Conotoxins

Specimen were collected in the central Philippines during several collection expeditions in 2011–2015. Specimen identification was initially performed by morphological examination and later verified by sequence analysis of the cytochrome oxidase c subunit 1 (COI) gene. Venom glands were dissected and stored in RNAlater at −80 °C until further processing. Total RNA was isolated from venom glands using TRIzol^®Reagent (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) or the RNeasy kit (Qiagen, Germantown, MD, USA) following the manufacturers’ instructions. RNA integrity, quantity, and purity were determined on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). cDNA libraries were prepared and sequenced on an Illumina HiSeq 2000 instrument (Sanger/Illumina 1.9 reads, 101 bp or 125 bp paired-end, Illumina, San Diego, CA, USA). Publicly available Illumina datasets were used for the venom gland transcriptomes of C. marmoreus (specimen 2), C. virgo (specimen 2), C. coronatus, and C. ebraeus [10 (link)].
Adapter clipping and quality trimming of raw reads were performed using fqtrim software (Version 0.9.4, http://ccb.jhu.edu/software/fqtrim/) and PRINSEQ (Version 0.20.4 [38 (link)]). After processing, sequences shorter than 70 bps and those containing more than 5% ambiguous bases (Ns) were discarded. De novo transcriptome assembly was performed using Trinity Version 2.0.5 [39 (link)] with a kmer size for building De Bruijn Graphs of 31, a minimum kmer coverage of 10, and a minimum glue of 10. Assembled transcripts were annotated using Blastx ((NCBI-Blast-2.2.28+, [40 (link)]) against conotoxin sequences extracted from the ConoServer [5 (link)] and UniProt databases [34 (link)].

U937 cells were obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). The cells were cultured in RPMI 1640 (Gibco by Life Technologies GmbH, Darmstadt, Germany) supplemented with 10% fetal calf serum (FCS; Biochrome, Berlin, Germany) and 2 mM L-glutamine (Gibco by Life Technologies GmbH) under 5% CO₂ atmosphere at 37 °C.
To investigate IL-1β release cells were transferred to 24-well plates (1 × 10⁶ cells/ml and per well). Cells were primed with 1 μg/ml LPS from Escherichia coli (L2654; 1 μg/ml; Sigma-Aldrich, Deisenhofen, Germany) for 5 h. After priming, the P2X7 receptor agonist BzATP (Sigma-Aldrich; 100 μM) was added for 30 min in presence or absence of different concentrations of cholinergic agonists and antagonists. Cho chloride (100 μM), PC chloride calcium salt tetrahydrate (100 μM), and Mec hydrochloride (100 μM) were purchased from Sigma-Aldrich. An analogue of α-conotoxin RgIA (RgIA4)22 was used in concentrations from 0.2 to 200 nM. After cell treatment, cells were spun down (500 g, 8 min) the supernatants were collected and stored at −20 °C. IL-1β concentrations were measured using a human Quantikine Immunoassays (R&D Systems, Minneapolis, MN) and LDH was determined.

We applied homology searches and an ab initio prediction method [83 (link)] to predict conotoxins from the five transcriptomes and one EST sequencing dataset.
For homology searches, all previously known conopeptides were downloaded from the ConoServer database [42 (link)] to construct a local reference dataset. We subsequently used BLASTX (with an E-value of 1e-5) to run our assembled sequences against the local dataset. Those unique genes/ESTs with the best hits in the BLASTX data were translated into peptide sequences.
In addition, an ab initio prediction method using a Hidden Markov Model (HMM) was adopted to discover new conopeptides. First, the reference dataset of known conopeptides from the ConoServer database [42 (link)] was grouped into different classes according to their published superfamily and family classifications. Second, sequences of each class were aligned with the ClustalW tool [84 (link)] and the ambiguous results were checked using the ConoPrec tool [42 (link)] and manual inspection. Finally, a profile HMM was built for the conserved-domain of each class using hmmbuild from the HMMER 3.0 package [68 (link)] to find the best HMM parameter, and the hmmsearch tool was then applied, using this trained HMM parameter, to scan every unique assembled gene/EST for identification of conopeptides.

As a typical model to study the development and function of the NMJ [43 (link)–45 (link)], diaphragm muscle was dissected with special care to preserve phrenic nerve connectivity. Isolated nerve–muscle preparations were immersed in Ringer’s solution and maintained at 26 °C.
One hemidiaphragm was used as a treatment, and the other served as its paired untreated control. All treatments were performed ex vivo. Muscles were stimulated through the phrenic nerve at 1 Hz, which allows the maintenance of different tonic functions without depleting synaptic vesicles, for 30 min using the A-M Systems 2100 isolated pulse generator (A-M System) as in previous studies [38 (link)–40 (link)]. We designed a protocol of stimulation that preserves the nerve stimulation and the associated neurotransmission mechanism. This method prevents other mechanisms associated with non-nerve-induced (direct) muscle contraction [46 –48 (link)]. To verify muscle contraction, a visual checking was done. Two main experiments were performed to distinguish the effects of synaptic activity from those of muscle activity (Fig. 1).

Presynaptic stimulation (Ctrl versus ES): to show the impact of the synaptic activity, we compared presynaptically stimulated muscles whose contraction was blocked by μ-CgTx-GIIIB with nonstimulated muscles also incubated with μ-CgTx-GIIIB to control for nonspecific effects of the blocker.

