Protein Painting Mass Spectrometry in the Discovery of Interaction Sites within the Acetylcholine Binding Protein.

Nicotinic acetylcholine receptors (nAChRs) are a family of ligand-gated ion channel receptors that contribute to cognition, memory, and motor control in many organisms. The pharmacological targeting of these receptors, using small molecules or peptides, presents an important strategy for the development of drugs that can treat important human diseases, including neurodegenerative disorders. The Aplysia californica acetylcholine binding protein (Ac-AChBP) is a structural surrogate of the nAChR with high homology to the extracellular ligand binding domain of homopentameric nAChRs. In this study, we optimized protein-painting-based mass spectrometry to identify regions of interaction between the Ac-AChBP and several nAChR ligands. Using molecular dyes that adhere to the surface of a solubilized Ac-AChBP complex, we identified amino acid residues that constitute a contact site within the Ac-AChBP for α-bungarotoxin, choline, nicotine, and amyloid-ß 1-42. By integrating innovation in protein painting mass spectrometry with computational structural modeling, we present a new experimental tool for analyzing protein interactions of the nAChR.

Development and optimization of a DNA aptamer to delay ß-bungarotoxin-induced lethality in a rodent model.

Current treatment of snakebite relies on immunoglobulin-rich antivenoms. However, production of these antivenoms is complicated and costly. Aptamers - single-stranded DNAs or RNAs with specific folding structures that bind to specific target molecules - represent excellent alternatives or complements to antibody-based therapeutics. However, no studies have systematically assessed the feasibility of using aptamers to mitigate venom-induced toxicity in vivo. ß-bungarotoxin is the predominant protein responsible for the toxicity of the venom of Bungarus multicinctus, a prominent venomous snake inhabiting Taiwan. In this study, we reported the screening and optimization of a DNA aptamer against ß-bungarotoxin and tested its utility in a mouse model. After 14 rounds of directed evolution of ligands by exponential enrichment, an aptamer, called BB3, displaying remarkable binding affinity and specificity for ß-bungarotoxin was obtained. Following structural prediction and point-modification experiments, BB3 underwent truncation and was modified with 2'-O-methylation and a 3'-inverted dT. This optimized aptamer showed sustained, high-affinity binding for ß-bungarotoxin and exhibited remarkable nuclease resistance in plasma. Importantly, administration of this optimized aptamer extended the survival time of mice treated with a lethal dose of ß-bungarotoxin. Collectively, our data provide a compelling illustration of the potential of aptamers as promising candidates for development of recombinant antivenom therapies.

Network Pharmacological Study on the Mechanism of Cynanchum paniculatum (Xuchangqing) in the Treatment of Bungarus multicinctus Bites.

Background: Bungarus multicinctus is one of the top ten venomous snakes in China. Its venom is mainly neurotoxin-based. Novel antivenom drugs need to be further researched and developed. Objective: This study aimed to explore the molecular mechanism of Cynanchum paniculatum in treating Bungarus multicinctus bites based on network pharmacology. Material and methods. The potential active ingredients of Cynanchum paniculatum were screened and their SDF structures were obtained using the PubChem database and imported into the SwissTargetPrediction database, and targets were obtained for the antitoxin effects of Cynanchum paniculatum in the treatment of Bungarus multicinctus bites. The Cynanchum paniculatum-active compound-potential target network and protein-protein interaction network were constructed by using Cytoscape software, and then biological function analysis and KEGG pathway enrichment analysis were performed using the DAVID. Results: Seven potential active components (cynapanoside C, cynatratoside B, tomentolide A, sitosterol, sarcostin, tomentogenin, and paeonol) and 286 drug targets were obtained, including 30 key targets for the treatment of bungarotoxin toxicity. The active components mainly acted on PIK3CA, MAPK1, MAP2K1, JAK2, FYN, ACHE, CHRNA7, CHRNA4, and CHRNB2, and they antagonized the inhibitory effect of bungarotoxin on the nervous system through cholinergic synapses and the neurotrophin signaling pathway. Conclusions: Cynanchum paniculatum exerts a therapeutic effect on Bungarus multicinctus bites through multiple active components, multiple targets, and multiple pathways. The findings provide a theoretical basis for the extraction of active components of Cynanchum paniculatum and for related antivenom experiments.

