Functional involvement of Lys-6 in the enzymatic activity of phospholipase A2 fromBungarus multicinctus (Taiwan banded krait) snake venom

Phospholipase A₂ (PLA₂) fromBungarus multicinctus snake venom was subjected to Lys modification with 4-chloro-3,5-dinitrobenzoate and trinitrobenzene sulfonic acid, and one major carboxydinitrophenylated (CDNP) PLA₂ and two trinitrophenylated (TNP) derivatives (TNP-1 and TNP-2) were separated by high-performance liquid chromatography. The results of amino acid analysis and sequence determination revealed that CDNP-PLA₂ and TNP-1 contained one modified Lys residue at position 6, and both Lys-6 and Lys-62 were modified in TNP-2. It seemed that the Lys-6 was more accessible to modified reagents than other Lys residues in PLA₂. Modification of Lys-6 caused a 94% drop in enzymatic activity as observed with CDNP-PLA₂ and TNP-1. Alternatively, the enzyme modified on both Lys-6 and Lys-62 retained little PLA₂ activity. Either carboxydinitrophenylation or trinitrophenylation did not significantly affect the secondary structure of the enzyme molecule as revealed by the CD spectra, and Ca²⁺ binding and antigenicity of Lys-6-modified PLA₂ were unaffected. Conversion of nitro groups to amino groups resulted in a partial restoration of enzymatic activity of CDNP-PLA₂ to 32% of that of PLA₂. It reflected that the positively charged side chain of Lys-6 might play an exclusive role in PLA₂ activity. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activity of the regenerated PLA₂ is almost the same as that of native PLA₂. These results suggest that the intact Lys-6 is essential for the enzymatic activity of PLA₂, and that incorporation of a bulky CDNP or TNP group on Lys-6 might give rise to a distortion of the interaction between substrate and the enzyme molecule, and the active conformation of PLA₂.     

Characterization and gene organization of Taiwan banded krait (Bungarus multicinctus) gamma-bungarotoxin

-Bungarotoxin was isolated from Bungarus multicinctus (Taiwan banded krait) venom using a combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase high-performance liquid chromatography column. Circular dichroism (CD) measurement revealed that its secondary structure was dominant with -sheet structure as is that of snake venom -neurotoxins and cardiotoxins. -Bungarotoxin exhibits activity on inhibiting the binding of [³H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine receptor subtype, and competes weakly with radioiodinated -bungarotoxin for binding to the Torpedo nicotinic acetylcholine receptor. Moreover, the toxin inhibits collagen-induced platelet aggregation, with an IC₅₀ of approximately 200 nM. The genomic DNA encoding the -bungarotoxin precursor is amplified by polymerase chain reaction (PCR). The gene is organized with three exons separated by two introns, and shares virtually identical overall organization with those reported for -neurotoxin and cardiotoxin genes, including similar intron insertions. The intron sequences of these genes share sequence identity up to 85%, but the exon sequences are highly variable. These observations suggest that -bungarotoxin, -neurotoxins, and cardiotoxins originate from a common ancestor, and the evolution of these genes shows a tendency to diversify the functions of snake venom proteins.     

Muscarinic toxin-like proteins from Taiwan banded krait (Bungarus multicinctus) venom: purification,characterization and gene organization

Two novel proteins, BM8 and BM14, were isolated from Bungarus multicinctus (Taiwan banded krait) venom using the combination of chromatography on a SP-Sephadex C-25 column and a reverse-phase HPLC column. Both proteins contained 82 amino acid residues including 10 cysteine residues, but there were two amino acid substitutions at positions 37 and 38 (Glu37-Ala38 in BM8; Lys37-Lys38 in BM14). CD spectra and acrylamide quenching studies revealed that the gross conformation of BM8 and BM14 differed. In contrast to BM8, BM14 inhibited the binding of [3H]quinuclidinyl benzilate to the M2 muscarinic acetylcholine (mAchR) receptor subtype. Trinitrophenylation of Lys residues abolished the mAchR-binding activity of BM14, indicating that the Lys substitutions at positions 37 and 38 played a crucial role in the activity of BM14. The genomic DNA encoding the precursor of BM14 was amplified by PCR. The gene shared virtually identical structural organization with alpha-neurotoxin and cardiotoxin genes. The intron sequences of these genes shared a sequence identity up to 84%, but the protein-coding regions were highly variable. These results suggest that BM8, BM14, neurotoxins and cardiotoxins may have originated from a common ancestor, and the evolution of snake venom proteins shows a tendency to diversify their functions.     

