Emerging Opportunities in Human Pluripotent Stem-Cells Based Assays to Explore the Diversity of Botulinum Neurotoxins as Future Therapeutics.

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and are responsible for botulism, a fatal disorder of the nervous system mostly induced by food poisoning. Despite being one of the most potent families of poisonous substances, BoNTs are used for both aesthetic and therapeutic indications from cosmetic reduction of wrinkles to treatment of movement disorders. The increasing understanding of the biology of BoNTs and the availability of distinct toxin serotypes and subtypes offer the prospect of expanding the range of indications for these toxins. Engineering of BoNTs is considered to provide a new avenue for improving safety and clinical benefit from these neurotoxins. Robust, high-throughput, and cost-effective assays for BoNTs activity, yet highly relevant to the human physiology, have become indispensable for a successful translation of engineered BoNTs to the clinic. This review presents an emerging family of cell-based assays that take advantage of newly developed human pluripotent stem cells and neuronal function analyses technologies.

Using Reduced Amino Acid Alphabet and Biological Properties to Analyze and Predict Animal Neurotoxin Protein.

AIMS: Because of the high affinity of these animal neurotoxin proteins for some special target site, they were usually used as pharmacological tools and therapeutic agents in medicine to gain deep insights into the function of the nervous system. BACKGROUND AND OBJECTIVE: The animal neurotoxin proteins are one of the most common functional groups among the animal toxin proteins. Thus, it was very important to characterize and predict the animal neurotoxin proteins. METHODS: In this study, the differences between the animal neurotoxin proteins and non-toxin proteins were analyzed. RESULT: Significant differences were found between them. In addition, the support vector machine was proposed to predict the animal neurotoxin proteins. The predictive results of our classifier achieved the overall accuracy of 96.46%. Furthermore, the random forest and k-nearest neighbors were applied to predict the animal neurotoxin proteins. CONCLUSION: The compared results indicated that the predictive performances of our classifier were better than other two algorithms.

Insertions within the Saxitoxin Biosynthetic Gene Cluster Result in Differential Toxin Profiles.

The neurotoxin saxitoxin and related paralytic shellfish toxins are produced by multiple species of cyanobacteria and dinoflagellates. This study investigates the two saxitoxin-producing strains of Scytonema crispum, CAWBG524 and CAWBG72, isolated in New Zealand. Each strain was previously reported to have a distinct paralytic shellfish toxin profile, a rare observation between strains within the same species. Sequencing of the saxitoxin biosynthetic clusters ( sxt) from S. crispum CAWBG524 and S. crispum CAWBG72 revealed the largest sxt gene clusters described to date. The distinct toxin profiles of each strain were correlated to genetic differences in sxt tailoring enzymes, specifically the open-reading frame disruption of the N-21 sulfotransferase sxtN, adenylylsulfate kinase sxtO, and the C-11 dioxygenase sxtDIOX within S. crispum CAWBG524 via genetic insertions. Heterologous overexpression of SxtN allowed for the proposal of saxitoxin and 3'-phosphoadenosine 5'-phosphosulfate as substrate and cofactor, respectively, using florescence binding assays. Further, catalytic activity of SxtN was confirmed by the in vitro conversion of saxitoxin to the N-21 sulfonated analog gonyautoxin 5, making this the first known report to biochemically confirm the function of a sxt tailoring enzyme. Further, SxtN could not convert neosaxitoxin to its N-21 sulfonated analog gonyautoxin 6, indicating paralytic shellfish toxin biosynthesis most likely occurs along a predefined route. In this study, we identified key steps toward the biosynthetic conversation of saxitoxin to other paralytic shellfish toxins.

Shotgun Proteomics Analysis of Saliva and Salivary Gland Tissue from the Common Octopus Octopus vulgaris.

