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Studies on cysteine-rich peptides from Nemertea and Violaceae: Proteomic and transcriptomic discovery and characterization
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, Farmakognosi. (Farmakognosi)
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The overall aims of the projects included in this thesis were to discover, synthesize and characterize disulphide-stabilized peptides from marine worms (Nemertea sp.) and plants (Viola sp.). 

One of the main outcomes of this thesis is the discovery of a new family of highly active cysteine-rich toxins, alpha nemertides, from nemertean worms (paper II). Functional characterization and production routes of nemertides were further explored (papers II-III). In addition, 12 new cyclotides from the bog violet were discovered (paper I). Finally, transcriptomes and mucus of the Antarctic nemertean Parborlasia corrugatus were investigated for toxin content (paper IV).

 In paper I wild-type leaf and callus tissue of the endangered bog violet, V. uliginosa, were analyzed using transcriptomics and LC-MS, resulting in the discovery of 12 new cyclotides (i.e. cysteine-rich cyclic peptides). In addition, cyclotide expression under different cell-growth conditions was monitored.

In paper II  the discovery and initial characterization of a new family of highly active peptides, the alpha nemertides, from the epidermal mucus of the world’s longest animal; Lineus longissimus is described. The most abundant alpha nemertide, alpha-1, was extracted in minute amounts, prompting the use solid phase peptide synthesis (SPPS) for further characterization. The tertiary structure of alpha-1 was elucidated and revealed an inhibitory cystine knot (ICK) framework. The knotted core-structure is similar to the cyclic cystine knot (CCK) motif, found in the cyclotides described in paper I.

In manuscript III, the production route established in paper II was used to produce nemertides alpha 1-7. These were tested in vivo in an Artemia microwell assay as well as on an extended panel of voltage-gated sodium channels (NaV1.1 – 1.8 and BgNaV1). All seven alpha nemertides were highly active in the in vivo Artemia assay with EC50 values in the sub to low µM range. The alpha nemertides were also active in the NaVs tested. However, differences in the activity profiles were observed, indicating an opportunity for future optimization of alpha nemertides to reach higher specificity to certain NaV subtypes.

In manuscript IV, the exploration of nemertide toxins was extended to include the Antarctic P. corrugatus. Resulting findings include a set of cysteine-rich peptides, some similar to the nemertides previously discovered in paper II. Two purified peptides and one fraction were evaluated for their membranolytic activity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. , p. 66
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 277
Keywords [en]
Peptide toxin, cystine knot, nemertide, cyclotide, nemertea.
National Category
Pharmacology and Toxicology
Research subject
Pharmacognosy
Identifiers
URN: urn:nbn:se:uu:diva-390885ISBN: 978-91-513-0719-0 (print)OAI: oai:DiVA.org:uu-390885DiVA, id: diva2:1343187
Public defence
2019-09-20, BMC A1:107a, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-08-30 Created: 2019-08-15 Last updated: 2019-09-17
List of papers
1. Exogenous plant hormones and cyclotide expression in Viola uliginosa (Violaceae)
Open this publication in new window or tab >>Exogenous plant hormones and cyclotide expression in Viola uliginosa (Violaceae)
2015 (English)In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 117, p. 527-536Article in journal (Refereed) Published
Abstract [en]

Plants from Violaceae produce cyclotides, peptides characterized by a circular peptide backbone and a cystine knot. This signature motif gives stability that can harness a wide spectrum of biological activities, with implications in plant defense and with applications in medicine and biotechnology. In the current work, cyclotide expressing in vitro cultures were established from Viola uliginosa. These cultures are useful models for studying biosynthesis of cyclotides and can also be used in their production. The cyclotide expression pattern is shown to be dependent on exogenous plant growth regulators, both on peptide and gene expression levels. The highest yields of cyclotides were obtained on media containing only a cytokinin and were correlated with storage material accumulation. Exposure to auxins decreased cyclotide production and caused shifting of the biosynthesis pattern to root specific cyclotides. The response to stimuli in terms of cyclotide expression pattern appears to be developmental, and related to polar auxin transportation and the auxin/cytokinin ratio regulating tissue differentiation. By the use of whole transcriptome shotgun sequencing (WTSS) and peptidomics, 20 cyclotide sequences from V. uliginosa (including 12 new) and 12 complete precursor proteins could be identified. The most abundant cyclotides were cycloviolacin O3 (CyO3), CyO8 and CyO13. A suspension culture was obtained that grew exponentially with a doubling time of approximately 3 days. After ten days of growth, the culture provided a yield of more than 4 mg CyO13 per gram dry mass.

