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.

MolProbity score is a value normalized to be in the scale of X-ray resolution and combines clashes, rotamers, and Ramachandran values; a lower value is better than a higher.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES
Collection of Lineus longissimus. Living specimens of L. longissimus were collected and identified at the west coast of Sweden (Kosterfjord, 35 m depth) by Dr Malin Strand, Tjärnö Lovén Center, who also identified the specimens. Mucus was collected by placing specimens in a small container containing seawater and gently agitating the animal with a glass rod. Mucus was then collected and lyophilized. One specimen was cut into pieces, which were either flash-frozen in liquid nitrogen or placed in RNA-later® solution. The flash-frozen samples were stored at -80°C and the RNA-later® preserved samples were stored at -20°C, after overnight storage at 4°C, until further processing.
Peptide Isolation. The lyophilized mucus from one collection was dissolved in 12.5 ml 30% acetonitrile (AcN) in water and 0.1% formic acid (FA). Aliquots of 2.5 ml were desalted using size exclusion chromatography (SEC; PD-10, GE Healthcare). The high molecular weight eluate was collected and lyophilized before being redissolved in 10% AcN, 0.1% FA in water, and subjected to RP-HPLC on a Phenomenex Jupiter column (5µ C18 300Å, 250x4.6 mm) using a Shimadzu LC20 system equipped with a UV-detector. The gradient ranged from 5% AcN, 0.05% trifluoroacetic acid (TFA) to 55% AcN over 25 minutes. The three main peptides were subjected to quantitative amino acid analysis at the Amino Acid Analysis Center, Department of Biochemistry, Uppsala University.

LC-MS/MSMS and Peptide Sequencing.
Peptides were reduced and alkylated using dithiothreitol (DTT) and iodoacetamide (IAM). Alkylated peptides were desalted using SEC, and digested with trypsin, chymotrypsin and endoproteinase Glu-C, in separate experiments, prior to MS-sequencing 5 . In short, dry, reduced and alkylated peptide was dissolved in 50 mM NH 4  After reduction and alkylation, peptide masses were increased with 348 Da for the two smaller compounds and 464 Da for the larger one. These increments in mass correspond to the presence of three and four disulfide bonds, respectively. In the MSMS analyses, some fragments of α-1 and α-2 showed identical masses and retention times, demonstrating homology between peptides. Combined, the m/z 463 2+ and 679 2+ fragments revealed identical 14-residue long sequences. Other fragments differed between peptides, including two ions with Δ 47.95: the tryptic 701 2+ fragment of α-1 and 677 2+ of α-2. MSMS sequencing of these fragments showed that these peptide fragments differ by a Phe to Val substitution (Δ 48.00), as shown in Figure 3. In total, MSMS sequencing revealed 20 out of 31 residues of α-1 and α-2. No sequence could be determined for β-1 by MSMS alone. The presence of the two Hyp-residues was unambiguously confirmed by comparing retention times and MS/MSMS spectra of reduced, and reduced and alkylated, peptides with and without modified Pro-residues.
Total RNA Extraction and Transcriptome Analyses. Total RNA was extracted from both flashfrozen and samples stored in RNAlater®, using Qiagen AllPrep DNA/RNA Mini Kit. The combined total RNA was sent to Macrogen (Korea) for Illumina HiSeq 2000 based RNA-seq paired end analysis, and assembled by Macrogen using Trinity (v 2011-11-26) 6 . The assembled transcriptome was either translated into protein sequences using the EMBOSS getorf tool as utilized in the graphic user interface eBioX (v. 1.5.1), or for preparation of local nucleotide NCBI BLAST+ databases through Unipro uGENE's (v. 1.14.0) 7 implementation of NCBI BLAST+. The sequenced tryptic/chymotryptic peptides were used as query in tBLASTn or BLASTp BLAST+ searches in the local L. longissimus transcriptome databases to confirm and complete the sequence. The α-1, α-2 and β-1 sequences were blasted against public generalistic databases (NCBI, UniProt) and the specialized databases Conoserver 8 , and Arachnoserver 9 . The latter two databases are focused on toxin-like