A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken α7 nicotinic acetylcholine receptors

Abstract

Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx-5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (K d ¼ 180 nM) with the muscular-type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 lM) with the neuronal a7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve-muscle preparations revealed no blocking activity of sWntx-5 at lM concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.Keywords: snake neurotoxins; nicotinic acetylcholine receptors.During the past three decades, the most ÔobviousÕ venom toxins have been uncovered either because they are present in large amounts and/or because they have been directly associated with the search for an important target. At present, two additional approaches may be considered to discover new toxin functions. One of them is a proteomictype approach, which aims at isolating all components of the ÔtoxinomeÕ [1,2]. The second approach involves investigation of the vast number of venom components that have already been isolated, and sometimes chemically characterized, but whose biological activity still remains mysterious. These functionally unknown components are often classified as miscellaneous types of toxins, even though they usually belong to well-identified structural families [3]. This is the case of the so-called Ôweak neurotoxinsÕ (Wntxs) found in elapid snakes and isolated for the first time 26 years ago from the venom of Naja melanoleuca [4]. Since then, more such toxins have been isolated [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19].The Wntxs possess 62-68 amino acids and belong to the structural family of Ôthree-fingeredÕ folded toxins, which includes the cardiotoxins, muscarinic toxins, acetylcholinesterase inhibitors and the a-neurotoxins that block muscular and/or neuronal nicotinic acetylcholine receptors (AChRs) [20][21][22]. The fold adopted by all these toxins is characterized by three adjacent loops rich in b-pleated sheet, tethered by four conserved disulphides. A fifth loop is sometimes observed in the second loop of the a/j-neurotoxins and j-neurotoxins [22], where it specifically contributes to the binding of the toxins to the neuronal AChR [23][24][25][26]. Wntxs also possess a fifth disulfide bond, but this is located in the first loop [16,27,28].Using Wntxs isolated from venom, it was shown that these molecules interact with AChRs but with low affinities [10,29]. Many ...

Exploring the Diversity and Novelty of Toxin Genes in Naja sumatrana, the Equatorial Spitting Cobra from Malaysia through De Novo Venom-Gland Transcriptomics

“…Prior to that, toxinologists seldom distinguished between the three species of spitting cobras ( N. sumatrana , N. sputatrix , and Naja siamensis ) distributed in this region. Gene cloning studies on N. naja sputatrix by Jeyaseelan and colleagues [24,25,26,27,28] were based on venom glands extracted from spitting cobra(s) that appeared to originate from the Peninsular Malaya or Singapore. In view of the identical match of the sequences to N. sumatrana transcripts in the present study, it is very likely that the spitting cobra labeled as “ N. naja sputatrix ” previously was actually an N. sumatrana .…”

“…In addition, the information obtained can be used to validate several toxin sequences annotated to “ Naja naja sputatrix ” available in the open database. These toxin sequences were reported from cloning studies in the late 1990’s and early 2000’s [24,25,26,27,28]. Unfortunately, the authenticity of the snake species used in the early days was difficult to be ascertained as the work was carried out at a time when confusion perhaps still existed in the systematics of spitting cobras.…”

A Decoy-Receptor Approach Using Nicotinic Acetylcholine Receptor Mimics Reveals Their Potential as Novel Therapeutics Against Neurotoxic Snakebite

“…One such example is candoxin (Nirthanan et al, 2002), a 66-amino-acid 3FTx neurotoxin from B. candidus that binds with high affinity to the α7-nAChR (IC 50 = 50 nM) via a fifth disulfide bond present in loop I. This is in contrast to other weak neurotoxins from N. kaouthia (WTX) (Utkin et al, 2001) and Naja sputatrix (Wtn-5) (Poh et al, 2002) that bind only weakly (micromolar affinity) to this receptor, suggesting that other residues beside the fifth disulfide bond located in loop I dictate the binding affinity of these toxins to the neuronal receptor. Moreover, haditoxin (Roy et al, 2010), a homodimeric 3FTx from the venom of the king cobra, Ophiophagus hannah , was the first reported dimeric toxin to bind to α7-nAChR (IC 50 = 180 nM).…”

From Synthetic Fragments of Endogenous Three-Finger Proteins to Potential Drugs

“…It should be mentioned that there are many publications on the activity of the synthetic three-finger toxins and of their fragments: synthetic fragments of the central loop II of α-neurotoxins have been already mentioned (Kasheverov and Tsetlin, 2017), there are also full-size synthetic α-neurotoxins (Rey-Suárez et al, 2012), non-conventional neurotoxins (Poh et al, 2002), TFP toxins inhibiting acetylcholinesterase (fasciculins) (Falkenstein and Peña, 1997), synthetic mambalgins, and their analogs attacking ASICs (Schroeder et al, 2014). Not always in the work on TFP toxins the interest was focused on the central loop: for the synthetic N-terminal loop of cardiotoxin I from the Naja atra cobra venom the authors reported antimicrobial activity against Gram-positive and Gram-negative bacteria (Sala et al, 2018).…”