Structure and gating mechanism of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. This ligand-gated ion channel assembles as a homopentamer, is exceptionally permeable to Ca2+, and desensitizes faster than any other Cys-loop receptor. The α7 receptor has served as a prototype for the Cys-loop superfamily yet has proven refractory to structural analysis. We present cryo-EM structures of the human α7 nicotinic receptor in a lipidic environment in resting, activated, and desensitized states, illuminating the principal steps in the gating cycle. The structures also reveal elements that contribute to its function, including a C-terminal latch that is permissive for channel opening, and an anionic ring in the extracellular vestibule that contributes to its high conductance and calcium permeability. Comparisons among the α7 structures provide a foundation for mapping the gating cycle and reveal divergence in gating mechanisms in the Cys-loop receptor superfamily.

A Wolf in Another Wolf's Clothing: Post-Genomic Regulation Dictates Venom Profiles of Medically-Important Cryptic Kraits in India.

The Common Krait (Bungarus caeruleus) shares a distribution range with many other 'phenotypically-similar' kraits across the Indian subcontinent. Despite several reports of fatal envenomings by other Bungarus species, commercial Indian antivenoms are only manufactured against B. caeruleus. It is, therefore, imperative to understand the distribution of genetically distinct lineages of kraits, the compositional differences in their venoms, and the consequent impact of venom variation on the (pre)clinical effectiveness of antivenom therapy. To address this knowledge gap, we conducted phylogenetic and comparative venomics investigations of kraits in Southern and Western India. Phylogenetic reconstructions using mitochondrial markers revealed a new species of krait, Romulus' krait (Bungarus romulusi sp. nov.), in Southern India. Additionally, we found that kraits with 17 mid-body dorsal scale rows in Western India do not represent a subspecies of the Sind Krait (B. sindanus walli) as previously believed, but are genetically very similar to B. sindanus in Pakistan. Furthermore, venom proteomics and comparative transcriptomics revealed completely contrasting venom profiles. While the venom gland transcriptomes of all three species were highly similar, venom proteomes and toxicity profiles differed significantly, suggesting the prominent role of post-genomic regulatory mechanisms in shaping the venoms of these cryptic kraits. In vitro venom recognition and in vivo neutralisation experiments revealed a strong negative impact of venom variability on the preclinical performance of commercial antivenoms. While the venom of B. caeruleus was neutralised as per the manufacturer's claim, performance against the venoms of B. sindanus and B. romulusi was poor, highlighting the need for regionally-effective antivenoms in India.

Three-finger proteins from snakes and humans acting on nicotinic receptors: Old and new.

The first toxin to give rise to the three-finger protein (TFP) family was α-bungarotoxin (α-Bgt) from Bungarus multicinctus krait venom. α-Bgt was crucial for research on nicotinic acetylcholine receptors (nAChRs), and in this Review article we focus on present data for snake venom TFPs and those of the Ly6/uPAR family from mammalians (membrane-bound Lynx1 and secreted SLURP-1) interacting with nAChRs. Recently isolated from Bungarus candidus venom, αδ-bungarotoxins differ from α-Bgt: they bind more reversibly and distinguish two binding sites in Torpedo californica nAChR. Naja kaouthia α-cobratoxin, classical blocker of nAChRs, was shown to inhibit certain GABA-A receptor subtypes, whereas α-cobratoxin dimer with 2 intermolecular disulfides has a novel type of 3D structure. Non-conventional toxin WTX has additional 5th disulfide not in the central loop, as α-Bgt, but in the N-terminal loop, like all Ly6/uPAR proteins, and inhibits α7 and Torpedo nAChRs. A water-soluble form of Lynx1, ws-Lynx1, was expressed in E. coli, its 1 H-NMR structure and binding to several nAChRs determined. For SLURP-1, similar information was obtained with its recombinant analogue rSLURP-1. A common feature of ws-Lynx1, rSLURP-1, and WTX is their activity against nAChRs and muscarinic acetylcholine receptors. Synthetic SLURP-1, identical to the natural protein, demonstrated some differences from rSLURP-1 in distinguishing nAChR subtypes. The loop II fragment of the Lynx1 was synthesized having the same µM affinity for the Torpedo nAChR as ws-Lynx1. This review illustrates the productivity of parallel research of nAChR interactions with the two TFP groups.

