cDNA cloning and characterization of vascular apoptosis-inducing protein 1

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells (VEC). In previous reports, we described the purification from crude venom of Crotalus atrox of two vascular apoptosis-inducing proteins (VAP1 and VAP2) that specifically induce apoptosis in vascular endothelial cells. We report here the cDNA cloning and characterization of VAP1. VAP1 cDNA encoded a protein with 610 amino acid residues. The amino acid sequence predicted from the cDNA indicated that VAP1 belongs to the metalloprotease/disintegrin family and that it is a multidomain polypeptide with a proprotein domain, a metalloprotease domain, a disintegrin-like domain, and a cysteine-rich domain. In the disintegrin-like domain, the sequence DECD replaces the RGD sequence that has frequently been found in such domains. We demonstrated that VAP1 has Zn(2+)-dependent metalloprotease activity and degrades fibrinogen. After incubation in the presence of either EDTA or EGTA, VAP1 was hardly able to degrade fibrinogen and to induce apoptosis in VEC. Our results indicated that VAP1 is a new type of snake venom metalloprotease/disintegrin and suggest that the metalloprotease activity of VAP1 might be involved in the induction of apoptosis by VAP1 in VEC.     

Isolation and structural characterization of a cytotoxic L-amino acid oxidase from Agkistrodon contortrix laticinctus snake venom: preliminary crystallographic data.

We have purified a cytotoxic L-amino acid oxidase (LAO) from Agkistrodon contortrix laticinctus snake venom by means of Superdex-200 gel filtration, followed by phenyl-Sepharose CL-4B chromatography. The purified enzyme (ACL LAO) is a dimer on gel filtration, with a M(r) of 60,000 for the monomer as estimated by SDS-PAGE. LAO activity was tested against 15 amino acids, but only 9 were oxidized by the enzyme, suggesting that it presents some degree of specificity. ACL LAO has apoptosis-inducing activity in an HL-60 cell culture assay. After 24 h treatment with 25 micrograms/ml of ACL LAO, the typical DNA fragmentation pattern of apoptotic cells was observed on agarose gel electrophoresis. NMR analysis showed the presence of a flavin mononucleotide prosthetic group. To solve its 3-D structure, crystals of the purified protein were grown in 0.1 M Tris-HCl, pH 8.5, and 2 M (NH(4))(2)SO(4). Diffraction data collected to 3.5 A showed that the protein crystallized in the tetragonal system, with unit cell a = b = 103.22 A, c = 183.45 A. This is the first report of preliminary crystallization data for a snake venom L-amino acid oxidase.     

Inhibition of Naja naja venom hyaluronidase: role in the management of poisonous bite

Hyaluronidase is present virtually in all snake venoms and has been known as a "spreading factor." The enzyme damages the extracellular matrix at the site of the bite, leading to severe morbidity. In this study, the benefits of inhibiting the hyaluronidase activity of Indian cobra (Naja naja) venom have been investigated. Anti-NNH1 and aristolochic acid both inhibited the in vitro activity of the purified hyaluronidase, (NNH1) and the hyaluronidase activity of whole venom in a dose-dependent manner. Both anti-NNH1 and aristolochic acid abolished the degradation of hyaluronan in human skin tissue sections by NNH1 and by whole venom. Aristolochic acid quenched the fluorescent emission of NNH1. A non-competitive mechanism of NNH1 inhibition was observed with aristolochic acid. NNH1 potentiates the toxicity of Daboia russellii VRV-PL-VIII myotoxin and hemorrhagic complex-I. However, the potentiation of toxicity was inhibited dose-dependently by anti-NNH1 and aristolochic acid. Further, mice injected with whole venom which had been preincubated with anti-NNH1/aristolochic acid, showed more than a two-fold increase in survival time, compared to mice injected with venom alone. A more moderate increase in survival time was observed when mice were injected with anti-NNH1/aristolochic acid 10 min after whole venom injection. This study illustrates the significance of venom hyaluronidase in the pathophysiology of snake venom poisoning and the therapeutic value of its inhibition.     

