Structure of myotoxin II, a catalytically inactive Lys49 phospholipase A2 homologue from Atropoides nummifer venom

Lys49 snake-venom phospholipase A2 (PLA2) homologues are highly myotoxic proteins which, although lacking catalytic activity, possess the ability to disrupt biological membranes, inducing significant muscle-tissue loss and permanent disability in severely envenomed patients. Since the structural basis for their toxic activity is still only partially understood, the structure of myotoxin II, a monomeric Lys49 PLA2 homologue from Atropoides nummifer, has been determined at 2.08 Å resolution and the anion-binding site has been characterized. © 2006 International Union of Crystallography. All rights reserved.

Modulation of the pharmacological effects of enzymatically-active PLA 2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

Background. An interaction between lectins from marine algae and PLA 2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA 2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA 2 and its complex. Results. This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA 2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion. The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules. © 2008 Oliveira et al; licensee BioMed Central Ltd.

Structure of a novel class II phospholipase D: Catalytic cleft is modified by a disulphide bridge

Phospholipases D (PLDs) are principally responsible for the local and systemic effects of Loxosceles envenomation including dermonecrosis and hemolysis. Despite their clinical relevance in loxoscelism, to date, only the SMase I from Loxosceles laeta, a class I member, has been structurally characterized. The crystal structure of a class II member from Loxosceles intermedia venom has been determined at 1.7. Å resolution. Structural comparison to the class I member showed that the presence of an additional disulphide bridge which links the catalytic loop to the flexible loop significantly changes the volume and shape of the catalytic cleft. An examination of the crystal structures of PLD homologues in the presence of low molecular weight compounds at their active sites suggests the existence of a ligand-dependent rotamer conformation of the highly conserved residue Trp230 (equivalent to Trp192 in the glycerophosphodiester phosphodiesterase from Thermus thermophofilus, PDB code: 1VD6) indicating its role in substrate binding in both enzymes. Sequence and structural analyses suggest that the reduced sphingomyelinase activity observed in some class IIb PLDs is probably due to point mutations which lead to a different substrate preference. © 2011 Elsevier Inc.

Crystallographic portrayal of different conformational states of a Lys49 phospholipase A2 homologue: Insights into structural determinants for myotoxicity and dimeric configuration

Catalytically inactive phospholipase A2 (PLA2) homologues play key roles in the pathogenesis induced by snake envenomation, causing extensive tissue damage via a mechanism still unknown. Although, the amino acid residues directly involved in catalysis are conserved, the substitution of Asp49 by Arg/Lys/Gln or Ser prevents the binding of the essential calcium ion and hence these proteins are incapable of hydrolyzing phospholipids. In this work, the crystal structure of a Lys49-PLA2 homologue from Bothrops brazili (MTX-II) was solved in two conformational states: (a) native, with Lys49 singly coordinated by the backbone oxygen atom of Val31 and (b) complexed with tetraethylene glycol (TTEG). Interestingly, the TTEG molecule was observed in two different coordination cages depending on the orientation of the nominal calcium-binding loop and of the residue Lys49. These structural observations indicate a direct role for the residue Lys49 in the functioning of a catalytically inactive PLA2 homologue suggesting a contribution of the active site-like region in the expression of pharmacological effects such as myotoxicity and edema formation. Despite the several crystal structures of Lys49-PLA2 homologues already determined, their biological assembly remains controversial with two possible conformations. The extended dimer with the hydrophobic channel exposed to the solvent and the compact dimer in which the active site-like region is occluded by the dimeric interface. In the MTX-II crystal packing analysis was found only the extended dimer as a possible stable quaternary arrangement. © 2012 Elsevier B.V.

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.

Identification of phospholipase C zeta in normospermic and teratospermic domestic cat sperm

In mammalian species, oocyte activation is initiated by oscillations in the intracellular concentration of free calcium ([Ca2+]i), which are also essential to allow embryonic development. To date, evidence supporting the hypothesis that a sperm factor is responsible for initiating oocyte activation has been presented in various mammalian species. Among the possible candidates to be the active sperm factor is the novel sperm-specific phospholipase C ζ (PLCζ), which besides its testis-specific expression is capable of initiating [Ca2+]i oscillations. In this study, we investigated the presence of PLCζ in the sperm of the domestic cat and whether normospermic and teratospermic cats differ in their PLCζ expression. Immunoblotting with anti-PLCζ antibodies confirmed the presence of an immunoreactive band of ~70 kDa in whole sperm lysates of domestic cat as well as in both soluble and insoluble fractions from this sperm. Additional immunoreactive bands, probably C- and N-terminal truncated versions of PLCζ, were also visualized in the soluble sperm fractions. Interestingly, immunoreactivity of PLCζ was detectable in teratospermic sperm, although with slightly less intensity than in normospermic sperm. In conclusion, domestic cat sperm express PLCζ in both cytosolic and high-pH fractions, which is consistent with data in other mammals. Sperm from teratospermic cats also express PLCζ, albeit at reduced concentrations, which may affect the fertility of these males. © 2013 Elsevier Inc..

Componentes do valor adaptativo e investigação do polimorfismo genético do gene Cyp6g1 (exons 1 e 2) em linhagens resistentes e suscetíveis de Drosophila melanogaster e de Drosophila simulans

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Skeletal muscle degeneration and regeneration after injection of bothropstoxin-II, a phospholipase A2 isolated from the venom of the snake Bothrops jararacussu

A myotoxic phospholipase A2, named bothropstoxin II (BthTX-II), was isolated from the venom of the South American snake Bothrops jararacussu and the pathogenesis of myonecrosis induced by this toxin was studied in mice. BthTX-II induced a rapid increase in plasma creatine kinase levels. Histological and ultrastructural observations demonstrate that this toxin affects muscle fibers by first disrupting the integrity of plasma membrane, as delta lesions were the earliest morphological alteration and since the plasma membrane was interrupted or absent in many portions. In agreement with this hypothesis, BthTX-II released peroxidase entrapped in negatively charged multilamellar liposomes and behaved as an amphiphilic protein in charge shift electrophoresis, an indication that its mechanism of action might be based on the interaction and disorganization of plasma membrane phospholipids. Membrane damage was followed by a complex series of morphological alterations in intracellular structures, most of which are probably related to an increase in cytosolic calcium levels. Myofilaments became hypercontracted into dense clumps which alternated with cellular spaces devoid of myofibrillar material. Later on, myofilaments changed to a hyaline appearance with a more uniform distribution. Mitochondria were drastically affected, showing high amplitude swelling, vesiculation of cristae, formation of flocculent densities, and membrane disruption. By 24 hr, abundant polymorphonuclear leucocytes and macrophages were observed in the interstitial space as well as inside necrotic fibers. Muscle regeneration proceeded normally, as abundant myotubes and regenerating myofibers were observed 7 days after BthTX-II injection. By 28 days regenerating fibers had a diameter similar to that of adult muscle fibers, although they presented two distinctive features: central location of nuclei and some fiber splitting. This good regenerative response may be explained by the observation that BthTX-II does not affect blood vessels, nerves, or basal laminae. © 1991.

Characterization of the phosphatidylinositol-specific phospholipase C-released form of rat osseous plate alkaline phosphatase and its possible significance on endochondral ossification

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