The salivary apparatus of the common octopus ( Octopus vulgaris) has been the subject of biochemical study for over a century. A combination of bioassays, behavioral studies and molecular analysis on O. vulgaris and related species suggests that its proteome should contain a mixture of highly potent neurotoxins and degradative proteins. However, a lack of genomic and transcriptomic data has meant that the amino acid sequences of these proteins remain almost entirely unknown. To address this, we assembled the posterior salivary gland transcriptome of O. vulgaris and combined it with high resolution mass spectrometry data from the posterior and anterior salivary glands of two adults, the posterior salivary glands of six paralarvae and the saliva from a single adult. We identified a total of 2810 protein groups from across this range of salivary tissues and age classes, including 84 with homology to known venom protein families. Additionally, we found 21 short secreted cysteine rich protein groups of which 12 were specific to cephalopods. By combining protein expression data with phylogenetic analysis we demonstrate that serine proteases expanded dramatically within the cephalopod lineage and that cephalopod specific proteins are strongly associated with the salivary apparatus.

Novel Botulinum Neurotoxins: Exploring Underneath the Iceberg Tip.

Botulinum neurotoxins (BoNTs), the etiological agents of botulism, are the deadliest toxins known to humans. Yet, thanks to their biological and toxicological features, BoNTs have become sophisticated tools to study neuronal physiology and valuable therapeutics for an increasing number of human disorders. BoNTs are produced by multiple bacteria of the genus Clostridium and, on the basis of their different immunological properties, were classified as seven distinct types of toxin. BoNT classification remained stagnant for the last 50 years until, via bioinformatics and high-throughput sequencing techniques, dozens of BoNT variants, novel serotypes as well as BoNT-like toxins within non-clostridial species have been discovered. Here, we discuss how the now “booming field” of botulinum neurotoxin may shed light on their evolutionary origin and open exciting avenues for future therapeutic applications.

Analysis and prediction of presynaptic and postsynaptic neurotoxins by Chou's general pseudo amino acid composition and motif features.

Presynaptic neurotoxins and postsynaptic neurotoxins are two important neurotoxins isolated from venoms of venomous animals and have been proven to be potential effective in neurosciences and pharmacology. With the number of toxin sequences appeared in the public databases, there was a need for developing a computational method for fast and accurate identification and classification of the novel presynaptic neurotoxins and postsynaptic neurotoxins in the large databases. In this study, the Multinomial Naive Bayes Classifier (MNBC) had been developed to discriminate the presynaptic neurotoxins and postsynaptic neurotoxins based on the different kinds of features. The Minimum Redundancy Maximum Relevance (MRMR) feature selection method was used for ranking 400 pseudo amino acid (PseAA) compositions and 50 top ranked PseAA compositions were selected for improving the prediction results. The motif features, 400 PseAA compositions and 50 PseAA compositions were combined together, and selected as the input parameters of MNBC. The best correlation coefficient (CC) value of 0.8213 was obtained when the prediction quality was evaluated by the jackknife test. It was anticipated that the algorithm presented in this study may become a useful tool for identification of presynaptic neurotoxin and postsynaptic neurotoxin sequences and may provide some useful help for in-depth investigation into the biological mechanism of presynaptic neurotoxins and postsynaptic neurotoxins.

Spider Neurotoxins, Short Linear Cationic Peptides and Venom Protein Classification Improved by an Automated Competition between Exhaustive Profile HMM Classifiers.

Spider venoms are rich cocktails of bioactive peptides, proteins, and enzymes that are being intensively investigated over the years. In order to provide a better comprehension of that richness, we propose a three-level family classification system for spider venom components. This classification is supported by an exhaustive set of 219 new profile hidden Markov models (HMMs) able to attribute a given peptide to its precise peptide type, family, and group. The proposed classification has the advantages of being totally independent from variable spider taxonomic names and can easily evolve. In addition to the new classifiers, we introduce and demonstrate the efficiency of hmmcompete, a new standalone tool that monitors HMM-based family classification and, after post-processing the result, reports the best classifier when multiple models produce significant scores towards given peptide queries. The combined used of hmmcompete and the new spider venom component-specific classifiers demonstrated 96% sensitivity to properly classify all known spider toxins from the UniProtKB database. These tools are timely regarding the important classification needs caused by the increasing number of peptides and proteins generated by transcriptomic projects.

