2- METHODS
2.1 MQs identification . Venoms were fractionated as described
(Ciolek et al., 2017). One gram of each Dendroaspis sp. venom (Latoxan,
Valence, France) was separated into primary fractions by cation exchange
liquid chromatography (5 x 40 cm) on the resin Source 15S using a
multi-step NaCl gradient at 20 mL·min-1 on an Akta
purifier (Pfizer, Quebec, Canada). The fractions were further purified
by reverse-phase chromatography on a C18 Vydac preparative column (19.6
mm, 5 µm, 25 cm,), using a linear gradient from 0 to 100% acetonitrile
in 0.1% trifluoroacetic acid in 100 min at a flow rate of 20 mL/min.
Analytical high-pressure liquid chromatography (HPLC) was finally
performed on a C18 Vydac column (4.6 mm, 5 mm, 15 cm) using a gradient
of 0.5% acetonitrile per min and a flow rate of 1 mL/min. Protein
concentrations were determined using the Bio-Rad protein assay with BSA
as a standard. Toxin sequences were determined by mass analysis.
Sequencing by in-source decay (ISD) MALDI-TOF was carried out with 15 µg
of each fraction treated with 2 µL of 100 mM Tris(carboxyethyl)phosphine
at 50°C and purified on a Zip-Tip C18 microcolumn. The matrix used for
ISD experiments was 1,5-diaminonaphthalene (Accros). For peptide mass
fingerprinting, 300 ng of purified toxins were reduced in 5 µL of 50 mM
NH4HCO3, pH 8, by 2 µL of 250 mM DTT for
30 min at 56 °C, followed by 2.2 µL of 500 mM iodoacetamide for 1 h in
the dark at room temperature. Mass analysis was made on a MALDI-TOF/TOF
(rapifleX, Bruker Daltonics). Tandem mass spectrometry experiments were
performed using LIFT-TOF/TOF technology. Data was acquired with Flex
Control 3.0. Resulting spectra were analyzed with Biotools 3.2 and
Sequence Editor 3.2 (Bruker Daltonics).
2.2 Binding assay with radioligand . 3H-AVP
was purchased from PerkinElmer (Courtaboeuf, France). Binding
experiments on self-prepared CHO cells overexpressing vasopressin hV2R
subtype or HEK cells transfected with a plasmid coding for the rat V2R,
were performed as described (Ciolek et al., 2017; Droctové et al.,
2020). Briefly, 1.5 nM 3H-AVP in a 100 μL reaction
mixture at room temperature in buffer composed of 50 mM Tris-HCl, pH
7.4, 10 mM MgCl2, 1 g/L BSA was incubated with
increasing concentrations of competitors for 3 hours. Non-specific
binding was measured in the presence of 1 μM vasopressin. Incubation was
stopped by filtration through 96 GF/C filter plates pre-incubated with
0.5% polyethylenimine. 25 μL of Microscint 0 were added onto each dry
filter and the radioactivity was quantified on a TopCount beta counter
with a 33% yield (PerkinElmer, Courtaboeuf, France). We fitted
competition binding data with the one-site/state inhibition mass action
curve using Kaleidagraph (Synergy software, Reading, USA).
IC50 values were converted to Ki using 1
nM as Kd in the Cheng-Prusoff equation. Data represents independent
experiments and are presented as the mean of pKi ± SEM.
2.3 cAMP cell-based assay. The CHO cell line expressing hV2R
was cultured at 37 °C in 5% CO2 in DMEM containing 10%
FCS and 100 units/ml penicillin/100 μg/ml streptomycin supplemented with
0.1 mM non-essential amino-acids and 0.4 mg/ml geneticin. We seeded CHO
cells (5000/well) into 96-well plates for cAMP quantification. For
stimulation curves (EC50 determinations), CHO-hV2R cells
were stimulated by increasing AVP concentrations in the absence (control
conditions) or in the presence of increasing competitor concentrations
in a total 50 μl volume incubation medium containing DMEM, 5% BSA and
0.1 mM RO201724. For competition curves, 1.77 nM AVP stimulated CHO-hV2R
cells in the absence (control conditions) or presence of increasing
competitor concentrations. Experiments lasted for 30 min at 37 °C and
were stopped by the addition of 25 μl of lysis buffer from the cAMP
Dynamic 2 kit (Cisbio-International) containing cAMP labeled with
acceptor entity first then by the addition of 25 μl of lysis buffer
containing donor fluorophore-labeled antibody against cAMP (100 μl total
volume per well). The fluorophores elicited a FRET signal (F% = 100 x
(Rpos - Rneg)/Rneg) with Rpos fluorescence ratio (665/620 nm) measured
in wells incubated with both donor- and acceptor-labeled entities, and
Rneg being the same ratio for the negative control with donor
fluorophore-labeled antibody only. This signal was then transformed into
cAMP concentration in wells using a calibration curve.
