Skeletal muscle fiber hypercontraction induced by Bothrops asper myotoxic phospholipases A2 ex vivo does not involve a direct action on the contractile apparatus.

Myonecrosis is a frequent clinical manifestation of envenomings by Viperidae snakes, mainly caused by the toxic actions of secreted phospholipase A2 (sPLA2) enzymes and sPLA2-like homologs on skeletal muscle fibers. A hallmark of the necrotic process induced by these myotoxins is the rapid appearance of hypercontracted muscle fibers, attributed to the massive influx of Ca2+ resulting from cell membrane damage. However, the possibility of myotoxins having, in addition, a direct effect on the contractile machinery of skeletal muscle fibers when internalized has not been investigated. This question is here addressed by using an ex vivo model of single-skinned muscle fibers, which lack membranes but retain an intact contractile apparatus. Rabbit psoas skinned fibers were exposed to two types of myotoxins of Bothrops asper venom: Mt-I, a catalytically active Asp49 sPLA2 enzyme, and Mt-II, a Lys49 sPLA2-like protein devoid of phospholipolytic activity. Neither of these myotoxins affected the main parameters of force development in striated muscle sarcomeres of the skinned fibers. Moreover, no microscopical alterations were evidenced after their exposure to Mt-I or Mt-II. In contrast to the lack of effects on skinned muscle fibers, both myotoxins induced a strong hypercontraction in myotubes differentiated from murine C2C12 myoblasts, with drastic morphological alterations that reproduce those described in myonecrotic tissue in vivo. As neither Mt-I nor Mt-II showed direct effects upon the contractile apparatus of skinned fibers, it is concluded that the mechanism of hypercontraction triggered by both myotoxins in patients involves indirect effects, i.e., the large cytosolic Ca2+ increase after sarcolemma permeabilization.

Secreted phospholipase A2 XIIA triggers a mitochondrial damage-induced senescence in chronic obstructive pulmonary disease fibroblasts.

RATIONALE: Lung fibroblast senescence is involved in the pathophysiology of chronic obstructive pulmonary disease (COPD). However, the mechanisms underlining this phenomenon are still poorly understood. Secreted phospholipases (sPLA2, a subclass of phospholipases) are secreted by senescent cells and can in turn induce senescence. However, their role in fibroblasts senescence in COPD is unknown. OBJECTIVES: The aim of this study was to analyze the role of sPLA2 in pulmonary fibroblast senescence. METHODS: Fibroblasts were isolated from patients with COPD and control subjects, and senescence markers and inflammatory profile was analyzed. sPLA2 levels were quantified in serum of COPD and controls. MAIN RESULTS: In comparison with non-smokers and smoker controls, senescent lung COPD fibroblasts exhibited a higher mRNA and protein expression of the sPLA2 isoform XIIA and of syndecan 4 (one of its receptors). sPLA2 XIIA induced in turn senescence of non-senescent pulmonary fibroblasts via a pathway involving consecutively syndecan 4, activation of MAPK and p-serine 727 STAT-3, increased mitochondrial ROS production, and activation of AMPK/p53. This pathway was associated with a specific inflammatory secretome (IL-10, IL-12 and TNFα), globally suggesting occurrence of a mitochondrial damage-induced senescence. COPD fibroblasts were more susceptible to this sPLA2 XIIA effect than cells from controls subjects. sPLA2 XIIA levels were significantly higher in serum from COPD patients as compared to controls. CONCLUSION: sPLA2 XIIA is involved in senescence in COPD and could be a potential target to dampen this process.

Potential neurotoxic activity of diverse molecules released by astrocytes.

