How does the neurotoxin ß-N-methylamino-L-alanine exist in biological matrices and cause toxicity?

The neurotoxin ß-N-methylamino-L-alanine (BMAA) has been deemed as a risk factor for some neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism dementia complex (ALS/PDC). This possible link has been proved in some primate models and cell cultures with the appearance that BMAA exposure can cause excitotoxicity, formation of protein aggregates, and/or oxidative stress. The neurotoxin BMAA extensively exists in the environment and can be transferred through the food web to human beings. In this review, the occurrence, toxicological mechanisms, and characteristics of BMAA were comprehensively summarized, and proteins and peptides were speculated as its possible binding substances in biological matrices. It is difficult to compare the published data from previous studies due to the inconsistent analytical methods and components of BMAA. The binding characteristics of BMAA should be focused on to improve our understanding of its health risk to human health in the future.

Botulinum neurotoxin X lacks potency in mice and in human neurons.

Botulinum neurotoxins (BoNTs) are a class of toxins produced by Clostridium botulinum (C. botulinum) and other species of Clostridia. BoNT/X is a putative novel botulinum neurotoxin identified through genome sequencing and capable of SNARE cleavage, but its neurotoxic potential in humans and vertebrates remained unclear. The C. botulinum strain producing BoNT/X, Strain 111, encodes both a plasmid-borne bont/b2 as well as the chromosomal putative bont/x. This study utilized C. botulinum Strain 111 from Japan as well as recombinantly produced full-length BoNT/X to more fully analyze this putative pathogenic toxin. We confirmed production of full-length, catalytically active native BoNT/X by C. botulinum Strain 111, produced as a disulfide-bonded dichain polypeptide similar to other BoNTs. Both the purified native and the recombinant BoNT/X had high enzymatic activity in vitro but displayed very low potency in human-induced pluripotent stem cell-derived neuronal cells and in mice. Intraperitoneal injection of up to 50 µg of native BoNT/X in mice did not result in botulism; however, mild local paralysis was observed after injection of 2 µg into the gastrocnemius muscle. We further demonstrate that the lack of toxicity by BoNT/X is due to inefficient neuronal cell association and entry, which can be rescued by replacing the receptor binding domain of BoNT/X with that of BoNT/A. These data demonstrate that BoNT/X is not a potent vertebrate neurotoxin like the classical seven serotypes of BoNTs. IMPORTANCE: The family of botulinum neurotoxins comprises the most potent toxins known to humankind. New members of this family of protein toxins as well as more distantly related homologs are being identified. The discovery of BoNT/X via bioinformatic screen in 2017 as a putative new BoNT serotype raised concern about its potential as a pathogenic agent with no available countermeasures. This study for the first time assessed both recombinantly produced and native purified BoNT/X for its vertebrate neurotoxicity.

An in vitro assay to investigate venom neurotoxin activity on muscle-type nicotinic acetylcholine receptor activation and for the discovery of toxin-inhibitory molecules.

Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by α-neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting α-neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the γ-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying α-neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.

Resistance Is Not Futile: Widespread Convergent Evolution of Resistance to Alpha-Neurotoxic Snake Venoms in Caecilians (Amphibia: Gymnophiona).

Predatory innovations impose reciprocal selection pressures upon prey. The evolution of snake venom alpha-neurotoxins has triggered the corresponding evolution of resistance in the post-synaptic nicotinic acetylcholine receptors of prey in a complex chemical arms race. All other things being equal, animals like caecilians (an Order of legless amphibians) are quite vulnerable to predation by fossorial elapid snakes and their powerful alpha-neurotoxic venoms; thus, they are under strong selective pressure. Here, we sequenced the nicotinic acetylcholine receptor alpha-1 subunit of 37 caecilian species, representing all currently known families of caecilians from across the Americas, Africa, and Asia, including species endemic to the Seychelles. Three types of resistance were identified: (1) steric hindrance from N-glycosylated asparagines; (2) secondary structural changes due to the replacement of proline by another amino acid; and (3) electrostatic charge repulsion of the positively charged neurotoxins, through the introduction of a positively charged amino acid into the toxin-binding site. We demonstrated that resistance to alpha-neurotoxins convergently evolved at least fifteen times across the caecilian tree (three times in Africa, seven times in the Americas, and five times in Asia). Additionally, as several species were shown to possess multiple resistance modifications acting synergistically, caecilians must have undergone at least 20 separate events involving the origin of toxin resistance. On the other hand, resistance in non-caecilian amphibians was found to be limited to five origins. Together, the mutations underlying resistance in caecilians constitute a robust signature of positive selection which strongly correlates with elapid presence through both space (sympatry with caecilian-eating elapids) and time (Cenozoic radiation of elapids). Our study demonstrates the extent of convergent evolution that can be expected when a single widespread predatory adaptation triggers parallel evolutionary arms races at a global scale.

