Bothrops moojeni myotoxin-II, a Lys49-phospholipase A(2) homologue: An example of function versatility of snake venom proteins

MjTX-II, a myotoxic phospholipase A(2) (PLA(2)) homologue from Bothrops moojeni venom, was functionally and structurally characterized. The MjTX-II characterization included: (i) functional characterization (antitumoral, antimicrobial and antiparasitic effects); (ii) effects of structural modifications by 4-bromophenacyl bromide (BPB), cyanogen bromide (CNBr), acetic anhydride and 2-nitrobenzenesulphonyl fluoride (NBSF); (iii) enzymatic characterization: inhibition by low molecular weight heparin and EDTA; and (iv) molecular characterization: cDNA sequence and molecular structure prediction. The results demonstrated that MjTX-II displayed antimicrobial activity by growth inhibition against Escherichia coli and Candida albicans, antitumoral activity against Erlich ascitic tumor (EAT), human breast adenocarcinoma (SK-BR-3) and human T leukemia cells (JURKAT) and antiparasitic effects against Schistosoma mansoni and Leishmania spp., which makes MjTX-II a promising molecular model for future therapeutic applications, as well as other multifunctional homologous Lys49-PLA(2)S or even derived peptides. This work provides useful insights into the structural determinants of the action of Lys49-PLA2 homologues and, together with additional strategies, supports the concept of the presence of others bioactive sites distinct from the catalytic site in snake venom myotoxic PLA(2)s. (c) 2005 Elsevier B.V. All rights reserved.

Purification and structural characterization of snake venom proteins

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Alkylation of Histidine Residues of Bothrops jararacussu Venom Proteins and Isolated Phospholipases A(2): A Biotechnological Tool to Improve the Production of Antibodies

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Venomics of the Australian eastern brown snake (Pseudonaja textilis): detection of new venom proteins and splicing variants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

PLGA microspheres containing bee venom proteins for preventive immunotherapy

Bee venom (BV) allergy is potentially dangerous for allergic individuals because a single bee sting may induce an anaphylactic reaction, eventually leading to death. Currently, venom immunotherapy (VIT) is the only treatment with long-lasting effect for this kind of allergy and its efficiency has been recognized worldwide. This therapy consists of subcutaneous injections of gradually increasing doses of the allergen. This causes patient lack of compliance due to a long time of treatment with a total of 30-80 injections administered over years. In this article we deal with the characterization of different MS-PLGA formulations containing BV proteins for VIT. The PLGA microspheres containing BV represent a strategy to replace the multiple injections, because they can control the solute release. Physical and biochemical methods were used to analyze and characterize their preparation. Microspheres with encapsulation efficiencies of 49-75% were obtained with a BV triphasic release profile. Among them, the MS-PLGA 34 kDa-COOH showed to be best for VIT because they presented a low initial burst (20%) and a slow BV release during lag phase. Furthermore, few conformational changes were observed in the released BV. Above all, the BV remained immunologically recognizable, which means that they could continuously stimulate the immune system. Those microspheres containing BV could replace sequential injections of traditional VIT with the remarkable advantage of reduced number of injections. (C) 2011 Elsevier B.V. All rights reserved.

Snake venom proteins affecting platelets and their applications to anti-thrombotic research

Snake venoms are very complex mixtures of biologically active proteins and peptides that may affect hemostasis in many ways, by activating or inhibiting coagulant factors or platelets, or by disrupting endothelium. They have been classified into various families, including serine proteases, metalloproteinases, C-type lectins, disintegrins and phospholipases. The various members of a particular family act selectively on different blood coagulation factors, blood cells or tissues. Venom proteins affect platelet function in particular by binding to and blocking or clustering and activating receptors or by cleaving receptors or von Willebrand factor. They may also activate protease-activated receptors or modulate ADP release or thromboxane A(2) formation. L-amino acid oxidases activate platelets by producing H(2)O(2). Many of these purified components are valuable tools in platelet research, providing new information about receptor function and signaling.