Astrocytes are the main support cells of the central nervous system. They also participate in neuroimmune reactions. In response to pathological and immune stimuli, astrocytes transform to reactive states characterized by increased release of inflammatory mediators. Some of these molecules are neuroprotective and inflammation resolving while others, including reactive oxygen species (ROS), nitric oxide (NO), matrix metalloproteinase (MMP)- 9, L-glutamate, and tumor necrosis factor α (TNF), are well-established toxins known to cause damage to surrounding cells and tissues. We hypothesized that similar to microglia, the brain immune cells, reactive astrocytes can release a broader set of diverse molecules that are potentially neurotoxic. A literature search was conducted to identify such molecules using the following two criteria: 1) evidence of their expression and secretion by astrocytes and 2) direct neurotoxic action. This review describes 14 structurally diverse molecules as less-established astrocyte neurotoxins, including C-X-C motif chemokine ligand (CXCL)10, CXCL12/CXCL12(5-67), FS-7-associated surface antigen ligand (FasL), macrophage inflammatory protein (MIP)- 2α, TNF-related apoptosis inducing ligand (TRAIL), pro-nerve growth factor (proNGF), pro-brain-derived neurotrophic factor (proBDNF), chondroitin sulfate proteoglycans (CSPGs), cathepsin (Cat)B, group IIA secretory phospholipase A2 (sPLA2-IIA), amyloid beta peptides (Aß), high mobility group box (HMGB)1, ceramides, and lipocalin (LCN)2. For some of these molecules, further studies are required to establish either their direct neurotoxic effects or the full spectrum of stimuli that induce their release by astrocytes. Only limited studies with human-derived astrocytes and neurons are available for most of these potential neurotoxins, which is a knowledge gap that should be addressed in the future. We also summarize available evidence of the role these molecules play in select neuropathologies where reactive astrocytes are a key feature. A comprehensive understanding of the full spectrum of neurotoxins released by reactive astrocytes is key to understanding neuroinflammatory diseases characterized by the adverse activation of these cells and may guide the development of novel treatment strategies.

Intestinal phospholipase A2 from Sparidae species: Functional properties and cytotoxic potential evaluation.

Marine species have gained significant attention as potential source for a broad spectrum of bioactive proteins. Fish phospholipases A2 (PLA2) have attracted renewed interest due to their excellent properties in lipid digestion. Herein, we report for the first time the catalytic properties of two intestinal secreted PLA2 (sPLA2) identified from Diplodus sargus (IDsPLA2) and Sparus aurata (ISaPLA2). The highest sequence identity was obtained with recently isolated Sparidae digestive PLA2 (45%) and Human pancreatic PLA2 (42%). IDsPLA2 and ISaPLA2 were overexpressed in E. coli as inclusion bodies, refolded and purified. Both enzymes have improved thermostability compared to mammalian pancreatic sPLA2 since they are active and stable at 55 °C, with specific activities of 320 and 190 U mg-1 measured on phosphatidylcholine, respectively. Interestingly, IDsPLA2, but not ISaPLA2, revealed weak toxicity towards macrophages and suggests its involvement in cell membrane degradation. ISaPLA2 was found to be more active than IDsPLA2 when using the monolayer technique at 20 mN m-1. Structural models of both enzymes revealed their differences. In silico docking of phospholipids with both models allowed proposing key amino-acids in substrate binding and selectivity. Overall, these results provide insight into the enzymatic and structural properties of two novel sPLA2 with potential for future applications.

The neurotoxic secreted phospholipase A2 from the Vipera a. ammodytes venom targets cytochrome c oxidase in neuronal mitochondria.

The ß-neurotoxic secreted phospholipases A2 (sPLA2s) block neuro-muscular transmission by poisoning nerve terminals. Damage inflicted by such sPLA2s (ß-ntx) on neuronal mitochondria is characteristic, very similar to that induced by structurally homologous endogenous group IIA sPLA2 when its activity is elevated, as, for example, in the early phase of Alzheimer's disease. Using ammodytoxin (Atx), the ß-ntx from the venom of the nose-horned viper (Vipera a. ammodytes), the sPLA2 receptor R25 has been detected in neuronal mitochondria. This receptor has been purified from porcine cerebral cortex mitochondria by a new Atx-affinity-based chromatographic procedure. Mass spectrometry analysis revealed R25 to be the subunit II of cytochrome c oxidase (CCOX), an essential constituent of the respiratory chain complex. CCOX was confirmed as being the first intracellular membrane receptor for sPLA2 by alternative Atx-affinity-labellings of purified CCOX, supported also by the encounter of Atx and CCOX in PC12 cells. This discovery suggests the explanation of the mechanism by which ß-ntx hinders production of ATP in poisoned nerve endings. It also provides a new insight into the potential function and dysfunction of endogenous GIIA sPLA2 in mitochondria.

