Tropical vibes from Sri Lanka - cyclotides from Viola betonicifolia by transcriptome and mass spectrometry analysis

Sanjeevan Rajendran; Blazej Slazak; Supun Mohotti; Adam A Strömstedt; Ulf Göransson; Chamari M Hettiarachchi; Sunithi Gunasekera

doi:10.1016/j.phytochem.2021.112749

Tropical vibes from Sri Lanka - cyclotides from Viola betonicifolia by transcriptome and mass spectrometry analysis

Phytochemistry. 2021 Jul:187:112749. doi: 10.1016/j.phytochem.2021.112749. Epub 2021 Apr 28.

Authors

Sanjeevan Rajendran¹, Blazej Slazak², Supun Mohotti³, Adam A Strömstedt⁴, Ulf Göransson⁴, Chamari M Hettiarachchi³, Sunithi Gunasekera⁵

Affiliations

¹ Pharmacognosy, Department of Pharmaceutical Biosciences, Uppsala University, 751 23, Uppsala, Sweden; Department of Chemistry, Faculty of Science, University of Colombo, Thurston Rd, Colombo 03, Sri Lanka.
² Pharmacognosy, Department of Pharmaceutical Biosciences, Uppsala University, 751 23, Uppsala, Sweden; W. Szafer Institute of Botany, Polish Academy of Science, 46 Lubicz St., 31-512, Cracow, Poland.
³ Department of Chemistry, Faculty of Science, University of Colombo, Thurston Rd, Colombo 03, Sri Lanka.
⁴ Pharmacognosy, Department of Pharmaceutical Biosciences, Uppsala University, 751 23, Uppsala, Sweden.
⁵ Pharmacognosy, Department of Pharmaceutical Biosciences, Uppsala University, 751 23, Uppsala, Sweden. Electronic address: sunithi.gunasekera@farmbio.uu.se.

PMID: 33932786
DOI: 10.1016/j.phytochem.2021.112749

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.

Keywords: Cyclisation enzyme; Cyclotides; LC-MS; Protein disulfide isomerase; Transcriptome de novo sequencing; Viola betonicifolia; Violaceae.

MeSH terms

Amino Acid Sequence
Cyclotides* / genetics
Mass Spectrometry
Plant Proteins / metabolism
Sri Lanka
Transcriptome
Viola* / genetics
Viola* / metabolism

Substances

Cyclotides
Plant Proteins