Primary structure of an inactive mutant of phospholipase A2 in the venom of Bungarus fasciatus (banded krait).

From the acidic components of Bungarus fasciatus venom, a very small amount (0.16%) of a novel phospholipase A2 was obtained. Both neurotoxicity and enzyme activity were found to be lacking. Amino acid sequence study showed that it has a normal backbone of group I snake venom phospholipase A2 with 118 amino acid residues. The lack of enzyme activity was attributed to its mutation of the indispensable Asp residue to an Ala residue, i.e., the usual His-Asp47 turned out to be His-Ala47. This is the eighth isoform of phospholipase A2 found from the venom of Bungarus fasciatus. Examination of structural homology with three other isoforms revealed 66% similarity at most.     

Chemical properties and amino acid composition of beta1-bungarotoxin from the venom of Bungarus multicinctus (Formosan banded krait)

beta-Bungarotoxin purified from the venom of Bungarus multicinctus (Formosan banded krait) contained no carbohydrate and behaved as a homogeneous protein on polyacrylamide gel electrophoresis at pH 4.1 and SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol treatment. Its molecular weight and isoelectric point were estimated to be about 21,000 by gel filtration and about 9.5 by isoelectric focusing, respectively. The toxin treated with the reducing agent was split into two polypeptide chains as revealed by SDS-polyacrylamide gel electrophoresis and their molecular weights were calculated to be about 13,000 and 7,000. The two polypeptide chains (the large one named the A chain and the small one the B chain) were isolated by gel filtration after reduction of disulfide bonds in the toxin followed by alkylation. The A chain contained 120 amino acid residues including 13 half-cystines and the B chain 60 residues including 7 half-cystines. The two chains were supposed to link by disulfide bond(s) in the intact toxin which contained no free sulfhydryl groups. The N-terminal residues of the A and B chains were asparagine and arginine and the C-terminal ones were glutamine and proline, respectively, in accordance with the results of the terminal analyses of the intact toxin.     

Therapeutic application of natural inhibitors against snake venom phospholipase A(2)

Natural inhibitors occupy an important place in the potential to neutralize the toxic effects caused by snake venom proteins and enzymes. It has been well recognized for several years that animal sera, some of the plant and marine extracts are the most potent in neutralizing snake venom phospholipase A(2) (svPLA(2)). The implication of this review to update the latest research work which has been accomplished with svPLA(2) inhibitors from various natural sources like animal, marine organisms presents a compilation of research in this field over the past decade and revisiting the previous research report including those found in plants. In addition to that the bioactive compounds/inhibitor molecules from diverse sources like aristolochic alkaloid, flavonoids and neoflavonoids from plants, hydrocarbones -2, 4 dimethyl hexane, 2 methylnonane, and 2, 6 dimethyl heptane obtained from traditional medicinal plants Tragia involucrata (Euphorbiaceae) member of natural products involved for the inhibitory potential of phospholipase A(2) (PLA(2)) enzymes in vitro and also decrease both oedema induced by snake venom as well as human synovial fluid PLA(2). Besides marine natural products that inhibit PLA(2) are manoalide and its derivatives such as scalaradial and related compounds, pseudopterosins and vidalols, tetracylne from synthetic chemicals etc. There is an overview of the role of PLA(2) in inflammation that provides a rationale for seeking inhibitors of PLA(2) as anti-inflammatory agents. However, more studies should be considered to evaluate antivenom efficiency of sera and other agents against a variety of snake venoms found in various parts of the world. The implications of these new groups of svPLA(2) toxin inhibitors in the context of our current understanding of snake biology as well as in the development of new novel antivenoms therapeutics agents in the efficient treatment of snake envenomations are discussed.     