Signaling pathways involved in zymosan phagocytosis induced by two secreted phospholipases A2 isolated from Bothrops asper snake venom in macrophages.

Phagocytosis, a process involved in host defense, requires coordination of a variety of signaling reactions. MT-II, a catalytically-inactive Lys49-PLA2¸ and MT-III, an active Asp49-PLA2 isolated from Bothrops asper snake venom, activate phagocytosis in macrophages. In this study the signal pathways mediating zymosan phagocytosis, focusing in lipidic second messengers, were investigated. Macrophages collected from male Swiss mouse peritoneum were obtained 96h after i.p. injection of thioglycollate. Phagocytosis was evaluated with non-opsonized zymosan in the presence or absence of specific inhibitors. Data showed that both venom PLA2s increased phagocytosis. Zileuton, Etoricoxib, PACOCF3 (5-LO, COX-2 and iPLA2 inhibitors, respectively), as well as WEB2170 (PAF receptor antagonist) significantly reduced phagocytosis induced by both venom PLA2s. However, Indomethacin (COX-1/COX-2 inhibitor) and Montelukast (CysL receptor antagonist) did not affect the toxins-induced phagocytosis. Moreover, while PACOCF3 (iPLA2 inhibitor), reduced the phagocytosis induced by MT-II and MT-III, AACOCF3 (cPLA2 inhibitor) significantly reduced the MT-II, but not MT-III-induced phagocytosis. These data suggest the effect of both sPLA2s depends on iPLA2 and that the effect of MT-II depends on activation of cPLA2. COX-2 and 5-LO-derived metabolites as well as PAF are involved in the signaling events required for phagocytosis induced by both venom sPLA2s.

Broad-spectrum antiviral agents: secreted phospholipase A2 targets viral envelope lipid bilayers derived from the endoplasmic reticulum membrane.

Hepatitis C virus (HCV), dengue virus (DENV) and Japanese encephalitis virus (JEV) belong to the family Flaviviridae. Their viral particles have the envelope composed of viral proteins and a lipid bilayer acquired from budding through the endoplasmic reticulum (ER). The phospholipid content of the ER membrane differs from that of the plasma membrane (PM). The phospholipase A2 (PLA2) superfamily consists of a large number of members that specifically catalyse the hydrolysis of phospholipids at a particular position. Here we show that the CM-II isoform of secreted PLA2 obtained from Naja mossambica mossambica snake venom (CM-II-sPLA2) possesses potent virucidal (neutralising) activity against HCV, DENV and JEV, with 50% inhibitory concentrations (IC50) of 0.036, 0.31 and 1.34 ng/ml, respectively. In contrast, the IC50 values of CM-II-sPLA2 against viruses that bud through the PM (Sindbis virus, influenza virus and Sendai virus) or trans-Golgi network (TGN) (herpes simplex virus) were >10,000 ng/ml. Moreover, the 50% cytotoxic (CC50) and haemolytic (HC50) concentrations of CM-II-sPLA2 were >10,000 ng/ml, implying that CM-II-sPLA2 did not significantly damage the PM. These results suggest that CM-II-sPLA2 and its derivatives are good candidates for the development of broad-spectrum antiviral drugs that target viral envelope lipid bilayers derived from the ER membrane.

Ammodytin L is the main cardiotoxic component of the Vipera ammodytes ammodytes venom.