Contraction (ES versus ES + C): to estimate the effect of nerve-induced muscle contraction, we compared stimulated/contracting muscles with stimulated/noncontracting muscles whose contraction was blocked by μ-CgTx-GIII. By comparing the presynaptic stimulation with or without postsynaptic activity, we separate the effect of contraction. However, one should consider that postsynaptic contraction experiments also contain presynaptic activity.

Fig. 1

Design of experimental treatment for the study of effects of presynaptic activity and nerve-induced muscle contraction. μ-CgTx-GIIIB, μ-conotoxin GIIIB

In the experiments that needed only stimulation without contraction, μ-CgTx-GIIIB was used (see “Reagents”). Nevertheless, before immersing these muscles in μ-CgTx-GIIIB, a visual checking of the correct contraction of the muscle was done [39 (link)].
Furthermore, to assess the effect of PKA blocking, three different experiments have been performed:

To estimate the effect of PKA inhibition under synaptic activity, we compared presynaptically stimulated muscles whose contraction was blocked by μ-CgTx-GIIIB with and without H-89: ES versus ES + H-89.

To show the impact of the PKA inhibition under muscle contraction, we compared stimulating and contracting muscles with and without H-89: (ES + C) versus (ES + C) + H-89.

To demonstrate if degradation or redistribution along the axon is involved, the diaphragm muscle was dissected with special care to preserve phrenic nerve connectivity. We compared stimulating and contracting muscles with and without protease inhibitor (Prot.Inh.) cocktail 1% (10 μl/ml; Sigma, Saint Louis, MO, USA): (ES + C) versus (ES + C) + Prot.Inh.

To block muscle stimulation μ-conotoxin GIIIB (#C-270, Alomone Labs Ltd, Jerusalem, Israel) was used. This toxin inhibits sarcolemmal voltage-dependent sodium channels (VSDCs) without affecting synaptic ACh release or ACh signaling [42 (link)]. It was supplied as lyophilized powder of > 99% purity. μ-conotoxin GIIIB was 150 μM stock, and working concentration was 1.5 μM in Ringer’s solution [mM: NaCl 137, KCl 5, CaCl₂ 2, MgSO₄ 1, NaH₂PO₄ 1, NaHCO₃ 12, glucose 12.1, and DMSO 0.1%, oxygenated with O₂:CO₂ (95:5)].
PKA activity was blocked with N-[2-((p-Bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H-89, Calbiochem). H-89 was made as 10 mM stock and used at 10 μM diluted in Ringer’s solution with DMSO.
All chemicals were diluted in Ringer’s solution, and both control and drug-containing solutions contained 0.1% dimethyl sulfoxide (DMSO) as the vehicle.

Native and synthetic conotoxins were individually separated using the following equipment: (i) capillary scale (Phenomenex; C18, 5 μm, 300 Å, 1.0 × 250 mm, flow 100 μL min⁻¹)—used for comparative RP-HPLC profiling to control the quality of peptide purity, to quantify the peptides and to perform peptide co-elution experiments, (ii) analytical scale (Vydac; C18, 5 μm, 300 Å, 4.2 × 250 mm, flow 1 mL min⁻¹)—used for the isolation and purification of native peptides for MS analysis, and (iii) preparative scale (Vydac; C18, 10 μm, 300 Å, 22 × 250 mm, flow 5 mL min⁻¹)—used for the preparative separation of synthetic peptides. Capillary and analytical scale separations utilized a Waters 2695 Alliance RP-HPLC System interfaced with a 996 Waters photo diode array detector. Data was acquired and analyzed using Waters Millennium32 (v3.2) software. Samples were eluted using a linear 1% min⁻¹ gradient of organic (90/10%/0.008% v/v CH₃CN/H₂O/TFA) Solvent B against aqueous (0.1% v/v TFA/H₂O) Solvent A for 65 min, terminating with a high organic wash (80% Solvent B for 5 min), and pre-equilibration step (5% Solvent B) for 10 min prior to sample injection. Elution from the column was monitored at 214 nm. The preparative scale RP-HPLC (iii) used a 625 Waters HPLC pump and controller interfaced with a 996 Waters photo diode array detector. Synthetic peptides and crude venom peptide extracts were filtered (Nylon 0.22 μm), manually loaded, and eluted from the preparative scale column using the same 1% gradient at 5 mL min⁻¹ and monitored at 214 and 280 nm. Fractions were collected manually and stored at −20 °C or freeze-dried until required.
LC-MS analysis of crude duct venom was performed on a C18 capillary-bore RP-HPLC (Phenomenex; 5 μm, 300 Å, 1.0 × 250 mm) column interfaced to a PerSeptive Biosystems Mariner MS, using a 1% gradient, 100 μL min⁻¹, 214 nm, Solvents (A) 0.1% formic acid/H₂O, (B) 0.65% formic acid/CH3CN, with only 20% of the flow directed into the MS ion source. To achieve total venom peptide reduction, materials were resuspended in 200 mM TCEP/25 mM NH₄OAc, pH 4.5, heated at 60 °C for 10 min, then centrifuged (12,000× g) prior to LC-MS analysis.