Amino acid sequence of a postsynaptic neurotoxin from the venom of the Australian tiger snake Notechis scutatus scutatus.

Although 60 percent of the protein in tiger snake (Notechis scutatus scutatus) venom consists of the basic per-synaptically neurotoxic and myotoxic phospholipases notexin and Notechis II-5 and other phospholipase homologs such as Notechis II-1, several post-synaptic "curaremimetic" neurotoxins are present in small amounts. The major one of these is a typical "long" neurotoxin containing 73 amino acids in a single peptide chain cross-linked by five disulfide bridges. The formula weight calculated from the amino acid sequence is 8,051. The LD50 for intravenous injection into mice is 125 micrograms/kg.     

Isolation and amino acid sequence of a neurotoxic phospholipase A from the venom of the Australian tiger snake Notechis scutatus scutatus.

The complete amino acid sequence of notechis 5, a neurotoxic phospholipase A from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The main fragmentation of the 119-residue peptide chain was accomplished by digesting the reduced and S-carboxymethylated derivative of the protein with a staphylococcal protease specific for glutamoyl bonds. Tryptic peptides were used to align and complete the sequence of the four staphylococcal protease peptides. The sequence was determined by Edman degradation by means of the direct phenylthiohydantoin method. Notechis 5 differs in seven positions from the recently elucidated sequence of the presynaptic neurotoxin notexin from the same venom. Notechis 5 has a 50% higher specific prospholipase A activity than notexin when assayed against egg yolk but is only one-third as toxic.     

Amino acid sequence of a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake).

The complete amino acid sequence of notexin, a presynaptic neurotoxin from the venom of Notechis scutatus scutatus (Australian tiger snake), has been elucidated. The protein consists of a single chain of 119 amino acids cross-linked by seven disulfide bridges and has a formula weight of 13,578. The main fragmentation of the peptide chain was accomplished with a staphylococcal protease specific for glutamoyl bonds. A cyanogen bromide fragment and tryptic peptides were used to align the five major staphylococcal protease peptides. The sequence was determined by Edman degradation using the direct phenylthiohydantoin method and with carboxypeptidase A. Notexin is shown to be homologous to both porcine pancreatic phospholipase A and a phospholipase A from the venom of Naja melanoleuca.     

Effect of presynaptic neurotoxin notechis II-5 from tiger snake venom on the motor nerve endings of mice]

The neurotoxin notechis II-5 (N-II-5) from tiger snake venom (Notechis scutatus) induces three-phasic changes in miniature end-plate potential (MEPP) frequency recorded in the mouse diaphragm muscle: an initial fall of frequency followed by increase and decrease in MEPP frequency up to complete blockade. The effect of N-II-5 was enhanced with rising of the solution temperature from 20 to 30 and 35 degrees C. Removal of Ca2+ from the solution prevented the presynaptic effect of N-II-5. After washing out of the muscle from N-II-5 with Ca-free solution, addition of Ca2+ to the solution provoked the development of the effect typical of the effect of typical of the toxin. In the presence of N-II-5 an increase in K+ concentration in the solution up to 20 mM did not result in a sharp rise of MEPP frequency characteristic of depolarized nerve terminals. The agents that raise Ca2+ axoplasmic concentration not on account of depolarization of nerve terminals (hypertonic solution, ionofor A23187) preserved the capacity for increasing MEPP frequecy. It is suggested that the presynaptic effect of N-II-5 is related to its phospholipase activity and can be explained by disturbance of the activity of release sites rather than by depletion of transmitter stores.     

Characterization of a human coagulation factor Xa-binding site on <Emphasis Type="Italic">Viperidae</Emphasis> snake venom phospholipases A<Subscript>2</Subscript> by affinity binding studies and molecular bioinformatics

Background  

The snake venom group IIA secreted phospholipases A₂ (SVPLA₂), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA₂-inhibitors from snake serum; and (iii) coagulation factors present in human blood.     

Amino-acid sequence of the beta 1 isosubunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan (Oxyuranus s. scutellatus)

The amino acid sequence of the beta 1 isosubunit of taipoxin, an extremely potent presynaptic neurotoxin from the Australian snake taipan, has been determined. The beta 1 isosubunit, which is neither toxic nor enzymatically active on its own, consists of a single polypeptide chain of 118 amino acids. The main fragmentation of the reduced and S-carboxymethylated derivative was accomplished by cleavage with Staphylococcus aureus V8 protease. Tryptic peptides were used to align and complete the sequence, which was determined by automated Edman degradation. The taipoxin beta 1 isosubunit is closely homologous to the taipoxin alpha and gamma subunits and to enzymatically active pancreatic and elapid snake venom phospholipases A2.     

