Journal of Biological Chemistry
Volume 295, Issue 32, 7 August 2020, Pages 10911-10925
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Protein Structure and Folding
Discovery and mechanistic studies of cytotoxic cyclotides from the medicinal herb Hybanthus enneaspermus

https://doi.org/10.1074/jbc.RA120.012627Get rights and content
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Cyclotides are plant-derived peptides characterized by an ∼30-amino acid–long cyclic backbone and a cystine knot motif. Cyclotides have diverse bioactivities, and their cytotoxicity has attracted significant attention for its potential anticancer applications. Hybanthus enneaspermus (Linn) F. Muell is a medicinal herb widely used in India as a libido enhancer, and a previous study has reported that it may contain cyclotides. In the current study, we isolated 11 novel cyclotides and 1 known cyclotide (cycloviolacin O2) from H. enneaspermus and used tandem MS to determine their amino acid sequences. We found that among these cyclotides, hyen C comprises a unique sequence in loops 1, 2, 3, 4, and 6 compared with known cyclotides. The most abundant cyclotide in this plant, hyen D, had anticancer activity comparable to that of cycloviolacin O2, one of the most cytotoxic known cyclotides. We also provide mechanistic insights into how these novel cyclotides interact with and permeabilize cell membranes. Results from surface plasmon resonance experiments revealed that hyen D, E, L, and M and cycloviolacin O2 preferentially interact with model lipid membranes that contain phospholipids with phosphatidyl-ethanolamine headgroups. The results of a lactate dehydrogenase assay indicated that exposure to these cyclotides compromises cell membrane integrity. Using live-cell imaging, we show that hyen D induces rapid membrane blebbing and cell necrosis. Cyclotide–membrane interactions correlated with the observed cytotoxicity, suggesting that membrane permeabilization and disintegration underpin cyclotide cytotoxicity. These findings broaden our knowledge on the indigenous Indian herb H. enneaspermus and have uncovered cyclotides with potential anticancer activity.

cyclic peptide
peptide chemical synthesis
cyclotide
nuclear magnetic resonance (NMR)
cancer cell cytotoxicity
peptide-membrane interaction
anticancer property
surface plasmon resonance (SPR)
flow cytometry
microscopy
cancer cell cytoxicity

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This article contains supporting information.

Author contributions—Q. D. and D. J. C. conceptualization; Q. D. and Q. K. data curation; Q. D., E. K. G., N. D. C., and Q. K. formal analysis; Q. D., L. Y. C., E. K. G., S. T. H., Q. K., Y-H. H., and D. J. C. investigation; Q. D. writing-original draft; L. Y. C., Q. K., Y-H. H., and D. J. C. supervision; L. Y. C., Y-H. H., E. K. G., and S. T. H. methodology; L. Y. C., E. K. G., S. T. H., N. D. C., A. S. R., Q. K., Y-H. H., and D. J. C. writing-review and editing; A. S. R. resources; D. J. C. funding acquisition; D. J. C. project administration.

Funding and additional information—This work was supported by the Australian Research Council (ARC) Grant DP150100443 (to E. K. G. and D. J. C) and National Health and Medical Research Council Grant APP1084965 (to S. T. H. and D. J. C.). This work was also supported by Advance Queensland Women's Academic Fund Grant WAF-6884942288 (to L. Y. C.). Q. D. is supported by the China Scholarship Council.

Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Present address for Anjaneya S. Ravipati: MWAC, Bioanalytical Mass Spectrometry Facility, The University of New South Wales, Sydney, New South Wales, Australia

Abbreviations—The abbreviations used are:

    ARC

    Australian Research Council

    PE

    phosphatidylethanolamine

    RP-HPLC

    reversed phase HPLC

    TOCSY

    total correlation spectroscopy

    kB1

    kalata B1

    RBC

    red blood cell

    POPC

    phospholipid 1-palmitoyloleoyl-phosphatidylcholine

    LDH

    lactate dehydrogenase

    TB

    trypan blue

    SPR

    surface plasmon resonance

    P/L max

    peptide-to-lipid maximum binding

    FA

    formic acid

    BMRB

    Biological Magnetic Resonance Bank

    HUVEC

    human umbilical vein endothelial cells

    POPS

    1-palmitoyloleoyl-phosphatidylserine.