In Planta Discovery and Chemical Synthesis of Bracelet Cystine Knot Peptides from Rinorea bengalensis

Abstract

Cyclotides are plant-derived peptides that have attracted interest as biocides and scaffolds for the development of stable peptide therapeutics. Cyclotides are characterized by their cyclic backbone and cystine knot framework, which engenders them with remarkably high stability. This study reports the cystine knotrelated peptidome of Rinorea bengalensis, a small rainforest tree in the Violaceae family that is distributed from Australia westward to India. Surprisingly, many more acyclic knotted peptides (acyclotides) were discovered than cyclic counterparts (cyclotides), with 32 acyclotides and 1 cyclotide sequenced using combined transcriptome and proteomic analyses. Nine acyclotides were isolated and screened against a panel of mammalian cell lines, showing they had the cytotoxic properties normally associated with cyclotide-like peptides. NMR analysis of the acyclotide ribes 21 and 22 and the cyclotide ribe 33 confirmed that these peptides contained the cystine knot structural motif. The bracelet-subfamily cyclotide ribe 33 was amenable to chemical synthesis in reasonable yield, an achievement that has long eluded previous attempts to synthetically produce bracelet cyclotides. Accordingly, ribe 33 represents an exciting new bracelet cyclotide scaffold that can be subject to chemical modification for future molecular engineering applications.

Plant derived cyclic peptides

“…cyclotide related peptides that do not contain a cyclic backbone, which have been referred to as acyclotides) [ 17 ]. In contrast, analysis of another Violaceae plant species, Rinorea bengalensis , showed a large number of acyclotides and only one cyclotide sequence [ 18 ]. Despite the lack of the cyclic backbone, several of these peptides have been shown to have cytotoxic activities similar to their cyclic counterparts [ 18 ].…”

“…In contrast, analysis of another Violaceae plant species, Rinorea bengalensis , showed a large number of acyclotides and only one cyclotide sequence [ 18 ]. Despite the lack of the cyclic backbone, several of these peptides have been shown to have cytotoxic activities similar to their cyclic counterparts [ 18 ]. New cyclotide sequences have also recently been found in Palicourea sessilis , that have potential as immunosuppressants [ 19 ].…”

The acyclotide ribe 31 from Rinorea bengalensis has selective cytotoxicity and potent insecticidal properties in Drosophila

“…Acyclotides have a similar sequence to cyclotides and also contain a cystine knot, but lack a head-to-tail cyclic backbone. The backbone cyclization is absent due to a stop codon occupying the position that would encode an Asx (Asn or Asp) residue in a typical cyclotide precursor (20). This key Asx residue and an adjacent C-terminal tail peptide in the precursor facilitates cyclization in cyclotides by asparaginyl endopeptidase (AEP) enzymes in plants (19,21).…”

“…This key Asx residue and an adjacent C-terminal tail peptide in the precursor facilitates cyclization in cyclotides by asparaginyl endopeptidase (AEP) enzymes in plants (19,21). Despite their acyclic backbone, acyclotides have comparable stability and structures to cyclotides, primarily due to the stabilizing effect of the cystine knot motif (20,22). Apart from local changes at the terminal residues, the NMR structures of two acyclotides, violacin A and MCoTI-V, have shown that linearization has negligible effects upon the structure of the cystine knot core (23,24).…”

“…In the current study, a family of acyclotides previously discovered from the stem and leaf of Rinorea bengalensis, named ribe peptides (20), were isolated and evaluated for their potential insecticidal activity. The toxicity of these peptides was examined against two insect cell lines in vitro and tested for in vivo insecticidal activity against D. melanogaster (Fig.…”