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.
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.
Phytochemistry. 2021 Jul;187:112749. doi: 10.1016/j.phytochem.2021.112749. Epub 2021 Apr 28.
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.
环肽是一类极其稳定的肽，广泛分布于紫堇科。本研究旨在利用转录组学和质谱联用的方法，研究斯里兰卡紫堇科植物中环肽的存在情况。首次在斯里兰卡野生植物中发现了新的环肽，这些环肽存在于作为抗菌剂用于传统医学的贝母堇菜 Viola betonicifolia 中。从小规模制备的贝母堇菜植物提取物首先进行 LC-MS 分析。随后对贝母堇菜进行转录组从头测序，发现了 25 个新的（vibe 1-25）和 3 个已知的（varv A/kalata S、viba 17、viba 11）肽序列，分别来自莫比乌斯和手链环肽亚科以及杂合环肽。在这些转录本中，还存在缺乏对环化至关重要的保守天冬酰胺或天冬氨酸的假定线性无环肽序列（vibe 4、vibe 10、vibe 11 和 vibe 22）。在贝母堇菜转录本中还发现了 4 种天冬酰胺内肽酶（AEPs），即 VbAEP1-4，包括一种肽天冬酰胺连接酶（PAL），可能参与环肽骨架环化，与 Viola yedoensis 肽天冬酰胺连接酶 VyPALs 的序列同源性>93%。此外，我们还鉴定了 2 种蛋白二硫键异构酶（PDIs），即 VbPDI1-2，可能参与环肽氧化折叠，与先前报道的茜草科和紫堇科 PDIs 的序列同源性>74%。本研究突出了环肽在紫堇科中的普遍性，以及转录组分析在环肽及其潜在加工酶发现中的应用。贝母堇菜中环肽序列在环大小和环内残基方面的高度变异性表明，环肽结构是一种适应性支架，作为一种组合文库，在植物防御中具有重要作用。