Isolation and enzymatic characterization of a basic phospholipase A2 from Bothrops jararacussu snake venom

A novel basic phospholipase A₂ (PLA2) isoform was isolated from Bothrops jararacussu snake venom and partially characterized. The venom was fractionated by HPLC ion-exchange chromatography in ammonium bicarbonate buffer, followed by reverse-phase HPLC to yield the protein Bj IV. Tricine SDS-PAGE in the presence or absence of dithiothreitol showed that Bj IV had a molecular mass of 15 and 30 kDa, respectively. This enzyme was able to form multimeric complexes (30, 45, and 60 kDa). Amino acid analysis showed a high content of hydrophobic and basic amino acids as well as 14 half-cysteine residues. The N-terminal sequence (DLWSWGQMIQETGLLPSYTTY . . .) showed a high degree of homology with basic D49 PLA₂ myotoxins from other Bothrops venoms. Bj IV had high PLA₂ activity and produced moderate myonecrosis in skeletal muscle, but showed no neuromuscular activity in mouse phrenic nerve-diaphragm preparations. Bj IV showed allosteric enzymatic behavior, with maximal activity at pH 8.2 and 35-45°C. Full PLA₂ activity required Ca²⁺ but was inhibited by Cu²⁺ and Zn²⁺, and by Cu²⁺ and Mg²⁺ in the presence and absence of Ca²⁺, respectively. Crotapotins from Crotalus durissus terrificus rattlesnake venom significantly inhibited the enzymatic activity of Bj IV. The latter observation suggested that the binding site for crotapotin in this PLA₂ was similar to that in the basic PLA₂ of the crotoxin complex from C. d. terrificus venom. The presence of crotapotin-like proteins capable of inhibiting the catalytic activity of D49 PLA₂ could partly explain the low PLA₂ activity of Bothrops venoms.     

日本蝮蛇蛇毒磷脂酶A2(Gln49-PLA2)的细胞毒性

从日本蝮蛇(Agkistrodonblomhoffiiussurens)蛇毒中分离得到PLA2同源物Gln49PLA2,该蛋白具有抗凝血活性、肌肉毒性,缺乏磷脂酶A2活性。分析了该蛋白质对不同培养细胞生长的影响,探讨其细胞毒性。结果表明:贴壁细胞的Gln49PLA2半致死量(LD50)明显低于悬浮细胞,肝素可以明显抑制Gln49PLA2的细胞毒性。将其作用于K562细胞,随着Gln49PLA2用量的增加乳酸脱氢酶(LDH)释放量增加,细胞凋亡率增大,线粒体膜电位下降,但Bcl2蛋白的表达量无明显变化。     

Studies on the status of lysine residues in phospholipase A2 from Naja naja atra (Taiwan cobra) snake venom.

Phospholipase A2 (PLA2) from Naja naja atra (Taiwan cobra) snake venom was subjected to lysine modification with trinitrobenzene sulphonic acid (TNBS), and two major trinitrophenylated (TNP) derivatives, TNP-1 and TNP-2, were separated by h.p.l.c. TNP-1 contained only one TNP group on Lys-6 and showed a marked decrease in enzymic activity, but still retained 45% of the lethal toxicity. Both Lys-6 and Lys-65 were modified in TNP-2, and modification of Lys-65 caused a further reduction of the lethal toxicity to 12.6%. However, the antigenicity of both TNP-1 and TNP-2 remained unchanged. The reactivity of Lys-6 and Lys-65 toward TNBS was greatly enhanced by Ca2+ and dihexanoyl-lecithin, suggesting that the two Lys residues are not directly involved in the binding of Ca2+ and substrate. The modified derivatives retained their affinity for Ca2+, indicating that Lys-6 and Lys-65 did not participate in the Ca2+ binding. The TNP derivatives could be regenerated with hydrazine hydrochloride. The biological activities of the regenerated PLA2 are almost the same as those of native PLA2. These results indicate that Lys-6 and Lys-65 are important for the biological activities of PLA2, and incorporation of a bulky TNP group on Lys-6 and Lys-65 might give rise to a distortion of the active conformation of PLA2.     

