Intracellular transport of proteins in active and resting secretory cells of the venom gland of Vipera palaestinae

The intracellular transport of venom proteins has been studied in active and resting venom glands of the snake Vipera palaestinae by electron microscope radioautography after an intra-arterial injection of [3H]leucine. In the active gland, most of the label is initially (10 min) found over the RER. By 30 min, the relative grain density of the Golgi complex reaches its maximum, with concomitant increase in the labeling of the condensing vacuoles. Later on, a steep increase in radioactivity of the secretory granules is observed. At 3 h, these granules, which comprise about 2% of the cell volume, contain 22% of the total grains. At the following hour, their labeling declines and at the same time the radioactivity of the secreted venom is increased. It is concluded that, in the active cell, venom proteins are transported via the Golgi apparatus into membrane-bounded granules which are the immediate source of the secreted venom. An alternative pathway, which involves the RER cisternae as a storage compartment, seems unlikely, since incorporated label does not accumulate in this compartment after prolonged postpulse intervals. The route of intracellular transport of proteins in the resting glands is similar to that of the active ones, but the rate of synthesis and transport is much slower. The present results and earlier data, thus, show that the increase in the rate of secretion after initiation of a new venom regeneration cycle is the result of accelerated rates of both synthesis and transport.     

Evaluation of the effect of gamma rays on the venom of Vipera lebetina by biochemical study

Snake bites represent a serious public health problem in many areas of the world. In Algeria, two widespread snakes are Vipera lebetina and Cerastes cerastes. Vipera lebetina venom causes local hemorrhage and necrosis, and it may lead to permanent limb loss. The principal causes of mortality after snakebites are acute renal failure and hemorrhage, which occur not only locally, at the site of the bite, but also systemically, contributing to the cardiovascular shock characteristic of severe envenomation. Gamma radiation has been shown to be effective for attenuating venom toxicity. Vipera lebetina venom was irradiated with two doses of gamma rays (1 and 2 kGy) from a 60Co source, and the venom's toxic, enzymatic, and structural properties were analyzed. Intraperitoneal injection of the irradiated venoms (100-500 microg/20 g mouse body mass) revealed a significant decrease of the toxicity. Irradiated venoms with 1 and 2 kGy doses were four and nine times less toxic, respectively, than the native venom. A biochemical characterization of in vitro enzymatic activities was performed. Vipera lebetina displayed in vitro caseinolytic, amidolytic, esterasic, coagulant, and phospholipase A2 activities. Caseinolytic, amidolytic, esterasic, and coagulative activities were reduced for the irradiated venoms; only phospholipase A2 activity was abolished in the irradiated venom with a dose of 2 kGy. The native and irradiated venoms were separated by gel filtration and electrophoresis. Chromatographic and electrophoretic profiles were drastically changed as compared with the native venom. Vipera lebetina venom detoxified by gamma rays was used for active immunization, and the presence of antibody in the immune sera was detected by ELISA. The immunogenic properties were preserved and the antisera obtained with the irradiated venoms could cross-react. Antisera were able to neutralize the toxic effect of V. lebetina native venom. These results indicate that irradiation of V. lebetina venom with a dose of 2 kGy can promote a significant detoxification, keeping the immunological properties intact.     

Comparative analysis of the venom proteomes of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis

The venom proteomics of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, snakes of public health significance and the most poisonous reptiles in Europe, were analyzed by FPLC, 2-D electrophoresis, sequence analysis, and MS/MS. FPLC analysis showed the presence of l-amino acid oxidase, monomeric and heterodimeric phospholipases A2, C-type lectin protein, and proteinases in the venom of V. a. ammodytes. Representatives of the same protein families were found in the venom of the other subspecies, V. a. meridionalis. N-terminally identical PLA2 neurotoxins were identified in both venoms. Difference in the PLA2 compositions of the venoms was also observed: a monomeric protein with phospholipase A2 activity, identical in the first 20 amino acid residues to the catalitically inactive acidic component of the heterodimeric PLA2 present in both venoms, was found only in that of V. a. meridionalis. Probably, this protein represents an intermediate form of the two components of the heterodimer. 2-D electrophoresis and MS/MS analysis showed that the two venoms shared a number of protein families: monomeric and heterodimeric Group II PLA2s, serine proteinases, Group I, II, and III metalloproteinases, l-amino acid oxidases (LAAOs), cysteine-rich secretory proteins, disintegrins, and growth factors. Totally, 38 venom components of the V. a. ammodytes, belonging to 9 protein families, and 67 components of the V. a. meridionalis venom belonging to 8 protein families were identified. The venom proteome of V. a. ammodytes shows larger diversity of proteins (139) in comparison to that of V. a. meridionalis (104 proteins). Most of the proteins are homologues of known representatives of the respective protein families. The protein compositions explain clinical effects of the V. ammodytes snakebites, such as difficulties in the breathing, paralysis, apoptosis, cloting disorders, hemorrhage, and tissue necrosis. The lists of secreted proteins by the two vipers can be used for further study of structure-function relationships in the toxins and for prediction and treatment of snakebite consequences.     