Nucleotide sequence of U-2 ribonucleic acid. The sequence of the 5'-terminal oligonucleotide.

The nucleotide sequences were determined for the 5'-oligonucleotides obtained by complete pancreatic RNase digestion (P25) and complete T1 RNase digestion (T27) of U-2 RNA. Complete digestion of oligonucleotide P25 with snake venom phosphodiesterase produced pm3 2,2,7G, pAm, pUm, and pCp in approximately equimolar ratios. Partial digestion of these oligonucleotides with snake venom phosphodiesterase produced -Um-C-Gp and pAm-Um, indicating the sequence of the 3'-terminal portion of the 5'-oligonucleotide is pAm-Um-C-Gp. The 5'-terminal oligonucleotide did not contain a 5'-phosphate and no free nucleoside was released from the 5' end by venom phosphodiesterase digestion. Since free pm3 2,2,7G was released by digestion with nucleotide pyrophosphatase and limited digestion with snake venom phosphodiesterase, this nucleotide is apparently linked to pAm in a pyrophosphate linkage. Mass spectrometry and thin layer chromatography in borate systems showed the ribose of m3 2, 2, 7G contains no 2'O-methyl residue. Moreover, the finding that the ribose of m3 2, 2, 7G was oxidized by NaIO4 and reduced by KB3H4 in intact U-2 RNA rules out other linkages involving the 2' and 3' positions. Accordingly, it is concluded that the structure of the 5'-terminal pentanucleotide of U-2 RNA is(see article).     

Molecular characterization of L-amino acid oxidase from Agkistrodon halys blomhoffii with special reference to platelet aggregation

L-Amino acid oxidase (LAO, EC 1.4.3.2) is widely distributed in snake venom, and induces apoptosis in vascular endothelial cells, causing prolonged bleeding from vessel walls at bite sites. The effect of snake venom LAOs on platelet function is controversial. Further, we have little information on their structural characterization. We purified M (mamushi)-LAO, a single-chain glycoprotein with a molecular mass of 60 kDa and a pI of 4.9, from Agkistrodon halys blomhoffii (Japanese mamushi) venom, and determined the N-terminal and several internal amino acid sequences of this enzyme. Molecular cloning based on these data was conducted to elucidate its full-length cDNA structure (2192 nucleotides), which includes a putative 18 amino acid residue signal peptide and a 504 residue mature subunit. The predicted M-LAO translation product shares 87.3% identity with that of Crotalus adamanteus (Southeastern diamondback rattlesnake) LAO. M-LAO, up to a final concentration of 2.6 microM, inhibited both agonist- and shear stress-induced platelet aggregation (SIPA) dose-dependently. In agonist-induced platelet aggregation, M-LAO predominantly inhibited the second aggregation, but with a marginal inhibition of the first. In SIPA, the inhibition was more dramatic under low-shear stress than high-shear stress, and was enhanced by the presence of L-leucine, a substrate of this enzyme. Catalase, a H2O2 scavenger, totally quenched such enhancement. These results suggest that M-LAO inhibits the interaction between activated platelet integrin alphaIIb/beta3 and fibrinogen through the continuous generation of H2O2, and may contribute to prolonged bleeding from the vessels at snake bite sites.     

Isolation, structural, and functional characterization of an apoptosis-inducing L-amino acid oxidase from leaf-nosed viper (Eristocophis macmahoni) snake venom