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.     

Inhibition of Nicotinic Acetylcholine Receptors,a Novel Facet in the Pleiotropic Activities of Snake Venom Phospholipases A2

Phospholipases A₂ represent the most abundant family of snake venom proteins. They manifest an array of biological activities, which is constantly expanding. We have recently shown that a protein bitanarin, isolated from the venom of the puff adder Bitis arietans and possessing high phospholipolytic activity, interacts with different types of nicotinic acetylcholine receptors and with the acetylcholine-binding protein. To check if this property is characteristic to all venom phospholipases A₂, we have studied the capability of these enzymes from other snakes to block the responses of Lymnaea stagnalis neurons to acetylcholine or cytisine and to inhibit α-bungarotoxin binding to nicotinic acetylcholine receptors and acetylcholine-binding proteins. Here we present the evidence that phospholipases A₂ from venoms of vipers Vipera ursinii and V. nikolskii, cobra Naja kaouthia, and krait Bungarus fasciatus from different snake families suppress the acetylcholine- or cytisine-elicited currents in L. stagnalis neurons and compete with α-bungarotoxin for binding to muscle- and neuronal α7-types of nicotinic acetylcholine receptor, as well as to acetylcholine-binding proteins. As the phospholipase A₂ content in venoms is quite high, under some conditions the activity found may contribute to the deleterious venom effects. The results obtained suggest that the ability to interact with nicotinic acetylcholine receptors may be a general property of snake venom phospholipases A₂, which add a new target to the numerous activities of these enzymes.     

Discrimination between the regioisomeric 1,2- and 1,3-diacylglycerophosphocholines by phospholipases

The artificial 1,3-diacyl-glycero-2-phosphocholines (1,3-PCs), which form similar aggregate structures as the naturally occurring 1,2-diacyl-sn-glycero-3-phosphocholines (1,2-PCs), were tested as substrates for different classes of phospholipases such as phospholipase A₂ (PLA₂) from porcine pancreas, bee and snake venom, and Arabidopsis thaliana, phospholipase C (PLC) from Bacillus cereus, and phospholipase D (PLD) from cabbage and Streptomyces species. The regioisomers of the natural phospholipids were shown to bind to all investigated phospholipases with an affinity similar to the corresponding naturally occurring phospholipids, however their hydrolysis was reduced to different degrees (PLA₂s and PLC) or even abolished (PLDs belonging to the PLD superfamily). The results are in accordance with binding models obtained by docking the substrates to the crystal structures or homology models of the phospholipases.     

Molecular dynamics simulations reveal structural insights into inhibitor binding modes and functionality in human Group IIA phospholipase A2

Human Group IIA phospholipase A₂ (hGIIA) promotes inflammation in immune‐mediated pathologies by regulating the arachidonic acid pathway through both catalysis‐dependent and ‐independent mechanisms. The hGIIA crystal structure, both alone and inhibitor‐bound, together with structures of closely related snake‐venom‐derived secreted phospholipase enzymes has been well described. However, differentiation of biological and nonbiological contacts and the relevance of structures determined from snake venom enzymes to human enzymes are not clear. We employed molecular dynamics (MD) and docking approaches to understand the binding of inhibitors that selectively or nonselectively block the catalysis‐independent mechanism of hGIIA. Our results indicate that hGIIA behaves as a monomer in the solution environment rather than a dimer arrangement that is in the asymmetric unit of some crystal structures. The binding mode of a nonselective inhibitor, KH064, was validated by a combination of the experimental electron density and MD simulations. The binding mode of the selective pentapeptide inhibitor FLSYK to hGIIA was stipulated to be different to that of the snake venom phospholipases A₂ of Daboia russelli pulchella (svPLA₂). Our data suggest that the application of MD approaches to crystal structure data is beneficial in evaluating the robustness of conclusions drawn based on crystal structure data alone. Proteins 2017; 85:827–842. © 2016 Wiley Periodicals, Inc.     