Spatial Structure and Activity of Synthetic Fragments of Lynx1 and of Nicotinic Receptor Loop C Models.

Lynx1, membrane-bound protein co-localized with the nicotinic acetylcholine receptors (nAChRs) and regulates their function, is a three-finger protein (TFP) made of three ß-structural loops, similarly to snake venom α-neurotoxin TFPs. Since the central loop II of α-neurotoxins is involved in binding to nAChRs, we have recently synthesized the fragments of Lynx1 central loop, including those with the disulfide between Cys residues introduced at N- and C-termini, some of them inhibiting muscle-type nAChR similarly to the whole-size water-soluble Lynx1 (ws-Lynx1). Literature shows that the main fragment interacting with TFPs is the C-loop of both nAChRs and acetylcholine binding proteins (AChBPs) while some ligand-binding capacity is preserved by analogs of this loop, for example, by high-affinity peptide HAP. Here we analyzed the structural organization of these peptide models of ligands and receptors and its role in binding. Thus, fragments of Lynx1 loop II, loop C from the Lymnaea stagnalis AChBP and HAP were synthesized in linear and Cys-cyclized forms and structurally (CD and NMR) and functionally (radioligand assay on Torpedo nAChR) characterized. Connecting the C- and N-termini by disulfide in the ws-Lynx1 fragment stabilized its conformation which became similar to the loop II within the 1H-NMR structure of ws-Lynx1, the activity being higher than for starting linear fragment but lower than for peptide with free cysteines. Introduced disulfides did not considerably change the structure of HAP and of loop C fragments, the former preserving high affinity for α-bungarotoxin, while, surprisingly, no binding was detected with loop C and its analogs.

Immunoreactivity and neutralization study of Chinese Bungarus multicinctus antivenin and lab-prepared anti-bungarotoxin antisera towards purified bungarotoxins and snake venoms.

Bungarus multicinctus is the most venomous snake distributed in China and neighboring countries of Myanmar, Laos, north Vietnam and Thailand. The high mortality rate of B. multicinctus envenomation is attributed to the lethal components of α-, ß-, γ- and κ- bungarotoxins contained in the venom. Although anti-B. multicinctus sera were produced in Shanghai, Taiwan and Vietnam, the most widely clinic used product was term as B. multicinctus antivenin and manufactured by Shanghai Serum Bio-technology Co. Ltd. In the present investigation, high purity α-, ß- and γ-bungarotoxins were separately isolated from B. multicinctus crude venom. Rabbit anti- α-, ß- and γ-bungarotoxin antisera were prepared by common methods, respectively. LD50 values of α-, ß- and γ-bungarotoxins were systematically determined via three administration pathways (intraperitoneal, intramuscular and intravenous injections) in Kunming mice. LD50 values of ß-bungarotoxin were closely related with injection routines but those of both α- and γ-bungarotoxins were not dependent on the injection routines. Commercial B. multicinctus antivenin showed strong immunoreaction with high molecular weight fractions of the B. multicinctus but weakly recognized low molecular weight fractions like α- and γ-bungarotoxins. Although B. multicinctus antivenin showed immunoreaction with high molecular weight fractions of Bungarus fasciatus, Naja atra, Ophiophagus hannah venoms but the antivenin only demonstrated animal protection efficacy against O. hannah venom. These results indicated that the high molecular weight fractions of the O. hannah played an important role in venom lethality but those of B. fasciatus and N. atra did not have such a role.

Efficient screening of ligand-receptor complex formation using fluorescence labeling and size-exclusion chromatography.

