Purification and Characterization of a Novel Ngf from Chinese Cobra (Naja Naja Atra) Venom

Abstract

Nerve Growth Factor (NGF) was demonstrated to play an important role in the neuron system; it was required for the development, maintenance, and differentiation of neuron. NGF was purified from the venom of Chinese cobra (Naja naja atra) by the gel filtration on a Sephadex G-50 column, followed by ion-exchange DEAE Cellulose D52 and CM Sepharose CL-6B column chromatography, and then by FPLC on Superose 12 column. The purified NGF was shown to be homogeneous in SDS-PAGE. In vitro, it possessed the biological activity on inducing the neurites growth of the cultured dorsal root ganglia of chicken embryos. Its molecular weight was estimated to be about 23,000 D. The isoelectric point was near 9.2. It was a glucoprotein containing 0.15% neutral hexose. We determined its terminal amino acid sequence, N-NVDFNSESTR, C-IIGNA. In this report, we also discussed the difference in characterizations between this NGF and other Elapidae venom NGFs.     

Effects of a cardiotoxin from Naja naja kaouthia venom on skeletal muscle: Involvement of calcium-induced calcium release, sodium ion currents and phospholipases A2 and C

Snake venom cardiotoxin (CTX) fractions induce contractures of skeletal muscle and hemolysis of red blood cells. The fractions also contain trace amounts of venom-derived phospholipase A₂ (PLA₂) contamination and activate tissue phospholipase C (PLC) activity. The present study examines the mechanisms of action of a CTX fraction from Naja naja kaouthia venom in skeletal muscle. Sphingosine competitively antagonized CTX-induced red blood cell hemolysis, but not skeletal muscle contractures. CTX rapidly lowered the threshold for Ca²⁺-induced Ca²⁺ release in heavy sarcoplasmic reticulum fractions, as monitored with arsenazo III. There was also a slower time-dependent reduction of Na⁺ currents, as assessed by whole cell patch-clamp techniques. The CTX fractions elevated levels of free fatty acids and diacylglycerol for 2 hr in primary cultures of human skeletal muscle by a combined action of venom-derived PLA₂ contamination in the fraction and activation of endogenous PLC activity. The activation of tissue PLC activity could be readily distinguished from the contribution of the venom PLA₂ by p-bromophenacyl bromide treatment of CTX fractions. The mechanism of action involved in contractures of skeletal muscle appears to be related to the immediate and specific effect of CTX (Ca²⁺ release by the sarcoplasmic reticulum), while the mechanisms involved in hemolysis of red blood cells and decreased Na⁺ currents in skeletal muscle most likely relate to long-term effects on lipid metabolism.     

Complete amino acid sequence of a phospholipase A2 from the venom of Naja naja atra (Taiwan cobra)

The acidic phospholipase A₂ has been isolated from Naja naja atra (Taiwan cobra) venom. The reduced and S-carboxymethylated enzyme was digested with trypsin, and eleven tryptic peptides which accounted for the whole molecule were isolated by a combination of chromatographic and gel filtration procedures, and their amino acid sequences were determined. The alignment of those eleven tryptic peptides was established by the analysis of nine major peptides obtained by Staphylococcus aureus protease digestion of the reduced and S-carboxymethylated enzyme. The phospholipase A₂ of Naja naja atra is a single polypeptide chain consisting of 120 amino acid residues including 14 intramolecularly linked half-cystines. It shows about 86% (103 out of 120) homology when compared with the Naja mossambica mossambica CM-II enzyme.     

Sequence characterization of venom toxins from Thailand cobra

Several toxins with distinct pharmacological properties were isolated from the venom of Thailand cobra (Naja naja siamensis) by cation-exchange chromatography. Two neurotoxins and one basic toxin with cardiotoxic activity were further purified and sequenced. The neurotoxins characterized were closely similar to the previously reported long- and short-chain neutrotoxins. The complete sequences of one minor neurotoxin and one cardiotoxin analogue were determined with the automatic protein sequencer in non-stop single runs of Edman degradation coupled with C-terminal sequence determination with carboxypeptidase digestion. The minor neurotoxin consists of 62 amino-acid residues with 8 cysteine residues and is found to be almost identical to cobrotoxin, a major toxic component of Formosa cobra (Naja naja atra). The sequence comparison of the 60-residue cardiotoxin with other reported cytotoxins of snake venoms indicates that 8 cysteine residues at the positions 3, 14, 21, 38, 42, 53, 54, and 59 are invariant among all sequences, with only two conservative changes at other positions along the sequence. The upshot of this report exemplified the facile sequence analysis of venom toxins by the application of pulsed-liquid phase protein sequencer and also revealed new analogues of a minor neurotoxin and one major cardiotoxin reported previously on the same species of Thailand cobra.     