The expression of vimentin in satellite cells of regenerating skeletal muscle in vivo

Summary The expression of the intermediate filament protein, vimentin, was studied in skeletal muscle during a cycle of degeneration and regeneration. Venom from the Australian tiger snake,Notechis scutatus scutatus, was used to initiate the breakdown of the soleus muscle of young, mature ratsin vivo. Cryosections and Western blots of muscle samples were labelled using antibodies to vimentin, and examined at fixed time points after venom injection. Vimentin was absent in control adult muscle fibres, but was identified in activated satellite cells 12 h after venom assault. The amount of this protein rose during the early stages of regeneration, reaching its peak at 2–3 days. At this time, the expression of muscle-specific intermediate filament protein, desmin, began. As the abundance of desmin increased with the maturation of the regenerating myofibres, the abundance of vimentin declined until it was no longer detectable in mature regenerated fibres. It is suggested that vimentin plays an important role during satellite cell activation in the early stages of regeneration, and that the expression of vimentin may act as a stimulus for the expression of desmin at later stages of regeneration.     

The variability of Vipera ammodytes ammodytes venoms from Croatia--biochemical properties and biological activity

Vipera ammodytes ammodytes venom has been used for many years in Croatia for immunization of horses and production of specific therapeutic anti-venoms. The neutralizing effectiveness of anti-venoms is directly dependent on the properties of the snake venom used for immunization. Therefore, appropriate characterization of the whole venom is necessary prior to use in the immunization procedure. In the course of such analyses, the variability in biochemical properties and biological activity was observed in venoms collected from snakes originating from different parts of Croatia. The venom pools also differed with respect to time of snake collection (1992-2003). Analyses of three samples of whole venom pools were carried out revealing differences in lethal activity (LD50), minimum haemorrhagic dose (MHD), minimum necrotizing dose (MND), phospholipase A2 activity and in anticomplementary activity. SDS-PAGE electrophoretic patterns were similar, but not identical, for all tested venom pools with respect to the number of protein bands detected, but intensity of particular components differed. Preliminary immunogenicity testing in terms of determination of specific antibodies revealed similar immunogenicity and high cross-reactivity for three samples tested.     

Venom regeneration in the centipede Scolopendra polymorpha: evidence for asynchronous venom component synthesis

Venom regeneration comprises a vital process in animals that rely on venom for prey capture and defense. Venom regeneration in scolopendromorph centipedes likely influences their ability to subdue prey and defend themselves, and may influence the quantity and quality of venom extracted by researchers investigating the venom's biochemistry. We investigated venom volume and total protein regeneration during the 14-day period subsequent to venom extraction in the North American centipede Scolopendra polymorpha. We further tested the hypothesis that venom protein components, separated by reversed-phase fast protein liquid chromatography (RP-FPLC), undergo asynchronous (non-parallel) synthesis. During the first 48 h, volume and protein mass increased linearly. Protein regeneration lagged behind volume regeneration, with 65–86% of venom volume and 29–47% of protein mass regenerated during the first 2 days. No additional regeneration occurred over the subsequent 12 days, and neither volume nor protein mass reached initial levels 7 months later (93% and 76%, respectively). Centipede body length was negatively associated with rate of venom regeneration. Analysis of chromatograms of individual venom samples revealed that 5 of 10 chromatographic regions and 12 of 28 peaks demonstrated changes in percent of total peak area (i.e., percent of total protein) among milking intervals, indicating that venom proteins are regenerated asynchronously. Moreover, specimens from Arizona and California differed in relative amounts of some venom components. The considerable regeneration of venom occurring within the first 48 h, despite the reduced protein content, suggests that predatory and defensive capacities are minimally constrained by the timing of venom replacement.     

