Abstract
Cyclotides are an extremely stable class of peptides, ubiquitously distributed in Violaceae. The aim of the present study was to investigate the presence of cyclotides in Sri Lankan Violaceae plants, using combined tools of transcriptomics and mass spectrometry. New cyclotides were discovered for the first time in the wild flora of Sri Lanka, within Viola betonicifolia, a plant used in traditional medicine as an antimicrobial. Plant extracts prepared in small scale from Viola betonicifolia were first subjected to LC-MS analysis. Subsequent transcriptome de novo sequencing of Viola betonicifolia uncovered 25 new (vibe 1–25) and three known (varv A/kalata S, viba 17, viba 11) peptide sequences from Möbius and bracelet cyclotide subfamilies as well as hybrid cyclotides. Among the transcripts, putative linear acyclotide sequences (vibe 4, vibe 10, vibe 11 and vibe 22) that lack a conserved asparagine or aspartic acid vital for cyclisation were also present. Four asparagine endopeptidases (AEPs), VbAEP1-4 were found within the Viola betonicifolia transcriptome, including a peptide asparaginyl ligase (PAL), potentially involved in cyclotide backbone cyclisation, showing >93% sequence homology to Viola yedoensis peptide asparaginyl ligases, VyPALs. In addition, we identified two protein disulfide isomerases (PDIs), VbPDI1-2, likely involved in cyclotide oxidative folding, having high sequence homology (>74%) with previously reported Rubiaceae and Violaceae PDIs. The current study highlights the ubiquity of cyclotides in Violaceae as well as the utility of transcriptomic analysis for cyclotides and their putative processing enzyme discovery. The high variability of cyclotide sequences in terms of loop sizes and residues in V. betonicifolia showcase the cyclotide structure as an adaptable scaffold as well as their importance as a combinatorial library, implicated in plant defense.
Highlights
IntroductionCyclotides show a diverse range of bioactivities including antimicrobial (Pranting et al., 2010; Tam et al, 1999), cytotoxic (Lindholm et al, 2002; Schopke et al, 1993), anti-HIV (Gustafson et al, 1994, 2000), insecticidal (Jennings et al, 2001) and uterotonic activities (Lorents Gran, 1973; Sletten and Gran, 1973)
Cyclotides are an exceptionally stable family of gene-encoded plant miniproteins
A total of 28 cyclotide precursor sequences were prepared as DNA sequences for the sequence alignment (Fig. 2, Supplementary Data S1), comprising selected domains of the precursor, i.e., N-terminal propep tide (NTPP), N-terminal repeat (NTR), and mature cyclotide domains
Summary
Cyclotides show a diverse range of bioactivities including antimicrobial (Pranting et al., 2010; Tam et al, 1999), cytotoxic (Lindholm et al, 2002; Schopke et al, 1993), anti-HIV (Gustafson et al, 1994, 2000), insecticidal (Jennings et al, 2001) and uterotonic activities (Lorents Gran, 1973; Sletten and Gran, 1973). Cyclotides are ribosomally synthesised and post-translationally modified peptides (RiPPs) (Van der Donk., 2013; Dutton et al, 2004; Jennings et al, 2001) This allows for their identification by genetic tools, presenting complementing tools for peptide discovery. Cyclotides grouped into the Mobius subfamily contain a Pro reside in loop 5 usually preceded by a Trp residue, facilitating a cis-Pro bond and resulting in a conceptual 180◦ twist in the backbone (Craik et al, 1999). Variants of naturally occurring cyclotides with an open backbone were identified in plants and termed acyclotides (Nguyen et al, 2012; Poth et al, 2012)
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