Preliminary X-ray crystallographic studies of a tetrameric phospholipase A(2) formed by two isoforms of crotoxin B from Crotalus durissus terrificus venom

Crotoxin B is a basic phospholipase A(2) found in the venom of Crotalus durissus terrificus and is one of the subunits that constitute crotoxin. This heterodimeric toxin, which is the main component of C. d. terrificus venom, is completed by an acidic, nontoxic and non-enzymatic component (crotoxin A) and is involved in important envenomation effects, such as neurological disorders, myotoxicity and renal failure. Although crotoxin was first crystallized in 1938, no crystal structure is currently available for crotoxin, crotoxin A or crotoxin B. In this work, the crystallization, X-ray diffraction data collection to 2.28 angstrom resolution and molecular-replacement solution of a novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2) is presented.

Insights into the role of oligomeric state on the biological activities of crotoxin: crystal structure of a tetrameric phospholipase A(2) formed by two isoforms of crotoxin B from Cratalus durissus terrificus venom

Crotoxin B (CB or Cdt PLA(2)) is a basic Asp49-PLA(2) found in the venom of Crotalus durissus terrificus and it is one of the subunits that constitute the crotoxin (Cro). This heterodimeric toxin, main component of the C. d. terrificus venom, is completed by an acidic, nontoxic, and nonenzymatic component (crotoxin A, CA or crotapotin), and it is related to important envenomation effects such as neurological disorders, myotoxicity, and renal failure. Although Cro has been crystallized since 1938, no crystal structure of this toxin or its subunits is currently available. In this work, the authors present the crystal structure of novel tetrameric complex formed by two dimers of crotoxin B isoforms (CB1 and CB2). The results suggest that these assemblies are stable in solution and show that Ser1 and Glu92 of CB1 and CB2, respectively, play an important role in the oligomerization. The tetrameric and dimeric conformations resulting from the association of the isoforms may increase the neurotoxicity of the toxin CB by the creation of new binding sites, which could improve the affinity of the molecular complexes to the presynaptic membrane.

Crystallization and preliminary X-ray diffraction analysis of crotoxin B from Crotalus durissus collilineatus venom

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

Secretory phospholipases A(2) isolated from Bothrops asper and from Crotalus durissus terrificus snake venoms induce distinct mechanisms for biosynthesis of prostaglandins E-2 and D-2 and expression of cyclooxygenases

The effects of myotoxin III (MT-III), a phospholipase A(2) (sPLA(2)) from Bothrops asper snake venom, and crotoxin B (CB), a neurotoxic and myotoxic sPLA2 from the venom of Crotalus durissus terrificus, on cyclooxygenases (COXs) expression and biosynthesis of prostaglandins (PGs) were evaluated, together with the mechanisms involved in these effects. Upon intraperitoneal injection in mice, both sPLA(2)s promoted the synthesis of PGD(2) and PGE(2), with a different time-course. MT-III, but not CB, induced COX-2 expression by peritoneal leukocytes without modification on COX-1 constitutive expression, whereas CB increased the constitutive activity of COX-1. MT-III increased the enzymatic activity of COX-1 and COX-2. Similar effects were observed when these sPLA(2)s were incubated with isolated macrophages, evidencing a direct effect on these inflammatory cells. Moreover, both toxins elicited the release of arachidonic acid from macrophages in vitro. inhibition of cPLA(2) by AACOCF(3), but not of iPLA(2) by PACOCF(3) or BEL, significantly reduced PGD2, PGE2 and arachidonic acid (AA) release promoted by MT-III. These inhibitors did not affect MT-III-induced COX-2 expression. In contrast, cPLA2 inhibition did not modify the effects of CB, whereas iPLA2 inhibition reduced PGD2 and AA production induced by CB. These findings imply that distinct regulatory mechanisms leading to PGs` synthesis are triggered by these snake venom sPLA(2)s. Such differences are likely to explain the dissimilar patterns of inflammatory reaction elicited by these sPLA(2)s in vivo. (C) 2008 Elsevier Ltd. All rights reserved.

Crotalus durissus collilineatus venom gland transcriptome: Analysis of gene expression profile