Precipitation of animal plasma proteins by puff-adder venom.

1. Plasma and serum samples obtained from various animals never previously exposed to snakes or snake venom were diffused against different concentrations of puff-adder, Bitis arietans, venom using the double immunodiffusion technique. 2. Depending upon venom concentration, two precipitin arcs could be produced in the case of all plasma samples used. No serum samples showed any arcs except pigeon serum, where one precipitin line was observed. 3. By altering the concentration of snake venom between 1% and 10% when immunodiffusing against plasma a change in position of the precipitin lines was observed and also the disappearance of one or both of the two bands at higher concentrations. This indicates that the arcs observed are in all probability due to precipitation of plasma protein fractions. 4. Previous results indicated that one of the two bands observed when diffusing venom against plasma was due to the precipitation of fibrinogen. By diffusing snake venom against heparin we have now shown that the second band involves this molecule and is not due to another coagulation factor as was suggested previously.     

Molecular cloning and expression of a functional snake venom vascular endothelium growth factor (VEGF) from the Bothrops insularis pit viper. A new member of the VEGF family of proteins

During the generation of abundant expressed sequence tags from the Viperidae snake Bothrops insularis venom glands, we identified for the first time a cDNA coding for a putative vascular endothelial growth factor-like (VEGF-like) protein. The deduced primary sequence, after complete sequencing of the longest snake venom VEGF (svVEGF) cDNA, displayed similarity with vertebrate VEGFs and with the hypotensive factor from Vipera aspis venom. Its cDNA was subcloned, expressed in Escherichia coli with a His(6) tag as an insoluble monomer, and purified by Ni(2+)-affinity chromatography after 8 m urea extraction. Antiserum against svVEGF was generated and tested in Western blot against proteins from snake venoms and cellular extracts. The mature svVEGF appears to be ubiquitously distributed throughout snake venoms and was also confirmed by Northern blot studies of other related Viperidae species and by cDNA cloning of svVEGF from Bothrops jararaca pit viper. The produced recombinant protein dimerizes after refolding processes and was biologically characterized, showing ability to increase vascular permeability. These results established that svVEGF is a novel and important active toxin during the early stages of bothropic snake bite envenoming and represents a new member of the VEGF family of proteins.     

REGULATION OF PROTEIN SYNTHESIS IN THE VENOM GLAND OF VIPERID SNAKES

Morphological changes in the venom gland of V. ammodytes were studied after the removal of the venom from the gland lumina (milking) It was found that the height of the secretory cells was changed during the secretory cycle. The patterns of the rough endoplasmic reticulum and of the Golgi complex were changed as well Milking induced an increased incorporation of [¹⁴C]amino acids into total and venom proteins In V ammodytes, during the first day after milking, 25% of the total counts in protein were precipitable by anti-venom serum, while at 8 days, 80% of the proteins synthesized were venom proteins At this stage, the incorporation was 10- and 20-fold that of unmilked glands for total and venom proteins, respectively. Venom was accumulated (secreted) in the gland lumina of V. ammodytes at a relatively high rate up to 2 wk after milking and leveled off afterwards. Intact glands and gland slices of V ammodytes and V palaestinae, taken from snakes a few days after milking, incorporated [¹⁴C]amino acids into proteins in vitro at a rate higher than that of unmilked glands. The activity of two exportable enzymes (phosphodiesterase and benzoyl arginyl ethyl esterase) was assayed in gland homogenates of V. ammodytes. It was found that 2–3 wk after milking, the intracellular level of these enzymes was up to 2-fold that of unmilked glands.     

Comparative characterisation of Russell's viper (Daboia/Vipera russelli) venoms from different regions of the Indian peninsula.