Toxins from Mamba Venoms that Facilitate Neuroiluscular Transmission

Abstract

Venoms from the four species of African mambas have many neurotoxins that are different from other snake neurotoxins. Postjunctional α-neurotoxins, which bind to nicotinic cholinoceptors, appear to be the only type of toxin that mambas have in common with other snakes with neurotoxic venoms. Typical mamba neurotoxins are prejunctional facilitatory toxins and anticholinesterase toxins or fasciculins.

The prejunctional facilitatory toxins enhance the amount of transmitter that is released in response to nerve stimulation. This activity was first demonstrated with dendrotoxin (from Dendroaspis anqusticeps) at the neuromuscular junction, but it can also be observed in both the sympathetic and parasympathetic branches of the autonomic nervous system and in the central nervous system. These facilitatory neurotoxins have 57-60 amino acids in a single polypeptide chain cross-linked by three disulphide bonds. They are structurally homologous to the Kunitz type protease inhibitors such as bovine pancreatic trypsin inhibitor.

The anticholinesterase toxins are specific and powerful in inhibitors (K_i of about 10^?10 M) of acetylcholinesterase from several sources (human erythrocytes, rat brain and muscle, eel electroplax) but they have no effect on acetylcholinesterase from cobra venom or chick brain and muscle. The first two of these inhibitors (from Dendroaspis angusticeps) were called fasciculins because of the long-lasting muscle fasciculations that they produced in mice. Fasciculins have 61 amino acids and four disulphides and show sequence homology with the postjunctional short neurotoxins.

Synergistic interactions between components are characteristic of mamba venoms. One of these components is always a member of the so-called “angusticeps-type” toxins (short neurotoxin homologues of 58-60 amino acids and four disulphides). These toxins are divided into four subgroups, the anticholinesterases constituting subgroup I. Angusticeps-type toxins interact with facilitatory toxins or with so-called “synergistic-type” proteins (toxins of two subunits, each with 62-63 amino acids and joined together by disulphide bonds). The pharmacology of these interactions is not known, only data on the increased lethality of the synergistic mixtures is available.     

Primary structure of a cytotoxin-like basic protein from Naja naja naja (Indian Cobra) venom

The complete amino acid sequence of a cytotoxin-like basic protein (CLBP) from the venom of Naja naja naja (Indian Cobra) was determined by manual degradation using a 4-dimethylaminoazobenzene-4′-isothiocyanate double-coupling method. Peptide fragments obtained by chemical cleavage with cyanogen bromide and enzymic cleavages with trypsin and Staphylococcus aureus proteases for sequence analysis were purified by reversed-phase chromatography. The total number of amino acid residues was 61, with leucine as the C-terminal residue.     

The multiplicity of cardiotoxins from Naja naja atra (Taiwan cobra) venom.

Four novel cardiotoxins were isolated from Naja naja atra (Taiwan cobra) venom by successive separation on a SP-Sephadex C-25 column and a reverse phase column. Amino acid sequences of the cardiotoxins were determined by Edman degradation and carboxypeptidase digestion. It shows that these cardiotoxins comprise 60 amino acid residues. Comparative analyses on the amino acid sequences of cardiotoxins from the venoms of N. naja atra and other Naja species indicated that amino acid substitutions of cardiotoxin isoforms frequently occurred at positions 7-11, 27-32 and 45-47. The hypervariable segments encoded by the second and third exon of cardiotoxin genes are located at or near the tips of loop structure of cardiotoxin molecules. These results, together with the suggestions that the residues at the tips of cardiotoxins' loop structure were involved in the manifestation of the biological activities of cardiotoxins, reflect that the preferential mutations may contribute to alterations in the function of cardiotoxin molecules. Analysis on the secondary structure of pre-mRNAs of N. naja atra cardiotoxin 4 gene and N. naja sputatrix cardiotoxin 3 gene has shown that the hypervariable regions of the exon 2 pertain to form intra-exon pairings and are not involved in the formation of intron-exon pairings. Since the pairings of splice sites and gene architecture were supposed to be associated with intron-exon recognition, it is likely that the preferred loci of mutations occurring with the evolution of cardiotoxin genes would not affect the processing of cardiotoxin precursors.     

