Structure of the snake-venom toxin convulxin

Snake venoms contain a number of proteins that interact with components of the haemostatic system that promote or inhibit events leading to blood- clot formation. The snake- venom protein convulxin ( Cvx) binds glycoprotein ( GP) VI, the platelet receptor for collagen, and triggers signal transduction. Here, the 2.7 Angstrom resolution crystal structure of Cvx is presented. In common with other members of this snake-venom protein family, Cvx is an alphabeta- heterodimer and conforms to the C- type lectin- fold topology. Comparison with other family members allows a set of Cvx residues that form a concave surface to be putatively implicated in GPVI binding. Unlike other family members, with the exception of flavocetin- A ( FL- A), Cvx forms an (alphabeta)(4) tetramer. This oligomeric structure is consistent with Cvx clustering GPVI molecules on the surface of platelets and as a result promoting signal transduction activity. The Cvx structure and the location of the putative binding sites suggest a model for this multimeric signalling assembly.

Effects of Spider Venom Toxin PWTX-I (6-Hydroxytrypargine) on the Central Nervous System of Rats

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

Isolation and chemical characterization of PwTx-II: A novel alkaloid toxin from the venom of the spider Parawixia bistriata (Araneidae, Araneae)

Brazil has many species of spiders belonging to Araneidae family however, very little is known about the composition, chemical structure and mechanisms of action of the main venom components of these spiders. The main objective of this work was to isolate and to perform the chemical characterization of a novel beta-carboline toxin from the venom of the spider Parawixia bistriata, a typical species of the Brazilian 'cerrado'. The toxin was purified by RP-HPLC and structurally elucidated by using a combination of different spectroscopic techniques (UV, ESI-MS/MS and H-1 NMR), which permitted the assignment of the molecular structure of a novel spider venom toxin, identified as 1-4-guanidinobutoxy-6-hydroxy-1,2,3,4-tetrahydro-beta-carboline, and referred to here as PwTx-II. This compound is toxic to insects (LD50 = 12 +/- 3 eta g/mg honeybee), neurotoxic, convulsive and lethal to rats (LD50 = 9.75 mg/kg of male Wistar rat). (c) 2005 Elsevier Ltd. All rights reserved.

Protonectin (1-6): A novel chemotactic peptide from the venom of the social wasp Agelaia pallipes pallipes

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

Proteome analysis of snake venom toxins: pharmacological insights

Snake venoms are an extremely rich source of pharmacologically active proteins with a considerable clinical and medical potential. To date, this potential has not been fully explored, mainly because of our incomplete knowledge of the venom proteome and the pharmacological properties of its components, in particular those devoid of enzymatic activity. This review summarizes the latest achievements in the determination of snake venom proteome, based primarily on the development of new strategies and techniques. Detailed knowledge of the venom toxin composition and biological properties of the protein constituents should provide the scaffold for the design of new more effective drugs for the treatment of the hemostatic system and heart disorders, inflammation, cancer and consequences of snake bites, as well as new tools for clinical diagnostic and assays of hemostatic parameters.

Peptide diversity in the venom of the social wasp Polybia paulista (Hymenoptera): A comparison of the intra- and inter-colony compositions

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

Venomics Profiling of Thamnodynastes strigatus Unveils Matrix Metalloproteinases and Other Novel Proteins Recruited to the Toxin Arsenal of Rear-Fanged Snakes

Rear-fanged and aglyphous snakes are usually considered not dangerous to humans because of their limited capacity of injecting venom. Therefore, only a few studies have been dedicated to characterizing the venom of the largest parcel of snake fauna. Here, we investigated the venom proteome of the rear-fanged snake Thamnodynastes strigatus, in combination with a transcriptomic evaluation of the venom gland. About 60% of all transcripts code for putative venom components. A striking finding is that the most abundant type of transcript (similar to 47%) and also the major protein type in the venom correspond to a new kind of matrix metalloproteinase (MMP) that is unrelated to the classical snake venom metalloproteinases found in all snake families. These enzymes were recently suggested as possible venom components, and we show here that they are proteolytically active and probably recruited to venom from a MMP-9 ancestor. Other unusual proteins were suggested to be venom components: a protein related to lactadherin and an EGF repeat-containing transcript. Despite these unusual molecules, seven toxin classes commonly found in typical venomous snakes are also present in the venom. These results support the evidence that the arsenals of these snakes are very diverse and harbor new types of biologically important molecules.

