Mechanisms of Vascular Damage by Hemorrhagic Snake Venom Metalloproteinases: Tissue Distribution and In Situ Hydrolysis

Snakebite accidents by vipers cause a massive disturbance in hemostasis and tissue damage at the snakebite area. The systemic effects are often prevented by antivenom therapy. However, the local symptoms are not neutralized by antivenoms and are related to the temporary or permanent disability observed in many patients. Although the mechanisms involved in coagulation or necrotic disturbances induced by snake venoms are well known, the disruption of capillary vessels by SVMPs leading to hemorrhage and consequent local tissue damage is not fully understood. In our study, we reveal the mechanisms involved in hemorrhage induced by SVMPs by comparing the action of high and low hemorrhagic toxins isolated from Bothrops venoms, in mouse skin. We show remarkable differences in the tissue distribution and hydrolysis of collagen within the hemorrhagic lesions induced by high and low hemorrhagic metalloproteinases. According to our data, tissue accumulation of hemorrhagic toxins near blood vessel walls allowing the hydrolysis of basement membrane components, preferably collagen IV. These observations unveil new mechanistic insights supporting the local administration of metalloproteinases inhibitors as an alternative to improve snakebite treatment besides antivenom therapy

Venom-related transcripts from Bothrops jararaca tissues provide novel molecular insights into the production and evolution of snake venom.

Attempts to reconstruct the evolutionary history of snake toxins in the context of their co-option to the venom gland rarely account for nonvenom snake genes that are paralogous to toxins, and which therefore represent important connectors to ancestral genes. In order to reevaluate this process, we conducted a comparative transcriptomic survey on body tissues from a venomous snake. A nonredundant set of 33,000 unigenes (assembled transcripts of reference genes) was independently assembled from six organs of the medically important viperid snake Bothrops jararaca, providing a reference list of 82 full-length toxins from the venom gland and specific products from other tissues, such as pancreatic digestive enzymes. Unigenes were then screened for nontoxin transcripts paralogous to toxins revealing 1) low level coexpression of approximately 20% of toxin genes (e.g., bradykinin-potentiating peptide, C-type lectin, snake venom metalloproteinase, snake venom nerve growth factor) in body tissues, 2) the identity of the closest paralogs to toxin genes in eight classes of toxins, 3) the location and level of paralog expression, indicating that, in general, co-expression occurs in a higher number of tissues and at lower levels than observed for toxin genes, and 4) strong evidence of a toxin gene reverting back to selective expression in a body tissue. In addition, our differential gene expression analyses identify specific cellular processes that make the venom gland a highly specialized secretory tissue. Our results demonstrate that the evolution and production of venom in snakes is a complex process that can only be understood in the context of comparative data from other snake tissues, including the identification of genes paralogous to venom toxins

A transcriptomic analysis of gene expression in the venom gland of the snake Bothrops alternatus (urutu)

<p>Abstract</p> <p>Background</p> <p>The genus <it>Bothrops </it>is widespread throughout Central and South America and is the principal cause of snakebite in these regions. Transcriptomic and proteomic studies have examined the venom composition of several species in this genus, but many others remain to be studied. In this work, we used a transcriptomic approach to examine the venom gland genes of <it>Bothrops alternatus</it>, a clinically important species found in southeastern and southern Brazil, Uruguay, northern Argentina and eastern Paraguay.</p> <p>Results</p> <p>A cDNA library of 5,350 expressed sequence tags (ESTs) was produced and assembled into 838 contigs and 4512 singletons. BLAST searches of relevant databases showed 30% hits and 70% no-hits, with toxin-related transcripts accounting for 23% and 78% of the total transcripts and hits, respectively. Gene ontology analysis identified non-toxin genes related to general metabolism, transcription and translation, processing and sorting, (polypeptide) degradation, structural functions and cell regulation. The major groups of toxin transcripts identified were metalloproteinases (81%), bradykinin-potentiating peptides/C-type natriuretic peptides (8.8%), phospholipases A₂(5.6%), serine proteinases (1.9%) and C-type lectins (1.5%). Metalloproteinases were almost exclusively type PIII proteins, with few type PII and no type PI proteins. Phospholipases A₂were essentially acidic; no basic PLA₂were detected. Minor toxin transcripts were related to L-amino acid oxidase, cysteine-rich secretory proteins, dipeptidylpeptidase IV, hyaluronidase, three-finger toxins and ohanin. Two non-toxic proteins, thioredoxin and double-specificity phosphatase Dusp6, showed high sequence identity to similar proteins from other snakes. In addition to the above features, single-nucleotide polymorphisms, microsatellites, transposable elements and inverted repeats that could contribute to toxin diversity were observed.</p> <p>Conclusions</p> <p><it>Bothrops alternatus </it>venom gland contains the major toxin classes described for other <it>Bothrops </it>venoms based on trancriptomic and proteomic studies. The predominance of type PIII metalloproteinases agrees with the well-known hemorrhagic activity of this venom, whereas the lower content of serine proteases and C-type lectins could contribute to less marked coagulopathy following envenoming by this species. The lack of basic PLA₂agrees with the lower myotoxicity of this venom compared to other <it>Bothrops </it>species with these toxins. Together, these results contribute to our understanding of the physiopathology of envenoming by this species.</p

The Indian cobra reference genome and transcriptome enables comprehensive identification of venom toxins