Adaptive evolution of insect selective excitatory ß-type sodium channel neurotoxins from scorpion venom.

Insect selective excitatory ß-type sodium channel neurotoxins from scorpion venom (ß-NaScTxs) are composed of about 70-76 amino acid residues and share a common scaffold stabilized by four unique disulfide bonds. The phylogenetic analysis of these toxins was hindered by limited sequence data. In our recent study, two new insect selective excitatory ß-NaScTxs, LmIT and ImIT, were isolated from Lychas mucronatus and Isometrus maculatus, respectively. With the sequences previously reported, we examined the adaptive molecular evolution of insect selective excitatory ß-NaScTxs by estimating the nonsynonymous-to-synonymous rate ratio (ω=dN/dS). The results revealed 12 positively selected sites in the genes of insect selective excitatory ß-NaScTxs. Moreover, these positively selected sites match well with the sites important for interacting with sodium channels, as demonstrated in previous mutagenesis study. These results reveal that adaptive evolution after gene duplication is one of the most important genetic mechanisms of scorpion neurotoxin diversification.

Heterologous expression, protein folding and antibody recognition of a neurotoxin from the Mexican coral snake Micrurus laticorallis

The cysteine-rich neurotoxins from elapid venoms are primarily responsible for human and animal envenomation; however, their low concentration in the venom may hamper the production of efficient elapid antivenoms. Therefore, the aim of the present study was to produce fully active elapid neurotoxic immunogens for elapid antivenom production. Method Cysteine-rich neurotoxins showed recombinant expression in two strains of E. coli, and were purified using affinity chromatography and reverse-phase HPLC (rpHPLC). Results The cDNA of the four disulfide-bridged peptide neurotoxin Mlat1 was cloned into a modified expression vector, pQE30, which was transfected into two different E. coli strains. The recombinant toxin (HisrMlat1) was found only in inclusion bodies in M15 strain cells, and in both inclusion bodies and cytoplasm in Origami strain cells. The HisrMlat1 from inclusion bodies from M15 cells was solubilized using guanidine hydrochloride, and then purified by rpHPLC. It showed various contiguous fractions having the same molecular mass, indicating that HisrMlat1 was oxidized after cell extraction forming different misfolded disulfide bridge arrangements without biological activity. In vitro folding conditions of the misfolded HisrMlat1 generated a biologically active HisrMlat1. On the other hand, the HisrMlat1 from the cytoplasm from Origami cells was already soluble, and then purified by HPLC. It showed a single fraction with neurotoxic activity; so, no folding steps were needed. The in vitro folded HisrMlat1 from M15 cells and the cytoplasmic soluble HisrMlat1from Origami cells were indistinguishable in their structure and neurotoxicity. Rabbit polyclonal antibodies raised up against biologically active HisrMlat1 recognized the native Mlat1 (nMlat1) from the whole venom of M. laticorallis. In addition, HisrMlat1 was recognized by horse polyclonal antibodies obtained from the immunization of elapid species from sub-Saharan Africa. Conclusion HisrMlat1 shows increased biological activities compared to the native peptide, and may be used as an immunizing agent in combination with other toxic components such phospholipases type A2 for elapid antivenom production.

Manual classification strategies in the ECOD database.

ECOD (Evolutionary Classification Of protein Domains) is a comprehensive and up-to-date protein structure classification database. The majority of new structures released from the PDB (Protein Data Bank) each week already have close homologs in the ECOD hierarchy and thus can be reliably partitioned into domains and classified by software without manual intervention. However, those proteins that lack confidently detectable homologs require careful analysis by experts. Although many bioinformatics resources rely on expert curation to some degree, specific examples of how this curation occurs and in what cases it is necessary are not always described. Here, we illustrate the manual classification strategy in ECOD by example, focusing on two major issues in protein classification: domain partitioning and the relationship between homology and similarity scores. Most examples show recently released and manually classified PDB structures. We discuss multi-domain proteins, discordance between sequence and structural similarities, difficulties with assessing homology with scores, and integral membrane proteins homologous to soluble proteins. By timely assimilation of newly available structures into its hierarchy, ECOD strives to provide a most accurate and updated view of the protein structure world as a result of combined computational and expert-driven analysis.