Evidence of a complex formation is a crucial step in the structural studies of ligand-receptor interactions. Here we presented a simple and fast approach for qualitative screening of the complex formation between the chimeric extracellular domain of the nicotinic acetylcholine receptor (α7-ECD) and three-finger proteins. Complex formation of snake toxins α-Bgtx and WTX, as well as of recombinant analogs of human proteins Lynx1 and SLURP-1, with α7-ECD was confirmed using fluorescently labeled ligands and size-exclusion chromatography with simultaneous absorbance and fluorescence detection. WTX/α7-ECD complex formation also was confirmed by cryo-EM. The proposed approach could easily be adopted to study the interaction of other receptors with their ligands.

Complex approach for analysis of snake venom α-neurotoxins binding to HAP, the high-affinity peptide.

Snake venom α-neurotoxins, invaluable pharmacological tools, bind with high affinity to distinct subtypes of nicotinic acetylcholine receptor. The combinatorial high-affinity peptide (HAP), homologous to the C-loop of α1 and α7 nAChR subunits, binds biotinylated α-bungarotoxin (αBgt) with nanomolar affinity and might be a protection against snake-bites. Since there are no data on HAP interaction with other toxins, we checked its binding of α-cobratoxin (αCtx), similar to αBgt in action on nAChRs. Using radioiodinated αBgt, we confirmed a high affinity of HAP for αBgt, the complex formation is supported by mass spectrometry and gel chromatography, but only weak binding was registered with αCtx. A combination of protein intrinsic fluorescence measurements with the principal component analysis of the spectra allowed us to measure the HAP-αBgt binding constant directly (29 nM). These methods also confirmed weak HAP interaction with αCtx (>10000 nM). We attempted to enhance it by modification of HAP structure relying on the known structures of α-neurotoxins with various targets and applying molecular dynamics. A series of HAP analogues have been synthesized, HAP[L9E] analogue being considerably more potent than HAP in αCtx binding (7000 nM). The proposed combination of experimental and computational approaches appears promising for analysis of various peptide-protein interactions.

Anticoagulant activity of krait, coral snake, and cobra neurotoxic venoms with diverse proteomes are inhibited by carbon monoxide.

BACKGROUND: A phenomena of interest is the in vitro anticoagulant effects of neurotoxins found in elapid venoms that kill by paralysis. These enzymes include phospholipase A2 (PLA2), and it has recently been demonstrated that carbon monoxide inhibits the PLA2-dependent neurotoxin contained in Mojave rattlesnake type A venom. The purpose of this investigation was to assess if the anticoagulant activity of elapid venoms containing PLA2 and/or three finger toxins could be inhibited by carbon monoxide. METHODS: Venoms collected from Bungarus multicinctus, Micrurus fulvius, and five Naja species were exposed to carbon monoxide via carbon monoxide releasing molecule-2 prior to placement into human plasma. Coagulation kinetics were assessed via thrombelastography. RESULTS: Compared with plasma without venom addition, all venoms had significant anticoagulant effects, with a 160-fold range of concentrations having similar anticoagulant effects in a species-specific manner. Carbon monoxide significantly inhibited the anticoagulant effect of all venoms tested, but inhibition was not complete in all cases. CONCLUSION: Given that individual neurotoxin activity often depends on intact activity that includes anticoagulant action, it may be possible that carbon monoxide inhibits neurotoxicity. Future investigation is justified to assess such carbon monoxide mediated inhibition with purified neurotoxins in vitro and in vivo.

Structural basis for α-bungarotoxin insensitivity of neuronal nicotinic acetylcholine receptors.

