Antitumor and antimetastatic effects of PLA2-BthTX-II from Bothrops jararacussu venom on human breast cancer cells.

This work shows the antitumor and antimetastatic effects of BthTX-II, an Asp-49 PLA2 from Bothrops jararacussu venom, on MDA-MB-231 human triple negative breast cancer cells. BthTX-II caused a dose-dependent cell death of MDA-MB-231 cells when compared with the non-tumorigenic breast cells by inducing apoptosis and autophagy. BthTX-II was also able to decrease the proliferation and to inhibit cell cycle progression. We also observed an upregulation of the ATM gene, which is responsible for cell-cycle arrest and DNA repair such as CCND1, CCNE1, CDC25A, E2F1, AKT1 and AKT3. Interestingly, BthTX-II inhibited invasion, migration and 3D cell growth of MDA-MB-231 cells, as well as inhibited the epithelial-mesenchymal transition (EMT) of this cell by increasing E-cadherin (CDH-1) and decreasing TWIST1, CTNNB1, vimentin and cytokeratin-5 expression. In conclusion, these results showed that BthTX-II displays antitumor and antimetastatic effects on MDA-MB-231 cells and may be useful for the development of new approaches and therapeutic strategies to manage triple negative breast cancer.

Purification and partial characterization of an anticoagulant PLA2 from the venom of Indian Daboia russelii that induces inflammation through upregulation of proinflammatory mediators.

The present study describes the purification and partial characterization of a basic anticoagulant PLA2 enzyme named as Rv(i) PLA2 from the venom of Indian Daboia russelii. The molecular mass of the protein was found to be 13,659.65 Da, and peptide mass fingerprinting revealed that it belongs to group II PLA2 family. The peptide sequence showed similarity to uncharacterized basic PLA2 enzyme having an accession no. of P86368 reported from Sri Lankan D. russelii. Rv(i) PLA2 exhibited strong phospholipase A2 and anticoagulant activity. It also induced expression of COX-2 and TNF-α mRNA in a dose-dependent manner in phorbol 12-myristate 13-acetate differentiated THP-1 cells, which play a crucial role during inflammation. Chemical modification of His residue in Rv(i) PLA2 with p-bromophenacyl bromide abolished the enzymatic, anticoagulant, and inflammatory activities. The result indicates that the catalytic site of Rv(i) PLA2 might play a vital role in inducing inflammation at the bite site during D. russelii envenomation.

PhTX-II a basic myotoxic phospholipase A2 from Porthidium hyoprora snake venom, pharmacological characterization and amino acid sequence by mass spectrometry.

A monomeric basic PLA2 (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA2 enzyme class and displays conserved domains as the catalytic network, Ca²âº-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA2 showed an allosteric behavior and its enzymatic activity was dependent on Ca²âº. Examination of PhTX-II PLA2 by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA2 causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA2 that contributes with toxic actions caused by P. hyoprora venom.

Protein content analysis and antimicrobial activity of the crude venom of Montivipera bornmuelleri; a viper from Lebanon.

Viperidae snakes venoms represent a source of efficient bioactive components that have already led to the development of several new drugs. In this work, we analyzed the protein content of the Montivipera bornmuelleri crude venom using LC-ESI-MS, sephadex G-75 gel filtration and SDS-PAGE and demonstrated the presence of proteins with molecular masses corresponding to metalloprotease III, serine-protease and PLA2 in three fractions collected after gel filtration. Equally, we examined the antimicrobial effect of the venom that showed an important potency, as bactericidal agent, based on MBC and MIC values obtained, against Staphylococcus aureus and Morganella morganii bacteria. However, no activity was registered against Enterococcus faecalis, being the most resistant bacteria, neither against Aspergillus flavus and Penicillium digitatum fungal. Furthermore, on eleven other bacterial strains and the Candida albicans fungus, the venom has shown an intermediate efficacy by slightly reducing the growth. Our data concerning the Montivipera bornmuelleri venom give evidence of a rich and complex content aiding the exploration of new bioactive molecules for biopharmaceuticals purposes.