Three enzymatically active neurotoxins of Clostridium botulinum strain Af84: BoNT/A2, /F4, and /F5.

Botulinum neurotoxins (BoNTs) are produced by various species of clostridia and are potent neurotoxins which cause the disease botulism, by cleaving proteins needed for successful nerve transmission. There are currently seven confirmed serotypes of BoNTs, labeled A-G, and toxin-producing clostridia typically only produce one serotype of BoNT. There are a few strains (bivalent strains) which are known to produce more than one serotype of BoNT, producing either both BoNT/A and /B, BoNT/A and /F, or BoNT/B and /F, designated as Ab, Ba, Af, or Bf. Recently, it was reported that Clostridium botulinum strain Af84 has three neurotoxin gene clusters: bont/A2, bont/F4, and bont/F5. This was the first report of a clostridial organism containing more than two neurotoxin gene clusters. Using a mass spectrometry based proteomics approach, we report here that all three neurotoxins, BoNT/A2, /F4, and /F5, are produced by C. botulinum Af84. Label free MS(E) quantification of the three toxins indicated that toxin composition is 88% BoNT/A2, 1% BoNT/F4, and 11% BoNT/F5. The enzymatic activity of all three neurotoxins was assessed by examining the enzymatic activity of the neurotoxins upon peptide substrates, which mimic the toxins' natural targets, and monitoring cleavage of the substrates by mass spectrometry. We determined that all three neurotoxins are enzymatically active. This is the first report of three enzymatically active neurotoxins produced in a single strain of Clostridium botulinum.

The genome of Mesobuthus martensii reveals a unique adaptation model of arthropods.

Representing a basal branch of arachnids, scorpions are known as 'living fossils' that maintain an ancient anatomy and are adapted to have survived extreme climate changes. Here we report the genome sequence of Mesobuthus martensii, containing 32,016 protein-coding genes, the most among sequenced arthropods. Although M. martensii appears to evolve conservatively, it has a greater gene family turnover than the insects that have undergone diverse morphological and physiological changes, suggesting the decoupling of the molecular and morphological evolution in scorpions. Underlying the long-term adaptation of scorpions is the expansion of the gene families enriched in basic metabolic pathways, signalling pathways, neurotoxins and cytochrome P450, and the different dynamics of expansion between the shared and the scorpion lineage-specific gene families. Genomic and transcriptomic analyses further illustrate the important genetic features associated with prey, nocturnal behaviour, feeding and detoxification. The M. martensii genome reveals a unique adaptation model of arthropods, offering new insights into the genetic bases of the living fossils.

Identification and characterization of a taxon-specific three-finger toxin from the venom of the Green Vinesnake (Oxybelis fulgidus; family Colubridae).

Snake venoms contain a variety of protein and peptide toxins, and the three-finger toxins (3FTxs) are among the best characterized family of venom proteins. The compact nature and highly conserved molecular fold of 3FTxs, together with their abundance in many venoms, has contributed to their utility in structure-function studies. Although many target the nicotinic acetylcholine receptor of vertebrate skeletal muscle, often binding with nanomolar Kds, several non-conventional 3FTxs show pronounced taxon-specific neurotoxic effects. Here we describe the purification and characterization of fulgimotoxin, a monomeric 3FTx from the venom of Oxybelis fulgidus, a neotropical rear-fanged snake. Fulgimotoxin retains the canonical 5 disulfides of the non-conventional 3FTxs and is highly neurotoxic to lizards; however, mice are unaffected, demonstrating that this toxin is taxon-specific in its effects. Analysis of structural features of fulgimotoxin and other colubrid venom 3FTxs indicate the presence of a "colubrid toxin motif" (CYTLY) and a second conserved segment (WAVK) found in Boiga and Oxybelis taxon-specific 3FTxs, both in loop II. Because specific residues in loop II conventional α-neurotoxic 3FTxs are intimately associated with receptor binding, we hypothesize that this loop, with its highly conserved substitutions, confers taxon-specific neurotoxicity. These findings underscore the importance of rear-fanged snake venoms for understanding the evolution of toxin molecules and demonstrate that even among well-characterized toxin families, novel structural and functional motifs may be found.