The enzyme L-amino acid oxidase (LAO) from the leaf-nosed viper (Eristocophis macmahoni) snake venom was purified to homogeneity in a single step using high performance liquid chromatography on a Nucleosil 7C18 reverse phase column. The molecular mass of the purified enzyme was 58734.0 Da, as determined by matrix-assisted laser desorption/ionization mass spectrometry. The N-terminal amino acid sequence (ADDKNPLEEAFREADYEVFLEIAKNGL) and the chemical composition of the purified LNV-LAO shows close structural homology with other L-amino acid oxidases isolated from different snake venoms. The secondary structural contents analysis of LAO, established by means of circular dichroism, revealed ca. 49% alpha-helix, 19% beta-sheet, 10% beta-turn, and 22% random coil structure. The purified LNV-LAO not only retained its specific enzymatic activity (73.46 U/mg), determined against L-leucine as a substrate, but also exhibited potent haemolytic (1-10 microg/ml), edema- (MED 4.8 microg/ml) and human platelet aggregation-inducing (ED50 33 microg/ml) properties. Unlike other haemorrhagic snake venom L-amino acid oxidases, the LNV-LAO does not produce haemorrhage. In addition to these local effects, the purified LNV-LAO showed apoptosis-inducing activity in the MM6 cell culture assay. After 18 h treatment with 25-100 microg/ml of LAO, the typical DNA fragmentation pattern of apoptotic cells was observed by means of fluorescent microscopy and agarose gel electrophoresis.     

Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A(2) from the venom of Protobothrops mucrosquamatus

Group IIA phospholipase A(2) (PLA(2)) are major components in Viperidae/Crotalidae venom. In the present study, a novel PLA(2) named promutoxin with Arg at the site 49 has been purified from the venom of Protobothrops mucrosquamatus by chromatography. It consists of 122 amino acid residues with a molecular mass of 13,656 Da assessed by MALDI-TOF. It has the structural features of snake venom group IIA PLA(2)s, but has no PLA(2) enzymatic activity. Promutoxin shows higher amino acid sequence identity to the K49 PLA(2)s (72-95%) than to D49 PLA(2)s (52-58%). Promutoxin exhibits potent myotoxicity in the animal model with as little as 1 microg of promutoxin causing myonecrosis and myoedema in the gastrocnemius muscle of mice. Promutoxin is also able to stimulate the release of IL-12, TNFalpha, IL-6 and IL-1beta from human monocytes, and induce IL-2, TNFalpha and IL-6 release from T cells, indicating that this snake venom group IIA PLA(2) is actively involved in the inflammatory process in man caused by snake venom poisoning.     

Lipolytic enzymes in bovine thyroid tissue. I. Subcellular localization, purification and characterization of acid phospholipase A1.

In mammalian cells the catabolism of membrane phosphoglycerides proceeds probably entirely through a deacylation pathway catalysed by phospholipase A and lysophospholipase (Wise & Elwyn, 1965). In the initial attack of diacylphosphoglycerides by phospholipase A two enzymatic activities with different positional specificities have been distinguished: phospholipase A1 (phosphatidate 1-acyl hydrolase EN 3.1.1.32) and phospholipase A2 (phosphatidate 2-acyl hydrolase EN 3.1.1.4) (Van Deenen & De Haas, 1966). Studies on these intracellular phospholipases were mainly concerned with their subcellular localization. Only occasionally more detailed enzymatic investigations have been conducted on them, in contrast to export phospholipases e.g. from snake venom, bee venom and porcine pancreas, which have been extensively investigated (Brockerhoff & Jensen 1974a). In a previous paper (De Wolf et al., 1976a), the presence of phospholipase A1 and phospholipase A2 activities in bovine thyroid was demonstrated, using 1-[9, 10-3H] stearoyl-2-[1-14C] linoleyl-sn-glycero-3-phosphocholine as a substrate. Optimal activity was observed in both instances at pH 4. Addition of the anionic detergent sodium taurocholate increased the A2 type activity and decreased the A1 type activity suggesting the presence of different enzymes. The lack of influence of Ca2+-ions and EDTA and the acid pH optima could suggest lysosomal localization. In this paper the subcellular distribution of both acid phospholipase activities is described as well as a purification scheme for phospholipase A1. Some characteristics of the purified enzyme preparation are discussed.     