Venom of the nose-horned viper (V. a. ammodytes) as also venoms of some related European viperids can induce also cardiotoxic effects in mammals. In this work we demonstrated that the protein in the V. a. ammodytes venom acting on heart is a myotoxic secreted phospholipase A2 analogue ammodytin L (AtnL). In the isolated perfused rat heart AtnL induced significant and irreversible cardiotoxicity characterized by atrioventricular (AV) blockade. This venom protein induced appearance of high levels of creatine kinase, lactate dehydrogenase, aspartate aminotransferase and troponin I in the sinus effluent of the isolated heart, indicative for myocardial damage, which is obviously the primary cause of its cardiotoxic action. Gel filtration chromatography subfractions C1 and C2 of the V. a. ammodytes venom harboured most of the venom cardiotoxicity. As we showed, just these two subfractions contained also AtnL. Subfraction C1 in the final CF concentration 11.3 µg/mL (containing 3.1 µg/mL AtnL) induced a complete cardiac arrest while subfraction C2 in the final CF concentration 6.0 µg/mL (containing 0.8 µg/mL AtnL) and the pure AtnL (1.0 µg/mL) did not. Contrary to AtnL, subfraction C1 at 11.3 µg/mL was not able to induce the AV blockade. This exposed the only other cardiotoxic subfractions-specific venom protein, a cysteine-rich secretory protein (CRISP), as an additional venom component potentially involved in modulation of the heart activity. Cardiotoxicity reported in some cases of the adder (V. berus) venom and the asp viper (V. aspis) venom poisonings may be assigned to AtnL in these venoms.

Secreted phospholipase A2 of Clonorchis sinensis activates hepatic stellate cells through a pathway involving JNK signalling.

BACKGROUND: Secreted phospholipase A2 (sPLA2) is a protein secreted by Clonorchis sinensis and is a component of excretory and secretory products (CsESPs). Phospholipase A2 is well known for its role in liver fibrosis and inhibition of tumour cells. The JNK signalling pathway is involved in hepatic stellate cells (HSCs) activation. Blocking JNK activity with SP600125 inhibits HSCs activation. In a previous study, the protein CssPLA2 was expressed in insoluble inclusion bodies. Therefore, it's necessary to express CssPLA2 in water-soluble form and determine whether the enzymatic activity of CssPLA2 or cell signalling pathways is involved in liver fibrosis caused by clonorchiasis. METHODS: Balb/C mice were given an abdominal injection of MBP-CssPLA2. Liver sections with HE and Masson staining were observed to detect accumulation of collagen. Western blot of mouse liver was done to detect the activation of JNK signalling pathway. In vitro, HSCs were incubated with MBP-CssPLA2 to detect the activation of HSCs as well as the activation of JNK signalling pathway. The mutant of MBP-CssPLA2 without enzymatic activity was constructed and was also incubated with HSCs to check whether activation of the HSCs was related to the enzymatic activity of MBP-CssPLA2. RESULTS: The recombinant protein MBP-CssPLA2 was expressed soluble and of good enzymatic activity. A mutant of CssPLA2, without enzymatic activity, was also constructed. In vivo liver sections of Balb/C mice that were given an abdominal injection of 50 µg/ml MBP-CssPLA2 showed an obvious accumulation of collagen and a clear band of P-JNK1 could be seen by western blot of the liver tissue. In vitro, MBP-CssPLA2, as well as the mutant, was incubated with HSCs and it was proved that activation of HSCs was related to activation of the JNK signalling pathway instead of the enzymatic activity of MBP-CssPLA2. CONCLUSIONS: Activation of HSCs by CssPLA2 is related to the activation of the JNK signalling pathway instead of the enzymatic activity of CssPLA2. This finding could provide a promising treatment strategy to interrupt the process of liver fibrosis caused by clonorchiasis.

Secreted Phospholipases A2 from Animal Venoms in Pain and Analgesia.

