Identification of unusual peptides with new Cys frameworks in the venom of the cold-water sea anemone Cnidopus japonicus

Sea anemones (Actiniaria) are intensely popular objects of study in venomics. Order Actiniaria includes more than 1,000 species, thus presenting almost unlimited opportunities for the discovery of novel biologically active molecules. The venoms of cold-water sea anemones are studied far less than the venoms of tropical sea anemones. In this work, we analysed the molecular venom composition of the cold-water sea anemone Cnidopus japonicus. Two sets of NGS data from two species revealed molecules belonging to a variety of structural classes, including neurotoxins, toxin-like molecules, linear polypeptides (Cys-free), enzymes, and cytolytics. High-throughput proteomic analyses identified 27 compounds that were present in the venoms. Some of the toxin-like polypeptides exhibited novel Cys frameworks. To characterise their function in the venom, we heterologously expressed 3 polypeptides with unusual Cys frameworks (designated CjTL7, CjTL8, and AnmTx Cj 1c-1) in E. coli. Toxicity tests revealed that the CjTL8 polypeptide displays strong crustacean-specific toxicity, while AnmTx Cj 1c-1 is toxic to both crustaceans and insects. Thus, an improved NGS data analysis algorithm assisted in the identification of toxins with unusual Cys frameworks showing no homology according to BLAST. Our study shows the advantage of combining omics analysis with functional tests for active polypeptide discovery.

: Phylogenetic analysis of Zn-dependent metalloproteinases from C. japonicus transcripts.
Trx -bacterial thioredoxin, PT7 -T7 promotor, term -T7 terminator, 6His -hexa histidine-tag, HindIII(729) and BamHI(579) -restriction sites for DNA fragments, encoding toxins cloning. A B Fig. S5: SDS-PAGE gel electrophoresis scan. TrxA-Toxin fusion proteins were applied on the gel as well as standard SDS-PAGE markers. Short names of tracks imply: Mstandard markers; 1c1fusion of AnmTX Cj 1c-1; TL7fusion of CjTL7; TL8fusion of CjTL8; X1 and X2two fusion proteins not used in this particular research; Cg -fusion of δ-actitoxin-Cgg1a; CgMfusion of Cys→Ser mutant of δactitoxin-Cgg1a.         Table S4: Polypeptide sequences of proteins from sea anemone C. japonicus venom. E-value is calculated by pBLAST for the closest homologs. Specie name from which the homolog is isolated and superfamily accessory by UniProt/CD are listed. (VPvenom protein; PVPpossible venom protein; LPVPlow possibility venom protein). If signal sequence is found, length is denoted for both precursor and mature proteins (for mature proteins numbers are shown in parentheses).    Paralysis is immediate, with no remission 6 Action of the negative control -Bovine Serum Albumin (BSA) solution on shrimps** 13 did not happen did not happen -6 260 did not happen did not happen -6 Action of the negative control -physiological salt solution on shrimps** -did not happen did not happen -6 Acute toxicity action of AnmTX Cj 1c-1 on insect larvae***  Paralysis is immediate, with no remission 6 Action of the negative control -Bovine Serum Albumin (BSA) solution on shrimps** 13 did not happen did not happen -6 260 did not happen did not happen -6 Action of the negative control -physiological salt solution on shrimps** did not happen did not happen -6 A cute toxicity action of CjTL8 on insect larvae***  Short-term paralysis, then active moving around vessel for 15 sec. Then convulsive movements with the paralysis frontal legs for 5-10 min. After that shrimps are behaving normally, they are undistinguishable from controls. This 'calm' behavior lasts for approximately 15 min, and then shrimps start to move extremely quick around the vessel with short periods when they stay put. During short 'stay put' periods (20-30 sec) intensive frontal legs convulsions are observed. Shrimps tend to exhibit this behavior for approximately 1 h, and then they finally calm down and stay motionless but alive in the bottom of the vessel. No lethal effect is observed during 24 hours after injection.   Proteins that are deduced from transcriptomic data only may be as interesting as those that are validated by proteomics. Full-length sequences of these venom proteins are presented in Table S3.
At first, 4 phospholipase sequences (CjPVP1 -CjPVP4) were present in both specimens. Amino acid sequences of phospholipases have a high degree of homology with known components of sea anemone venoms such as CgPLA2 (Condylactis gigantea), AcPLA2 (Adamsia palliata), and UcPLA2 (Urticina crassicornis). One of the sequences we found is similar to Phospholipase A2 from the gila monster (Heloderma suspectum, a venomous lizard). All the listed phospholipases belong to the PLA2 family. Representatives of this family are components of venoms of different animals, including Arthropoda (scorpions 75-77 , hymenoptera [78][79][80], Gastropoda (cone snails), 81 sea anemones, [81][82][83][84][85][86] and Reptilia (snakes [87][88][89][90][91][92][93] and lizards 87 ). These molecules facilitate the functions of prey capture and digestion and defence against other animals. However, phospholipases can also induce multiple types of responses, such as irritation and systemic envenomation in humans. 94 For phospholipases PLA2 (detected in different natural sources), quite different types of functional activities are revealed: anticoagulatory, myotoxicity, lytic activity towards plasma membrane of the affected muscle cell, 95 inflammatory, antioxidant, anti-inflammatory, phagocytic function regulation, CNS regulation, membrane trafficking, and leukocyte chemotaxis. Therefore, the activities of PLA2 phospholipases are diverse and might be important for the overall actions of the venom.
Secondly, C-lectin-like proteins (CjPVP11 -CjPVP13), which may play a wide spectrum of roles in multicellular organisms, are also found. Immune response, 96-98 intercellular interactions, 99 and endocytosis and apoptosis 100 are the processes in which C-lectin-like proteins participate, and they are promising for anti-cancer therapy. 101 It is known that C-lectin-like proteins may be important components of snake venoms. [102][103][104][105] Calcium-dependent lectins may induce hemaglutination, edemas, hyperpermeability of vessels, and reduction of arterial tension. 106,107 We have deduced several proteins homologs of lectins, which may potentially be acting components of venoms.

