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.

Keywords: Violaceae; cyclotide; plant peptide; plant protein; stable proteins.

Figure 1

Schematic structure of a Möbius and bracelet cyclotide, together with typical cyclotide sequences from both subfamilies. The structures are based on the PDB files 1NB1 and 2KNM. The abbreviated notation Cy O2 and Cy O19 stand for the cycloviolacins O2 and O19, respectively. Note the unique features of the CCK motif: a cyclic backbone with sequence loops (1–6) and three stabilizing disulfide bonds. These disulfides are arranged in a cystine knot: that is, two of the disulfides form a ring structure together with the backbone connecting the four cysteines (I–IV; II–V), while the third disulfide is threaded through the ring (III–VI).

Figure 2

Classification and distribution of species in Violaceae. (A) Classification of Violaceae in accordance with the phylogenetic analysis by Wahlert et al. (2014) with additional taxonomical changes (de Paula-Souza and Ballard, ; de Paula-Souza and Pirani, ; Wahlert et al., 2015). The number of recognized species positively identified to contain cyclotides is tabulated next to the total number of species. The genera containing isolated cyclotides are indicated by bullet points (•). (B) Geographical origins of herbarium specimens sampled for mapping cyclotide diversity and occurrence in the Violaceae. Although the family has a cosmopolitan distribution, nearly all genera are tropical. The only exceptions are species from the genus Viola and the species from the genus Cubelium.

Figure 3

Base peak chromatograms (m/z = 800–1900) from six representative species in the Violaceae. The cyclotide region spanning from 25 to 40 min is shown, and major components are labeled with molecular weights (Da). Note that the LC-MS traces of Viola kiangsiensis and Viola sepincola contain more cyclotides, and also express varv A (mass 2877 eluting at 35 min), which is found in 2/3 of all Viola species.

Figure 4

Scatter plot data of all detected cyclotides. Each symbol represents one of the 1043 cyclotides detected in the 143 species. Some cyclotides were found in more than one species. The vertical clusters of peaks around 2900 Da reflect the more common cyclotides such as varv A, varv F, and kalata B1. Masses range from 2800 to 3700 Da and retention times range from 15 to 60 min.

Figure 5

Detection of glycosylated cyclotides. Cyclotides with additions of 162 mass units were observed for several of the cyclotides in the screening. (A) Shows representative MS-data for three cyclotides and their glycosylation products from three species. At the top, Viola odorata containing cycloviolacin O2 (cyO2) as demonstrated by the doubly and triply charged ions (1570.6²⁺/1047.7³⁺) and the corresponding ions for cyO2(gly) (marked with ^*). Cyclotides mram 1 from Melicytus ramiflorus (1610.4²⁺/1073.6³⁺) and mema A from Melicytus macrophyllus (1653.0²⁺/1102.5³⁺) and their respective glycosylated derivates show the same patterns of ions. Note that the cyO2 and mram 1 show signs of double glycosylations (^**). The cyclotide derivates with additional 162 Da (corresponding to one sugar residue) shown in the figure have also been sequenced by MS-MS, demonstrating that the glycosylation is localized in loop 5. Cyclotides and their glycosylated derivates elute closely (or co-elute) on RP-HPLC, but may be separated. (B) Shows the separation of cyO2 and cyO2(gly), which unambiguously define them as two separate molecular species. The separation was done using a Phenomenex Kinetex C18 column (150 × 4.6 mm, 2.6 μm) operated at a flow rate of 1 ml/min and a 2%/min gradient of acetonitrile in water, containing 0.1% formic acid.

Figure 6

Stability of cyclotides shown in an almost 200-year old sample. Base peak chromatogram (m/z = 800–1900) from five specimens of Viola odorata collected in the years 1820, 1849, 1886, 1948, and a commercially available Viola odorata (2006). The cyclotide region from 25 to 40 min is shown, and the major components are labeled. Note that the major cyclotide components—the cycloviolacins O2, O19, and varv A—are present in all samples.

Figure 7

Intercysteine loops of Möbius and bracelet cyclotides illustrating the plasticity of the cyclotide scaffold. The total number of different loops between each of the cysteines is shown as bars in upwards direction for the bracelet (including the hybrids) and downwards for Möbius cyclotide (including linear variants). The loops with longer sequences (2, 3, 5, and 6) clearly have more variants than the loops with few amino acids (1 and 4).