Potent tetravalent replicon vaccines against botulinum neurotoxins using DNA-based Semliki Forest virus replicon vectors.

Human botulism is commonly associated with botulinum neurotoxin (BoNT) serotypes A, B, E and F. This suggests that the greatest need is for a tetravalent vaccine that provides protection against all four of these serotypes. In current study, we investigated the feasibility of generating several tetravalent vaccines that protected mice against the four serotypes. Firstly, monovalent replicon vaccine against BoNT induced better antibody response and protection than that of corresponding conventional DNA vaccine. Secondly, dual-expression DNA replicon pSCARSE/FHc or replicon particle VRP-E/FHc vaccine was well resistant to the challenge of BoNT/E and BoNT/F mixture as a combination vaccine composed of two monovalent replicon vaccines. Finally, the dual-expression DNA replicon or replicon particle tetravalent vaccine could simultaneously and effectively neutralize and protect the four BoNT serotypes. Protection correlated directly with serum ELISA titers and neutralization antibody levels to BoNTs. Therefore, replicon-based DNA or particle might be effective vector to develop BoNT vaccines, which might be more desirable for use in clinical application than the conventional DNA vaccines. Our studies demonstrate the utility of combining dual-expression DNA replicon or replicon particle vaccines into multi-agent formulations as potent tetravalent vaccines for eliciting protective responses to four serotypes of BoNTs.

Isolation and characterization of an anti-insect ß-toxin from the venom of the scorpion Isometrus maculatus.

Im-3 was isolated from the venom of the scorpion Isometrus maculatus through several steps of HPLC fractionation based on the insect paralytic activity. Injecting Im-3 into crickets induced paralysis, but no toxicity was apparent in mice after an intracerebroventricular injection. Im-3 shares sequence similarity to scorpion ß-toxins that specifically affect insect sodium channels.

Molecular evolution of α-latrotoxin, the exceptionally potent vertebrate neurotoxin in black widow spider venom.

Black widow spiders (members of the genus Latrodectus) are widely feared because of their potent neurotoxic venom. α-Latrotoxin is the vertebrate-specific toxin responsible for the dramatic effects of black widow envenomation. The evolution of this toxin is enigmatic because only two α-latrotoxin sequences are known. In this study, ~4 kb α-latrotoxin sequences and their homologs were characterized from a diversity of Latrodectus species, and representatives of Steatoda and Parasteatoda, establishing the wide distribution of latrotoxins across the mega-diverse spider family Theridiidae. Across black widow species, α-latrotoxin shows ≥ 94% nucleotide identity and variability consistent with purifying selection. Multiple codon and branch-specific estimates of the nonsynonymous/synonymous substitution rate ratio also suggest a long history of purifying selection has acted on α-latrotoxin across Latrodectus and Steatoda. However, α-latrotoxin is highly divergent in amino acid sequence between these genera, with 68.7% of protein differences involving non-conservative substitutions, evidence for positive selection on its physiochemical properties and particular codons, and an elevated rate of nonsynonymous substitutions along α-latrotoxin's Latrodectus branch. Such variation likely explains the efficacy of red-back spider, L. hasselti, antivenom in treating bites from other Latrodectus species, and the weaker neurotoxic symptoms associated with Steatoda and Parasteatoda bites. Long-term purifying selection on α-latrotoxin indicates its functional importance in black widow venom, even though vertebrates are a small fraction of their diet. The greater differences between Latrodectus and Steatoda α-latrotoxin, and their relationships to invertebrate-specific latrotoxins, suggest a shift in α-latrotoxin toward increased vertebrate toxicity coincident with the evolution of widow spiders.

