Novel Cyclotides and Uncyclotides with Highly Shortened Precursors from Chassalia chartacea and Effects of Methionine Oxidation on Bioactivities

Cyclotides are a new class of plant biologics that display a diverse range of bioactivities with therapeutic potentials. They possess an unusual end-to-end cyclic backbone combined with a cystine knot arrangement, making them exceptionally stable to heat, chemical and enzymatic degradation. Currently, >200 cyclotides have been discovered but only three naturally occurring linear variants (also known as uncyclotides) have been isolated. In this study, we report the discovery of 18 novel peptides, chassatides C1 to C18, composed of 14 new cyclotides and four uncyclotides from Chassalia chartacea (Rubiaceae family). Thus far, this is the largest number of uncyclotides being reported in a single species. Activity testing showed that the uncyclotides not only retain the effectiveness but also are the most potent chassatides in the assays for antimicrobial, cytotoxic, and hemolytic activities. Genetic characterization of novel chassatides revealed that they have the shortest precursors of all known cyclotides hitherto isolated, which represents a new class of cyclotide precursors. This is the first report of cyclotide genes in a second genus, the Chassalia, other than the Hedyotis (Oldenlandia) of the Rubiaceae family. In addition, we also report the characterization of two Met-oxidized derivatives of chassatides C2 and C11. The oxidation of Met residue causes loss of bioactivities, strengthening the importance of the hydrophobic patch for membrane interaction. Cyclotides are biologically active, plant-derived macrocyclic peptides. Only three naturally occurring linear variants have been discovered. We discovered 14 novel cyclotides and four uncyclotides from Chassalia chartacea. Their precursors are the shortest of all known cyclotides. Uncyclotides have comparable activities to cyclotides. Met oxidation disrupts the hydrophobic patch causing loss of activity. Our study broadens knowledge of cyclotides and uncyclotides.