Mammalian neurotoxins, Blarina paralytic peptides, cause hyperpolarization of human T-type Ca channel hCav3.2 activation.

Among the rare venomous mammals, the short-tailed shrew Blarina brevicauda has been suggested to produce potent neurotoxins in its saliva to effectively capture prey. Several kallikrein-like lethal proteases have been identified, but the active substances of B. brevicauda remained unclear. Here, we report Blarina paralytic peptides (BPPs) 1 and 2 isolated from its submaxillary glands. Synthetic BPP2 showed mealworm paralysis and a hyperpolarization shift (-11 mV) of a human T-type Ca2+ channel (hCav3.2) activation. The amino acid sequences of BPPs were similar to those of synenkephalins, which are precursors of brain opioid peptide hormones that are highly conserved among mammals. However, BPPs rather resembled centipede neurotoxic peptides SLPTXs in terms of disulfide bond connectivity and stereostructure. Our results suggested that the neurotoxin BPPs were the result of convergent evolution as homologs of nontoxic endogenous peptides that are widely conserved in mammals. This finding is of great interest from the viewpoint of the chemical evolution of vertebrate venoms.

SDS-induced hexameric oligomerization of myotoxin-II from Bothrops asper assessed by sedimentation velocity and nuclear magnetic resonance.

We report the solution behavior, oligomerization state, and structural details of myotoxin-II purified from the venom of Bothrops asper in the presence and absence of sodium dodecyl sulfate (SDS) and multiple lipids, as examined by analytical ultracentrifugation and nuclear magnetic resonance. Molecular functional and structural details of the myotoxic mechanism of group II Lys-49 phospholipase A2 homologues have been only partially elucidated so far, and conflicting observations have been reported in the literature regarding the monomeric vs. oligomeric state of these toxins in solution. We observed the formation of a stable and discrete, hexameric form of myotoxin-II, but only in the presence of small amounts of SDS. In SDS-free medium, myotoxin-II was insensitive to mass action and remained monomeric at all concentrations examined (up to 3 mg/ml, 218.2 µM). At SDS concentrations above the critical micelle concentration, only dimers and trimers were observed, and at intermediate SDS concentrations, aggregates larger than hexamers were observed. We found that the amount of SDS required to form a stable hexamer varies with protein concentration, suggesting the need for a precise stoichiometry of free SDS molecules. The discovery of a stable hexameric species in the presence of a phospholipid mimetic suggests a possible physiological role for this oligomeric form, and may shed light on the poorly understood membrane-disrupting mechanism of this myotoxic protein class.

Lys49 myotoxins, secreted phospholipase A2-like proteins of viperid venoms: A comprehensive review.

Muscle necrosis is a potential clinical complication of snakebite envenomings, which in severe cases can lead to functional or physical sequelae such as disability or amputation. Snake venom proteins with the ability to directly damage skeletal muscle fibers are collectively referred to as myotoxins, and include three main types: cytolysins of the "three-finger toxin" protein family expressed in many elapid venoms, the so-called "small" myotoxins found in a number of rattlesnake venoms, and the widespread secreted phospholipase A2 (sPLA2) molecules. Among the latter, protein variants that conserve the sPLA2 structure, but lack such enzymatic activity, have been increasingly found in the venoms of many viperid species. Intriguingly, these sPLA2-like proteins are able to induce muscle necrosis by a mechanism independent of phospholipid hydrolysis. They are commonly referred to as "Lys49 myotoxins" since they most often present, among other substitutions, the replacement of the otherwise invariant residue Asp49 of sPLA2s by Lys. This work comprehensively reviews the historical developments and current knowledge towards deciphering the mechanism of action of Lys49 sPLA2-like myotoxins, and points out main gaps to be filled for a better understanding of these multifaceted snake venom proteins, to hopefully lead to improved treatments for snakebites.