Snakebite envenoming is a serious and neglected tropical disease that kills ~100,000 people annually. High-quality, genome-enabled comprehensive characterization of toxin genes will facilitate development of effective humanized recombinant antivenom. We report a de novo near-chromosomal genome assembly of Naja naja, the Indian cobra, a highly venomous, medically important snake. Our assembly has a scaffold N50 of 223.35 Mb, with 19 scaffolds containing 95% of the genome. Of the 23,248 predicted protein-coding genes, 12,346 venom-gland-expressed genes constitute the \u27venom-ome\u27 and this included 139 genes from 33 toxin families. Among the 139 toxin genes were 19 \u27venom-ome-specific toxins\u27 (VSTs) that showed venom-gland-specific expression, and these probably encode the minimal core venom effector proteins. Synthetic venom reconstituted through recombinant VST expression will aid in the rapid development of safe and effective synthetic antivenom. Additionally, our genome could serve as a reference for snake genomes, support evolutionary studies and enable venom-driven drug discovery

Brown Spider (Loxosceles genus) Venom Toxins: Tools for Biological Purposes

Venomous animals use their venoms as tools for defense or predation. These venoms are complex mixtures, mainly enriched of proteic toxins or peptides with several, and different, biological activities. In general, spider venom is rich in biologically active molecules that are useful in experimental protocols for pharmacology, biochemistry, cell biology and immunology, as well as putative tools for biotechnology and industries. Spider venoms have recently garnered much attention from several research groups worldwide. Brown spider (Loxosceles genus) venom is enriched in low molecular mass proteins (5–40 kDa). Although their venom is produced in minute volumes (a few microliters), and contain only tens of micrograms of protein, the use of techniques based on molecular biology and proteomic analysis has afforded rational projects in the area and permitted the discovery and identification of a great number of novel toxins. The brown spider phospholipase-D family is undoubtedly the most investigated and characterized, although other important toxins, such as low molecular mass insecticidal peptides, metalloproteases and hyaluronidases have also been identified and featured in literature. The molecular pathways of the action of these toxins have been reported and brought new insights in the field of biotechnology. Herein, we shall see how recent reports describing discoveries in the area of brown spider venom have expanded biotechnological uses of molecules identified in these venoms, with special emphasis on the construction of a cDNA library for venom glands, transcriptome analysis, proteomic projects, recombinant expression of different proteic toxins, and finally structural descriptions based on crystallography of toxins

Sympathetic outflow activates the venom gland of the snake Bothrops jararaca by regulating the activation of transcription factors and the synthesis of venom gland proteins

The venom gland of viperid snakes has a central lumen where the venom produced by secretory cells is stored. When the venom is lost from the gland, the secretory cells are activated and new venom is produced. The production of new venom is triggered by the action of noradrenaline on both alpha(1)- and beta-adrenoceptors in the venom gland. In this study, we show that venom removal leads to the activation of transcription factors NF kappa B and AP-1 in the venom gland. In dispersed secretory cells, noradrenaline activated both NF kappa B and AP-1. Activation of NF kappa B and AP-1 depended on phospholipase C and protein kinase A. Activation of NF kappa B also depended on protein kinase C. Isoprenaline activated both NF kappa B and AP-1, and phenylephrine activated NF kappa B and later AP-1. We also show that the protein composition of the venom gland changes during the venom production cycle. Striking changes occurred 4 and 7 days after venom removal in female and male snakes, respectively. Reserpine blocks this change, and the administration of alpha(1)- and beta-adrenoceptor agonists to reserpine-treated snakes largely restores the protein composition of the venom gland. However, the protein composition of the venom from reserpinized snakes treated with alpha(1)- or beta-adrenoceptor agonists appears normal, judging from SDS-PAGE electrophoresis. A sexual dimorphism in activating transcription factors and activating venom gland was observed. Our data suggest that the release of noradrenaline after biting is necessary to activate the venom gland by regulating the activation of transcription factors and consequently regulating the synthesis of proteins in the venom gland for venom production.FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)[02/00422-8]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)[07/50083-9]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)FAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)[04/11611-1]Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Characteristics of neural and humoral systems involved in the regulation of blood pressure in snakes

Cardiovascular function is affected by many mechanisms, including the autonomic system, the kallikrein-kinin system (KKS), the reninangiotensin system (RAS) and the endothelin system. the function of these systems seems to be fairly well preserved throughout the vertebrate scale, but evolution required several adaptations. Snakes are particularly interesting for studies related to the cardiovascular fanction because of their elongated shape, their wide variation in size and length, and because they had to adapt to extremely different habitats and gravitational influences. To keep the normal cardiovascular control the snakes developed anatomical and functional adaptations and interesting structural peculiarities are found in their autonomic, KKS, RAS and endothelin systems. Our laboratory has characterized some biochemical, pharmacological and physiological properties of these systems in South American snakes. This review compares the components and function of these systems in snakes and other vertebrates, and focuses on differences found in snakes, related with receptor or ligand structure and/or function in autonomic system, RAS and KKS, absence of components in KKS and the intriguing identity between a venom and a plasma component in the endothelin system. (c) 2006 Elsevier Inc. All rights reserved.Inst Butantan, Farmacol Lab, BR-05503900 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Farmacol Setor Endocrinol Expt, BR-04044020 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Farmacol Setor Endocrinol Expt, BR-04044020 São Paulo, BrazilWeb of Scienc