Genetic diversity within Clostridium botulinum serotypes, botulinum neurotoxin gene clusters and toxin subtypes.

Clostridium botulinum is a species of spore-forming anaerobic bacteria defined by the expression of any one or two of seven serologically distinct botulinum neurotoxins (BoNTs) designated BoNT/A-G. This Gram-positive bacterium was first identified in 1897 and since then the paralyzing and lethal effects of its toxin have resulted in the recognition of different forms of the intoxication known as food-borne, infant, or wound botulism. Early microbiological and biochemical characterization of C. botulinum isolates revealed that the bacteria within the species had different characteristics and expressed different toxin types. To organize the variable bacterial traits within the species, Group I-IV designations were created. Interestingly, it was observed that isolates within different Groups could express the same toxin type and conversely a single Group could express different toxin types. This discordant phylogeny between the toxin and the host bacteria indicated that horizontal gene transfer of the toxin was responsible for the variation observed within the species. The recent availability of multiple C. botulinum genomic sequences has offered the ability to bioinformatically analyze the locations of the bont genes, the composition of their toxin gene clusters, and the genes flanking these regions to understand their variation. Comparison of the genomic sequences representing multiple serotypes indicates that the bont genes are not in random locations. Instead the analyses revealed specific regions where the toxin genes occur within the genomes representing serotype A, B, C, E, and F C. botulinum strains and C. butyricum type E strains. The genomic analyses have provided evidence of horizontal gene transfer, site-specific insertion, and recombination events. These events have contributed to the variation observed among the neurotoxins, the toxin gene clusters and the bacteria that contain them, and has supported the historical microbiological, and biochemical characterization of the Group classification within the species.

Effects of snake venom polypeptides on central nervous system.

The nervous system is a primary target for animal venoms as the impairment of its function results in the fast and efficient immobilization or death of a prey. There are numerous evidences about effects of crude snake venoms or isolated toxins on peripheral nervous system. However, the data on their interactions with the central nervous system (CNS) are not abundant, as the blood-brain barrier (BBB) impedes penetration of these compounds into brain. This updated review presents the data about interaction of snake venom polypeptides with CNS. Such data will be described according to three main modes of interactions: - Direct in vivo interaction of CNS with venom polypeptides either capable to penetrate BBB or injected into the brain. - In vitro interactions of cell or sub-cellular fractions of CNS with crude venoms or purified toxins. - Indirect effects of snake venoms or their components on functioning of CNS under different conditions. Although the venom components penetrating BBB are not numerous, they seem to be the most suitable candidates for the leads in drug design. The compounds with other modes of action are more abundant and better studied, but the lack of the data about their ability to penetrate BBB may substantially aggravate the potentials for their medical perspectives. Nevertheless, many such compounds are used for research of CNS in vitro. These investigations may give invaluable information for understanding the molecular basis of CNS diseases and thus lay the basis for targeted drug design. This aspect also will be outlined in the review.

Emerging opportunities for serotypes of botulinum neurotoxins.

BACKGROUND: Two decades ago, botulinum neurotoxin (BoNT) type A was introduced to the commercial market. Subsequently, the toxin was approved by the FDA to address several neurological syndromes, involving muscle, nerve, and gland hyperactivity. These syndromes have typically been associated with abnormalities in cholinergic transmission. Despite the multiplicity of botulinal serotypes (designated as types A through G), therapeutic preparations are currently only available for BoNT types A and B. However, other BoNT serotypes are under study for possible clinical use and new clinical indications; OBJECTIVE: To review the current research on botulinum neurotoxin serotypes A-G, and to analyze potential applications within basic science and clinical settings; CONCLUSIONS: The increasing understanding of botulinal neurotoxin pathophysiology, including the neurotoxin's effects on specific neuronal populations, will help us in tailoring treatments for specific diagnoses, symptoms and patients. Scientists and clinicians should be aware of the full range of available data involving neurotoxin subtypes A-G.

Unique biological activity of botulinum D/C mosaic neurotoxin in murine species.

Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.