Crotalus durissus rattlesnakes are responsible for the most lethal cases of snakebites in Brazil. Crotalus durissus collilineatus subspecies is related to a great number of accidents in Southeast and Central West regions, but few studies on its venom composition have been carried out to date. In an attempt to describe the transcriptional profile of the C. durissus collilineatus venom gland, we generated a cDNA library and the sequences obtained could be identified by similarity searches on existing databases. Out of 673 expressed sequence tags (ESTs) 489 produced readable sequences comprising 201 singletons and 47 clusters of two or more ESTs. One hundred and fifty reads (60.5%) produced significant hits to known sequences. The results showed a predominance of toxin-coding ESTs instead of transcripts coding for proteins involved in all cellular functions. The most frequent toxin was crotoxin, comprising 88% of toxin-coding sequences. Crotoxin B, a basic phospholipase A(2) (PLA(2)) subunit of crotoxin, was represented in more variable forms comparing to the non-enzymatic subunit (crotoxin A), and most sequences coding this molecule were identified as CB1 isoform from Crotalus durissus terrificus venom. Four percent of toxin-related sequences in this study were identified as growth factors, comprising five sequences for vascular endothelial growth factor (VEGF) and one for nerve growth factor (NGF) that showed 100% of identity with C. durissus terrificus NGF. We also identified two clusters for metalloprotease from PII class comprising 3% of the toxins, and two for serine proteases, including gyroxin (2.5%). The remaining 2.5% of toxin-coding ESTs represent singletons identified as homologue sequences to cardiotoxin, convulxin, angiotensin-converting enzyme inhibitor and C-type natriuretic peptide, Ohanin, crotamin and PLA(2) inhibitor. These results allowed the identification of the most common classes of toxins in C. durissus collilineatus snake venom, also showing some unknown classes for this subspecies and even for C. durissus species, such as cardiotoxins and VEGF. (C) 2009 Published by Elsevier Masson SAS.

An alpha-type phospholipase A(2) inhibitor from Bothrops jararacussu snake plasma: Structural and functional characterization

An inhibitory protein that neutralizes the enzymatic, toxic and pharmacological activities of several phospholipases A(2) from Bothrops venoms was isolated from B. jararacussu snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on Sepharose gel. Biochemical characterization of this inhibitory protein, denominated alpha BjussuMIP, showed it to be an oligomeric glycoprotein with M-r of 24,000 for the monomeric subunit. Secondary structural analysis by circular dichroism revealed 44% alpha-helix, 18% beta-sheet, 10% beta-turn and 28% random coil structures. Circular dichroism spectroscopy indicated that no significant alterations in the secondary structure of either alpha BjussuMIP or the target protein occur following their interaction. The product from the reaction with reverse transcriptase produced a cDNA fragment of 432 bp that codifies for a mature protein of 144 amino acid residues. The first 21 amino acid residues from the N-terminal and five tryptic peptides were characterized by mass spectrometry of the mature protein and confirmed by the nucleotide sequence. Alignment of alpha BjussuMIP with other snake inhibitors showed a sequence similarity of 73-92% with these alpha PLIs. alpha BjussuMIP was relatively stable within the pH range of 6-12 and temperatures from 0 degrees C to 80 degrees C, even after deglycosylation. The results showed effects against Bothrops phospholipase A(2) activities (enzymatic, edema inducing, myotoxic, cytotoxic and bactericidal), suggesting that alpha BjussuMIP may prove useful in the treatment of snakebite envenomations. (C) 2008 Elsevier Masson SAS. All rights reserved.

Biochemical, functional, structural and phylogenetic studies on Intercro, a new isoform phospholipase A2 from Crotalus durissus terrificus snake venom

Crotoxin is a neurotoxin from Crotalus durissus terrificus venom that shows immunomodulatory, anti-inflammatory, antimicrobial, antitumor and analgesic activities. Structurally, this toxin is a heterodimeric complex composed by a toxic basic PLA2 (Crotoxin B or CB) non-covalently linked to an atoxic non-enzymatic and acidic component (Crotapotin, Crotoxin A or CA). Several CA and CB isoforms have been isolated and characterized, showing that the crotoxin venom fraction is, in fact, a mixture of different molecules derived from the combination of distinct subunit isoforms. Intercro (IC) is a protein from the same snake venom which presents high similarity in primary structure to CB, indicating that it could be an another isoform of this toxin. In this work, we compare IC to the crotoxin complex (CA/CB) and/or CB in order to understand its functional aspects. The experiments with IC revealed that it is a new toxin with different biological activities from CB, keeping its catalytic activity but presenting low myotoxicity and absence of neurotoxic activity. The results also indicated that IC is structurally similar to CB isoforms, but probably it is not able to form a neurotoxic active complex with crotoxin A as observed for CB. Moreover, structural and phylogenetic data suggest that IC is a new toxin with possible toxic effects not related to the typical CB neurotoxin. © 2013.

Estudos estruturais com fosfolipases A2 isoladas de venenos de serpentes dos gêneros Bothrops e Crotalus

Pós-graduação em Ciências Biológicas (Genética) - IBB

Um novo protocolo in silico para a predição de complexos flexíveis entre proteínas: estudo de caso para inibidores plasmáticos de fosfolipase A2 de serpentes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Crotoxin alters lymphocyte distribution in rats: Involvement of adhesion molecules and lipoxygenase-derived mediators