Antioxidant potentials and rosmarinic acid levels of the methanolic extracts of Salvia virgata (Jacq), Salvia staminea (Montbret & Aucher ex Bentham) and Salvia verbenaca (L.) from Turkey

This study was designed to examine the in vitro antioxidant activities and rosmarinic acid levels of the methanol extracts of Salvia virgata, Salvia staminea and Salvia verbenaca. The extracts were screened for their possible antioxidant activity by two complementary test systems, namely DPPH free radical scavenging and beta-carotene/linoleic acid systems. In the first case, the most active plant was S. verbenaca (14.30+/-1.42 microg mg(-1)), followed by S. virgata (65.70+/-2.12 microg mg(-1)). S. staminae exhibited the weakest antioxidant activity in this test system of which IC(50) value is 75.40+/-0.57 microg mg(-1). In beta-carotene/linoleic acid test system, S. verbenaca extract was superior to the other extracts studied (inhibition value is 77.03%+/-0.42). Antioxidant activities of BHT, ascorbic acid, curcumin and alpha-tocopherol were determined in parallel experiments. Activity of rosmarinic acid was also screened for better establishing the relationship between rosmarinic acid level and antioxidant activity for the plant extracts. According to the results obtained by spectrophotometric analysis and further supported by HPLC, S. verbenaca has the highest rosmarinic acid level with a value of 29.30+/-0.24 microg mg(-1). Our results showed that the rosmarinic acid and its derivatives are more likely to be responsible for most of the observed antioxidant activities of Salvia species.     

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle alpha(1)betagammadelta-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 A by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snake's diet.     

Cloning and characterization of novel snake venom proteins that block smooth muscle contraction.

In this study, we isolated a 25-kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino-acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage-gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K+-induced depolarization in the 0.1-1 microm range, but did not block caffeine-stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K+-induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity.     

Influence of nucleic acid base aromaticity on substrate reactivity with enzymes acting on single-stranded DNA.

Stacking between aromatic amino acids and nucleic acid bases may play an important role in the interaction of enzymes with nucleic acid substrates. In such circumstances, disruption of base aromaticity would be expected to decrease enzyme activity on the modified substrates. We have examined the requirement for DNA base aromaticity of five enzymes that act on single-stranded DNA, T4 polynucleotide kinase, nucleases P1 and S1, and snake venom and calf spleen phosphodiesterases, by comparing their kinetics of reaction with a series of dinucleoside monophosphates containing thymidine or a ring-saturated derivative. The modified substrates contained either cis-5R,6S-di-hydro-5,6-dihydroxythymidine (thymidine glycol) or a mixture of the 5R and 5S isomers of 5,6-dihydrothymidine. It was observed that for all the enzymes, except snake venom phosphodiesterase, the parent molecules were better substrates than the dihydrothymidine derivatives, while the thymidine glycol compounds were significantly poorer substrates. Snake venom phosphodiesterase acted on the unmodified and dihydrothymidine molecules at almost the same rate. These results imply that for all the remaining enzymes base aromaticity is a factor in enzyme-substrate interaction, but that additional factors must contribute to the poorer substrate capacity of the thymidine glycol compounds. The influence of the stereochemistry of the dihydrothymidine derivatives was also investigated. We observed that nuclease P1 and S1 hydrolysed the molecules containing 5R-dihydrothymidine approximately 50-times faster than those containing the S-isomer. The other enzymes displayed no measurable stereospecificity.     

Amino acid sequence of a long-chain neurotoxin homologue, Pa ID, from the venom of an Australian elapid snake, Pseudechis australis

Pa ID, a long-chain neurotoxin homologue, was isolated from the venom of an Australian elapid snake, Pseudechis australis, and its amino acid sequence was determined by conventional methods. Pa ID was an acidic protein (pI = 6.2) and consisted of 68 amino acid residues. It did not show binding activity to the acetylcholine receptor of an electric ray (Narke japonica) nor lethal effect on mice, though the amino acid sequence is homologous with those of long-chain neurotoxins isolated from other elapid snakes (homology, 39-51%). In the sequence of Pa ID, a structurally invariant residue (Tyr-22) and two functionally invariant residues (Val/Ala-49 and Lys/Arg-50) in snake venom neurotoxins are replaced by a cysteine, an arginine, and a methionine residue, respectively, and furthermore, four common residues in long-chain neurotoxins, Gly-17, Ala-43, Ser-59, and Phe/His-66 are replaced by a glutamic acid, a threonine, a threonine, and a valine residue, respectively. The conformational change of the protein molecule caused by these replacements and the removal of a positive charge at position 50 are probably the reasons why Pa ID has lost the lethality.     

