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Macrocyclic polypeptides from plants
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
2002 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of this work was to explore the structural and functional diversity of polypeptides that are found in plants. Expanding knowledge of simililarities between plant use of these compound and animal use promises exceptional opportunities for finding, from plant research, new structures with biomedical and biotechnological potential.

A fractionation protocol was developed and applied to many plant species, providing fractions enriched in polypeptides, amenable to chemical and biological evaluation. From one species, the common field pansy (Viola arvensis), a 29-amino-acid residue polypeptide was isolated, named varv A, which revealed a remarkable macrocyclic structure (i.e., N- and C-termini are joined) stabilised by three knotted disulfides.

Varv A, together with an increasing number of homologous peptides, form the currently known peptide family of cyclotides. Their stable structure makes them an attractive scaffold for protein engineering. In addition, they display a wide range of biological activities (e.g., antimicrobial, cytotoxic, and insecticidal). As a part of this work, the cytotoxic effects of varv A and two other isolated cyclotides were evaluated in a human cell-line panel: all were active in the low µM range. Most likely, these effects involve pore formation through cell membranes.

Cyclotides were found to be common in the plant family Violaceae; with eleven cyclotides isolated and sequenced from V. arvensis, V. cotyledon, and Hybanthus parviflorus. For six members of the genus Viola, cyclotide expression profiles were examined by liquid chromatography-mass spectrometry (LC-MS): all expressed notably complex mixtures, with single species containing more than 50 cyclotides. These profiles reflect the evolution of the genus.

To assess these mixtures, a rational strategy for MS based amino acid sequencing of cyclotides was developed, circumventing inherent structural problems, such as low content of positively charged amino acids and the macrocyclic structure. This was achieved by aminoethylation of cysteines, which, following tryptic digestion, produced fragments of size and charge amenable to MS analysis. This method was also modified and used for mapping of disulfide bonds.

Methods for isolation and characterisation developed in this work may prove useful not only for further studies on macrocyclic polypeptides from plants, but also for other plant peptides and disulfide-rich peptides from animals.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis , 2002. , p. 59
Series
Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 0282-7484 ; 270
Keywords [en]
Pharmaceutical chemistry
Keywords [sv]
Farmaceutisk kemi
National Category
Medicinal Chemistry
Research subject
Pharmacognosy
Identifiers
URN: urn:nbn:se:uu:diva-1956ISBN: 91-554-5279-5 (print)OAI: oai:DiVA.org:uu-1956DiVA, id: diva2:161550
Public defence
2002-04-26, BMC sal B22, Uppsala, 10:15
Opponent
Available from: 2002-04-05 Created: 2002-04-05 Last updated: 2018-01-13Bibliographically approved
List of papers
1. Fractionation protocol for the isolation of polypeptides from plant biomass
Open this publication in new window or tab >>Fractionation protocol for the isolation of polypeptides from plant biomass
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1998 (English)In: Journal of natural products (Print), ISSN 0163-3864, E-ISSN 1520-6025, Vol. 61, no 1, p. 77-81Article in journal (Refereed) Published
Abstract [en]

A fractionation protocol for the isolation of a highly purified polypeptide fraction from plant biomass is described. The procedure dereplicates ubiquitous substance classes known to interfere with bioassays often used in natural product-based drug discovery programs. The protocol involves pre-extraction with dichloromethane, extraction with ethanol (50%), removal of tannins with polyamide, removal of low-molecular-weight components with size-exclusion chromatography over Sephadex G-10, and final removal of salts and polysaccharides with solid-phase extraction using reversed-phase cartridges. The method has been applied to the aerial parts of Viola arvensis, resulting in the isolation of a peptide fraction that on further separation yielded a novel 29-residue macrocyclic polypeptide named varv peptide A, cyclo(-TCVGGTCNTPGCSCSWPVCTRNGLPVCGE-).

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-89791 (URN)10.1021/np970342r (DOI)9548831 (PubMedID)
Available from: 2002-04-05 Created: 2002-04-05 Last updated: 2017-12-14Bibliographically approved
2. Seven novel macrocyclic polypeptides from Viola arvensis
Open this publication in new window or tab >>Seven novel macrocyclic polypeptides from Viola arvensis
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1999 In: J Nat Prod, Vol. 62, no 2, p. 283-286Article in journal (Refereed) Published
Identifiers
urn:nbn:se:uu:diva-89792 (URN)
Available from: 2002-04-05 Created: 2002-04-05Bibliographically approved
3. First cyclotide from Hybanthus (Violaceae)
Open this publication in new window or tab >>First cyclotide from Hybanthus (Violaceae)
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2001 (English)In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 58, no 1, p. 47-51Article in journal (Refereed) Published
Abstract [en]

Hypa A, a novel macrocyclic polypeptide containing 30 amino acid residues, has been isolated from the n-butanol extract of the Argentine plant Hybanthus parviflorus. The sequence, cyclo-(SCVYIPCTITALLGCSCKNKVCYNGIPCAE), was determined by automated Edman degradation, quantitative amino acid analysis and nanospray MS/MS(2). Three intramolecular disulfide bridges stabilize the cyclic peptide backbone of hypa A. Using these structural features to classify the peptide as a cyclotide, we extended the distribution of that substance class to a new genus, and now propose a uniform nomenclature for cyclotides.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-89793 (URN)10.1016/S0031-9422(01)00173-X (DOI)11524112 (PubMedID)
Available from: 2002-04-05 Created: 2002-04-05 Last updated: 2017-12-14Bibliographically approved
4. Cyclotides: a novel type of cytotoxic agents
Open this publication in new window or tab >>Cyclotides: a novel type of cytotoxic agents
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2002 (English)In: Molecular Cancer Therapeutics, ISSN 1535-7163, E-ISSN 1538-8514, Vol. 1, no 6, p. 365-369Article in journal (Refereed) Published
Abstract [en]

Cytotoxic activities of three naturally occurring macrocyclic peptides (cyclotides) isolated from the two violets, Viola arvensis Murr. and Viola odorata L., were investigated. A nonclonogenic fluorometric microculture assay was used to examine cytotoxicity in a panel of 10 human tumor cell lines representing defined types of cytotoxic drug resistance. Additionally, primary cultures of tumor cells from patients, and for comparison normal lymphocytes, were used to quantify cytotoxic activity. All three cyclotides, varv A, varv F, and cycloviolacin O2, exhibited strong cytotoxic activities, which varied in a dose-dependent manner. Cycloviolacin O2 was the most potent in all cell lines (IC50 0.1– 0.3 _M), followed by varv A (IC50 2.7–6.35 _M) and varv F (IC50 2.6 –7.4 _M), respectively. Activity profiles of the cyclotides differed significantly from those of antitumor drugs in clinical use, which may indicate a new mode of action. This, together with the exceptional chemical and biological stability of cyclotides, makes them interesting in particular for their potential as pharmacological tools and possibly as leads to antitumor agents.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-92805 (URN)000178770300001 ()12477048 (PubMedID)
Note

De två första författarna delar på förstaförfattarskapet.

Available from: 2005-04-01 Created: 2005-04-01 Last updated: 2017-12-14Bibliographically approved
5. Peptide profiling by LC-MS and MS sequencing of intercysteine loops: Expression profiles of Viola cyclotides
Open this publication in new window or tab >>Peptide profiling by LC-MS and MS sequencing of intercysteine loops: Expression profiles of Viola cyclotides
Article in journal (Refereed) Submitted
Identifiers
urn:nbn:se:uu:diva-89795 (URN)
Available from: 2002-04-05 Created: 2002-04-05Bibliographically approved

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