Crystal structure determination of alkaline haemorrhagin AaHⅢ from snake venom of Agkistrodon acutus

The haemorrhagin AaH III isolated from the snake venom ofAgkistrodon acutus is one of the few alkaline ones in snake venoms. Its crystals belong to space group P2₁2₁2₁ witha = 9. 573 4 nm,b = 4. 996 7 nm andc = 4.728 8 nm. Its crystal structure was determined by the molecular replacement method according to the model of metalloproteinase Adamalysin II from eastern rattlesnake venom. The AaH III structure has been refined by PROLSQ. The finalR factor was 0.254 and the RMS deviations of bond lengths and angles were 0.001 8 nm and 1.5°. The structure comparison suggested that AaH III has a similar structure to other snake venom zinc-metalloproteinases. They all belong to matrix metalloproteinases super-family. Project supported by the Chinese Academy of Sciences, State Key Laboratory of Biomacromolecules and State Education Commission of China.     

An examination of structural interactions presumed to be of importance in the stabilization of phospholipase A2 dimers based upon comparative protein sequence analysis of a monomeric and dimeric enzyme from the venom ofAgkistrodon p. piscivorus

Phospholipases A₂ may exist in solution both as monomers and dimers, but enzymes that form strong dimers (K _D approximately 10^–9 M) have been found, thus far, only in venoms of the snake family Crotilidae. The complete amino acid sequences of a basic monomeric and an acidic dimeric phospholipase A₂ fromAgkistrodon piscivorus piscivorus (American cotton-mouth water moccasin) venom have been determined by protein sequencing methods as part of a search for aspects of structure contributing to formation of stable dimers. Both the monomeric and dimeric phospholipases A₂ are highly homologous to the dimeric phospholipases A₂ fromCrotalus atrox andCrotalus adamanteus venoms, and both have the seven residue carboxy-terminal extension characteristic of the crotalid and viperid enzymes. Thus, it is clear that the extension is not a prerequisite for dimerization. Studies to date have revealed two characteristic features of phosphilipases A₂ that exist in solution as strong dimers. One is the presence in the dimers of a Pro-Pro sequence at position 112 and 113 which just precedes the seven residue carboxy-terminal extension (residues 116–122). The other is a low isoelectric point; only the acidic phospholipases A₂ have been observed, thus far, to form stable dimers. These, alone or together, may be necessary, though not sufficient conditions for phospholipase A₂ dimer formation. Ideas regarding subunit interactions based upon crystallographic data are evaluated relative to the new sequence information on the monomeric and dimeric phospholipases A₂ fromA. p. piscivorus venom.     

Inhibition of red cell Ca2+-dependent K+ channels by snake venoms

We have investigated the effects of several snake venoms on the Ca2+-dependent K+ channels of human red cells. A heat-resistant component of the venom of the snake Notechis scutatus irreversibly inhibited Ca2+-dependent K+ transport with a Ki value of 0.1-0.2 micrograms/ml. Metabolic changes of the cells modified the maximal effect of the venom. Binding of the venom required extracellular Ca2+ and was quick, but development of full inhibition required additional time. The effects of the venoms from Notechis scutatus and Leiurus quinquestriatus were additive, suggesting that both venoms act through different mechanisms. Venoms of the snakes Vipera russelli russelli and Oxyuranus scutellatus also inhibited Ca2+-dependent K+ transport with the same characteristics as the Notechis scutatus venom.     

On the purification of notexin. Isolation of a single amino acid variant from the venom of Notechis scutatus scutatus

Venom of the Australian tiger snake, Notechis scutatus scutatus was fractionated by conventional ion-exchange chromatography. The fraction containing notexin, a well-known single-chain toxic phospholipase A2, was further purified by reverse-phase high-performance liquid chromatography. Two main components were isolated and the major one corresponded to notexin. The other component, designated as notechis Ns, was an isoform of notexin. Notechis Ns and notexin possessed similar in vitro esterase activity, in vitro neuromuscular activity and in vivo lethality. Amino acid composition and sequence of the Staphylococcus aureus V8-protease peptides demonstrated that primary structures of notechis Ns and notexin differed from each other by a single substitution amongst 119 amino acids: Lys----Arg at position 16.     