Renaturation and one step purification of the chicken GIIA secreted phospholipase A2 from inclusion bodies.

The cDNA coding for a mature protein of 123 amino acids, containing all of the structural features of catalytically active group IIA sPLA2, has been amplified from chicken intestine. The gene has been cloned into the bacterial expression vector pET-21a(+), which allows protein over-expression as inclusion bodies and enables about 3mg/l of pure refolded fully active enzyme to be obtained. Recombinant expression of chicken intestinal sPLA2-IIA (ChPLA2-IIA) in Escherichia coli shows that the enzyme is Ca(2+) dependent, maximally active at pH 8-9, and hydrolyses phosphatidylglycerol versus phosphatidylcholine with a 10-fold preference. Indeed, we report in this work, a comparative kinetic study between the wild type and the recombinant ChPLA2-IIA, on zwitterionic head group phospholipids (DDPC) and negatively charged phospholipids (POPG) using the monomolecular film technique. The ability to express reasonably large amounts of the sPLA2 Group IIA, compared to that obtained with the classical purification will provide a basis for future site directed mutagenesis studies of this important enzyme.

Chip electrophoretic separation of highly homologous ammodytoxin isoforms: three neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom.

Ammodytoxins (Atxs), a group of Ca(2+) -dependent neurotoxic phospholipases A2 of Vipera ammodytes ammodytes venom, are mainly responsible for venom toxicity. Within the Atx group, LD50 values between three isoforms, A, B, and C are differing with AtxA exhibiting an LD50 value by an order of magnitude lower (more toxic) than the other two isoforms. This difference in toxicity justifies the necessity to prepare suitable antibodies and thus isoform separation to characterize the Atx content of Vipera ammodytes ammodytes venom is of importance. However, a high homology between the three Atx isoforms (differences in only two, respectively, three residues within the last 18 amino acids at the C-terminus, total length 122 residues) hindered the successful separation of isoforms to date. As the investigated phospholipases A2 were reported to exhibit differences in pI values, we concentrate with the current work on the separation of Atx isoforms after fluorescence labeling via chip electrophoresis on a commercially available instrument to build the basis for a fast and easy to handle screening method. In the course of our work, we were able to show that samples of AtxA, AtxB, and AtxC declared to be homogenous by standard analytical techniques consisted indeed of more than one isoform of which the relative amounts were calculated by using the newly developed method.

Purification and characterization of a phospholipase A2-IIA from common stingray (Dasyatis pastinaca) intestine.

A phospholipase A2 belonging to IIA group secretory PLA2 was isolated and purified to homogeneity from the intestine of common stingray (Dasyatis pastinaca) using acidic treatment (pH 1.5) and ammonium sulphate precipitation methods combined with single-column ion-exchange chromatography. The purified enzyme was found to be a glycosylated monomeric protein with a molecular mass of about 14 kDa. The stingray sPLA2-IIA had optimum activity at 45 degrees C, unlike known mammalian PLA2-IIAs, which show optimum activity at 37 degrees C. The purified enzyme exhibited a specific activity of 290 U/mg at optimal conditions (pH 9.5 and 45 degrees C) in the presence of 6 mM NaDC and 8 mM CaCl2 with egg yolk as substrate. The NH2-terminal sequence of the enzyme and some protein fragments obtained from its tryptic digestion were also determined. All sequences obtained were similar to those of sPLA2-IIA. The enzyme also showed good stability in the presence of organic solvents, acidic and alkaline pH media and high temperature conditions. Thus, the purified enzyme exhibited a number of unique and promising properties, making it a potential possible candidate for future applications in the treatment of phospholipid-rich industrial effluents and synthesis of useful preparations for the food production and processing industry.

Secretory phospholipase A2 in dromedary tears: a host defense against staphylococci and other gram-positive bacteria.