The ten types of nicotinic acetylcholine receptor α-subunits show substantial sequence homology, yet some types confer high affinity for α-bungarotoxin, whereas others confer negligible affinity. Combining sequence alignments with structural data reveals three residues unique to α-toxin-refractory α-subunits that coalesce within the 3D structure of the α4ß2 receptor and are predicted to fit between loops I and II of α-bungarotoxin. Mutating any one of these residues, Lys189, Ile196 or Lys153, to the α-toxin-permissive counterpart fails to confer α-bungarotoxin binding. However, mutating both Lys189 and Ile196 affords α-bungarotoxin binding with an apparent dissociation constant of 104 nM, while combining mutation of Lys153 reduces the dissociation constant to 22 nM. Analogous residue substitutions also confer high affinity α-bungarotoxin binding upon α-toxin-refractory α2 and α3 subunits. α4ß2 receptors engineered to bind α-bungarotoxin exhibit slow rates of α-toxin association and dissociation, and competition by cholinergic ligands typical of muscle nicotinic receptors. Receptors engineered to bind α-bungarotoxin co-sediment with muscle nicotinic receptors on sucrose gradients, and mirror single channel signatures of their α-toxin-refractory counterparts. Thus the inability of α-bungarotoxin to bind to neuronal nicotinic receptors arises from three unique and interdependent residues that coalesce within the receptor's 3D structure.

Toxins for decoding interface selectivity in nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are pentameric ligand-gated ion channels that play crucial roles in neurotransmission and regulate complex processes in brain functions, including anxiety, learning and memory, food intake, drug addiction, cognition and nociception. To perform these and other functions, a diverse array of nAChR subtypes are generated by homomeric or heteromeric assembly of 17 homologous nAChR subunits. Agonists, acetylcholine and nicotine, bind to the interface formed between two α subunits and between α and non-α subunits to activate the nAChR and allow cation influx. The diversity of subunit interfaces determines the channel properties, the responses to different agonists/antagonists, desensitization and downstream signaling and thus, define specialized properties and functions. Over the last several decades, snake venom neurotoxins have contributed to the purification, localization and characterization of molecular details of various nAChRs. Utkin et al. have described the purification and characterization of αδ-bungarotoxins, a novel class of neurotoxins in a recent paper published in the Biochemical Journal [Biochem. J. (2019) 476, 1285-1302]. These toxins from Bungarus candidus venom preferably bind to α-δ site with two orders of magnitude higher affinity compared with α-γ or α-ε sites. The subtle changes in the structure of αδ-bungarotoxins led to variation in interface selectivity. Such new classes of antagonists will offer us great opportunity to delineate the pharmacophores and design new highly selective antagonists. Thus, their findings provide new impetus to re-evaluate molecular details of pharmacological properties of α-neurotoxins with careful consideration towards subtype-, interface- and species-selectivity.

Novel long-chain neurotoxins from Bungarus candidus distinguish the two binding sites in muscle-type nicotinic acetylcholine receptors.

αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α-δ subunits. Three isoforms (αδ-BgTx-1-3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17-0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve-muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α-δ site as compared with α-ε or α-γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs.

Polymer Cancerostatics Targeted by Recombinant Antibody Fragments to GD2-Positive Tumor Cells.

A water-soluble polymer cancerostatic actively targeted against cancer cells expressing a disialoganglioside antigen GD2 was designed, synthesized and characterized. A polymer conjugate of an antitumor drug doxorubicin with a N-(2-hydroxypropyl)methacrylamide-based copolymer was specifically targeted against GD2 antigen-positive tumor cells using a recombinant single chain fragment (scFv) of an anti-GD2 monoclonal antibody. The targeting protein ligand was attached to the polymer-drug conjugate either via a covalent bond between the amino groups of the protein using a traditional nonspecific aminolytic reaction with a reactive polymer precursor or via a noncovalent but highly specific interaction between bungarotoxin covalently linked to the polymer and the recombinant scFv modified with a C-terminal bungarotoxin-binding peptide. The GD2 antigen binding activity and GD2-specific cytotoxicity of the targeted noncovalent polymer-scFv complex proved to be superior to the covalent polymer-scFv conjugate.

Proteomic Investigation of Murine Neuronal α7-Nicotinic Acetylcholine Receptor Interacting Proteins.