长白山白眉蝮蛇乌苏里亚种类凝血酶（TLE）基因克隆及分析

目的为了获得长白山白眉蝮蛇乌苏里亚种类凝血酶基因。方法根据GeneBank自眉类凝血酶eDNA5．和3保守序列设计了引物,通过RT-PCR从白眉蝮蛇乌苏里亚种毒腺TotalRNA中扩增得到1条长714bp的特异cDNA片段,将该cDNA片段重组到SimpleTvector,转化进E．coliJM109competentcell,阳性克隆委托生物公司测序,利用生物信息学方法对测序结果进行分析。结果该特异性片段与蛇毒类凝血酶同源性为95％,它为一个开发阅读框架,其编码的蛋白质序列与其他蛇毒类凝血酶序列同源性为94％,与其他蛇毒类凝血亲缘关系非常近。结论本实验获得了一种新型白眉蝮蛇乌苏里亚种类凝血酶基因。     

Rosmarinic acid and siRNA combined therapy represses Hsp27 (HSPB1) expression and induces apoptosis in human glioma cells

High expression of Hsp27 in glioma cells has been closely associated with tumor cell proliferation and apoptosis inhibition. The aim of the present study was to asses the effects of rosmarinic acid (RA) on Hsp27 expression and apoptosis in non-transfected and transfected human U-87 MG cells. The effect of rosmarinic acid was compared to quercetin, which is known to be a good Hsp27 inhibitor. In order to block the expression of Hsp27 gene (HSPB1), transfection with specific siRNAs was performed. Western blotting technique was used to assess the Hsp27 expression, and caspase-3 colorimetric activity assay was performed to determine apoptosis induction. According to the results, it was found that RA and quercetin effectively silenced Hsp27 and both agents induced apoptosis by activating the caspase-3 pathway. Eighty and 215 μM RA decreased the level of Hsp27 by 28.8 and 46.7% and induced apoptosis by 30 and 54%, respectively. For the first time, we reported that rosmarinic acid has the ability to trigger caspase-3 induced apoptosis in human glioma cells. As a result of siRNA transfection, the Hsp27 gene was silenced by ~?50% but did not cause a statistically significant change in caspase-3 activation. It was also observed that apoptosis was induced at a higher level as a result of Hsp27 siRNA and subsequent quercetin or RA treatment. siRNA transfection and 215 μM RA treatment suppressed Hsp27 expression level by 90.5% and increased caspase-3 activity by 58%. Herein, we demonstrated that RA administered with siRNA seems to be a potent combination for glioblastoma therapy.     

Complete amino acid sequence of kaouthiagin, a novel cobra venom metalloproteinase with two disintegrin-like sequences

The primary structure of kaouthiagin, a metalloproteinase from the venom of the cobra snake Naja kaouthia which specifically cleaves human von Willebrand factor (VWF), was determined by amino acid sequencing. Kaouthiagin is composed of 401 amino acid residues and one Asn-linked sugar chain. The sequence is highly similar to those of high-molecular mass snake venom metalloproteinases from viperid and crotalid venoms comprised of metalloproteinase, disintegrin-like, and Cys-rich domains. The metalloproteinase domain had a zinc-binding motif (HEXXHXXGXXH), which is highly conserved in the metzincin family. Kaouthiagin had an HDCD sequence in the disintegrin-like domain and uniquely had an RGD sequence in the Cys-rich domain. Metalloproteinase-inactivated kaouthiagin had no effect on VWF-induced platelet aggregation but still had an inhibitory effect on the collagen-induced platelet aggregation with an IC(50) of 0.2 microM, suggesting the presence of disintegrin-like activity in kaouthiagin. To examine the effects of these HDCD and RGD sequences, we prepared synthetic peptides cyclized by an S-S linkage. Both the synthetic cyclized peptides from the disintegrin-like domain and from the Cys-rich domain) had an inhibitory effect on collagen-induced platelet aggregation with IC(50) values of approximately 90 and approximately 4.5 microM, respectively. The linear peptide (RAAKHDCDLPELC) and the cyclized peptide had little effect on collagen-induced platelet aggregation. These results suggest that kaouthiagin not only inhibits VWF-induced platelet aggregation by cleaving VWF but also disturbs the agonist-induced platelet aggregation by both the disintegrin-like domain and the RGD sequence in the Cys-rich domain. Furthermore, our results imply that the corresponding part of the Cys-rich domain in other snake venom metalloproteinases also has a synergistic disturbing effect on platelet aggregation, serving as a second disintegrin-like domain. This is the first report of an elapid venom metalloproteinase with two disintegrin-like sequences.     