The best known physiologic function of secreted phospholipase A2 (sPLA2) group IIA (sPLA2-IIA) is defense against bacterial infection through hydrolytic degradation of bacterial membrane phospholipids. In fact, sPLA2-IIA effectively kills Gram-positive bacteria and to a lesser extent Gram-negative bacteria and is considered a major component of the eye's innate immune defense system. The antibacterial properties of sPLA2 have been demonstrated in rabbit and human tears. In this report, we have analyzed the bactericidal activity of dromedary tears and the subsequently purified sPLA2 on several Gram-positive bacteria. Our results showed that the sPLA2 displays a potent bactericidal activity against all the tested bacteria particularly against the Staphylococcus strains when tested in the ionic environment of tears. There is a synergic action of the sPLA2 with lysozyme when added to the bacteria culture prior to sPLA2. Interestingly, lysozyme purified from dromedary tears showed a significant bactericidal activity against Listeria monocytogene and Staphylococcus epidermidis, whereas the one purified from human tears displayed no activity against these two strains. We have also demonstrated that Ca(2+) is crucial for the activity of dromedary tear sPLA2 and to a less extent Mg(2+) ions. Given the presence of sPLA2 in tears and intestinal secretions, this enzyme may play a substantial role in innate mucosal and systemic bactericidal defenses against Gram-positive bacteria.

Why myotoxin-containing snake venoms possess powerful nucleotidases?

The venom of the snake Bothrops asper causes muscle necrosis, pain and inflammation. This venom contains myotoxins which cause an increase in intracellular Ca(2+) concentration and release of K(+) and ATP from myotubes. ATP is a key danger molecule that triggers a variety of reactions, including activation of the innate immune response. Here, using ATP-luciferase bioluminescence imaging technique, we show for the first time in vivo, that the purified myotoxins induce rapid release of ATP, whilst the complete venom of B. asper does at a very small extent. This apparent contradiction is explained by the finding that the venom contains powerful nucleotidases that in vivo convert ATP into ADP, AMP and Adenosine. These findings indicate that high concentrations of adenosine are generated by the double action of the venom and provide the experimental basis to the suggestion that in situ generated adenosine plays an important role in envenomation via its hypotensive, paralyzing and anti-coagulant activities.

Daboia russelli venom hyaluronidase: purification, characterization and inhibition by ß-3-(3-hydroxy-4-oxopyridyl) α-amino-propionic Acid.

The present study describes the purification and characterization of a hyaluronidase (DRHyal-II) from Daboia/Vipera russelli venom and its inhibition by ß-3-(3-hydroxy-4-oxopyridyl) α-amino-propionic acid, the mimosine. Gel permeation and ion exchange chromatography were employed to isolate DRHyal-II. The molecular mass by MALDITOF mass spectrometry was found to be 28.3 kDa. Single band in reduced SDS-PAGE suggested the monomeric nature. It was optimally active at pH 5.5 and at 37C and require 150 mM NaCl in the reaction mixture. It was specific to hyaluronan substrate and belongs to class-I or the neutral active enzymes. DRHyal-II was non-toxic by itself but, it potentiated the myotoxicity of VRV-PL-VIII myotoxin and hemorrhagic activity of hemorrhagic complex (HC). In in vitro experiments, mimosine inhibited the activity of DRHyal-II and the hyaluronidase activity of whole venom dose dependently. In in vivo experiments, mimosine inhibited the DRHyal-II potentiated myotoxicity of VRV-PL-VIII myotoxin and hemorrhagic activity of HC. The inhibition was due to the formation of DRHyal-II-mimosine inhibitory complex that resulted in significant structural changes at secondary and tertiary levels as evidenced by fluorescence emission and CD spectral studies. Hence, in this study an attempt was made to establish the possible role of hyaluronidase activity in the pathology of Daboia/Vipera russelli venom and the beneficial effects of its inhibition with special emphasis on the management of local toxicity.

Expression, purification, and refolding of active human and mouse secreted group IIE phospholipase A2.