Expression of the collagen receptor glycoprotein VI during megakaryocyte differentiation

This study examined the expression of the platelet collagen receptor glycoprotein VI (GPVI) in megakaryocyte cell lines and primary megakaryocytes by reverse transcriptase-polymerase chain reaction and by flow cytometry and ligand blotting using the snake venom toxin convulxin. Expression of GPVI is increased in the megakaryoblastic cell lines HEL and CMK on differentiation with the phorbol ester phorbol 12-myristate 13-acetate (PMA), along with the Fc receptor gamma-chain (FcR gamma-chain). The increase in GPVI expression is associated with marked potentiation of tyrosine phosphorylation and Ca(++) elevation in response to convulxin. Syk, linker for activated T cells, and phospholipase C gamma 2 (PLC gamma 2) are among the proteins tyrosine phosphorylated on convulxin stimulation in PMA-differentiated HEL cells. Studies on primary murine megakaryocytes grown in vitro confirmed that GPVI is up-regulated in parallel with functional activation, assessed by measurement of [Ca(++)](i), during differentiation. The results demonstrate that expression of GPVI is up-regulated along with the FcR gamma-chain during differentiation of megakaryocytes. (Blood. 2000;96:2740-2745)

CLEC-2 is not required for platelet aggregation at arteriolar shear

The C-type lectin receptor CLEC-2 is expressed primarily on the surface of platelets, where it is present as a dimer, and is found at low level on a subpopulation of other hematopoietic cells, including mouse neutrophils [1–4] Clustering of CLEC-2 by the snake venom toxin rhodocytin, specific antibodies or its endogenous ligand, podoplanin, elicits powerful activation of platelets through a pathway that is similar to that used by the collagen receptor glycoprotein VI (GPVI) [4–6]. The cytosolic tail of CLEC-2 contains a conserved YxxL sequence preceded by three upstream acidic amino acid residues, which together form a novel motif known as a hemITAM. Ligand engagement induces tyrosine phosphorylation of the hemITAM sequence providing docking sites for the tandem-SH2 domains of the tyrosine kinase Syk across a CLEC-2 receptor dimer [3]. Tyrosine phosphorylation of Syk by Src family kinases and through autophosphorylation leads to stimulation of a downstream signaling cascade that culminates in activation of phospholipase C γ2 (PLCγ2) [4,6]. Recently, CLEC-2 has been proposed to play a major role in supporting activation of platelets at arteriolar rates of flow [1]. Injection of a CLEC-2 antibody into mice causes a sustained depletion of the C-type lectin receptor from the platelet surface [1]. The CLEC-2-depleted platelets were unresponsive to rhodocytin but underwent normal aggregation and secretion responses after stimulation of other platelet receptors, including GPVI [1]. In contrast, there was a marked decrease in aggregate formation relative to controls when CLEC-2-depleted blood was flowed at arteriolar rates of shear over collagen (1000 s−1 and 1700 s−1) [1]. Furthermore, antibody treatment significantly increased tail bleeding times and mice were unable to occlude their vessels after ferric chloride injury [1]. These data provide evidence for a critical role for CLEC-2 in supporting platelet aggregation at arteriolar rates of flow. The underlying mechanism is unclear as platelets do not express podoplanin, the only known endogenous ligand of CLEC-2. In the present study, we have investigated the role of CLEC-2 in platelet aggregation and thrombus formation using platelets from a novel mutant mouse model that lacks functional CLEC-2.

Renal cells activate the platelet receptor CLEC-2 through podoplanin.

We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.