Crotoxin is the main neurotoxic component of Crotalus durissus terrificus snake venom and modulates immune and inflammatory responses, interfering with the activity of leukocytes. In the present work, the effects of crotoxin on the number of blood and lymphatic leukocytes and on lymph nodes and spleen lymphocytes population were investigated. The toxin s.c. administered to male Wistar rats, decreases the number of lymphocytes in blood and lymph circulation and increases the content of B and T-lymphocytes in lymph nodes. These effects were detected 1-2 h after treatment. The crotoxin molecule is composed of two subunits, an acidic non-toxic polypeptide, named crotapotin and a toxic basic phospholipase A(2) (PLA(2)). PLA(2), but not crotapotin, decreased the number of circulating blood and lymph lymphocytes. Crotoxin promotes leukocyte adherence to endothelial cells of blood microcirculation and to lymph node high endothelial venules, which might contribute to the drop in the number of circulating lymphocytes. Crotoxin increases expression of the adhesion molecule LFA-1 in lymphocytes. The changes in the expression of the adhesion molecule might contribute, at least in part, for the increased leukocyte adhesion to endothelium. Zileuton, a 5-lipoxygenase inhibitor, blocked the decrease in the number of circulating leukocytes induced by crotoxin and also abolished the changes observed in leukocyte-endothelial interactions, suggesting the involvement of lipoxygenase-derived mediators in the effects of the toxin. (c) 2008 Elsevier Ltd. All rights reserved.

Immunomodulatory effects of crotoxin isolated from Crotalus durissus terrificus venom in mice immunised with human serum albumin

Crotalus durissus terrificus venom and its main component, crotoxin (CTX), have the ability to down-modulate the immune system. Certain mechanisms mediated by cells and soluble factors of the immune system are responsible for the elimination of pathogenic molecules to ensure the specific protection against subsequent antigen contact. Accordingly, we evaluated the immunomodulatory effects of CTX on the immune response of mice that had been previously primed by immunisation with human serum albumin (HSA). CTX inoculation after HSA immunisation, along with complete Freund`s adjuvant (CFA) or Aluminium hydroxide (Alum) immunisation, was able to suppress anti-HSA IgG1 and IgG2a antibody production. We showed that the inhibitory effects of this toxin are not mediated by necrosis or apoptosis of any lymphoid cell population. Lower proliferation of T lymphocytes from mice immunised with HSA/CFA or HSA/Alum that received the toxin was observed in comparison to the mice that were only immunised. In conclusion, CTX is able to exert potent inhibitory effects on humoural and cellular responses induced by HSA immunisation, even when injected after an innate immune response has been initiated. (C) 2011 Elsevier Ltd. All rights reserved.

Reduction of crotoxin-induced neuromuscular blockade by gamma radiation

A comparative study between crotoxin and gamma irradiated crotoxin was performed on the indirectly evoked twitches and tetani of sciatic nerve-extensor digitorum longus muscle of rats. Crotoxin (3 to 14 mu g/ml) decreased the amplitude of twitches and induced a slight tetanic fade, and irradiated crotoxin did not significantly affect either twitch amplitude or tetanic tension. Since gamma radiation reduced the neurotoxicity of crotoxin it may be useful for the production of anticrotalic serum. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Crotoxin: Novel activities for a classic beta-neurotoxin

Crotoxin, the main toxin of South American rattlesnake (Crotalus durissus terrificus) venom, was the first snake venom protein to be purified and crystallized. Crotoxin is a heterodimeric beta-neurotoxin that consists of a weakly toxic basic phospholipase A(2) and a nonenzymatic, non-toxic acidic component (crotapotin). The classic biological activities normally attributed to crotoxin include neurotoxicity, myotoxicity, nephrotoxicity and cardiotoxicity. However, numerous studies in recent years have shown that crotoxin also has immunomodulatory, anti-inflammatory, anti-microbial, anti-tumor and analgesic actions. In this review, we describe the historical background to the discovery of crotoxin and its main toxic activities and then discuss recent structure-function studies and investigations that have led to the identification of novel pharmacological activities for the toxin. (C) 2010 Elsevier Ltd. All rights reserved.

Crystallization and preliminary X-ray crystallographic analysis of the heterodimeric crotoxin complex and the isolated subunits crotapotin and phospholipase A(2)

Crotoxin, a potent neurotoxin from the venom of the South American rattlesnake Crotalus durissus terrificus, exists as a heterodimer formed between a phospholipase A(2) and a catalytically inactive acidic phospholipase A(2) analogue (crotapotin). Large single crystals of the crotoxin complex and of the isolated subunits have been obtained. The crotoxin complex crystal belongs to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 38.2, b = 68.7, c = 84.2 angstrom, and diffracted to 1.75 angstrom resolution. The crystal of the phospholipase A(2) domain belongs to the hexagonal space group P6(1)22 (or its enantiomorph P6(5)22), with unit-cell parameters a = b = 38.7, c = 286.7 angstrom, and diffracted to 2.6 angstrom resolution. The crotapotin crystal diffracted to 2.3 angstrom resolution; however, the highly diffuse diffraction pattern did not permit unambiguous assignment of the unit-cell parameters.