Isolation and characterization of two toxins from the venom of the Malayan cobra (Naja naja sputatrix)

Two principal toxins of the Malayan cobra (Naja naja sputatrix) venom have been purified to electrophoretic homogeneity by successive SP-Sephadex C-25 ion exchange chromatography and Sephadex G-50 gel filtration chromatography. They are designated as sputa-neurotoxin 1 (SN1) and sputa-neurotoxin 2 (SN2), respectively. Both toxins belong to the group of short neurotoxins which are composed of approximately 60 amino acid residues. The ld₅₀ values following i.p. injection of the purified toxins are 91 (SN1) and 71 (SN2) μg/kg mouse. Amino acid compositions of the two toxins show close similarities to other postsynaptically acting curaremimetic cobra neurotoxins.     

Biochemical characterization of a toxin from Indian cobra (Naja naja naja) venom

A major toxic component was isolated from the venom of Indian cobra (Naja naja naja) by ammonium sulfate fractional precipitation followed by carboxymethyl cellulose column chromatography and Sephadex gel filtration. This component constituted 2% of the venom and produced a block of neuromuscular transmission in nerve muscle preparations. Three other toxic fractions comprising 3% of the venom were also detected. The major toxic component was homogeneous on starch and polyacrylamide gel electrophoresis and on rechromatography on CM-cellulose. Its molecular weight was approximately 6300. This toxin contained 61 amino acid residues including 8 half-cystine residues whereas alanine, methionine and phenylalanine were totally absent. Its ld₅₀, as determined by i.p. injection in mice, was 0·2 mg per kg body weight. The fraction did not possess any enzymatic, hemolytic or hemagglutinin activities of crude venom but showed a close resemblance to the major neurotoxin of Formosan cobra venom.     

Comparison of two Elapidae venoms: Naja naja and Naja nigricollis

In our study on the identification of snake venoms by electrofocusing technics, the protein profiles of Naja naja naja, Naja nigricollis pallida and Naja nigricollis West Africa were compared. The typical profile of venoms of Elapidae was easily identified in this comparison. The two species could easily be differentiated, whereas the differences between the two sub-species were more difficult to evidence, since they are geographic variants of the same type.     

Conus Venoms: A Rich Source of Neuroactive Peptides

Abstract

Purification of toxins from the venoms of two fish-hunting gastropod cone snails, Conus geographus and Conus magus has revealed the presence of three classes of paralytic peptide toxins. These are: 1) the w-conotoxins, which block voltage activated calcium channels at the presynaptic terminus; 2) the α-conotoxins, which block the acetylcholine receptor and 3) the ω-conotoxins, which inhibit muscle sodium channels, and therefore prevent propagation of the muscle action potential. These toxins are basic peptides from 13–27 amino acids long, rich in cystine residues which are present as disulfides. A number of α-conotoxins and one a-conotoxin have been chemically synthesized.

In addition to the paralytic conotoxins, the venoms of Conus have other toxins which have not yet been completely characterized. A large number of neuroactive peptides and proteins have also been found. Since there are approximately 300 species of Conus, all of which produce venoms, the cone snails promise to be a rich source of neuroactive peptides in the years ahead.     

Neurotoxic Proteins in Scorpion Venom

Abstract

Scorpion venoms are complex mixtures of mucous, low molecular weight components (salts and organic compounds) and many basic, neurotoxic proteins (M_R < 8000). These neurotoxins alter the properties of Na channels in excitable cells in three ways. One group of toxins delays inactivation of the Na channel, thereby greatly prolonging the action potential. A second effect, produced by other scorpion toxins, is a transient shift in the voltage dependence of activation, which is demonstrated upon application of a depolarizing pulse. This effect, shown thus far only by toxins from New World species of scorpions, results in an increased tendency of the cell to fire spontaneously and repetitively. The third effect produced by some of the toxins is a reduction of ionic currents (both Na and K currents) with no changes in the kinetics of activation and inactivation. Many observations from pathophysiological studies with scorpion venoms and isolated toxins can be explained on the basis of their effects on Na channels.