Russell's viper (Daboia/Vipera russelli) venom from different regions of India was subjected to chromatographic, electrophoretic, biochemical and immunological analysis. The elution profiles from ion-exchange chromatography and protein banding pattern from SDS-PAGE showed a significant variation in the constituents of venoms. The acidic proteins are found to be predominant in the venoms of eastern and western regions while basic proteins are the major contributors of the northern and southern regional venoms. The major variation of phospholipases A(2) in the venom samples of India may be described as: southern regional venom is rich in basic, toxic PLA(2) while this activity showed a dramatic decrease as one moves towards west, north and eastern regions of India. In addition, the caseinolytic, TAME-hydrolytic, anticoagulant, oedema-inducing and haemorrhagic activities of the venoms have also varied from one region to another. The muscle specimens of mice injected with venoms of different regions showed variable change in the muscle fibre damage and cell morphology. The eastern regional venom is most lethal among all the venoms. The lethal potencies for four regional venoms vary as: eastern>western>southern>northern. The polyclonal antibodies prepared against the venom of southern region showed cross-reaction with the venoms of other regions, but the extent of cross-reaction and diffusion patterns are different. However, the polyclonal antibodies prepared against southern regional venom showed no protection against lethal toxicity of other regional venoms.     

Primary culture of epithelial secretory cells from venom gland of the common adder Vipera berus

A primary culture of epithelial secretory cells from the venom gland of Vipera berus was obtained. The cells adhered to collagen 1 and to a mixture of adhesion proteins (Matrigel), proliferated and retained the features of differentiation. Electron microscopy demonstrated the presence of all ultrastructures typical of these cells in vivo, a full complex of intercellular junctions, and cellular membrane polarity. The immunohistochemistry confirmed the capacity of secretory cells to synthesize venom in culture. We have studied the role of carbochole, an agonist of M-cholinoreceptor, in the initiation of the secretory cycle in cells in vitro. We propose that M-cholinoreceptors may play an important role in the initiation of the secretory cycle in vivo.     

Amino-acid sequence of ammodytoxin B partially reveals the location of the site of toxicity of ammodytoxins

The complete amino-acid sequence of ammodytoxin B, a presynaptically toxic phospholipase A2 isolated from Vipera ammodytes ammodytes venom, was determined by manual and automated protein sequencing. Ammodytoxin B (i.v. LD50 = 0.58 mg/kg for white mice) is 30-fold less toxic than ammodytoxin A, the most toxic phospholipase isolated from the same venom. The two proteins (each 122 residues long) differ in only 3 residues located in positions 115, 118 and 119 (numbering according to R. Renetseder et al. (1985) J. Biol. Chem. 260, 11627-11634) suggesting that an exposed hydrophobic residue in position 115 and a basic residue in position 118 may be responsible for the increased toxicity of ammodytoxin A and should form at least one part of the site of toxicity in ammodytoxins.     

Molecular cloning and analysis of cDNA sequences encoding serine proteinase and Kunitz type inhibitor in venom gland of Vipera nikolskii viper

Serine proteinases and Kunitz type inhibitors are widely represented in venoms of snakes from different genera. During the study of the venoms from snakes inhabiting Russia we have cloned cDNAs encoding new proteins belonging to these protein families. Thus, a new serine proteinase called nikobin was identified in the venom gland of Vipera nikolskii viper. By amino acid sequence deduced from the cDNA sequence, nikobin differs from serine proteinases identified in other snake species. Nikobin amino acid sequence contains 15 unique substitutions. This is the first serine proteinase of viper from Vipera genus for which a complete amino acid sequence established. The cDNA encoding Kunitz type inhibitor was also cloned. The deduced amino acid sequence of inhibitor is homologous to those of other proteins from that snakes of Vipera genus. However there are several unusual amino acid substitutions that might result in the change of biological activity of inhibitor.     

Cross-reactivities of polyclonal antibodies against factor V activating enzyme, a serine proteinase from Vipera lebetina (snake) venom

Antibodies to the factor V activating enzyme from Vipera lebetina venom were produced by immunizing a rabbit with chromatographically purified factor V activating enzyme probes. The antibodies cross-reacted with different protein fractions in 23 snake venoms (ten viperid, eight crotalid, and five elapid venoms) as demonstrated by western immunoblotting. In the venom of Vipera russelli the antibodies recognized only one protein band which probably belonged to factor V activating enzyme.     