Critical analysis in the advancement of cell-based assays for botulinum neurotoxin.

The study on botulinum neurotoxins (BoNTs) has rapidly evolved for their structure and functions as opposed to them being poisons or cures. Since their discoveries, the scientific community has come a long way in understanding BoNTs' structure and biological activity. Given its current application as a tool for understanding neurocellular activity and as a drug against over 800 neurological disorders, relevant and sensitive assays have become critical for biochemical, physiological, and pharmacological studies. The natural entry of the toxin being ingestion, it has also become important to examine its mechanism while crossing the epithelial cell barrier. Several techniques and methodologies have been developed, for its entry, pharmacokinetics, and biological activity for identification, and drug efficacy both in vivo and in vitro conditions. However, each of them presents its own challenges. The cell-based assay is a platform that exceeds the sensitivity of mouse bioassay while encompassing all the steps of intoxication including cell binding, transcytosis, endocytosis, translocation and proteolytic activity. In this article we review in detail both the neuronal and nonneuronal based cellular interaction of BoNT involving its transportation, and interaction with the targeted cells, and intracellular activities.

A secretory system for extracellular production of spider neurotoxin huwentoxin-I in Escherichia coli.

Due to their advantages in structural stability and versatility, cysteine-rich peptides, which are secreted from the venom glands of venomous animals, constitute a naturally occurring pharmaceutical arsenal. However, the correct folding of disulfide bonds is a challenging task in the prokaryotic expression system like Escherichia coli due to the reducing environment. Here, a secretory expression plasmid pSE-G1M5-SUMO-HWTX-I for the spider neurotoxin huwentoxin-I (HWTX-I) with three disulfides as a model of cysteine-rich peptides was constructed. By utilizing the signal peptide G1M5, the fusion protein 6 × His-SUMO-HWTX-I was successfully secreted into extracellular medium of BL21(DE3). After enrichment using cation-exchange chromatography and purification utilizing the Ni-NTA column, 6 × His-SUMO-HWTX-I was digested via Ulp1 kinase to release recombinant HWTX-I (rHWTX-I), which was further purified utilizing RP-HPLC. Finally, both impurities with low and high molecular weights were completely removed. The molecular mass of rHWTX-I was identified as being 3750.8 Da, which was identical to natural HWTX-I with three disulfide bridges. Furthermore, by utilizing whole-cell patch clamp, the sodium currents of hNav1.7 could be inhibited by rHWTX-I and the IC50 value was 419 nmol/L.

Utilize a few features to classify presynaptic and postsynaptic neurotoxins.

Neurotoxins are a class of proteins that have a significant damaging effect on nerve tissue. Neurotoxins are classified into presynaptic neurotoxins and postsynaptic neurotoxins, and accurate identification of neurotoxins plays a key role in drug development. In this study, 90 presynaptic neurotoxins and 165 postsynaptic neurotoxins were classified. The features of the presynaptic and postsynaptic neurotoxin sequences were extracted using the AutoProp feature extraction method and feature selection was performed using the maximum relevance maximum distance (MRMD) program, Finally, only two features were retained to achieve 84.7% classification accuracy. Moreover, it was found that the two retained features were present in the conserved sites and motifs of presynaptic neurotoxins and could represent the critical structures of presynaptic neurotoxins. This method demonstrates that using a few key features to classify proteins can effectively identify critical protein structures.

Structural Basis of Botulinum Toxin Type F Binding to Glycosylated Human SV2A: In Silico Studies at the Periphery of a Lipid Raft.