The three-dimensional structure of one scorpion toxin (Toxin Var3) is known. Because of homology in the amino acid sequences of many different scorpion toxins, it is believed that the structure proposed for Toxin Var3 from Centruroides sculpturatus (Fig. 2 and 3) is typical of the scorpion toxins in general. Featured in this structure are a dense core of protein secondary structure and a surface hydrophobic patch. Many of the conserved amino acids in the scorpion toxins are associated with this patch thus suggesting that this part of the structure is associated with the biological action of the toxins.     

Effects of cardiotoxin D from Naja naja siamensis snake venom upon murine splenic lymphocytes

Cardiotoxin D from Naja naja siamensis is cytotoxic to T-lymphocytes above 150 femtomoles/10(6) cells. Equivalent lysis of macrophages or B-lymphocytes requires at least 1000 times more toxin. Reduction and carboxamidomethylation of cardiotoxin D does not effect T cell lysis. At higher toxin concentrations, 50% T-cell lysis occurs within 10 min. Splenocytes cultured with mitogens are up to five times more susceptible to toxin than unstimulated cells. Cardiotoxin D may directly disrupt the plasma membrane, since lysis is unaltered at 4 degrees C.     

Structure and Function of Cobrotoxin

Abstract

In this review I describe the current status of the study of postsynaptic neurotoxins with special emphasis on the extensive studies of cobrotoxin which I started more than three decades ago. Cobrotoxin, is a neurotoxic protein isolated in crystalline state from the venom of Taiwan cobra Naja naja atra. It is a small basic protein consisting of a single peptide chain of 62 amino acid residues, crosslinked by four disulfide bonds. Cobrotoxin binds specifically to the nicotinic acetylcholine receptor on the postsynaptic membrane and thus blocks the neuromuscular transmission. The disulfide bonds and Tyr-25 which are buried in the molecule form a central core to maintain and stabilize the active conformation of the toxin. Selective and stepwise chemical modifications of cobrotoxin indicate that at least two cationic groups, an ? -amino group of Lys-47 and a guanidino group of Arg-33, both of which are common to all known postsynaptic snake neurotoxins, held at a certain critical distance in the molecule, are functionally important for its neuromuscular blocking activity.     

Phospholipid hydrolysis and loss of membrane integrity following treatment of rat brain synaptosomes with beta-bungarotoxin, notexin, and Naja naja atra and Naja nigricollis phospholipase A2

The effects of the phospholipase A2 (PLA2) toxins, beta-bungarotoxin and notexin, and the PLA2 enzymes from Naja naja atra and Naja nigricollis snake venoms on the plasma membrane integrity of synaptosomes were examined. Synaptosomes were isolated from rat brain cerebral cortex, corpus striatum and hippocampus. Osmotic activity, lactate dehydrogenase leakage, and leakage of 2-deoxy-D-(1-3H)-glucose-6-phosphate were monitored (37 degrees C, 10-120 min) following incubation with 0.5, 5 and 50 nM concentrations of toxins and enzymes. Damage to the synaptosomal plasma membrane was time and concentration but not tissue dependent. The potencies of the treatments were as follows: N. n. atra PLA2 greater than or equal to N. nigricollis PLA2 greater than notexin greater than beta-bungarotoxin. Chelation of Ca2+ with 5 mM EDTA completely inhibited plasma membrane disruption caused by beta-bungarotoxin and N. n. atra PLA2. One mg/ml of bovine serum albumin also blocked the disruptive action of N. n. atra PLA2, while 8 mg/ml was required to antagonize beta-bungarotoxin. A correlation between phospholipid hydrolysis and loss of membrane integrity was also observed. The generation of phospholipid hydrolytic products may be critical in the permeabilization of synaptic plasma membranes by these toxins and enzymes, however, they do not explain the presynaptic specificity and potency of beta-bungarotoxin and notexin.     

眼镜蛇毒细胞毒素CTX-d的分离纯化及抗肿瘤活性

目的 分离纯化眼镜蛇毒细胞毒素,测定其体内外抗癌作用.方法 应用柱层析及RP-HPLC,从眼镜蛇毒粗毒中分离纯化细胞素素(CTX).体内外抗癌作用利用噻唑兰(MTT)法及对荷瘤小鼠U14瘤的抑瘤作用.结果 分离纯化获得的CTX-d不混有PLA2,在体内外实验中显示明显的抗肿瘤作用.结论 结合阳离子交换层析和RP-HPLC可从眼镜蛇毒中高效分离获得不含PLA2的CTX.     