Venom proteins from polydnavirus-producing endoparasitoids: Their role in host-parasite interactions

Endoporasitoid wasps have evolved various mechanisms to ensure successful development of their progeny, including co-injection of a cocktail of maternal secretions into the host hemocoel, including venom, calyx fluid, and polydnoviruses. The components of each type of secretion may influence host physiology and development independently or in a synergistic fashion. For example, venom fluid consists of several peptides and proteins that promote expression of polydnavirus genes in addition to other activities, such as inhibition of prophenoloxidase activation, inhibition of hemocytes spreading and aggregation, and inhibition of development. This review provides a brief overview of advances and prospects in the study of venom proteins from polydnavirus-producing endoparositoid wasps with a special emphasis on the role of C. rubecula venom proteins in host-parositoid interactions.

Molecular Characterization of Three Novel Phospholipase A2 Proteins from the Venom of Atheris chlorechis, Atheris nitschei and Atheris squamigera

Secretory phospholipase A2 (sPLA2) is known as a major component of snake venoms and displays higher-order catalytic hydrolysis functions as well as a wide range of pathological effects. Atheris is not a notoriously dangerous genus of snakes although there are some reports of fatal cases after envenomation due to the effects of coagulation disturbances and hemorrhaging. Molecular characterization of Atheris venom enzymes is incomplete and there are only a few reports in the literature. Here, we report, for the first time, the cloning and characterization of three novel cDNAs encoding phospholipase A2 precursors (one each) from the venoms of the Western bush viper (Atheris chlorechis), the Great Lakes bush viper (Atheris nitschei) and the Variable bush viper (Atheris squamigera), using a “shotgun cloning” strategy. Open-reading frames of respective cloned cDNAs contained putative 16 residue signal peptides and mature proteins composed of 121 to 123 amino acid residues. Alignment of mature protein sequences revealed high degrees of structural conservation and identity with Group II venom PLA2 proteins from other taxa within the Viperidae. Reverse-phase High Performance Liquid Chromatography (HPLC) profiles of these three snake venoms were obtained separately and chromatographic fractions were assessed for phospholipase activity using an egg yolk suspension assay. The molecular masses of mature proteins were all identified as approximately 14 kDa. Mass spectrometric analyses of the fractionated oligopeptides arising from tryptic digestion of intact venom proteins, was performed for further structural characterization.

Anti-snake venom properties of Schizolobium parahyba (Caesalpinoideae) aqueous leaves extract

Many medicinal plants have been recommended for the treatment of snakebites. The aqueous extracts prepared from the leaves of Schizolobium parahyba (a plant found in Mata Atlantica in Southeastern Brazil) were assayed for their ability to inhibit some enzymatic and biological activities induced by Bothropspauloensis and Crotalus durissus terrificus venoms as well as by their isolated toxins neuwiedase (metalloproteinase), BnSP-7 (basic Lys49 PLA(2)) and CB (PLA(2) from crotoxin complex). Phospholipase A(2), coagulant, fibrinogenolytic, hemorrhagic and myotoxic activities induced by R pauloensis and C. d. terrificus venoms, as well as by their isolated toxins were significantly inhibited when different amounts of S. parahyba were incubated previously with these venoms and toxins before assays. However, when S. parahyba was administered at the same route as the venoms or toxins injections, the tissue local damage, such as hemorrhage and myotoxicity was only partially inhibited. The study also evaluated the inhibitory effect of S. parahyba upon the spreading of venom proteins from the injected area into the systemic circulation. The neutralization of systemic alterations induced by i.m. injection of R pauloensis venom was evaluated by measuring platelet and plasma fibrinogen levels which were significantly maintained when S. parahyba extract inoculation occurred at the same route after R pauloensis venom injection. In conclusion, the observations confirmed that the aqueous extract of S. parahyba possesses potent snake venom neutralizing properties. It may be used as an alternative treatment to serum therapy and as a rich source of potential inhibitors of toxins involved in several physiopathological human and animal diseases. Copyright (c) 2008 John Wiley & Sons, Ltd.

Isolation and characterization of a novel venom protein from an endoparasitoid, Cotesia rubecula (Hym : Braconidae)

Insects are important vectors of diseases with remarkable immune defense capabilities. Hymenopteran endoparasitoids are adapted to overcome the host defense system and, therefore, are useful sources of immune-suppressing proteins. Not much is known about venom proteins in endoparasitoids, especially those that have a functional relationship with polydnaviruses (PDVs). Here, we describe the isolation and characterization of a small venom protein (Vn4.6) from an endoparositoid, Cotesia rubecula, which interferes with the activation of the host hemolymph prophenoloxidose. The coding region for Vn4.6 is located upstream in the opposite direction of a gene coding for a C rubecula PDV-protein (Crp32). Arch. Insect Biochem. Physiol. 53:92-100, 2003. (C) 2003 Wiley-Liss, Inc.

