Peptide Toxins from Antarctica: The Nemertean Predator and Scavenger Parborlasia corrugatus (McIntosh, 1876).

Peptide toxins from marine invertebrates have found use as drugs and in biotechnological applications. Many marine habitats, however, remain underexplored for natural products, and the Southern Ocean is among them. Here, we report toxins from one of the top predators in Antarctic waters: the nemertean worm Parborlasia corrugatus (McIntosh, 1876). Transcriptome mining revealed a total of ten putative toxins with a cysteine pattern similar to that of alpha nemertides, four nemertide-beta-type sequences, and two novel full-length parborlysins. Nemertean worms express toxins in the epidermal mucus. Here, the expression was determined by liquid chromatography combined with mass spectrometry. The findings include a new type of nemertide, 8750 Da, containing eight cysteines. In addition, we report the presence of six cysteine-containing peptides. The toxicity of tissue extracts and mucus fractions was tested in an Artemia assay. Notably, significant activity was observed both in tissue and the high-molecular-weight mucus fraction, as well as in a parborlysin fraction. Membrane permeabilization experiments display the membranolytic activity of some peptides, most prominently the parborlysin fraction, with an estimated EC50 of 70 nM.

Spatial Distribution and Stability of Cholinesterase Inhibitory Protoberberine Alkaloids from Papaver setiferum.

During a research program to identify new cholinesterase inhibitors of natural origin, two new 7,8-didehydroprotoberberine alkaloids (1 and 2) and nine known compounds (3-11) were isolated from the capsules of the common ornamental poppy, Papaver setiferum (previously P. pseudo-orientale). Despite their reported instability, the 7,8-didehydroprotoberberines isolated herein appeared relatively stable, particularly as their trifluoroacetic acid salts. The spatial distributions of the isolated alkaloids were also analyzed using desorption electrospray ionization imaging mass spectrometry. The alkaloids were localized predominantly within the walls and vascular bundles of the capsules, with the highest relative abundances occurring in the lower half of the capsules toward the peduncle. The relative abundances of the alkaloids were also compared across plant development stages. Although most alkaloids did not show clear patterns in their concentration across development stages, the concentration of suspected oxidation products clearly spiked upon plant death. Finally, all isolated natural products were screened for inhibitory activities against a panel of cholinesterases, from both human and animal sources. These studies identified several competitive inhibitors of cholinesterases with potency in the low micromolar range (1-4, 6, 7), offering new lead compounds for the development of cholinesterase inhibitory drugs.

Functional Characterization of the Nemertide α Family of Peptide Toxins.

Peptide toxins find use in medicine, biotechnology, and agriculture. They are exploited as pharmaceutical tools, particularly for the investigation of ion channels. Here, we report the synthesis and activity of a novel family of peptide toxins: the cystine-knotted α nemertides. Following the prototypic α-1 and -2 (1 and 2), six more nemertides were discovered by mining of available nemertean transcriptomes. Here, we describe their synthesis using solid phase peptide chemistry and their oxidative folding by using an improved protocol. Nemertides α-2 to α-7 (2-7) were produced to characterize their effect on voltage-gated sodium channels (Blatella germanica BgNaV1 and mammalian NaVs1.1-1.8). In addition, ion channel activities were matched to in vivo tests using an Artemia microwell assay. Although nemertides demonstrate high sequence similarity, they display variability in activity on the tested NaVs. The nemertides are all highly toxic to Artemia, with EC50 values in the sub-low micromolar range, and all manifest preference for the insect BgNaV1 channel. Structure-activity relationship analysis revealed key residues for NaV-subtype selectivity. Combined with low EC50 values (e.g., NaV1.1: 7.9 nM (α-6); NaV1.3: 9.4 nM (α-5); NaV1.4: 14.6 nM (α-4)) this underscores the potential utility of α-nemertides for rational optimization to improve selectivity.

The Toxins of Nemertean Worms.

Most ribbon worms (phylum: Nemertea) are found in marine environments, where they act as predators and scavengers. They are characterized by an eversible proboscis that isused to hunt for prey and thick mucus covering their skin. Both proboscis and epidermal mucus mediate toxicity to predators and preys. Research into the chemical nature of the substances that render toxicity has not been extensive, but it has nevertheless led to the identification of several compounds of potential medicinal use or for application in biotechnology. This review provides a complete account of the current status of research into nemertean toxins.

Peptide ion channel toxins from the bootlace worm, the longest animal on Earth.

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 α-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 α-1, was isolated, synthesized, and its 3D NMR structure determined. Transcriptome analysis including 17 species revealed eight α-nemertides, mainly distributed in the genus Lineus. α-1 caused paralysis and death in green crabs (Carcinus maenas) at 1 µg/kg (~300 pmol/kg). It showed profound effect on invertebrate voltage-gated sodium channels (e.g. Blattella germanica Nav1) at low nanomolar concentrations. Strong selectivity for insect over human sodium channels indicates that α-nemertides can be promising candidates for development of bioinsecticidal agents.

Cyclotide Evolution: Insights from the Analyses of Their Precursor Sequences, Structures and Distribution in Violets (Viola).

Cyclotides are a family of plant proteins that are characterized by a cyclic backbone and a knotted disulfide topology. Their cyclic cystine knot (CCK) motif makes them exceptionally resistant to thermal, chemical, and enzymatic degradation. By disrupting cell membranes, the cyclotides function as host defense peptides by exhibiting insecticidal, anthelmintic, antifouling, and molluscicidal activities. In this work, we provide the first insight into the evolution of this family of plant proteins by studying the Violaceae, in particular species of the genus Viola. We discovered 157 novel precursor sequences by the transcriptomic analysis of six Viola species: V. albida var. takahashii, V. mandshurica, V. orientalis, V. verecunda, V. acuminata, and V. canadensis. By combining these precursor sequences with the phylogenetic classification of Viola, we infer the distribution of cyclotides across 63% of the species in the genus (i.e., ~380 species). Using full precursor sequences from transcriptomes, we show an evolutionary link to the structural diversity of the cyclotides, and further classify the cyclotides by sequence signatures from the non-cyclotide domain. Also, transcriptomes were compared to cyclotide expression on a peptide level determined using liquid chromatography-mass spectrometry. Furthermore, the novel cyclotides discovered were associated with the emergence of new biological functions.

The Bacterial (Vibrio alginolyticus) Production of Tetrodotoxin in the Ribbon Worm Lineus longissimus-Just a False Positive?

We test previous claims that the bacteria Vibrio alginolyticus produces tetrodotoxin (TTX) when living in symbiosis with the nemertean Lineus longissimus by a setup with bacteria cultivation for TTX production. Toxicity experiments on the shore crab, Carcinus maenas, demonstrated the presence of a paralytic toxin, but evidence from LC-MS and electrophysiological measurements of voltage-gated sodium channel-dependent nerve conductance in male Wistar rat tissue showed conclusively that this effect did not originate from TTX. However, a compound of similar molecular weight was found, albeit apparently non-toxic, and with different LC retention time and MS/MS fragmentation pattern than those of TTX. We conclude that C. maenas paralysis and death likely emanate from a compound <5 kDa, and via a different mechanism of action than that of TTX. The similarity in mass between TTX and the Vibrio-produced low-molecular-weight, non-toxic compound invokes that thorough analysis is required when assessing TTX production. Based on our findings, we suggest that re-examination of some published claims of TTX production may be warranted.

Exogenous plant hormones and cyclotide expression in Viola uliginosa (Violaceae).

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.