Structural analysis of trimeric phospholipase A(2) neurotoxin from the Australian taipan snake venom

Snake pre-synaptic neurotoxins endowed with phospholipase A(2) activity are potent inducers of paralysis through the specific disruption of the neuromuscular junction pre-synaptic membrane and represent a valuable tool for investigating neuronal degeneration and recovery. They have different structural complexity and a wide range of lethal potency and enzymatic activity, although they share a similar mechanism of action. Although no correlation has been reported between neurotoxicity and enzymatic activity, toxicity increases with structural complexity and phospholipase A(2) oligomers show 10-fold lower LD(50) values compared to their monomeric counterparts. To date, no structural study has been performed on multimeric SPANs with the aim of shedding light on the correlation between structural complexity and neurotoxicity. In the present study, we investigated the structure of taipoxin, a trimeric phospholipase A(2) neurotoxin, as well as that of its subunits, by X-ray crystallography and small angle X-ray scattering analysis. We present the high-resolution structure of two isoforms of the taipoxin β subunit, which show no neurotoxic activity but enhance the activity of the other subunits in the complex. One isoform shows no structural change that could justify the lack of activity. The other displays three point mutations in critical positions for the catalytic activity. Moreover, we designed a model for the quaternary structure of taipoxin under physiological conditions, in which the three subunits are organized into a flat holotoxin with the substrate binding sockets exposed on the same side of the complex, which suggests a role for this interface in the toxin-membrane interaction. Database The coordinates and structure factors have been deposited in the RCSB Protein Data Bank (http://www.rcsb.org) under accession numbers 3VBZ and 3VC0, corresponding to β isoforms 1 and 2 respectively Structure digital abstract ? taipoxin beta isoform 2 and?taipoxin beta isoform 2?bind?by?x-ray crystallography?(View interaction).     

Purification of a nontoxic phospholipase A2 from the venom of Indian krait (Bungarus caeruleus).

A nontoxic phospholipase A2 was purified from the venom of Indian krait (Bungarus caeruleus) by a four-step procedure involving electrophoresis, gel filtration and ion-exchange chromatography. The recovery of the enzyme activity was 37% and the purified preparation was 38 times as active as the crude venom. The purified enzyme had a molecular weight of 12,500 and the optimum pH of 7.2. The enzyme showed higher specificity toward phosphatidylethanolamine than phosphatidylcholine. The preparation was not very labile to heat and its activity was dependent on the presence of divalent cations, calcium ions being the most effective activators. The enzyme was completely inhibited by iodoacetic acid but showed high stability against 8 M urea. Purified phospholipase A2 was nontoxic at an iv dose of 5 microgram/g mouse. The high specific activity, the high yield and the nontoxic nature of the enzyme indicate that the major form of phospholipase A2 in Bungarus caeruleus venom is not associated with any toxicity and has properties somewhat similar to that of phospholipase A2 from some other venoms.     

Role of the N-terminal region of the A chain in beta 1-bungarotoxin from the venom of Bungarus multicinctus (Taiwan-banded krait)

The N-terminal -amino groups of ₁-bungarotoxin (₁-Bgt) fromBungarus multicinctus venom were modified with trinitrobenzene sulfonic acid and the modified derivative was separated by high performance liquid chromatography. The trinitrophenylated (TNP) derivative contained two TNP groups at the -amino groups of A chain and B chain and showed a marked decrease in enzymatic activity. Methionine residues at positions 6 and 8 of the A chain were oxidized with chloramine T or cleaved with cyanogen bromide to remove the N-terminal octapeptide. Oxidation of methionine residues and removal of the N-terminal octapeptide caused a precipitous decrease in enzymatic activity, whereas antigenicity remained unchanged. The presence of dihexanoyllecithin influenced the interaction between ₁-Bgt and 8-antilinonaphthalene sulfonate (ANS) and revealed that ₁-Bgt consists of two types of ANS-binding sites, one at the substrate binding site of the A chain and the other might be at the B chain. The modified derivatives still retained their affinity for Ca²⁺ and ANS, indicating that the N-terminal region is not involved in Ca²⁺ and substrate binding. A fluorescence study revealed that the -amino group of the A chain was in the vicinity of substrate binding site and that the TNP -amino groups were in proximity to Trp-19 of the A chain. In addition, the study showed that the N-terminal region is important for stabilizing the architectural environment of Trp-19. The results, together with the proposal that Trp-19 of the A chain is involved in substrate binding, suggest that the N-terminal region of the A chain plays a crucial role in maintaining a functional active site for ₁-Bgt.     

