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.

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.

Isolation and biochemical characterization of a gamma-type phospholipase A2 inhibitor from Crotalus durissus collilineatus snake serum

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Inhibition of inducible nitric oxide synthase-derived nitric oxide as a therapeutical target for acute pancreatitis induced by secretory phospholipase A(2)

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

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.

Biological characterization of the Amazon coral Micrurus spixii snake venom: isolation of a new neurotoxic phospholipase A2

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Structural and functional evidence for membrane docking and disruption sites on phospholipase A2-like proteins revealed by complexation with the inhibitor suramin

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

Structural basis for the inhibition of a phospholipase A2-like toxin by caffeic and aristolochic acids

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

Ac2-26 mimetic peptide of annexin A1 inhibits local and systemic inflammatory processes induced by bothrops moojeni venom and the lys-49 phospholipase A2 in a rat model

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

Production of Human Antibody Fragments Binding to Melittin and Phospholipase A2 in Africanised Bee Venom: Minimising Venom Toxicity

The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.

Antioxidant and inflammatory aspects of lipoprotein-associated phospholipase A2 (Lp-PLA2 ): a review

The association of cardiovascular events with Lp-PLA2 has been studied continuously today. The enzyme has been strongly associated with several cardiovascular risk markers and events. Its discovery was directly related to the hydrolysis of the platelet-activating factor and oxidized phospholipids, which are considered protective functions. However, the hydrolysis of bioactive lipids generates lysophospholipids, compounds that have a pro-inflammatory function. Therefore, the evaluation of the distribution of Lp-PLA2 in the lipid fractions emphasized the dual role of the enzyme in the inflammatory process, since the HDL-Lp-PLA2 enzyme contributes to the reduction of atherosclerosis, while LDL-Lp-PLA2 stimulates this process. Recently, it has been verified that diet components and drugs can influence the enzyme activity and concentration. Thus, the effects of these treatments on Lp-PLA2 may represent a new kind of prevention of cardiovascular disease. Therefore, the association of the enzyme with the traditional assessment of cardiovascular risk may help to predict more accurately these diseases.

Influence of obesity and cardiometabolic makers on lipoprotein-associated phospholipase A2 (Lp-PLA2) activity in adolescents: the healthy young cross-sectional study

Abstract Background Lipoprotein-associated phospholipase A2 activity (Lp-PLA2) is a good marker of cardiovascular risk in adults. It is strongly associated with stroke and many others cardiovascular events. Despite this, the impact of obesity on this enzyme activity and its relation to biomarkers of cardiovascular disease in adolescents is not very well investigated. The purpose of this article is to evaluate the influence of obesity and cardiometabolic markers on Lp-PLA2 activity in adolescents. Results This cross-sectional study included 242 adolescents (10–19 years) of both gender. These subjects were classified in Healthy Weight (n = 77), Overweight (n = 82) and Obese (n = 83) groups. Lipid profile, glucose, insulin, HDL size, LDL(−) and anti-LDL(−) antibodies were analyzed. The Lp-PLA2 activity was determined by a colorimetric commercial kit. Body mass index (BMI), waist circumference and body composition were monitored. Food intake was evaluated using three 24-hour diet recalls. The Lp-PLA2 activity changed in function to high BMI, waist circumference and fat mass percentage. It was also positively associated with HOMA-IR, glucose, insulin and almost all variables of lipid profile. Furthermore, it was negatively related to Apo AI (β = −0.137; P = 0.038) and strongly positively associated with Apo B (β = 0.293; P < 0.001) and with Apo B/Apo AI ratio (β = 0.343; P < 0.001). The better predictor model for enzyme activity, on multivariate analysis, included Apo B/Apo AI (β = 0.327; P < 0.001), HDL size (β = −0.326; P < 0.001), WC (β = 0.171; P = 0.006) and glucose (β = 0.119; P = 0.038). Logistic regression analysis demonstrated that changes in Apo B/Apo AI ratio were associated with a 73.5 times higher risk to elevated Lp-PLA2 activity. Conclusions Lp-PLA2 changes in function of obesity, and that it shows important associations with markers of cardiovascular risk, in particular with waist circumference, glucose, HDL size and Apo B/Apo AI ratio. These results suggest that Lp-PLA2 activity can be a cardiovascular biomarker in adolescence.

Human group II 14 kDa phospholipase A2 activates human platelets

Recombinant human group II phospholipase A2 (sPLA2) added to human platelets in the low microg/ml range induced platelet activation, as demonstrated by measurement of platelet aggregation, thromboxane A2 generation and influx of intracellular free Ca2+ concentration and by detection of time-dependent tyrosine phosphorylation of platelet proteins. The presence of Ca2+ at low millimolar concentrations is a prerequisite for the activation of platelets by sPLA2. Mg2+ cannot replace Ca2+. Mg2+, given in addition to the necessary Ca2+, inhibits sPLA2-induced platelet activation. Pre-exposure to sPLA2 completely blocked the aggregating effect of a second dose of sPLA2. Albumin or indomethacin inhibited sPLA2-induced aggregation, similarly to the inhibition of arachidonic acid-induced aggregation. Platelets pre-treated with heparitinase or phosphatidylinositol-specific phospholipase C lost their ability to aggregate in response to sPLA2, although they still responded to other agonists. This suggests that a glycophosphatidylinositol-anchored platelet-membrane heparan sulphate proteoglycan is the binding site for sPLA2 on platelets. Previous reports have stated that sPLA2 is unable to activate platelets. The inhibitory effect of albumin and Mg2+, frequently used in aggregation studies, and the fact that isolated platelets lose their responsiveness to sPLA2 relatively quickly, may explain why the platelet-activating effects of sPLA2 have not been reported earlier.