Secreted phospholipase A2s form a large family of proteins involved in diverse biological and pathophysiological processes. Group IIE secreted phospholipase A2 (sPLA2-IIE) is one of the latest discovered members of this family. Previous studies revealed that the expression profile of sPLA2-IIE was restricted to a few tissue types including brain, heart, lung and placenta compared to the broad expression profile of other isoforms. Accumulating evidence suggests that sPLA2-IIE might play a pivotal role in the progression of inflammatory processes. However, functional study of sPLA2-IIE was hindered by the low yield of soluble expressed protein. In this study, we have expressed human and mouse sPLA2-IIE in Escherichia coli in the form of inclusion bodies. The inclusion bodies were dissolved, purified and refolded in a step-wise dialysis approach and further purified. We obtained soluble and active proteins for human and mouse sPLA2-IIE with a final yield of 1.1 and 1.2 mg/500 mL bacterial culture, respectively. The refolded sPLA2-IIEs exhibited similar calcium and pH dependence of their enzymatic activity with those expressed in COS cells. This protein expression and purification protocol will facilitate the further structural and functional studies of human and mouse sPLA2-IIEs.

Application of isothermal titration calorimetry and column chromatography for identification of biomolecular targets.

This protocol describes a method for identifying unknown target proteins from a mixture of biomolecules for a given drug or a lead compound. This method is based on a combination of chromatography and isothermal titration calorimetry (ITC) where ITC is used as a tracking tool. The first step involves the use of ITC to confirm the binding of ligand to a component in the biomolecular mixture. Subsequently, the biomolecular mixture is fractionated by chromatography, and the binding of the ligand with individual fractions (or subfractions) is verified by ITC. The iteration of chromatographic purification on the fractions combined with ITC results in identifying the target protein. This method is useful when the target protein or ligand is unknown and/or not amenable to labeling, chemical modification or immobilization. This protocol has been successfully used by our team and by others to identify both low-abundance and highly abundant target proteins present in biomolecular mixtures. With this protocol, it takes approximately 3-5 d to identify the target protein from a mixture.

Induction of inflammatory cell accumulation by TM-N49 and promutoxin, two novel phospholipase A(2).

Local inflammation is a prominent characteristic of snakebite wound. Snake venom phospholipase A(2)s (PLA(2)s) are one of the main components which contribute to accumulation of inflammatory cells. We have isolated TM-N49 and promutoxin from Protobothrops mucrosquamatus venom and investigated their ability in induction of cell accumulation by using an in vivo mouse model. The results showed that both TM-N49 and promutoxin are potent stimuli for induction of neutrophil, lymphocyte, macrophage and eosinophil accumulation in the mouse peritoneum. The TM-N49- and promutoxin-induced inflammatory cell accumulation was inhibited by pretreatment of animals with cyproheptadine, terfenadine and Ginkgolide B, indicating that histamine and PAF is likely to contribute to the cells accumulation. Pre-injection of antibodies against adhesion molecules ICAM-1, CD18, CD11a and L-selectin showed that ICAM-1 is a key adhesion molecule of TM-N49- and promutoxin-induced lymphocyte, macrophage and eosinophil accumulation; CD18 and CD11a plays an important role in the migration of neutrophils, eosinophils and macrophages; and L-selectin is involved in the neutrophil and eosinophil accumulation. In conclusion, induction of inflammatory cell accumulation by TM-N49 and promutoxin confirms that group II PLA(2)s is pivotal stimulus for cell infiltration, through which they participate in the formation of snakebite inflammation.

Induction of mast cell accumulation, histamine release and skin edema by N49 phospholipase A2.