Animal venoms comprise a complex mixture of components that affect several biological systems. Based on the high selectivity for their molecular targets, these components are also a rich source of potential therapeutic agents. Among the main components of animal venoms are the secreted phospholipases A2 (sPLA2s). These PLA2 belong to distinct PLA2s groups. For example, snake venom sPLA2s from Elapidae and Viperidae families, the most important families when considering envenomation, belong, respectively, to the IA and IIA/IIB groups, whereas bee venom PLA2 belongs to group III of sPLA2s. It is well known that PLA2, due to its hydrolytic activity on phospholipids, takes part in many pathophysiological processes, including inflammation and pain. Therefore, secreted PLA2s obtained from animal venoms have been widely used as tools to (a) modulate inflammation and pain, uncovering molecular targets that are implicated in the control of inflammatory (including painful) and neurodegenerative diseases; (b) shed light on the pathophysiology of inflammation and pain observed in human envenomation by poisonous animals; and, (c) characterize molecular mechanisms involved in inflammatory diseases. The present review summarizes the knowledge on the nociceptive and antinociceptive actions of sPLA2s from animal venoms, particularly snake venoms.

Diverse activity of human secretory phospholipases A2 on the migration of human vascular smooth muscle cells.

OBJECTIVE: Investigation of the diversity of human secretory phospholipases A2 (sPLA2) on the migration of human vascular smooth muscle cells (VSMC). MATERIAL: We investigated the impact of sPLA2 IIA, V, and X and of oleic acid, linoleic acid and lysophosphatidylcholine on the migration of human VSMC. METHODS: Recombinant human sPLA2's and Boyden's chamber method were applied. RESULTS: sPLA2, IIA but not V or X enhanced migration of VSMC in a dose/time dependent manner. Oleic and linoleic acids, and lysophosphatidylcholine markedly enhanced migration. CONCLUSIONS: These results imply that sPLA2 IIA, which is known to be present in the arterial wall in the vicinity of VSMC, as well as products of lipid hydrolysis induced by sPLA2, enhance the migration of VSMC, and thus may contribute to atherogenic process.

Sphingomyelin Regulates the Activity of Secretory Phospholipase A2 in the Plasma Membrane.

We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and the activity of secretory phospholipase A2 (sPLA2 ) using two Chinese hamster ovary (CHO)-K1 cell mutants, LY-B and LY-A cells, deficient in sphingolipid synthesis. In LY-B cells, deficiency of sphingolipids enhanced the release of AA induced by bee venom sPLA2-III or human sPLA2-V. These alterations were reversed by replenishment of exogenous sphingomyelin (SM). In LY-A cells, deficiency of SM increased the release of AA induced by sPLA2. In CHO-K1 cells, decrease and increase of SM level in the plasma membrane by pharmacological methods increased and inhibited the release of AA, respectively. SM inhibited the activity of sPLA2 in vitro. Niemann-Pick disease type C (NPC) is a lysosomal storage disorder caused by mutation of either the NPC1 or NPC2 gene, and is characterized by accumulation of cholesterol and sphingolipids including SM in late endosomes/lysosomes. Increased levels of AA and sPLA2 activity are involved in various neurodegenerative diseases. In CHO cells lacking NPC1 (A101 cells), SM level was lower in the plasma membrane, while it was higher in late endosomes/lysosomes. The release of AA induced by sPLA2 was increased in A101 cells than that in parental cells (JP17 cells), which was attenuated by adding exogenous SM. In addition, sPLA2 -III-induced cytotoxicity in A101 cells was much higher than that in JP17 cells. These results suggest that SM in the plasma membrane plays important roles in regulating sPLA2 activity and the enzyme-induced cytotoxicity in A101 cells.

Effect of chlorogenic acid (5-Caffeoylquinic Acid) isolated from Baccharis oxyodonta on the structure and pharmacological activities of secretory phospholipase A2 from Crotalus durissus terrificus.

