Distribution of circular proteins in plants: large-scale mapping of cyclotides in the Violaceae

Abstract

During the last decade there has been increasing interest in small circular proteins found in plants of the violet family (Violaceae). These so-called cyclotides consist of a circular chain of approximately 30 amino acids, including six cysteines forming three disulfide bonds, arranged in a cyclic cystine knot (CCK) motif. In this study we map the occurrence and distribution of cyclotides throughout the Violaceae. Plant material was obtained from herbarium sheets containing samples up to 200 years of age. Even the oldest specimens contained cyclotides in the preserved leaves, with no degradation products observable, confirming their place as one of the most stable proteins in nature. Over 200 samples covering 17 of the 23–31 genera in Violaceae were analyzed, and cyclotides were positively identified in 150 species. Each species contained a unique set of between one and 25 cyclotides, with many exclusive to individual plant species. We estimate the number of different cyclotides in the Violaceae to be 5000–25,000, and propose that cyclotides are ubiquitous among all Violaceae species. Twelve new cyclotides from six phylogenetically dispersed genera were sequenced. Furthermore, the first glycosylated derivatives of cyclotides were identified and characterized, further increasing the diversity and complexity of this unique protein family.

Improved method for quantitative analysis of the cyclotide kalata B1 in plasma and brain homogenate

“…They appear to play a role in plant defence, not the least demonstrated by the insecticidal effect of kalata B1 . The cyclotides can be found in a number of plant families, including Violaceae, Rubiaceae and Fabaceae, with reports of large numbers of cyclotides found in single plant species . Kalata B1 has shown bioactivity in several in vitro assays including, but not limited to, nematocidal activity, haemolysis, insecticidal activity and membrane binding .…”

Thermodynamic instability of viral proteins is a pathogen-associated molecular pattern targeted by human defensins

“…The cyclotides cyO2 and cyO19 were isolated from a Viola odorata L. extract, while the cyclotides kB1 and kB7 were isolated from an Oldenlandia affinis D.C. extract according to the methods previously described4567. The peptides were of >95% purity as assessed by mass spectrometry and reversed-phase HPLC.…”

Unveiling the diversity of cyclotides by combining peptidome and transcriptome analysis

“…It has to be noted that the optimised database used for the automated search included nucleotide‐derived sequences as will be described later. Recently the detection of PTMs using MS technologies revealed for the first time the presence of glycosylated forms of cyclotides within species of the violet family and thus further expanding the pool of cyclotides to be discovered. Although MS‐based analysis is indispensable for cyclotide characterization it faces limitations with regard to fully unveiling their sequence diversity among flowering plants.…”

“…Although MS‐based analysis is indispensable for cyclotide characterization it faces limitations with regard to fully unveiling their sequence diversity among flowering plants. This becomes apparent with estimated numbers of different sequences ranging between 9000 and 150 000 for individual plant families and up to a theoretical library of 600 million sequences . Thus it is evident that discovery solely based on MS is not able to fully harness the potential of this vast sequence variability.…”