Effect of alkylation of tryptophan residues on the enzymatic and pharmacological properties of snake venom phospholipase A2

Snake venom phospholipases A2 show a remarkable degree of amino acid sequence homology yet differ markedly in enzymatic and pharmacological activities. The basic phospholipase A2 from Naja nigricollis venom has much greater lethal potency, cardiotoxicity, hemolytic and anticoagulant activity than the acidic or neutral enzymes from Naja naja atra or Hemachatus haemachatus venoms, respectively, even though it has lower enzymatic activity than the latter two enzymes. Previous studies in which we selectively modified lysine and free carboxyl groups suggested that the pharmacological and enzymatic active sites are not identical. Tryptophan residues have been suggested as being involved in substrate binding although some phospholipases have no tryptophan. We investigated the effect of alkylating the tryptophans in N. nigricollis, N. n. atra, and H. haemachatus phospholipases A2 with 2-hydroxy-5-nitrobenzyl bromide. Chemical modification caused decreases in enzymatic activity, although the extent of inactivation varied with the enzyme and with the substrate (lecithin micelles, egg yolk, heart homogenates). The specificity of the enzymes for individual phospholipid substrates was not affected. Alkylation of the tryptophans also caused decreases in lethal, hemolytic, anticoagulant, and cardiotoxic potencies, which were similar to the extents of decrease in enzymatic activity. Our results suggest that tryptophans are not specifically associated with either the enzymatic or the pharmacological active site nor are essential for either activity.     

The essentiality of B chain in stabilizing the structure of the A chain in β1-bungarotoxin fromBungarus multicinctus venom

The dynamic of Trp residue inΒ ₁-bungarotoxin (gb ₁-Bgt), the A chain ofΒ ₁-Bgt and phospholipase A₂ (PLA₂) was assessed by fluorescence measurement. Acrylamide quenching studies showed that the exposure degree of the Trp in PLA₂ is higher than the Trp inΒ ₁-Bgt. The Trp ofΒ ₁-Bgt had a higher accessibility for iodide, reflecting that the basic nature of the B chain might exert an attractive electrostatic force for iodide and increase the susceptibility of Trp in the A chain to iodide. Removal of the B chain ofΒ ₁-Bgt did not significantly affect the exposure degree of Trp in the A chain. Alternatively, the polarity of the environment around the Trp and the hydrophobic character of ANS and substrate binding sites in the separated A chain changed. Measurement of Trp fluorescence with increasing temperature showed that the stability of structure ofΒ ₁-Bgt was higher than those of the separated A chain and PLA₂. These results suggest that the B chain might interact with the A chain and stabilize the conformation of the A chain inΒ ₁-Bgt.     

Neurotoxicity and other pharmacological activities of the snake venom phospholipase A2 OS2: the N-terminal region is more important than enzymatic activity

Several snake venom secreted phospholipases A2 (sPLA2s) including OS2 exert a variety of pharmacological effects ranging from central neurotoxicity to anti-HIV activity by mechanisms that are not yet fully understood. To conclusively address the role of enzymatic activity and map the key structural elements of OS2 responsible for its pharmacological properties, we have prepared single point OS2 mutants at the catalytic site and large chimeras between OS2 and OS1, a homologous but nontoxic sPLA2. Most importantly, we found that the enzymatic activity of the active site mutant H48Q is 500-fold lower than that of the wild-type protein, while central neurotoxicity is only 16-fold lower, providing convincing evidence that catalytic activity is at most a minor factor that determines central neurotoxicity. The chimera approach has identified the N-terminal region (residues 1-22) of OS2, but not the central one (residues 58-89), as crucial for both enzymatic activity and pharmacological effects. The C-terminal region of OS2 (residues 102-119) was found to be critical for enzymatic activity, but not for central neurotoxicity and anti-HIV activity, allowing us to further dissociate enzymatic activity and pharmacological effects. Finally, direct binding studies with the C-terminal chimera, which poorly binds to phospholipids while it is still neurotoxic, led to the identification of a subset of brain N-type receptors which may be directly involved in central neurotoxicity.     