Isolation and biochemical characterization of a fibrinolytic proteinase from Bothrops leucurus (white-tailed jararaca) snake venom

In investigations aimed at characterizing snake venom clot-dissolving enzymes, we have purified a fibrinolytic proteinase from the venom of Bothrops leucurus (white-tailed jararaca). The proteinase was purified to homogeneity by a combination of molecular sieve chromatography on Sephacryl S-200 and ion-exchange chromatography on CM Sepharose. The enzyme called leucurolysin-a (leuc-a), is a 23 kDa metalloendopeptidase since it is inhibited by EDTA. PMSF, a specific serine proteinase inhibitor had no effect on leuc-a activity. The amino acid sequence was established by Edman degradation of overlapping peptides generated by a variety of selective cleavage procedures. Leuc-a is related in amino acid sequence to reprolysins. The protein is composed of 200 amino acid residues in a single polypeptide chain, possessing a blocked NH2-terminus and containing no carbohydrate. The proteinase showed proteolytic activity on dimethylcasein and on fibrin (specific activity=21.6 units/mg and 17.5 units/microg, respectively; crude venom=8.0 units/mg and 9.5 units/microg). Leuc-a degrades fibrin and fibrinogen by hydrolysis of the alpha chains. Moreover, the enzyme was capable of cleaving plasma fibronectin but not the basement membrane protein laminin. Leuc-a cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B chain. The pH optimum of the proteolysis of dimethylcasein by leuc-a was about pH 7.0. Antibody raised in rabbit against the purified enzyme reacted with leuc-a and with the crude venom of B. leucurus. In vitro studies revealed that leuc-a dissolves clots made either from purified fibrinogen or from whole blood, and unlike some other venom fibrinolytic metallopeptidases, leuc-a is devoid of hemorrhagic activity when injected (up to 100 microg) subcutaneously into mice.     

Purification and Functional Characterisation of Rhiminopeptidase A,a Novel Aminopeptidase from the Venom of Bitis gabonica rhinoceros

Background

Snake bite is a major neglected public health issue within poor communities living in the rural areas of several countries throughout the world. An estimated 2.5 million people are bitten by snakes each year and the cost and lack of efficacy of current anti-venom therapy, together with the lack of detailed knowledge about toxic components of venom and their modes of action, and the unavailability of treatments in rural areas mean that annually there are around 125,000 deaths worldwide. In order to develop cheaper and more effective therapeutics, the toxic components of snake venom and their modes of action need to be clearly understood. One particularly poorly understood component of snake venom is aminopeptidases. These are exo-metalloproteases, which, in mammals, are involved in important physiological functions such as the maintenance of blood pressure and brain function. Although aminopeptidase activities have been reported in some snake venoms, no detailed analysis of any individual snake venom aminopeptidases has been performed so far. As is the case for mammals, snake venom aminopeptidases may also play important roles in altering the physiological functions of victims during envenomation. In order to further understand this important group of snake venom enzymes we have isolated, functionally characterised and analysed the sequence-structure relationships of an aminopeptidase from the venom of the large, highly venomous West African gaboon viper, Bitis gabonica rhinoceros.