Botulinum neurotoxins are the deadliest microbial neurotoxins in humans, with a lethal dose of 1 ng/kg. Incidentally, these neurotoxins are also widely used for medical and cosmetic purposes. However, little is known about the molecular mechanisms that control binding of botulinum neurotoxin type F1 (BoNT/F1) to its membrane receptor, glycosylated human synaptic vesicle glycoprotein A (hSV2Ag). To elucidate these mechanisms, we performed a molecular dynamics simulation (MDS) study of initial binding kinetics of BoNT/F1 to SV2A. Since this toxin also interacts with gangliosides, the simulations were performed at the periphery of a lipid raft in the presence of both SV2A and gangliosides. Our study suggested that interaction of BoNT/F1 with SV2A is exclusively mediated by N-glycan moiety of SV2A, which interacts with aromatic residues Y898, Y910, F946, Y1059 and H1273 of this toxin. Thus, in contrast with botulinum neurotoxin A1 (BoNT/A1), BoNT/F1 does not interact with protein content of SV2A. We attributed this incapability to a barrage effect exerted by neurotoxin residues Y1132, Q1133 and K1134, which prevent formation of long-lasting intermolecular hydrogen bonds. We also provided structural elements that suggest that BoNT/F1 uses the strategy of BoNT/A1 combined with the strategy of botulinum neurotoxin type E to bind N-glycan of its glycoprotein receptor. Overall, our study opened a gate for design of a universal inhibitor aimed at disrupting N-glycan-toxin interactions and for bioengineering of a BoNT/F1 protein that may be able to bind protein content of synaptic vesicle glycoprotein for therapeutic purposes.

Neuronal Cellular Nanosponges for Effective Detoxification of Neurotoxins.

Neurotoxins attack and destruct the nervous system, which can cause serious health problems and security threats. Existing detoxification approaches, such as antibodies and small molecule antidotes, rely on neurotoxin's molecular structure as design cues and require toxin-specific development for each type of toxins. However, the enormous diversity of neurotoxins makes such structure-based development of antitoxin particularly challenging and inefficient. Here, we report on the development and use of neuronal membrane-coated nanosponges (denoted "Neuron-NS") as an effective approach to detoxifying neurotoxins. Specifically, Neuron-NS act as neuron decoys to lure neurotoxins, bind with and neutralize the toxins, and thus block them from attacking the host neuron cells. These nanosponges detoxify neurotoxins regardless of their molecular structures and therefore can overcome the challenge posed by toxin structural diversity. In the study, we fabricate Neuron-NS by coating the membrane of Neuro-2a cells onto polymeric cores. Meanwhile, we select tetrodotoxin (TTX) as a model neurotoxin and demonstrate the detoxification efficacy of the Neuron-NS in a cytotoxicity assay, a calcium flux assay, and a cell osmotic swelling assay in vitro. Additionally, in mouse models of TTX intoxication, the Neuron-NS significantly enhance mouse survival in therapeutic and prophylactic regimens without showing acute toxicity. Overall, the Neuron-NS contribute to the current detoxification arsenal with the potential to treat various injuries and diseases caused by neurotoxins.

The molecular mechanism of snake short-chain α-neurotoxin binding to muscle-type nicotinic acetylcholine receptors.

Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors. Here, we determine the cryo-EM structure of the muscle-type Torpedo receptor in complex with ScNtx, a recombinant short-chain α-neurotoxin. ScNtx is pinched between loop C on the principal subunit and a unique hairpin in loop F on the complementary subunit, thereby blocking access to the neurotransmitter binding site. ScNtx adopts a binding mode that is tilted toward the complementary subunit, forming a wider network of interactions than those seen in the long-chain α-Bungarotoxin complex. Certain mutations in ScNtx at the toxin-receptor interface eliminate inhibition of neuronal α7 nAChRs, but not of human muscle-type receptors. These observations explain why ScNtx binds more tightly to muscle-type receptors than neuronal receptors. Together, these data offer a framework for understanding subtype-specific actions of short-chain α-neurotoxins and inspire strategies for design of new snake antivenoms.

Two types of peptides derived from the neurotoxin GsMTx4 inhibit a mechanosensitive potassium channel by modifying the mechanogate.

Atrial fibrillation is the most common sustained cardiac arrhythmia in humans. Current atrial fibrillation antiarrhythmic drugs have limited efficacy and carry the risk of ventricular proarrhythmia. GsMTx4, a mechanosensitive channel-selective inhibitor, has been shown to suppress arrhythmias through the inhibition of stretch-activated channels (SACs) in the heart. The cost of synthesizing this peptide is a major obstacle to clinical use. Here, we studied two types of short peptides derived from GsMTx4 for their effects on a stretch-activated big potassium channel (SAKcaC) from the heart. Type I, a 17-residue peptide (referred to as Pept 01), showed comparable efficacy, whereas type II (i.e., Pept 02), a 10-residue peptide, exerted even more potent inhibitory efficacy on SAKcaC compared with GsMTx4. We identified through mutagenesis important sequences required for peptide functions. In addition, molecular dynamics simulations revealed common structural features with a hydrophobic head followed by a positively charged protrusion that may be involved in peptide channel-lipid interactions. Furthermore, we suggest that these short peptides may inhibit SAKcaC through a specific modification to the mechanogate, as the inhibitory effects for both types of peptides were mostly abolished when tested with a mechano-insensitive channel variant (STREX-del) and a nonmechanosensitive big potassium (mouse Slo1) channel. These findings may offer an opportunity for the development of a new class of drugs in the treatment of cardiac arrhythmia generated by excitatory SACs in the heart.