Keywords
Viola uliginosa (Violaceae), Cyclotides, In vitro culture, Plant growth regulators, Whole transcriptome shotgun sequencing, Mass spectrometry
National Category
Botany Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-264668 (URN)10.1016/j.phytochem.2015.07.016 (DOI)000361253300055 ()26246035 (PubMedID)
Funder
Swedish Research Council, 621-2007-5167Swedish Foundation for Strategic Research , F06-0058
Available from: 2015-10-16 Created: 2015-10-15 Last updated: 2019-08-15
2. Peptide ion channel toxins from the bootlace worm, the longest animal on Earth
Open this publication in new window or tab >>Peptide ion channel toxins from the bootlace worm, the longest animal on Earth
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 4596Article in journal (Refereed) Published
Abstract [en]

Polypeptides from animal venoms have found important uses as drugs, pharmacological tools, and within biotechnological and agricultural applications. We here report a novel family of cystine knot peptides from nemertean worms, with potent activity on voltage-gated sodium channels. These toxins, named the alpha-nemertides, were discovered in the epidermal mucus of Lineus longissimus, the 'bootlace worm' known as the longest animal on earth. The most abundant peptide, the 31-residue long alpha-1, was isolated, synthesized, and its 3D NMR structure determined. Transcriptome analysis including 17 species revealed eight alpha-nemertides, mainly distributed in the genus Lineus. alpha-1 caused paralysis and death in green crabs (Carcinus maenas) at 1 mu g/kg (similar to 300 pmol/kg). It showed profound effect on invertebrate voltage-gated sodium channels (e.g. Blattella germanica Na(v)1) at low nanomolar concentrations. Strong selectivity for insect over human sodium channels indicates that a-nemertides can be promising candidates for development of bioinsecticidal agents.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-351585 (URN)10.1038/s41598-018-22305-w (DOI)000428029600001 ()29567943 (PubMedID)
Funder
Swedish Research Council, 2014-3327]
Available from: 2018-05-29 Created: 2018-05-29 Last updated: 2019-08-15Bibliographically approved
3. Functional characterization of the nemertide alpha family of peptide toxins
Open this publication in new window or tab >>Functional characterization of the nemertide alpha family of peptide toxins
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(English)Manuscript (preprint) (Other academic)
National Category
Pharmacology and Toxicology
Research subject
Pharmacognosy
Identifiers
urn:nbn:se:uu:diva-390850 (URN)
Funder
Swedish Research Council, 2014-3327Swedish Research Council, 2018-005403Swedish Research Council Formas, 2018-00613
Available from: 2019-08-15 Created: 2019-08-15 Last updated: 2019-08-15
4. Peptide toxins from the Antarctica: the nemertean predator and scavenger Parborlasia corrugatus
Open this publication in new window or tab >>Peptide toxins from the Antarctica: the nemertean predator and scavenger Parborlasia corrugatus
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(English)Manuscript (preprint) (Other academic)
National Category
Pharmacology and Toxicology
Research subject
Pharmacognosy; Pharmacognosy
Identifiers
urn:nbn:se:uu:diva-390880 (URN)
Funder
Swedish Research Council, 2014-3327Swedish Research Council, 2018-005403Swedish Research Council Formas, 2018-00613
Available from: 2019-08-15 Created: 2019-08-15 Last updated: 2019-08-15

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