The aim of this paper was to investigate the effect of chlorogenic acid (5-caffeoylquinic acid, 5CQA), isolated from Baccharis oxyodonta, on the structure and pharmacological effect of secretory phospholipase A2 (sPLA2) from Crotalus durissus terrificus. All in vitro and in vivo experiments were conducted using a purified sPLA2 compared under the same experimental conditions with sPLA2 : 5CQA. 5CQA induced several discrete modifications in the secondary structure and the hydrophobic characteristics of native sPLA2 that induced slight changes in the α-helical content, increase in the random coil structure, and decrease of fluorescence of native sPLA2. Moreover, 5CQA significantly decreased the enzymatic activity and the oedema and myonecrosis induced by native sPLA2. As the catalytic activity of sPLA2 plays an important role in several of its biological and pharmacological properties, antibacterial activity was used to confirm the decrease in its enzymatic activity by 5CQA, which induced massive bacterial cell destruction. We found that 5CQA specifically abolished the enzymatic activity of sPLA2 and induced discrete protein unfolding that mainly involved the pharmacological site of sPLA2. These results showed the potential application of 5CQA in the snake poisoning treatment and modulation of the pathological effect of inflammation induced by secretory PLA2.

Roles of secreted phospholipases A2 in the mammalian immune system.

Secreted phospholipase A2 (sPLA2) molecules constitute a family of proteins that are involved functionally in many biological processes. In particular, they participate in diverse pathophysiological settings as enzymes that release free fatty acids and lysophospholipids from phospholipids in biological membranes, or as ligands for various cellular receptors. In this review the confirmed or expected functions of sPLA2s in the mammalian immune system are surveyed. Some of the twelve mammalian sPLA2 molecules constitute part of the so-called innate immune system by virtue of their antibacterial, antiviral and antifungal activities. They are also involved in acute inflammation, a protective reaction of the body to infection or injury. The acute inflammation sometimes escapes regulation, becomes chronic and can evolve into a severe pathology. One or more types of sPLA2 are involved in asthma, rheumatoid arthritis, sepsis, atherosclerosis, myocardial infarction, Crohn's disease, ulcerative colitis and cancer. sPLA2s are thus important therapeutic targets as well as biotherapeutic molecules. Improving the selectivity of inhibitors of sPLA2s to be able to target a particular sPLA2 could therefore be one of the most important tasks for future research.

Pharmacological study of a new Asp49 phospholipase A(2) (Bbil-TX) isolated from Bothriopsis bilineata smargadina (forest viper) venom in vertebrate neuromuscular preparations.

The neuromuscular activity of Bbil-TX, a PLA2 with catalytic activity isolated from Bothriopsis bilineata smargadina venom, was examined in chick biventer cervicis (BC) and mouse phrenic nerve-diaphragm (PND) preparations. In BC preparations, Bbil-TX (0.5-10 µg/ml) caused time- and concentration-dependent blockade that was not reversed by washing; the times for 50% blockade were 87 ± 7, 41 ± 7 and 19 ± 2 min (mean ± SEM; n = 4-6) for 1, 5 and 10 µg/ml, respectively. Muscle contractures to exogenous ACh and KCl were unaffected. The toxin (10 µg/ml) also did not affect the twitch-tension of directly-stimulated, curarized (10 µg/ml) BC preparations. However, Bbil-TX (10 µg/ml) produced mild morphological alterations (edematous and/or hyperchromic fibers) in BC; there was also a progressive release of CK (from 116 ± 17 IU/ml (basal) to 710 ± 91 IU/ml after 45 min). Bbil-TX (5 µg/ml)-induced blockade was markedly inhibited at 22-24 °C and pretreatment with p-bromophenacyl bromide (p-BPB) abolished the neuromuscular blockade. Bbil-TX (3-30 µg/ml, n = 4-6) caused partial time- and concentration-dependent blockade in PND preparations (52 ± 2% at the highest concentration). Bbil-TX (30 µg/ml) also markedly reduced the MEPPs frequency [from 26 ± 2.5 (basal) to 10 ± 1 after 60 min; n = 5; p < 0.05] and the quantal content [from 94 ± 14 (basal) to 24 ± 3 after 60 min; n = 5; p < 0.05] of PND preparations, but caused only minor depolarization of the membrane resting potential [from -80 ± 1 mV (basal) to -66 ± 2 mV after 120 min; n = 5; p < 0.05], with no significant change in the depolarizing response to exogenous carbachol. These results show that Bbil-TX is a presynaptic PLA2 that contributes to the neuromuscular blockade caused by B. b. smargadina venom.