The α7-nicotinic acetylcholine receptor (α7-nAChR) is a ligand-gated ion channel that is expressed widely in vertebrates and is the principal high-affinity α-bungarotoxin (α-bgtx) binding protein in the mammalian CNS. α7-nAChRs associate with proteins that can modulate its properties. The α7-nAChR interactome is the summation of proteins interacting or associating with α7-nAChRs in a protein complex. To identify an α7-nAChR interactome in neural tissue, we isolated α-bgtx-affinity protein complexes from wild-type and α7-nAChR knockout (α7 KO) mouse whole brain tissue homogenates using α-bgtx-affinity beads. Affinity precipitated proteins were trypsinized and analyzed with an Orbitrap Fusion mass spectrometer. Proteins isolated with the α7-nAChR specific ligand, α-bgtx, were determined to be α7-nAChR associated proteins. The α7-nAChR subunit and 120 additional proteins were identified. Additionally, 369 proteins were identified as binding to α-bgtx in the absence of α7-nAChR expression, thereby identifying nonspecific proteins for α7-nAChR investigations using α-bgtx enrichment. These results expand on our previous investigations of α7-nAChR interacting proteins using α-bgtx-affinity bead isolation by controlling for differences between α7-nAChR and α-bgtx-specific proteins, developing an improved protein isolation methodology, and incorporating the latest technology in mass spectrometry. The α7-nAChR interactome identified in this study includes proteins associated with the expression, localization, function, or modulation of α7-nAChRs, and it provides a foundation for future studies to elucidate how these interactions contribute to human disease.

Engineering varied serine protease inhibitors by converting P1 site of BF9, a weakly active Kunitz-type animal toxin.

Although there were a lot of weakly active animal toxins in the venoms, their values and applications are still mysterious, such as BF9, which is a Kunitz-type toxin isolated from the venom of the elapid snake Bungarus fasciatus. Here, we used BF9 to be a molecular scaffold, and engineered eight BF9-derived peptides by changing P1 site Asn17 of BF9, such as BF9-N17Y and BF9-N17T designed from the polar subfamily, BF9-N17L and BF9-N17G designed from the Non-polar subfamily, BF9-N17D designed from acidic subfamily, and BF9-N17H, BF9-N17K and BF9-N17R designed from basic subfamily. Through enzyme inhibitor experiment assays, we found a potent and selective chymotrypsin inhibitor BF9-N17Y, a potent and selective coagulation factor XIa inhibitor BF9-N17H, and two highly potent coagulation factor XIa inhibitors BF9-N17K and BF9-N17. APTT and PT assays further showed that BF9-N17H, BF9-N17K and BF9-N17R were three novel anticoagulants with selectively intrinsic coagulation pathway inhibitory activity. Considering that natural weakly active animal toxins are also a huge peptide resource, our present work might open a new window about pharmacological applications of weakly active animal toxins, which might be good templates for potent and selective molecular probe and lead drug designs.

Venomics of Bungarus caeruleus (Indian krait): Comparable venom profiles, variable immunoreactivities among specimens from Sri Lanka, India and Pakistan.