Non-Proteinogenic Amino Acid ß-N-Methylamino-L-Alanine (BMAA): Bioactivity and Ecological Significance.

Research interest in a non-protein amino acid ß-N-methylamino-L-alanine (BMAA) arose due to the discovery of a connection between exposure to BMAA and the occurrence of neurodegenerative diseases. Previous reviews on this topic either considered BMAA as a risk factor for neurodegenerative diseases or focused on the problems of detecting BMAA in various environmental samples. Our review is devoted to a wide range of fundamental biological problems related to BMAA, including the molecular mechanisms of biological activity of BMAA and the complex relationships between producers of BMAA and the environment in various natural ecosystems. At the beginning, we briefly recall the most important facts about the producers of BMAA (cyanobacteria, microalgae, and bacteria), the pathways of BMAA biosynthesis, and reliable methods of identification of BMAA. The main distinctive feature of our review is a detailed examination of the molecular mechanisms underlying the toxicity of BMAA to living cells. A brand new aspect, not previously discussed in any reviews, is the effect of BMAA on cyanobacterial cells. These recent studies, conducted using transcriptomics and proteomics, revealed potent regulatory effects of BMAA on the basic metabolism and cell development of these ancient photoautotrophic prokaryotes. Exogenous BMAA strongly influences cell differentiation and primary metabolic processes in cyanobacteria, such as nitrogen fixation, photosynthesis, carbon fixation, and various biosynthetic processes involving 2-oxoglutarate and glutamate. Cyanobacteria were found to be more sensitive to exogenous BMAA under nitrogen-limited growth conditions. We suggest a hypothesis that this toxic diaminoacid can be used by phytoplankton organisms as a possible allelopathic tool for controlling the population of cyanobacterial cells during a period of intense competition for nitrogen and other resources in various ecosystems.

Anti-epileptic/pro-epileptic effects of sodium channel modulators from Buthus martensii Karsch.

The East Asian scorpion Buthus martensii Karsch (BmK) is one of the classical traditional Chinese medicines for treating epilepsy for over a thousand years. Neurotoxins purified from BmK venom are considered as the main active ingredients, acting on membrane ion channels. Voltage-gated sodium channels (VGSCs) play a crucial role in the occurrence of epilepsy, which make them become important drug targets for epilepsy. Long chain toxins of BmK, composed of 60-70 amino acid residues, could specifically recognize VGSCs. Among them, α-like neurotoxins, binding to the receptor site-3 of VGSC, induce epilepsy in rodents and can be used to establish seizure models. The ß or ß-like neurotoxins, binding to the receptor site-4 of VGSC, have significant anticonvulsant effects in epileptic models. This review aims to illuminate the anticonvulsant/convulsant effects of BmK polypeptides by acting on VGSCs, and provide potential frameworks for the anti-epileptic drug-design.

Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes.

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins in vivo. Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-α-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites. Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-clamp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long α-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.

Neurotoxic and cytotoxic peptides underlie the painful stings of the tree nettle Urtica ferox.

The stinging hairs of plants from the family Urticaceae inject compounds that inflict pain to deter herbivores. The sting of the New Zealand tree nettle (Urtica ferox) is among the most painful of these and can cause systemic symptoms that can even be life-threatening; however, the molecular species effecting this response have not been elucidated. Here we reveal that two classes of peptide toxin are responsible for the symptoms of U. ferox stings: Δ-Uf1a is a cytotoxic thionin that causes pain via disruption of cell membranes, while ß/δ-Uf2a defines a new class of neurotoxin that causes pain and systemic symptoms via modulation of voltage-gated sodium (NaV) channels. We demonstrate using whole-cell patch-clamp electrophysiology experiments that ß/δ-Uf2a is a potent modulator of human NaV1.5 (EC50: 55 nM), NaV1.6 (EC50: 0.86 nM), and NaV1.7 (EC50: 208 nM), where it shifts the activation threshold to more negative potentials and slows fast inactivation. We further found that both toxin classes are widespread among members of the Urticeae tribe within Urticaceae, suggesting that they are likely to be pain-causing agents underlying the stings of other Urtica species. Comparative analysis of nettles of Urtica, and the recently described pain-causing peptides from nettles of another genus, Dendrocnide, indicates that members of tribe Urticeae have developed a diverse arsenal of pain-causing peptides.