BACKGROUND: It has been recognized that phospholipase A2 (PLA2) is a crucial component of snake venom, which contributes greatly to snake venom induced inflammation in man. However, the mechanisms through which N49 PLA2 provoke inflammation remain unclear. Recently, a N49 PLA2, TM-N49 from Protobothrops mucrosquamatus crude venom was characterized in our laboratory. Since the purification procedure developed is able to supply us with relatively large quantity of highly purified TM-N49, we investigated the ability of TM-N49 in induction of inflammation. RESULTS: The results showed that TM-N49 provoked a dose dependent increase in microvascular leakage in the skin of rats. The potency of TM-N49 in induction of skin edema appeared similar potency of bradykinin and histamine. Pretreatment of rats with compound 48/80 diminished TM-N49 induced skin reaction and reduced mast cell numbers in rats. Ginkgolide B and cyproheptadine, but not terfenadine and quinacrine, inhibited TM-N49 elicited microvascular leakage when they were co-injected with the stimulus to rat skin. Moreover, TM-N49 was found to induce histamine release from human colon, lung and tonsil mast cells, and both metabolic inhibitors and pertussis toxin were capable of inhibiting TM-N49 elicited histamine release. TM-N49 induced mast cell accumulation in the peritoneum of mice, which was inhibited by co-injection of ginkgolide B, cyproheptadine and terfenadine. Intravenous injection of monoclonal antibodies against CD18, ICAM-1 and CD11a also blocked TM-N49 induced mast cell accumulation. CONCLUSION: TM-N49 is a potent stimulus for skin edema, mast cell activation and accumulation.

Toxicity of phospholipases A2 D49 (6-1 and 6-2) and K49 (Bj-VII) from Bothrops jararacussu venom.

Purified phospholipase A2 (PLA2) enzymes from Bothrops jararacussu snake venom were examined to evaluate NIH 3T3 and COS7 fibroblast cytotoxicity, as well as muscle myotoxic and inflammatory activities. Separation of fractions Bj-VII (from BthTX-I; a Lys49 PLA2 homolog) and 6-1 and 6-2 (from BthTX-II; an Asp49 PLA2) from B. jararacussu snake venom by SDS-PAGE in tricine buffer in the absence and presence of dithiothreitol revealed a homodimer with an estimated molecular mass of approximately 30 kDa (monomer mass approximately 15 kDa). This finding indicates that these toxins form dimeric complexes-a previously reported tendency among PLA2s. These toxins were assayed for viability with the MTT assay, which is used to examine the effects of phospholipases on the mitochondrial viability of cells. The toxins were also assayed for cytolysis of the fibroblast cell lines NIH 3T3 and COS7 by quantification of lactate dehydrogenase released into the medium. The results indicate that the PLA2s 6-1, 6-2 and the Bj-VII PLA2 homolog studied here induce moderate footpad edema and local myotoxicity. Moreover, exposure to these phospholipases led to a reduction in fibroblast viability; at the 1 muM dose of PLA2 tested, a reduction of 50% in cell viability was observed. The present findings indicate that the inflammatory activity observed in envenomation may be correlated with the cytotoxicity observed in fibroblasts.

Structural characterization and neuromuscular activity of a new Lys49 phospholipase A(2) homologous (Bp-12) isolated from Bothrops pauloensis snake venom.

Bp-12 was isolated from Bothrops pauloensis snake venom in only one chromatographic step in reverse phase HPLC on micro-Bondapack C-18. The molecular mass of 13,789.56 Da was determined by mass spectrometry. The amino acids composition showed that Bp-12 presented high content of Lys, Tyr, Gly, Pro, and 14 half-Cys residues, typical of a basic PLA(2). The sequence of Bp-12 contains 122 amino acid residues: SLFELGKMIL QETGKNPAKS LGAFYCYCGW GSQGQPKDAV DRCCYVHKCC YKKITGCNPK KDRYSYSWKD KTLVCGEDNS CLKELCECDK AVAICLRENL NTYNKKYRYF LKPLCKKADA AC, with a pI value of 8.55 and with a high homology with Lys49 PLA(2) from other snake venoms. In mouse phrenic nerve-diaphragm, the time needed for 50% paralysis was: 45 +/- 6 min (1.4 microM) and 16 +/- 6 min (3.6 microM). Bp-12 can induce indirect and directly blocked evoked twitches, even in the preparations in which Ca(2+) is replaced by Sr(2+), being the addition of d-tubocurarine required for direct blocking. These results identify Bp-12 as a new member of the Lys49 PLA(2) family and shows that this toxin might contribute to the effects of the crude venom on the neuromuscular junction.