Reads assembly and annotation
Basic filters recommended for qualitative analysis of Ion Torrent PGM were applied to the raw reads, and the adapters used for cDNA synthesis were trimmed. For de novo transcriptome assembly, Newbler (Roche Diagnostics, Basel, Switzerland) was used in cDNA assembly mode. CLC Genomic Workbench (CLCbio a Qiagen Company, Aarhus, Denmark) was used in default mode. Reads with lengths less than 30 bp were not used for assembly. To group and annotate all the unigenes, we used local BLAST (BlastX algorithm threshold value of e = 1 × 10 -6 , matrix BLOSUM-62) against the protein databases NR and SWISS-PROT. Blast2GO was used to analyse gene ontology and to functionally annotate contigs and isotigs. Contig taxonomic distribution visualisation (KEGG; KOG/EGGNOG classifications) was conducted using MEGAN5 software. To identify ORF, mark up, and annotate, TransDecoder (Broad Institute; CSIRO) was used.

Other computational tools
Cutadapt v1.9 was used for trimming, which eliminated adapter sequences used for cDNA synthesis. Generation of the DNA fragments encoding toxin and plasmid construction for the expression in E.coli.

Expression and purification
We used laboratory fermenter Brunswick BioFlo 110 Fermentor/Bioreactor (Fisher Scientific) a volume of 5 liters. The target plasmids were introduced into E. coli BL21 (DE3) gold by heat-shock transformation and seeded on Petri dishes with LB agar and ampicillin. Pre-cultures of E. coli were grown overnight in 300 mL LB2x medium supplemented with ampicillin (100 µg/mL) at 37°C with shaking at 180 rpm. Overnight cultures were transferred to 3L of fresh medium LB2х and were grown at 37ºC until an OD value of 0.6-0.8 at 600 nm was reached. Isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, cultures were further grown 4 hours at 37 ºC. Cells were harvested by centrifugation and resuspended in 150 mL of distilled water. The cells were disrupted by gentle sonication, the precipitate was separated by centrifugation at 15000g 15min. To the lysate was added 1/7 part of 8x buffer A (final concentration -20 mM monosodium phosphate, 0.5 M NaCl, 10 mM imidazole, рН 7,5), re-centrifuged at 15000g 15min. The resulting solutions were placed in a chromatographic column (XK-16/20; GE Healthcare, USA) filled with 10 ml of sorbent Ni Sepharose High Performance (GE Healthcare, USA) and equilibrated with the same buffer. After application, the columns were washed with at least 50 ml of starting buffer and then washed again with 20 mM Na-phosphate buffer (pH 7.5) containing 25 mM imidazole, and 0.5 M NaCl. Subsequently, the protein was eluted with 20 mM Na-phosphate buffer (pH 7.5) containing, 0.5 M NaCl, and 500 mM imidazole. The flow rate was 2 ml/min. The inspection of the process and the collection of the fractions were performed via measurements of the eluate absorbance at 280 nm. The target fused proteins were purified using a chromatograph AKTA FPLC (GE Healthcare, USA).

Recombinant toxins production
Fusion proteins (see Figure S5 and S6) cleavage for target toxins release was performed using direct cyanogen bromide cleavage protocol (with omission of the desalting step). 62 All fusion protein solutions were diluted to a concentration of 0.1 mg/ml. HCl to a final concentration of 0.2 M and CNBr with a molar ratio to a fusion protein of 600:1 were added. Lowering cleavage temperature along with increasing time of the reaction was found to be beneficial for obtaining particular toxins of this research work. Cleavage reaction was performed at 14ºC during 20-22 h, whereupon cyanogen bromide was evaporated using Savant SpeedVac SVC 100H centrifugal evaporator.
For isolation of recombinant toxins from reaction mixture RP-HPLC method was utilised. Chromatographic separation for each toxin was conducted stepwise. Preliminary rough purification on semi-preparative Phenomenex Jupiter C5 (21.20x250 mm) 300Å 10µm column was performed using a linear gradient of acetonitrile (0.1% v/v TFA containing buffers) at a flow rate of 5 ml/min. For the final purification either Grace Davison Discovery Sciences Vydac HPLC column #218TP54 (C18, 4.6x250 mm,