Discovery and Characterization of Novel Cyclotides Originated from Chimeric Precursors Consisting of Albumin-1 Chain a and Cyclotide Domains in the Fabaceae Family

Abstract

The tropical plant Clitoria ternatea is a member of the Fabaceae family well known for its medicinal values. Heat extraction of C. ternatea revealed that the bioactive fractions contained heat-stable cysteine-rich peptides (CRPs). The CRP family of A1b (Albumin-1 chain b/leginsulins), which is a linear cystine knot CRP, has been shown to present abundantly in the Fabaceae. In contrast, the cyclotide family, which also belongs to the cystine knot CRPs but with a cyclic structure, is commonly found in the Rubiaceae, Violaceae, and Cucurbitaceae families. In this study, we report the discovery of a panel of 15 heat-stable CRPs, of which 12 sequences (cliotide T1-T12) are novel. We show unambiguously that the cliotides are cyclotides and not A1bs, as determined by their sequence homology, disulfide connectivity, and membrane active properties indicated by their antimicrobial activities against Escherichia coli and cytotoxicities to HeLa cells. We also show that cliotides are prevalent in C. ternatea and are found in every plant tissue examined, including flowers, seeds, and nodules. In addition, we demonstrate that their precursors are chimeras, half from cyclotide and the other half from Albumin-1, with the cyclotide domain displacing the A1b domain in the precursor. Their chimeric structures likely originate from either horizontal gene transfer or convergent evolution in plant nuclear genomes, which are exceedingly rare events. Such atypical genetic arrangement also implies a different mechanism of biosynthetic processing of cyclotides in the Fabaceae and provides new understanding of their evolution in plants.

Discovery of Linear Cyclotides in Monocot Plant Panicum laxum of Poaceae Family Provides New Insights into Evolution and Distribution of Cyclotides in Plants

“…It provides new insights in understanding their distribution and evolution in plants, particularly in agronomically important food crops. This finding is also timely because of the recent discovery of cyclotides in other food crops such as Fabaceae (legume) and Solanaceae (potato) families (9,15). The Poaceae is the fifth largest plant family with over 10,000 species divided into 12 subfamilies and over 700 genera (28).…”

“…In the Fabaceae, the architecture of the cyclotide-containing precursor is distinctly different. The N-terminal proto-domain is totally absent in their precursors, which contain only the ER signal sequence followed directly by a mature cyclotide domain, a short linker region, and an albumin-1 chain a domain (9,17).…”

“…Cyclotides exhibit a diverse range of biological activities, including insecticidal (4), antimicrobial (5), anti-HIV (6), neurotensin inhibition (7), and cytotoxicity (8,9). They are found in a variety of dicot plants, including the Cucurbitaceae (10), Rubiaceae (11), Violaceae (12,13), Fabaceae (9,14), and Solanaceae families (15).…”

“…They are ribosomally synthesized as linear precursors, excised at the N and C termini of their mature cyclotide domains, and then head-to-tail ligated to produce mature cyclic peptides (4). Their encoding genes were first characterized from the Rubiaceae plant Oldenlandia affinis (4) and subsequently in several other plant species (9,(15)(16)(17)(18)(19). The predicted precursors of cyclotides display low sequence homology among the known cyclotide-producing families, and sometimes even between species of the same genus.…”

“…They are found in a variety of dicot plants, including the Cucurbitaceae (10), Rubiaceae (11), Violaceae (12,13), Fabaceae (9,14), and Solanaceae families (15). They are ribosomally synthesized as linear precursors, excised at the N and C termini of their mature cyclotide domains, and then head-to-tail ligated to produce mature cyclic peptides (4).…”