颜氏大疣蛛蛛毒中多肽和蛋白质的多样性分析

对颜氏大疣蛛Macrothele yani蛛毒所富含的多肽与蛋白质多样性进行探索,采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和超高效液相色谱-电喷雾-四极杆-飞行时间质谱技术分离和鉴定颜氏大疣蛛蛛毒中的蛋白质和多肽,并对其相对分子质量分布多样性进行分析。结果显示:粗毒中所含蛋白质的相对分子质量主要分布在35kDa以上。在17~135kDa分离度较佳的共有11条电泳条带,主要集中于40~120kDa附近;在75kDa附近弥散着高丰度的蛋白条带,75kDa以上的蛋白质最丰富。粗毒经色谱分离后得到超过50个色谱峰,经质谱鉴定得到121个物质成分,其中,多肽类物质的相对分子质量呈双峰式分布,21%分布在500~2000 Da,76%分布在3000~5000Da,为粗毒中多肽含量最丰富的部分,且集中于35~60min的保留时间内被洗脱。研究结果表明,颜氏大疣蛛蛛毒中含有较为丰富的多肽和蛋白类物质,这些物质的相对分子质量分布特征与已报道的其他蜘蛛既有相似性又存在具体差异。本文展示了颜氏大疣蛛蛛毒的分子多样性,为后续该毒素的物质基础研究及药用价值开发提供参考。     

Intraspecies variability in Vipera ammodytes ammodytes venom related to its toxicity and immunogenic potential

Vipera ammodytes is the most venomous European snake, whose venom has been used as antigen for immunization of antivenom-producing animals. Same as venom of any other snake, it is a complex mixture of proteins, peptides and other compounds which biochemical and pharmacological variability has been demonstrated at interspecies and intraspecies level. In this work we demonstrated intraspecific variability between 8 venom production batches using both the conventional and the new methodology. Moreover, in contrast to the literature on different venoms' variability, for the first time we were able to select those biochemical differences that are related to and give information on the venom's toxicity and immunogenicity. We have shown that methods quantifying ammodytoxin (the most toxic compound identified so far in the Vipera ammodytes ammodytes venom) content of the venom clearly distinguish between high and low immunogenic venoms.     

Toxicity and Immunogenic Properties of Liposomal form of Vipera Libetina Venom

Abstract

The incorporation of Vipera libetina venom into liposomes obtained from pure egg phosphatidyl choline by the reverse phase evaporation method decreases its toxicity by 3-fold - in mice LD₅₀ for the native toxin is 2.22 mg/kg body weight and for the liposomal toxin 6.9 mg/kg. Subcutaneous injection of liposomal preparation into mice stimulates the development of cellular immunity and reproduces the reaction of the delayed-type hypersensitivity. It is. also, shown that after a single dose immunization of mice with liposomes containing 1xLD₅₀ dose of the venom, the.titer.of antibodies increases at the early postinfection period and is maintained on high level longer than after the injection of the native venom. Thus, liposom.es can be succesfully used for antiserum production and protective immunization against Vipera libetina venom.     

Isolation and characterization of nerve growth factor from Vipera lebetina (snake) venom

Nerve growth factor from Vipera lebetina venom was purified by gel filtration and ion exchange chromatography steps. The NGF preparation obtained is a glycoprotein with weak arginine esterase activity. It hydrolyzes benzoylarginine ethyl ester (BAEE). Vipera lebetina NGF consists of multiple forms of protein with pI in the interval 9-10.5. All isoforms have identical molecular weights of 32,500.     

A permanent magnetic field alters the phospholipase activity in the snake venom from Vipera lebetina

It was found that at the exposure of Vipera lebetina snakes (during 10 days for 30 min daily) to SMF (0.15 T1) the specific activity of venom phospholipase A1, A2 and phosphodiesterase C increased by 20.6 +/- 2.8; 31.7 +/- 3.2 and 32.7 +/- 1.3% correspondingly. The above mentioned changes of venom enzyme activity were accompanied with the decrease of its total protein amount by 31.6 +/- 2.2%. It could be supposed that the described changes are able to cause significant changes in the total metabolic activity of cells and the organism as a whole.