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The alpine violet, Viola biflora, is a rich source of novel cyclotides with potent cytotoxic cytotoxicity
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry, Division of Pharmacognosy.
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2008 (English)In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 69, no 4, p. 939-952Article in journal (Refereed) Published
Abstract [en]

The cyclotides are currently the largest known family of head-to-tail cyclic proteins. The complex structure of these small plant proteins, which consist of approximately 30 amino acid residues, contains both a circular peptide backbone and a cystine knot, the combination of which produces the cyclic cystine knot motif. To date, cyclotides have been found in plants from the Rubiaceae, Violaceace and Cucurbitaceae families, and are believed to be part of the host defence system. In addition to their insecticidal effect, cyclotides have also been shown to be cytotoxic, anti-HIV, antimicrobial and haemolytic agents. In this study, we show that the alpine violet Viola biflora (Violaceae) is a rich source of cyclotides. The sequences of 11 cyclotides, vibi A-K, were determined by isolation and MS/MS sequencing of proteins and screening of a cDNA library of V. biflora in parallel. For the cDNA screening, a degenerate primer against a conserved (AAFALPA) motif in the cyclotide precursor ER signal sequence yielded a series of predicted cyclotide sequences that were correlated to those of the isolated proteins. There was an apparent discrepancy between the results of the two strategies as only one of the isolated proteins could be identified as a cDNA clone. Finally, to correlate amino acid sequence to cytotoxic potency, vibi D, E, G and H were analysed using a fluorometric microculture cytotoxicity assay using a lymphoma cell line. The IC50-values of the bracelet cyclotides vibi E, G and H ranged between 0.96 and 5.0 mu M while the Mobius cyclotide vibi D was not cytotoxic at 30 mu M.

Place, publisher, year, edition, pages
2008. Vol. 69, no 4, p. 939-952
Keywords [en]
Viola biflora L., Violaceae, arctic yellow-violet, two-flower violet, cyclotides, circular proteins, cDNA screening, cytotoxicity, MS/MS, anticancer
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-96355DOI: 10.1016/j.phytochem.2007.10.023ISI: 000254149800011OAI: oai:DiVA.org:uu-96355DiVA, id: diva2:170904
Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2018-01-13Bibliographically approved
In thesis
1. Structure and Activity of Circular Plant Proteins: Cytotoxic Effects of Viola Cyclotides
Open this publication in new window or tab >>Structure and Activity of Circular Plant Proteins: Cytotoxic Effects of Viola Cyclotides
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Cyclotides are a family of small and macrocyclic proteins that have been found in Violacaee and Rubiaceae plant species. These proteins contain a cystine knot: two disulfides bonds together with their connecting peptide backbone form an embedded ring which is penetrated by a third disulfide bond. The cyclotides have been attributed a wide range of biological activities, which in combination with their chemical stability and structural plasticity have made them attractive tools for pharmaceutical applications.

The sequence of eleven novel cyclotides, vibi A-K, from Viola biflora was determined by the use of both chemical (extraction and characterization) and molecular biology (cDNA analyses) approaches. A clear discrepancy in the results from the two methods was observed. Additionally, one novel cyclotide, vodo O, was isolated from Viola odorata. To correlate cytotoxic potency to sequence, vodo O and vibi D, E, G and H were tested on a lymphoma cell line.

Based on the presence or absence of a cis-Pro bond, the cyclotides are divided into the Möbius and bracelet subfamilies. The bracelet proteins have a higher net charge and are more cytotoxic potent than the Möbius ones. To explore these differences, charged and hydrophobic residues in varv A (Möbius) and cycloviolacin O2 (bracelet) were chemically modified and tested for their cytotoxicity. The net-charge of the two proteins was not important for the potency. The Glu residue in cycloviolacin O2 was crucial, while this residue was of minor importance in varv A. Oxidation of the single Trp residue declined the potency significantly in both proteins. To evaluate how the surface properties correlate to the degree of cytotoxic potency, models of all cyclotides hitherto tested were constructed by homology modelling. Calculations showed that the membrane orientation of varv A and cycloviolacin O2 differed significantly, which might explain their difference in potency

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2007. p. 64
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 64
Keywords
Pharmacognosy, cyclotide, cytotoxic, anti-tumour, macrocyclic, cyclic cystine knot, Alpine violet, Sweet violet, Viola, Violaceae, chemical modifications, structure-activity studies, mass spectroscopy, cDNA clones, Farmakognosi
Identifiers
urn:nbn:se:uu:diva-8283 (URN)978-91-554-7003-6 (ISBN)
Public defence
2007-11-30, B22, BMC, Husargatan 3, Uppsala, 10:15 (English)
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Available from: 2007-11-08 Created: 2007-11-08 Last updated: 2009-10-07Bibliographically approved

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