Methodology and Principal Findings

The venom of B. g. rhinoceros was fractionated by size exclusion chromatography and fractions with aminopeptidase activities were isolated. Fractions with aminopeptidase activities showed a pure protein with a molecular weight of 150 kDa on SDS-PAGE. In the absence of calcium, this purified protein had broad aminopeptidase activities against acidic, basic and neutral amino acids but in the presence of calcium, it had only acidic aminopeptidase activity (APA). Together with the functional data, mass spectrometry analysis of the purified protein confirmed this as an aminopeptidase A and thus this has been named as rhiminopeptidase A. The complete gene sequence of rhiminopeptidase A was obtained by sequencing the PCR amplified aminopeptidase A gene from the venom gland cDNA of B. g. rhinoceros. The gene codes for a predicted protein of 955 amino acids (110 kDa), which contains the key amino acids necessary for functioning as an aminopeptidase A. A structural model of rhiminopeptidase A shows the structure to consist of 4 domains: an N-terminal saddle-shaped β domain, a mixed α and β catalytic domain, a β-sandwich domain and a C-terminal α helical domain.

Conclusions

This study describes the discovery and characterisation of a novel aminopeptidase A from the venom of B. g. rhinoceros and highlights its potential biological importance. Similar to mammalian aminopeptidases, rhiminopeptidase A might be capable of playing roles in altering the blood pressure and brain function of victims. Furthermore, it could have additional effects on the biological functions of other host proteins by cleaving their N-terminal amino acids. This study points towards the importance of complete analysis of individual components of snake venom in order to develop effective therapies for snake bites.     

Purification and amino acid sequence of basic protein II, a lysine-49-phospholipase A2 with low activity, from Trimeresurus flavoviridis venom

A basic protein (pI 10.3), named basic protein II, was purified to homogeneity from the venom of Trimeresurus flavoviridis (Habu snake) after four chromatographic steps. The amino acid sequence of this protein was determined by sequencing the S-pyridylethylated derivative and its peptides produced by chemical (cyanogen bromide) and enzymatic (chymotrypsin, clostripain, and Staphylococcus aureus V8 protease) cleavages. The protein consisted of 122 amino acid residues and was found to be identical in sequence to basic protein I from the same source except that Asp-58 of basic protein I is replaced by asparagine. Like basic protein I, the structural feature of basic protein II is that Tyr-28 and Asp-49 common in phospholipases A2 from snake venoms and mammalian pancreas are replaced by asparagine and lysine, respectively. Thus, basic protein II belongs to the category of lysine-49-phospholipase A2. The action of basic protein II on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine released only oleic acid, indicating that it has phospholipase A2 activity. Its molar activity toward 1,2-dilauroyl-sn-glycero-3-phosphorylcholine, however, was only 1.7% of that of T. flavoviridis phospholipase A2 isolated previously. Affinity for Ca2+ and reactivity toward p-bromophenacyl bromide of basic protein II were 8 and 5.3 times, respectively, smaller than those of phospholipase A2 from the same source, substantiating the low phospholipase A2 activity of basic protein II.     

Antisnake venom activity of ethanolic seed extract of Strychnos nux vomica Linn

The whole seed extract of S. nux vomica (in low doses) effectively neutralized Daboia russelii venom induced lethal, haemorrhage, defibrinogenating, PLA2 enzyme activity and Naja kaouthia venom induced lethal, cardiotoxic, neurotoxic, PLA2 enzyme activity. The seed extract potentiated polyvalent snake venom antiserum action in experimental animals. An active compound (SNVNF) was isolated and purified by thin layer chromatography and silica gel column chromatography, which effectively antagonised D. russelii venom induced lethal, haemorrhagic, defibrinogenating, oedema, PLA2 enzyme activity and N. kaouthia induced lethal, cardiotoxic, neurotoxic, PLA, enzyme activity. Polyvalent snake venom antiserum action was significantly potentiated by the active compound. Spectral studies revealed it to be a small, straight chain compound containing methyl and amide radicals. Detailed structure elucidation of the compound (SNVNF) is warranted before its clinical trials as a snake venom antagonist.