Novel genes encoding six kinds of three-finger toxins in Ophiophagus hannah (king cobra) and function characterization of two recombinant long-chain neurotoxins

Three-finger toxins are a family of low-molecular-mass toxins (<10 kDa) having very similar three-dimensional structures. In the present study, 19 novel cDNAs coding three-finger toxins were cloned from the venom gland of Ophiophagus hannah (king cobra). Alignment analysis showed that the putative peptides could be divided into six kinds of three-finger toxins: LNTXs (long-chain neurotoxins), short-chain neurotoxins, cardiotoxins (CTXs), weak neurotoxins, muscarinic toxins and a toxin with a free SH group. Furthermore, a phylogenetic tree was established on the basis of the toxin cDNAs and the previously reported similar nucleotide sequences from the same source venom. It indicated that three-finger-toxin genes in O. hannah diverged early in the course of evolution by long- and short-type pathways. Two LNTXs, namely rLNTX1 (recombinant LNTX1) and rLNTX3, were expressed and showed cytolytic activity in addition to their neurotoxic function. By comparing the functional residues, we offer some possible explanations for the differences in their neurotoxic function. Moreover, a plausible elucidation of the additonal cytolytic activity was achieved by hydropathy-profile analysis. This, to our knowledge, is the first observation that recombinant long chain alpha-neurotoxins have a CTX-like cytolytic activity.     

Specialisation of the venom gland proteome in predatory cone snails reveals functional diversification of the conotoxin biosynthetic pathway

Conotoxins, venom peptides from marine cone snails, diversify rapidly as speciation occurs. It has been suggested that each species can synthesize between 1000 and 1900 different toxins with little to no interspecies overlap. Conotoxins exhibit an unprecedented degree of post-translational modifications, the most common one being the formation of disulfide bonds. Despite the great diversity of structurally complex peptides, little is known about the glandular proteins responsible for their biosynthesis and maturation. Here, proteomic interrogations on the Conus venom gland led to the identification of novel glandular proteins of potential importance for toxin synthesis and secretion. A total of 161 and 157 proteins and protein isoforms were identified in the venom glands of Conus novaehollandiae and Conus victoriae, respectively. Interspecies differences in the venom gland proteomes were apparent. A large proportion of the proteins identified function in protein/peptide translation, folding, and protection events. Most intriguingly, however, we demonstrate the presence of a multitude of isoforms of protein disulfide isomerase (PDI), the enzyme catalyzing the formation and isomerization of the native disulfide bond. Investigating whether different PDI isoforms interact with distinct toxin families will greatly advance our knowledge on the generation of cone snail toxins and disulfide-rich peptides in general.     

A synthetic weak neurotoxin binds with low affinity to Torpedo and chicken alpha7 nicotinic acetylcholine receptors.

Weak neurotoxins from snake venom are small proteins with five disulfide bonds, which have been shown to be poor binders of nicotinic acetylcholine receptors. We report on the cloning and sequencing of four cDNAs encoding weak neurotoxins from Naja sputatrix venom glands. The protein encoded by one of them, Wntx-5, has been synthesized by solid-phase synthesis and characterized. The physicochemical properties of the synthetic toxin (sWntx-5) agree with those anticipated for the natural toxin. We show that this toxin interacts with relatively low affinity (K(d) = 180 nm) with the muscular-type acetylcholine receptor of the electric organ of T. marmorata, and with an even weaker affinity (90 microm) with the neuronal alpha7 receptor of chicken. Electrophysiological recordings using isolated mouse hemidiaphragm and frog cutaneous pectoris nerve-muscle preparations revealed no blocking activity of sWntx-5 at microm concentrations. Our data confirm previous observations that natural weak neurotoxins from cobras have poor affinity for nicotinic acetylcholine receptors.     

Were arachnids the first to use combinatorial peptide libraries?

Spiders, scorpions, and cone snails are remarkable for the extent and diversity of gene-encoded peptide neurotoxins that are expressed in their venom glands. These toxins are produced in the form of structurally constrained combinatorial peptide libraries in which there is hypermutation of essentially all residues in the mature-toxin sequence with the exception of a handful of strictly conserved cysteines that direct the three-dimensional fold of the toxin. This gene-based combinatorial peptide library strategy appears to have been first implemented by arachnids almost 400 million years ago, long before cone snails evolved a similar mechanism for generating peptide diversity.     

Morphology and ultrastructure of the venom glands of the northern pacific rattlesnake Crotalus viridis oreganus

The venom gland of Crotalus viridis oreganus is composed of two discrete secretory regions: a small anterior portion, the accessory gland, and a much larger main gland. These two glands are joined by a short primary duct consisting of simple columnar secretory cells and basal horizontal cells. The main gland has at least four morphologically distinct cell types: secretory cells, the dominant cell of the gland, mitochondria-rich cells, horizontal cells, and “dark” cells. Scanning electron microscopy shows that the mitochondria-rich cells are recessed into pits of varying depth; these cells do not secrete. Horizontal cells may serve as secretory stem cells, and “dark” cells may be myoepithelial cells. The accessory gland contains at least six distinct cell types: mucosecretory cells with large mucous granules, mitochondria-rich cells with apical vesicles, mitochondria-rich cells with electron-dense secretory granules, mitochondria-rich cells with numerous cilia, horizontal cells, and “dark” cells. Mitochondria-rich cells with apical vesicles or cilia cover much of the apical surface of mucosecretory cells and these three cell types are found in the anterior distal tubules of the accessory gland. The posterior regions of the accessory gland lack mucosecretory cells and do not appear to secrete. Ciliated cells have not been noted previously in snake venom glands. Release of secretory products (venom) into the lumen of the main gland is by exocytosis of granules and by release of intact membrane-bound vesicles. Following venom extraction, main gland secretory and mitochondria-rich cells increase in height, and protein synthesis (as suggested by rough endoplasmic reticulum proliferation) increases dramatically. No new cell types or alterations in morphology were noted among glands taken from either adult or juvenile snakes, even though the venom of each is quite distinct. In general, the glands of C. v. oreganus share structural similarities with those of crotalids and viperids previously described.     