Comparative study on extracts from the tissue covering the stingers of freshwater (Potamotrygon falkneri) and marine (Dasyatis guttata) stingrays

Stingrays are elasmobranchs found along the seacoast and in some rivers of Brazil. Pain is the most conspicuous symptom observed in patients wounded by the bilaterally retroserrate stingers located in the tail, which are covered by glandular and integument tissues. In addition, cutaneous necrosis is commonly observed in injuries caused by freshwater stingrays. The aim of this work was to characterize and compare certain properties of tissue extracts obtained from the glandular tissues covering the stinger apparatus of Potamotrygon falkneri and Dasyatis guttata stingrays. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), tissue extracts have similar bands above 80kDa, but most differences were observed below this molecular mass. Lethal, dermonecrotic and myotoxic activities were detected only in P. falkneri tissue extract. Edematogenic activity was similar and dose dependent in both tissue extracts. Nociceptive activity was verified in both tissue extracts, but P. falkneri presented a two-fold higher activity than D. guttata tissue extract. No direct hemolysis, phospholipase A(2) and coagulant activities were observed in both tissue extracts. Antigenic cross-reactivity was noticed by ELISA and Western blotting, using antisera raised in rabbits. Species-specific sera reacted with several components of both tissue extracts, noticeably above 22 kDa. Both tissue extracts presented gelatinolytic, caseinolytic and fibrinogenolytic activities, which were not caused by the action of metalloproteinases. Hyaluronidase activity was detected only in P. falkneri tissue extract. Our experimental observations suggest that P. falkneri tissue extract is more toxic than D. guttata tissue extract. These results may explain why injuries caused by freshwater stingrays are more severe in human accidents. (c) 2007 Elsevier Ltd. All rights reserved.

Crystallographic and spectroscopic characterization of a molecular hinge: Conformational changes in bothropstoxin I, a dimeric Lys49 phospholipase A2 homologue

Bothropstoxin I(BthTX-I) from the venom of Bothrops jararacussu is a myotoxic phospholipase A2 (PLA2) homologue which, although catalytically inactive due to an Asp49-->Lys substitution, disrupts the integrity of lipid membranes by a Ca2+-independent mechanism, the crystal structures of two dimeric farms of BthLTX-I which diffract X-rays eo resolutions of 3.1 and 2.1 Angstrom have been determined, the monomers in both structures are related by an almost perfect twofold axis of rotation and the dimer interfaces are defined by contacts between the N-terminal alpha-helical regions and the tips of the beta-wings of partner monomers. Significant differences in the relative orientation of the monomers in the two crystal forms results in open and closed dimer conformations, Spectroscopic Investigations of BthTX-I in solution have correlated these conformational differences with changes in the intrinsic fluorescence emission of the single tryptophan residues located at the dimer interface, the possible relevance of this structural transition in the Ca2+-independent membrane damaging activity is discussed. (C) 1998 Wiley-Liss, Inc.

Snake Venomic of Crotalus durissus terrificus-Correlation with Pharmacological Activities

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

Snake venomics of the Siamese Russell's viper (Daboia russelli siamensis) - Relation to pharmacological activities

The venom proteome of Daboia russelli siamensis, a snake of medical importance in several Asian countries, was analysed by 2-D electrophoresis, subsequent MS/MS and enzymatic assays. The proteome comprises toxins from six protein families: serine proteinases, metalloproteinases, phospholipases A(2), L-amino acid oxidases, vascular endothelial growth factors and C-type lectin-like proteins. The venom toxin composition correlates with the clinical manifestation of the Russell's viper bite and explains pathological effects of the venom such as coagulopathy, oedema, hypotensive, necrotic and tissue damaging effects. The vast majority of toxins are potentially involved in coagulopathy and neurotoxic effects. The predominant venom components are proteinases capable of activating blood coagulation factors and promoting a rapid clotting of the blood, and neurotoxic phospholipase A(2)s. The analysis of the venom protein composition provides a catalogue of secreted toxins. The proteome of D. r. siamensis exhibits a lower level of toxin diversity than the proteomes of other viperid snakes. In comparison to the venoms of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, the venom from D. r. siamensis showed quantitative differences in the proteolytic, phospholipase A2, L-amino acid oxidase and alkaline phosphatase activities. (c) 2009 Published by Elsevier B.V.

Histopathological changes induced by extracts from the tissue covering the stingers of Potamotrygon falkneri freshwater stingrays

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