Biochemical characterization and pharmacological properties of new basic PLA2 BrTX-I isolated from Bothrops roedingeri (Roedinger's Lancehead) Mertens, 1942, snake venom.

BrTX-I, a PLA2, was purified from Bothrops roedingeri venom after only one chromatographic step using reverse-phase HPLC on µ-Bondapak C-18 column. A molecular mass of 14358.69 Da was determined by MALDI-TOF mass spectrometry. Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The total amino acid sequence was obtained using SwissProt database and showed high amino acid sequence identity with other PLA2 from snake venom. The amino acid composition showed that BrTX-I has a high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA2. BrTX-I presented PLA2 activity and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0, 35-45°C, and required Ca(2+). In vitro, the whole venom and BrTX-I caused a neuromuscular blockade in biventer cervicis preparations in a similar way to other Bothrops species. BrTX-I induced myonecrosis and oedema-forming activity analyzed through injection of the purified BrTX-I in mice. Since BrTX-I exerts a strong proinflammatory effect, the enzymatic phospholipid hydrolysis might be relevant for these phenomena; incrementing levels of IL-1, IL-6, and TNF α were observed at 15 min, 30 min, one, two, and six hours postinjection, respectively.

Biochemical, pharmacological, and structural characterization of new basic PLA2 Bbil-TX from Bothriopsis bilineata snake venom.

Bbil-TX, a PLA2, was purified from Bothriopsis bilineata snake venom after only one chromatographic step using RP-HPLC on µ-Bondapak C-18 column. A molecular mass of 14243.8 Da was confirmed by Q-Tof Ultima API ESI/MS (TOF MS mode) mass spectrometry. The partial protein sequence obtained was then submitted to BLASTp, with the search restricted to PLA2 from snakes and shows high identity values when compared to other PLA2s. PLA2 activity was presented in the presence of a synthetic substrate and showed a minimum sigmoidal behavior, reaching its maximal activity at pH 8.0 and 25-37°C. Maximum PLA2 activity required Ca(2+) and in the presence of Cd(2+), Zn(2+), Mn(2+), and Mg(2+) it was reduced in the presence or absence of Ca(2+). Crotapotin from Crotalus durissus cascavella rattlesnake venom and antihemorrhagic factor DA2-II from Didelphis albiventris opossum sera under optimal conditions significantly inhibit the enzymatic activity. Bbil-TX induces myonecrosis in mice. The fraction does not show a significant cytotoxic activity in myotubes and myoblasts (C2C12). The inflammatory events induced in the serum of mice by Bbil-TX isolated from Bothriopsis bilineata snake venom were investigated. An increase in vascular permeability and in the levels of TNF-a, IL-6, and IL-1 was was induced. Since Bbil-TX exerts a stronger proinflammatory effect, the phospholipid hydrolysis may be relevant for these phenomena.

Secreted phospholipase A2-IIA-induced a phenotype of activated microglia in BV-2 cells requires epidermal growth factor receptor transactivation and proHB-EGF shedding.