Activation/inhibition curves were plotted to the log of AVP
concentrations and fitted to the Hill equation to extract the
EC50/IC50 using Graphpad Prism software.
The corresponding Arunlakshana-Shild plots allowed to determine pA2 from
the Schild equation. Experiments were done in triplicate. Data are
presented as mean ± SEM.
2.4 In vivo experiments . The CEA animal experiment ethic
committee approved the use of animals and experimental protocols
(reference: APAFIS#1496-2015082111349702 v1). Six-week-old male
Sprague-Dawley rats were obtained from Janvier Labs (Le Genest Saint
Isle, France) and hosted in CEA Saclay animal facilities under stable
and controlled conditions of temperature and pressure. They had free
access to the standard rodent diet and tap water. Metabolic cages were
purchased from Tecniplast (Buguggiate, Italie). After 3 days in
metabolic cages for habituation, rats were intraperitoneally injected
with 0.5 ml/kg of the toxin or vehicle dissolved in 0.9% NaCl solution.
Urine outputs were collected 24 hours post-injection and weighed. Animal
weight, food and water intakes were also measured for control. Urinary
pH was measured using a pH-meter SevenEasy™ (Mettler Toledo, Viroflay,
France). Urinary osmolality was measured using a Type 13 automatic
osmometer (Roebling, Berlin, Germany). Urinary flow (UF) and Osmole
excretion were reported to animal weight to avoid bias. UF = urinary
volume/(time × body weight). Osmole excretion = (urinary osmolality ×
urinary volume)/(time × body weight).
2.5 Peptide production . All the peptides were synthesized on a
Prelude synthesizer (Protein Technologies®, Tucson, USA), purified and
folded according to the method already described (Ciolek et al., 2017).
Briefly, the solid-phase synthesis using a Fmoc strategy was done on 25
μmol of ChemMatrix®, peptide cleavage and purification. Linear peptides
were folded in the presence of oxidized and reduced glutathione (1 mM)
and glycerol 20% (K10A) or guanidine 0.5 M (MQ2) in Tris buffer at pH 8
overnight at room temperature. All the other peptides were folded in
presence of oxidized and reduced cysteine (1 and 0.1 mM, respectively),
in Hepes 100 mM at pH 7.5 (R44A, MQ4, MQ5, MQ6, B4ESA3, C1IC51, D5J9Q8,
F8J2F6 and Q5ZPJ7) and acetonitrile 20% (F18A, E7FL11 and P19859) or
guanidine 0.5 M (MQ3, N-ter, N&C-ter, N6E, F21A, S24G, Q25A, K26A,
K26E, K29A, H31F, V9S, N15A, F18A, S19A, T34F, K39A, K39E, K39W, N41A,
S46R) or in Tris at pH 7.2 and guanidine 0.5 M (F17A and K10E),
overnight at room temperature.
2.6 Phylogenetic reconstruction. We used mafft v7.388 -auto
option 4 to align eight MQ with 40 dendrotoxin full mature sequences
from VenomZone (https://venomzone.expasy.org/) and three bovine protease
inhibitors with the Kunitz fold included as outgroup (UniProt IDs:
P00974, P00975, and P04815). With this multi-sequence alignment, we
obtained a Maximum Likelihood (ML) tree with RAxML version 8.2.7 5 under
the PROTGAMMAJTT substitution model and the rapid hill climbing option
(-f d). We calculate 15,000,000 trees with MrBayes v3.2.6 6 in three
independent runs with four chains each under the JTT substitution model
and rates = invgamma. We merged the 1,000 trees with best posteriors
from each run and annotated the branches of the ML tree with RAxML -b
option.
2.7 Statistical analysis. All statistical tests were performed
with Kaleidagraph (Synergy software, Reading, USA). Multiple group
comparisons were performed using a one-way ANOVA with post hoc test
according to Dunnett. P < 0.05 was considered statistically
significant. Values are expressed as means ± SEM.