Identification of a novel snake peptide toxin displaying high affinity and antagonist behaviour for the α2-adrenoceptors

Abstract

BACKGROUND AND PURPOSE Muscarinic and adrenergic G protein-coupled receptors (GPCRs) are the targets of rare peptide toxins isolated from snake or cone snail venoms. We used a screen to identify novel toxins from Dendroaspis angusticeps targeting aminergic GPCRs. These toxins may offer new candidates for the development of new tools and drugs. EXPERIMENTAL APPROACH In binding experiments with (3) H-rauwolscine, we studied the interactions of green mamba venom fractions with α(2) -adrenoceptors from rat brain synaptosomes. We isolated, sequenced and chemically synthesized a novel peptide, ρ-Da1b. This peptide was pharmacologically characterized using binding experiments and functional tests on human α(2)-adrenoceptors expressed in mammalian cells. KEY RESULTS ρ-Da1b, a 66-amino acid peptide stabilized by four disulphide bridges, belongs to the three-finger-fold peptide family. Its synthetic homologue inhibited 80% of (3) H-rauwolscine binding to the three α(2)-adrenoceptor subtypes, with an affinity between 14 and 73 nM and Hill slopes close to unity. Functional experiments on α(2A) -adrenoceptor demonstrated that ρ-Da1b is an antagonist, shifting adrenaline activation curves to the right. Schild regression revealed slopes of 0.97 and 0.67 and pA(2) values of 5.93 and 5.32 for yohimbine and ρ-Da1b, respectively. CONCLUSIONS AND IMPLICATIONS ρ-Da1b is the first toxin identified to specifically interact with α(2)-adrenoceptors, extending the list of class A GPCRs sensitive to toxins. Additionally, its affinity and atypical mode of interaction open up the possibility of its use as a new pharmacological tool, in the study of the physiological roles of α(2)-adrenoceptor subtypes.

Figure 3

Analytical reverse-phase chromatography of the reduced synthetic ρ-Da1b (line a), oxidized synthetic ρ-Da1b (line b) and natural ρ-Da1b (line c). Line d shows the co-elution of the natural and synthetic oxidized ρ-Da1b.

Figure 2

Mass and sequence analysis of ρ-Da1b. (A) Isotopic profile of ρ-Da1b. (B) Peptide mass fingerprint of ρ-Da1b after trypsin treatment. (C) Fragmentation by MALDI-LIFT-TOF/TOF of the m/z 1680.68 ion (red arrow) obtained after trypsin digestion. b- and y-ion types used for the sequencing are indicated in red and blue, respectively.

Figure 1

Purification and preliminary pharmacological characterization of ρ-Da1b. (A) Ion-exchange chromatography of Dendroaspis angusticeps crude venom. Labelled peaks were collected (13 fractions). (B) Reverse-phase chromatography of fraction H on a Vydac C18 preparative column. Labelled peaks were collected (20 fractions). Fraction D was eluted at around 27% acetonitrile. (C) Reverse-phase chromatography of fraction D on a Vydac C18 analytical column. Arrows show active peaks. (D) Inhibition of ³H-rauwolscine (1 nM) binding on rat brain synaptosomes by the minor peak purified in C.

Figure 4

Inhibition binding curves for human adrenoceptors of ³H-rauwolscine (1 nM) in CHO membranes expressing, hα_2A-adrenoceptor (12 µg) with yohimbine (▴) and ρ-Da1b (○) and membranes expressing hα_2B (11 µg, ) or hα_2C-adrenoceptor (3.1 µg, •) with ρ-Da1b. Inhibition of ³H-prazosin (1 nM) by ρ-Da1b in CHO membranes expressing hα_1A-adrenoceptor (3.8 µg).

Figure 5

ρ-Da1b prevented α_2A-adrenoceptor mediated Gi-inhibition. (A) Construction of the trimeric G protein with Renilla luciferase (RLuc) as a donor and GFP as acceptor link to the αi1 and γ2 subunits, respectively. (B) Cells were first treated directly with 10 µM ρ-Da1b or yohimbine to evaluate their agonistic property. Results are expressed as the difference in bioluminescence resonance energy transfer (BRET) signals measured before and after application of the compounds. Then, cells were stimulated with 10 µM UK14304 alone or after a pre-incubation with 10 µM of ρ-Da1b or yohimbine. Results are expressed as the difference in BRET signals measured before and after the α₂-agonist UK14304 stimulation. Data represent the mean ± SD of three independent experiments.

Figure 6

Functional characterization of yohimbine and ρ-Da1b on COS cells co-expressing the hα_2A-adrenoceptor and the chimeric G protein GqTop. Concentration–response curves for epinephrine were obtained in the presence of increasing concentrations of yohimbine (A) or ρ-Da1b (B). Error bars have been omitted for clarity. (C) Schild plot representations of the evolution of epinephrine EC₅₀ in the presence of yohimbine (○) or ρ-Da1b (•) were fitted by a linear regression.

Figure 7

Characterization of the mode of action of ρ-Da1b on α_2A-adrenoceptor expressed on CHO cells. (A) Saturation binding experimentof ³H-rauwolscine in the absence (•) or presence of ρ-Da1b (○, 10 µM). (B) Dissociation kinetic rate of ³H-rauwolscine in the presence of rauwolscine (black), rauwolscine plus ρ-Da1b (red), ρ-Da1b (green) and rauwolscine + 5-(N-ethyl-N-isopropyl)-amiloride (EPA) (purple).