Toxicity domain in presynaptically toxic phospholipase A2 of snake venom

About 42 complete amino-acid sequences of phospholipases A2 (phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) are known, including those of 13 presynaptically toxic enzymes, but the structural features responsible for the neurotoxicity and distinguishing the toxins from the non-neurotoxic enzymes are far from being clear. In this study, we examined the charged-residue distributions and hydrophobic characteristics based on the sequence data and the predicted tertiary structure and proposed a possible toxicity domain. We found that the presynaptically toxic enzymes have three or four more basic amino-acid residues than the non-neurotoxic enzymes at positions 59, 60, 65, 70-73 and 97 or 98. These residues appear to cluster near the surface region at the N-terminal side. The cationic nature of this basic cluster in the toxin is enhanced by the alpha-amino group of the N-terminus and the dipole moment of helices 96-110 and 1-10. Moreover, these toxic-site residues are usually associated with hydrophobic regions at 1-7, 64-81 and 97-109.     

Preliminary crystallographic study of phospholipase A2 from the venom of Trimeresurus flavoviridis (habu snake)

Crystallization and a preliminary crystallographic study of Trimeresurus flavoviridis (habu snake) phospholipase A2 (PLA2) were carried out. Although crystals were obtained from various solutions, crystals suitable for X-ray analysis could be obtained from polyethylene glycol solutions only when a repeated seeding technique was applied starting from twinned crystals. The crystal is monoclinic with space group P21, with a = 44.1, b = 55.7, c = 48.8 A, and beta = 92.4 degrees. An asymmetric unit contains a dimer consisting of two identical subunits made of 122 amino acids. The crystal reflects X-rays beyond 2.5 A. A Pt derivative gave a good isomorphous crystal.     

Genetic organization of alpha-bungarotoxins from Bungarus multicinctus (Taiwan banded krait): evidence showing that the production of alpha-bungarotoxin isotoxins is not derived from edited mRNAs.

Two genomic DNAs with a size of approximately 2.8 kb, isolated from the liver of Bungarus multicinctus (Taiwan banded krait), encode the precursors of the long neurotoxins, alpha-Bgt(A31) and alpha-Bgt(V31), respectively. Both genes share virtually identical overall organization with three exons separated by two introns, which were inserted in the same positions in the coding regions of the genes. Moreover, their nucleotide sequences share approximately 98% identity. This result indicates that the two genes co-exist in the genome of B.multicinctus, and probably arose from gene duplication. The exon/intron structures of the alpha-Bgt genes were essentially the same as those reported for the short neurotoxins. This reflects that the long and short neurotoxins should share a common evolutionary origin. Comparative analyses on long neurotoxin and short neurotoxin genes showed that the protein coding regions of the exons were more diverse than the introns except for the signal peptide domain. This implies that the protein coding regions of the neurotoxins may have evolved via accelerated evolution. PCR amplification of venom gland cDNA mixtures revealed that only two amino acid sequences corresponding to alpha-Bgt(A31) and alpha-Bgt(V31) could be deduced from the cDNAs. The results of chromatographic analyses and protein sequencing again emphasized the view that, with the exception of alpha-Bgt(A31) and alpha-Bgt(V31), no other alpha-Bgt isotoxins with amino acid substitutions were present in B.multicinctus venom. In contrast to the proposition of Liu et al. ( Nucleic Acids Res., 1998,26, 5624-5629), our findings strongly suggest that each alpha-Bgt isotoxin is derived from the respective gene, and that alpha-Bgt RNA polymorphism does not originate from one single, intronless gene by the mechanism of RNA editing.