigiana psypoe 1 Psychotria solitudinum psysol 2 305 Psychotria suterella PS-1 c Cucurbitaceae Momordica clarkeana TI peptides 315 These trypsin inhibitor cyclotides are also referred to as cyclic knottins; typically, these peptides are found only in seeds. M. cochinchinensis MCoTI peptides 24 M. denticulate TI peptides 315 M. diocia Modi peptides 424 M. gilgiana TI peptides 315 M. macrophylla TI peptides 315 M. subangulata TI peptides 315 Fabaceae Clitoria ternatea Cter peptides; 204 cliotides 206 These cyclotides are biosynthetically processed from 'hijacked' genes (i.e. from albumin genes in which an ancestral albumin subun...

Cysteines and Disulfide Bonds as Structure-Forming Units: Insights From Different Domains of Life and the Potential for Characterization by NMR

“…Thus, nature has evolved a multitude of proteins with specialized biological functions based on molecular architectures involving different numbers of cystines. Only a few examples are Kunitz-type trypsin inhibitors (Otting et al, 1993;Cohen et al, 2019), multi-domain Kazal-type thrombin inhibitors like rhodniin (Van De Locht et al, 1995) and dipetalin (Schlott et al, 2002), growth factors (Christinger et al, 2004;Sitar et al, 2006), defensins (Szyk et al, 2006), neuropeptides like oxytocin (Bhaskaran et al, 1992) and vasopressin (Schmidt et al, 1991) or peptidic toxins (Elnahriry et al, 2019) and cyclotides (Park et al, 2017;De Veer et al, 2019;Huang et al, 2019).…”

Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus

“…Cyclotides are a large family of cysteine-rich peptides, ~30 amino acids in size, derived from plants (1).They have attracted great attention from both academia and industry since they were first reported in the 1990s. In 2017, Sero-X, which is a cyclotide-based eco-friendly pesticide extracted from Clitoria ternatea (2), was approved to market in Australia for the protection of cotton and macadamia nut crops (https://innovateag.com.au/).…”

Discovery of Cyclotides from Australasian Plants

“…Cyclotides have been extensively reviewed in the past [62][63][64][65][66][67][68][69][82][83][84][85][86][87][88][89][90][91] and readers are referred to some of these articles and a recent book [92] for a more detailed background on their discovery, biosynthesis and applications. In the current article our focus is on just those plants that grow in Australasia and are thus accessible to researchers in the region.…”