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

The study reported here is a classical bottom-up proteomic approach where proteins from wasp venom were extracted and separated by 2-DE; the individual protein spots were proteolytically digested and subsequently identified by using tandem mass spectrometry and database query with the protein search engine MASCOT. Eighty-four venom proteins belonging to 12 different molecular functions were identified. These proteins were classified into three groups; the first is constituted of typical venom proteins: antigens-5, hyaluronidases, phospholipases, heat shock proteins, metalloproteinases, metalloproteinase-desintegrin like proteins, serine proteinases, proteinase inhibitors, vascular endothelial growth factor-related protein, arginine kinases, Sol i-II and -II like proteins, alpha-glucosidase, and superoxide dismutases. The second contained proteins structurally related to the muscles that involves the venom reservoir. The third group, associated with the housekeeping of cells from venom glands, was composed of enzymes, membrane proteins of different types, and transcriptional factors. The composition of P. paulista venom permits us to hypothesize about a general envenoming mechanism based on five actions: (i) diffusion of venom through the tissues and to the blood, (ii) tissue, (iii) hemolysis, (iv) inflammation, and (v) allergy-played by antigen-5, PLA1, hyaluronidase, HSP 60, HSP 90, and arginine kinases.

The UniProtKB/Swiss-Prot Tox-Prot program: A central hub of integrated venom protein data.

Animal toxins are of interest to a wide range of scientists, due to their numerous applications in pharmacology, neurology, hematology, medicine, and drug research. This, and to a lesser extent the development of new performing tools in transcriptomics and proteomics, has led to an increase in toxin discovery. In this context, providing publicly available data on animal toxins has become essential. The UniProtKB/Swiss-Prot Tox-Prot program (http://www.uniprot.org/program/Toxins) plays a crucial role by providing such an access to venom protein sequences and functions from all venomous species. This program has up to now curated more than 5000 venom proteins to the high-quality standards of UniProtKB/Swiss-Prot (release 2012_02). Proteins targeted by these toxins are also available in the knowledgebase. This paper describes in details the type of information provided by UniProtKB/Swiss-Prot for toxins, as well as the structured format of the knowledgebase.

Purification and functional characterisation of Rhinocerase, a novel serine protease from the venom of Bitis gabonica rhinoceros

Background: Serine proteases are a major component of viper venoms and are thought to disrupt several distinct elements of the blood coagulation system of envenomed victims. A detailed understanding of the functions of these enzymes is important both for acquiring a fuller understanding of the pathology of envenoming and because these venom proteins have shown potential in treating blood coagulation disorders. Methodology/Principal Findings: In this study a novel, highly abundant serine protease, which we have named rhinocerase, has been isolated and characterised from the venom of Bitis gabonica rhinoceros using liquid phase isoelectric focusing and gel filtration. Like many viper venom serine proteases, this enzyme is glycosylated; the estimated molecular mass of the native enzyme is approximately 36kDa, which reduces to 31kDa after deglycosylation. The partial amino acid sequence shows similarity to other viper venom serine proteases, but is clearly distinct from the sequence of the only other sequenced serine protease from Bitis gabonica. Other viper venom serine proteases have been shown to exert distinct biological effects, and our preliminary functional characterization of rhinocerase suggest it to be multifunctional. It is capable of degrading α and β chains of fibrinogen, dissolving plasma clots and of hydrolysing a kallikrein substrate. Conclusions/Significance: A novel multifunctional viper venom serine protease has been isolated and characterised. The activities of the enzyme are consistent with the known in vivo effects of Bitis gabonica envenoming, including bleeding disorders, clotting disorders and hypotension. This study will form the basis for future research to understand the mechanisms of serine protease action, and examine the potential for rhinocerase to be used clinically to reduce the risk of human haemostatic disorders such as heart attacks and strokes.

Profiling the Proteome of the Venom from the Social Wasp Polybia paulista: A Clue to Understand the Envenoming Mechanism

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)