Matrix Effect of Diverse Biological Samples Extracted with Different Extraction Ratios on the Detection of ß-N-Methylamino-L-Alanine by Two Common LC-MS/MS Analysis Methods.

Neurotoxin ß-N-methylamino-L-alanine (BMAA) is hypothesized as an important pathogenic factor for neurodegenerative diseases such as amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC). Comparative study on the accuracy of BMAA analyzed by the regular LC-MS/MS methods is still limited for different biological matrices. In this study, a free-BMAA sample of cyanobacterium and BMAA-containing positive samples of diatom, mussel, scallop, and oyster were extracted with varied extraction ratios (ER) ranging from 1:20 to 1:2000. These extracts were then purified by MCX cartridges. After SPE purification, these different biological samples were analyzed by two common LC-MS/MS analysis methods, a direct analysis without derivatization by a hydrophilic interaction liquid chromatography (HILIC)-MS/MS and pre-column 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization combined with a C18 column. The results suggested that the recoveries of BMAA spiked in the cyanobacterial sample were close to 100% in the total soluble form extracts with the ER of 1:100 (g/mL) and the precipitated bound form extracts with the ER of 1:500. The recommended ER for the precipitated bound form of BMAA in diatoms and the total soluble form of BMAA in mollusks are 1:500 and 1:50, respectively. The quantitative results determined by the AQC derivatization method were lower than those determined by the direct analysis of the HILIC method in diatom and mollusk samples. The results of the HILIC method without the derivatization process were closer to the true value of BMAA in cyanobacteria. This work contributes to the performance of the solid-phase extraction (SPE) purification protocol and the accuracy of BMAA analysis by LC-MS/MS in diverse biological samples.

Immunogenicity of snake α-neurotoxins and the CD4 T cell epitopes.

Snakebite envenomation is an important medical problem in numerous parts of the world causing about 2.7 million envenomations and between 81,000 and 138,000 deaths ayear. Antivenoms (AVs) are time proven effective therapeutics for snakebite envenomation. However, AVs, especially those against elapid neurotoxic venoms (cobras, kraits and mambas), are difficult to produce and are generally of low neutralizing potency. The most lethal component of most elapid venoms is the postsynaptic neurotoxins or the α-neurotoxins, which are responsible for death in most victims. It is generally believed that the low neutralizing potency of the AVs is due to the small molecular sizes, and thus the low immunogenicity, of the α-neurotoxins. Therefore, modifications of the toxins have been made to increase their size, and/or to detoxify them, hoping to improve the toxin's immunogenicity and AV potency. However, these maneuvers have not been applied to commercial AV production. The α-neurotoxins belong to a group of small proteins called three-finger toxins (3FTxs). The 3FTxs contain about 60-77 amino acid residues with four to five disulfide linkages and three anti-parallel ß-sheets, which extend from a globular hydrophobic core resembling three fingers. The members of the 3FTxs exhibit a number of important pharmacological activities, e.g., inhibition of neuromuscular transmission and acetyl cholinesterase activities. Recent immunization experiments with a 26 amino acid peptide containing the consensus sequence of the α-neurotoxins, and a mixture of elapid α-neurotoxins using highly effective adjuvants and immunization protocols have resulted in neutralizing antibodies in rabbit and horse, respectively. In the present report using bioinformatics, we show that 23 3FTxs which include α-neurotoxins, cardiotoxins and non-conventional toxins, and the 26 amino acid peptide, were all predicted to contain high to medium score CD4 T-cell epitopes for human and mouse MHC IIs. This information corroborates the results obtained from animal experiments that the α-neurotoxins, in spite of their small sizes and toxicity, are in fact immunogenic. Thus, the uses of effective adjuvants and immunization procedures, rather than chemical/physical modifications of the toxin structures, are crucial to the production of potent AVs against elapid neurotoxic venoms.