Neurotoxin localization to ectodermal gland cells uncovers an alternative mechanism of venom delivery in sea anemones

Jellyfish, hydras, corals and sea anemones (phylum Cnidaria) are known for their venomous stinging cells, nematocytes, used for prey and defence. Here we show, however, that the potent Type I neurotoxin of the sea anemone Nematostella vectensis, Nv1, is confined to ectodermal gland cells rather than nematocytes. We demonstrate massive Nv1 secretion upon encounter with a crustacean prey. Concomitant discharge of nematocysts probably pierces the prey, expediting toxin penetration. Toxin efficiency in sea water is further demonstrated by the rapid paralysis of fish or crustacean larvae upon application of recombinant Nv1 into their medium. Analysis of other anemone species reveals that in Anthopleura elegantissima, Type I neurotoxins also appear in gland cells, whereas in the common species Anemonia viridis, Type I toxins are localized to both nematocytes and ectodermal gland cells. The nematocyte-based and gland cell-based envenomation mechanisms may reflect substantial differences in the ecology and feeding habits of sea anemone species. Overall, the immunolocalization of neurotoxins to gland cells changes the common view in the literature that sea anemone neurotoxins are produced and delivered only by stinging nematocytes, and raises the possibility that this toxin-secretion mechanism is an ancestral evolutionary state of the venom delivery machinery in sea anemones.     

Immunolocalization of venom metalloproteases in venom glands of adult and of newborn snakes of Bothrops jararaca

Using immunoelectronmicroscopy we analyzed qualitative and quantitatively the intracellular distribution of bothropasin, hemorrhagic factor 2 (HF2) and hemorrhagic factor 3 (HF3) in the venom secretory cells from adult snakes in the active (7 days after venom extraction) and in the resting (without venom extraction for 40 days) stages of protein synthesis. Glands from the newborn Bothrops jararaca were also studied. The results lead to the conclusion that all the secretory cells and the secretory pathway in the cells are qualitatively alike in regard to their content of the three metalloproteases. Secretory cells from the resting glands, unlike the active ones and the newborn glands, did not present immunolabeling in the narrow intracisternal spaces of the rough endoplasmic reticulum (RER). The label intensity for bothropasin was greater than that for the other proteins in the adults. HF3 and HF2 labeling densities in the newborn were higher than in the adults and HF3 labeling was not different from that of bothropasin. Co-localization of the three metalloproteases was detected in the RER cisternae of the active gland secretory cells, implying that mixing of the proteases before co-packaging into secretory vesicles occurs at the beginning of protein synthesis in the RER cisternae.     

Long-term primary culture of secretory cells of Bothrops jararaca venom gland for venom production in vitro

This protocol details the optimal conditions to establish a long-term primary culture of secretory cells from the venom gland of the Bothrops jararaca snake. Furthermore, these conditions allow the production and secretion of venom into the culture medium. Snake venom is a rich source of active molecules and has been used for bioprospection studies. However, obtaining enough venom from snakes is a major obstacle. Secretory cells of venom glands are capable of producing active toxins. Therefore, a culture of secretory cells is a good in vitro system to acquire the venom of snakes without capturing the animal from the wild. The protocol described here provides a rapid (approximately 4 h) and reproducible means of producing sufficient amounts of snake venom for biological investigations.     

Cytochemical analysis of acid phosphatase activity in the venom secretory cells of Bothrops jararaca

A study of the histochemical reaction for acid phosphatase (AcPase) in venom gland secretory cells from Bothrops jararaca was done to investigate the distribution of lysosomes and related structures in stages of high- and low-protein synthesis. From this analysis, it was expected to gain insight into the cellular pathway by which AcPase is secreted into the venom. Two subtypes of AcPase reactivities were detected in the venom gland secretory cells: one was found in lysosomes and related structures and in some trans-Golgi network (TGN) elements and reacts with beta-glycerophosphate (betaGP) as substrate; the other was found in secretory vesicles, apical plasmalemma, lysosomes and related structures, and in some TGN elements, and reacts with cytidine monophosphate (CMP). The results are compatible with the possibility that there is a secretory via for AcPase in the venom gland of B. jararaca and that the elements composing this pathway are noted only when CMP is used as substrate. Large autophagosomes reactive to both betaGP and to CMP were commonly observed in the basal region of the secretory cells, and they were more abundant in the glands during the stage of low activity of protein synthesis.     

间斑寇蛛毒素的蛋白质化学与蛋白质组学研究新进展

间斑寇蛛Latrodectus tredecimguttatus俗称"黑寡妇"蜘蛛。其毒素不仅存在于毒腺中,而且存在于其身体的其他部分、卵粒甚至新生幼蛛体内。研究间斑寇蛛毒腺和毒腺外材料中的毒素成分,探明它们之间的异同、进化关系和生物学作用,具有重要的理论和实际意义。现代蛋白质化学和蛋白质组学技术的发展为间斑寇蛛蛋白质和多肽毒素的研究提供了有效手段,从而可以同时从单一纯化蛋白质和组学的层面探究毒素作用的分子基础和作用机制。到目前为止,间斑寇蛛毒素的蛋白质化学与蛋白质组学研究已取得一定的进展,但相关研究尤其是毒腺外材料来源的毒素研究还有待进一步深入。