BACKGROUND: Activation of microglia, the primary component of the innate immune response in the brain, is a hallmark of neuroinflammation in neurodegenerative disorders, including Alzheimer's disease (AD) and other pathological conditions such as stroke or CNS infection. In response to a variety of insults, microglial cells produce high levels of inflammatory cytokines that are often involved in neuronal injury, and play an important role in the recognition, engulfment, and clearance of apoptotic cells and/or invading microbes. Secreted phospholipase A2-IIA (sPLA2-IIA), an enzyme that interacts with cells involved in the systemic immune/inflammatory response, has been found up-regulated in the cerebrospinal fluid and brain of AD patients. However, despite several approaches, its functions in mediating CNS inflammation remain unknown. In the present study, the role of sPLA2-IIA was examined by investigating its direct effects on microglial cells. METHODS: Primary and immortalized microglial cells were stimulated by sPLA2-IIA in order to characterize the cytokine-like actions of the phospholipase. The hallmarks of activated microglia analyzed include: mitogenic response, phagocytic capabilities and induction of inflammatory mediators. In addition, we studied several of the potential molecular mechanisms involved in those events. RESULTS: The direct exposure of microglial cells to sPLA2-IIA stimulated, in a time- and dose-dependent manner, their phagocytic and proliferative capabilities. sPLA2-IIA also triggered the synthesis of the inflammatory proteins COX-2 and TNFα. In addition, EGFR phosphorylation and shedding of the membrane-anchored heparin-binding EGF-like growth factor (pro-HB-EGF) ectodomain, as well as a rapid activation/phosphorylation of the classical survival proteins ERK, P70S6K and rS6 were induced upon sPLA2-IIA treatment. We further demonstrated that the presence of an EGFR inhibitor (AG1478), a matrix metalloproteinase inhibitor (GM6001), an ADAM inhibitor (TAPI-1), and a HB-EGF neutralizing antibody abrogated the phenotype of activated microglia induced by the sPLA2-IIA. CONCLUSION: These results support the hypothesis that sPLA2-IIA may act as a potent modulator of microglial functions through its ability to induce EGFR transactivation and HB-EGF release. Accordingly, pharmacological modulation of EGFR might be a useful tool for treating neuroinflammatory diseases characterized by sPLA2-IIA accumulation.

A catalytically inactive Lys49 PLA2 isoform from Bothrops jararacussu venom that stimulates insulin secretion in pancreatic beta cells.

A new secretory phospholipase A2 (sPLA2) isoform from Bothrops jararacussu venom (BjVIII) has been characterized by causing platelet aggregation, an absent activity in BthTx-I, Prtx-I and PrTx-II sPLA2s. According to our results, BjVIII also enhances insulin release by the pancreatic beta cells. The complete amino acid sequence of the new isoform was determined by Edman degradation and de novo peptide sequencing. These analyses showed a G35K amino acid modification for BjVIII in comparison with BthTx-I, PrTx-I and Prtx-II, a structural difference that has been related to the conflicting biological activities among BjVIII and other Lys49 sPLA2s. The whole set of evidences collected in this work indicates that, besides the C-terminal region and B-wing of PLA2, the calcium binding loop in BjVIII should be considered as an important region, involved in the pharmacological effects of Lys49-sPLA2 isoforms from the Bothrops genus.

Abdominal hyperalgesia in secretory phospholipase A2-induced rat pancreatitis: distinct roles of NK1 receptors.

We investigated the potential of secretory phospholipase A(2) (sPLA(2))-induced pancreatitis to promote abdominal hyperalgesia, as well as to depolarize sensory fibres in vitro using a grease-gap technique. Pancreatitis was induced by the injection of sPLA(2) from Crotalus durissus terrificus (sPLA(2)Cdt, 300µgkg(-1)) venom into the common bile duct of rats. Pancreatic inflammatory signs, serum amylase levels and abdominal hyperalgesia were evaluated in rats treated or not with SR140333, a tachykinin NK(1) receptor antagonist. Injection of sPLA(2)Cdt caused pancreatic oedema formation and increased pancreatic neutrophil infiltration and serum amylase at 4h, which returned to normality by 24h, except for the neutrophil infiltration, which was still increased at this time point. Animals injected with sPLA(2) exhibited a lower withdrawal threshold to electronic von Frey stimulation in the upper abdominal region at 4h, but not 24h, post-injection when compared with saline-injected rats. Pre-treatment of animals with SR140333 significantly reduced the sPLA(2)Cdt-induced abdominal hyperalgesia, without affecting the other parameters. Neither sPLA(2)Cdt nor sPLA(2) from Naja mocambique mocambique venom depolarized capsaicin-sensitive sensory fibres from rat vagus nerve, but they decreased the propagated compound action potentials in both A and C fibres. These data show for the first time that NK(1) receptors play an important role in the early abdominal hyperalgesia in a rat model of sPLA(2)-induced pancreatitis, suggesting that these receptors are of importance in the development of pain in the pancreatitis condition. We also provide evidence that sPLA(2)s do not directly depolarize sensory fibres in vitro.