Venom glands and some associated muscles in sea snakes

The venom glands and related muscles of sea snakes conform in their general structure to those of the terrestrial elapids. The venom gland, however, is smaller in size and the accessory gland is considerably reduced. A similar pattern is found in the Australian elapid Notechis. The musculus compressor glandulae is well developed in the sea snakes and in some species its posterior-medial portion runs uninterruptedly from the origin to the insertion of the muscle. This might be considered as a primitive condition suggesting an early divergence of the sea snakes from an ancestral elapid stock. Three species of sea snakes, Aipysurus eydouxi, Emydocephalus annulatus, and E. ijimae, feed on fish eggs and have very small, but still functioning, venom glands. The reduced accessory gland of the sea snakes is apparently connected with their aquatic environment, as a similar condition is found also in the elapine Boulengerina annulata which lives in large lakes of Central Africa. The similarity in structure of the venom gland between sea snakes and Notechis scutatus may point to a possible phylogenetic relationship between this group of Australian elapids and hydrophiine snakes.     

The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: Evolution of venom group II phospholipase A2's may imply gene duplications

The sequence coding for a snake venom phospholipase A₂ (PLA₂), BJUPLA₂, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA₂'s. A striking feature of the cDNA is the high sequence conservation of the 5 and 3 untranslated regions in cDNAs coding for PLA₂'s from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA₂ with group II PLA₂'s in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA₂'s.The nucleotide sequence reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number X76289.Correspondence to: A.M. Moura-da-Silva     

Isolation and characterization of novel microsatellite markers from the Australian tiger snakes (Elapidae: Notechis) and amplification in the closely related genus Hoplocephalus

We provide details of seven microsatellite loci (out of 20 primer pairs designed) that exhibited the cleanest and strongest banding patterns for the Eastern tiger snake Notechis scutatus. These loci were used to screen 76 individuals from across the geographical range of N. scutatus and a further 14 individuals of the closely related and endangered broadheaded snake Hoplocephalus bungaroides. We observed large numbers of alleles per locus (14–52) and relatively high levels of heterozygosity (0.270–0.696) within the N. scutatus sample. These markers are also likely to be informative for work on H. bungaroides.     

Structures of genes encoding phospholipase A2 inhibitors from the serum of Trimeresurus flavoviridis snake

Inhibitors (PLIs) against snake venom gland phospholipases A₂ (PLA₂s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA₂ isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region.     

Hydrolysis of 1-palmitoyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero-3-phospholipids by phospholipase A2: effect of the polar head-group

The effect of the phospholipid polar head-group on the porcine pancreatic phospholipase A₂ (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) reaction was studied using 1-palmitoyl-2-[6-(pyren-1-yl)]hexanoyl-sn-glycero-3-phosphatidylcholine,-ethanolamine, -glycerol, -monomethylester and -serine as substrates. Except for the monomethylester analogue, which was maximally activated by 3.5 mM CaCl₂, maximal enhancement of hydrolysis of the other pyrenephospholipids was obtained at 2 mM Ca₂₊. Sodium cholate inhibited hydrolysis of the ethanolamine and serine lipids, whereas a slight (1.4–2.0-fold) activation was observed for the -choline, -glycerol and -monomethylester derivatives. Arrhenius plots of hydrolysis of pyrenephospholipids by porcine pancreatic phospholipase A₂ revealed no discontinuities, thus indicating the absence of phase transition for these lipids in the temperature range 15–45°C. Specific activities of porcine and bovine pancreatic, porcine intestinal and snake venom (Crotalus atrox) phospholipases A₂ towards pyrenephospholipid liposomes were then compared. Whereas the snake venom phospholipase A₂ preferred phosphatidylcholine as a substrate, the other phospholipases A₂ preferred acidic phospholipids in the order monomethylester ⩾ glycerol ⩾ serine.     

Studies on the subunit structure of textilotoxin, a potent presynaptic neurotoxin from the venom of the Australian common brown snake (Pseudonaja textilis). 2. The amino acid sequence and toxicity studies of subunit D

Textilotoxin is a presynaptic neurotoxin in the venom of the Australian common brown snake, Pseudonaja textilis. It has the highest lethality and is structurally the most complex of any known snake venom neurotoxin. Reverse-phase HPLC was used to resolve textilotoxin into subunits A, B, C and D. Subunit D consists of two identical covalently linked polypeptide chains. Its sequence is now reported. It is an acidic, slightly glycosylated polypeptide of 133 amino acid residues in each chain. Although it is not itself neurotoxic, it was found to be essential for the neurotoxicity of textilotoxin.