The Indian krait (Bungarus caeruleus) is one of the "Big Four" venomous snakes widely distributed in South Asia. The present venomic study reveals that its venom (Sri Lankan origin) is predominated by phospholipases A2 (64.5% of total proteins), in which at least 4.6% are presynaptically-acting ß-bungarotoxin A-chains. Three-finger toxins (19.0%) are the second most abundant, comprising 15.6% κ-neurotoxins, the potent postsynaptically-acting long neurotoxins. Comparative chromatography showed that venom samples from Sri Lanka, India and Pakistan did not exhibit significant variation. These venoms exhibited high immunoreactivity toward VINS Indian Polyvalent Antivenom (VPAV). The Pakistani krait venom, however, had a relatively lower degree of binding, consistent with its moderate neutralization by VPAV (potency=0.3mg venom neutralized per ml antivenom) while the Sri Lankan and Indian venoms were more effectively neutralized (potency of 0.44 mg/ml and 0.48 mg/ml, respectively). Importantly, VPAV was able to neutralize the Sri Lankan and Indian venoms to a comparable extent, supporting its use in Sri Lanka especially in the current situation where Sri Lanka-specific antivenom is unavailable against this species. The findings also indicate that the Pakistani B. caeruleus venom is immunologically less comparable and should be incorporated in the production of a pan-regional, polyspecific antivenom. BIOLOGICAL SIGNIFICANCE: The Indian krait or blue krait, Bungarus caeruleus, is a highly venomous snake that contributes to the snakebite envenoming problem in South Asia. This is a less aggressive snake species but its accidental bite can cause rapid and severe neurotoxicity, in which the patient may succumb to paralysis, respiratory failure and death within a short frame of time. The proteomic analysis of its venom (sourced from Sri Lanka) unveils its content that well correlates to its envenoming pathophysiology, driven primarily by the abundant presynaptic and postsynaptic neurotoxins (ß-bungarotoxins and κ-neurotoxins, respectively). The absence of cytotoxins in the venom proteome also correlates with the lack of local envenoming sign (pain, swelling), and explains why the bite may be insidious until later stage when paralysis sets in. The muscarinic toxin-like proteins in the venom may be the cause of severe abdominal pain that precedes paralysis in many cases, and justifies the need of closely monitoring this symptom in suspected cases. Venom samples from Sri Lanka, India and Pakistan exhibited no remarkable variation in protein profiling and reacted immunologically toward the VINS Indian Polyvalent Antivenom, though to a varying extent. The antivenom is effective in neutralizing the Sri Lankan and Indian venoms, confirming its clinical use in the countries. The antivenom efficacy against the Pakistani venom, however, may be further optimized by incorporating the Pakistani venom in the antivenom production.

NACHO Mediates Nicotinic Acetylcholine Receptor Function throughout the Brain.

Neuronal nicotinic acetylcholine receptors (nAChRs) participate in diverse aspects of brain function and mediate behavioral and addictive properties of nicotine. Neuronal nAChRs derive from combinations of α and ß subunits, whose assembly is tightly regulated. NACHO was recently identified as a chaperone for α7-type nAChRs. Here, we find NACHO mediates assembly of all major classes of presynaptic and postsynaptic nAChR tested. NACHO acts at early intracellular stages of nAChR subunit assembly and then synergizes with RIC-3 for receptor surface expression. NACHO knockout mice show profound deficits in binding sites for α-bungarotoxin, epibatidine, and conotoxin MII, illustrating essential roles for NACHO in proper assembly of α7-, α4ß2-, and α6-containing nAChRs, respectively. By contrast, GABAA receptors are unaffected consistent with NACHO specifically modulating nAChRs. NACHO knockout mice show abnormalities in locomotor and cognitive behaviors compatible with nAChR deficiency and underscore the importance of this chaperone for physiology and disease associated with nAChRs.

Molecular modeling and structural analysis of nAChR variants uncovers the mechanism of resistance to snake toxins.

Nicotinic acetylcholine receptors (nAChRs) are neuromuscular proteins responsible for muscle contraction upon binding with chemical stimulant acetylcholine (ACh). The α-neurotoxins of snake mimic the structure of ACh and attacks nAChRs, which block the flow of ACh and leads to numbness and paralysis. The toxin-binding site of alpha subunit in the nAChRs is highly conserved throughout chordate lineages with few exceptions in resistance organisms. In this study, we have analyzed the sequence and structures of toxin-binding/resistant nAChRs and their interaction stability with toxins through molecular docking and molecular dynamics simulation (MDS). We have reported the potential glycosylation residues within the toxin-binding cleft adding sugar moieties through N-linked glycosylation in resistant organisms. Residue variations at key positions alter the secondary structure of binding cleft, which might interfere with toxin binding and it could be one of the possible explanations for the resistance to snake venoms. Analysis of nAChR-α-neurotoxin complexes has confirmed the key interacting residues. In addition, drastic variation in the binding stability of Mongoose nAChR-α-Bungarotoxin (α-BTX) and human nAChR-α-BTX complexes were found at specific phase of MDS. Our findings suggest that specific mutations in the binding site of toxin are potentially preventing the formation of stable complex of receptor-toxin, which might lead to mechanism of resistance. This in silico study on the binding cleft of nAChR and the findings of interacting residues will assist in designing potential inhibitors as therapeutic targets.