Effect of calcium and phosphate ions on hemolysis induced by Pyrularia thionin and Naja naja kaouthia cardiotoxin.

Pyrularia thionin is a strongly basic bioactive peptide of 47 amino acids isolated from nuts of Pyrularia pubera. It is hemolytic, cytotoxic and activates an endogenous phospholipase A2 in 3T3 cells. Earlier studies have shown that the cardiotoxin from Naja naja kaouthia has similar activities and binds to the same site as Pyrularia thionin. Since the peptides appear to bind to the phospholipids of cell membranes to elicit their cellular responses, the effect of modifying the electrostatic environment was studied by separately adding phosphate ion and Ca2+, and by removing Ca2+ from the membrane by treatment with EGTA. Analysis of erythrocyte hemolysis for both Pyrularia thionin and cardiotoxin shows that the reactions follow Michaelis-Menten kinetics, with the peptides serving as the substrate. The basal rate of hemolysis in physiological saline is markedly increased by the addition of phosphate in the 5-10 mM range and also by removing membrane-bound Ca2+ by incubation of the cells with 10 mM EGTA. These treatments do not change the apparent K(m) values, but increase the V(max), indicating that more binding sites are made available by these treatments. On the other hand, added Ca2+ in the 5-10 mM range competitively inhibits the reaction by inhibiting the binding of the peptide to the membrane.     

Sensitivity of cultured embryonic heart cells to cardiotoxin obtained from Naja naja siamensis venom.

Clumps of embryonic chick heart cells in culture were exposed to cardiotoxin prepared by Sephadex CM-50 fractionation from snake (Naja naja siamensis) venom. Cardiotoxin irreversibly depolarized these cells. More of the cells cultured from 4-day embryos, than from 12–13-day embryos, continued to beat spontaneously after 30 min exposure to the toxin at a concentration of 15–20 μg per ml.     

Purification and characterization of a cytotoxic neurotoxin-like protein from Naja haje haje venom that induces mitochondrial apoptosis pathway

This study reported the purification and characterization of a cytotoxic, neurotoxin-like protein derived from the venom of the Egyptian cobra Naja haje haje, Elapidae family, and explored their mechanistic role in the cell death. The protein purification was performed through ion-exchange chromatography and gel-filtration chromatography and was characterized by SDS?CPAGE, amino acid sequencing, and mass spectrum analysis. The antitumor activity of Naja haje venom (NHV) and its fractions (NHVI, NHV-Ia, NHV-Ib, NHV-Ic, NHV-II, NHV-III, and NHV-IV) were tested against different human cancer cell lines. The molecular cascade of cell death was explored through evaluation of apoptosis/necrosis ratio, DNA fragmentation, histone deacetylase (HDAC) activity, mitochondrial transmembrane potential (????_m), cytochrome c release, total caspases, caspase-3, caspase-9, and cell cycle analysis by flow cytometry. Most of the separated fractions possessed variable cytotoxic effect against different cancer cells. The most potent antitumor fraction was NHV-Ic due to its ability to induce DNA damaging and fragmentation that was associated with a significant induction of apoptosis via mitochondrial pathway and disturbed cell cycle phases as well as an inhibition of HDAC activity. NHV-Ic induced the mitochondrial pathway initially by the impairment of ????_m besides the DNA damage and in response to that the mitochondria-released cytochrome c that may in turn activated total caspases, caspase-3 and caspase-9 in lymphoblastic leukemia 1301 cells. The partial amino acid sequencing of NHV-Ic revealed 100, 95.65, and 91.3% homology with the Long neurotoxin 1 from Naja haje anchietae (Angolan cobra), Naja haje haje (Egyptian cobra), and Boulengerina annulata annulata (banded water cobra), respectively.     

Use of HPLC to demonstrate variation of venom toxin composition in the Thailand cobra venoms Naja naja kaouthia and Naja naja siamensis.

The composition of the venoms of Naja naja kaouthia and Naja naja siamensis from different commercial sources has been investigated using both ion-exchange and reverse-phase high-pressure liquid chromatography (RP-HPLC) in order to investigate variation in toxin contents. The venoms contained identical major toxin components, although in different relative concentrations. The venom collected separately from the left and right glands of individual snakes were virtually the same as judged by RP-HPLC. The cytotoxin CT-II, which was previously only reported to be present in Naja naja siamensis venom, was detected in all the venoms investigated. Two long neurotoxin homologues have also been isolated.