Acetylcholinesterase from a Non-membrane Source (Bungarus fasciatus Venom)

Abstract

Acetylcholinesterase from Bungarus fasciatus venom has been purified by a conventional procedure with a specific activity of 230 millimoles acetylcholine hydrolysed mg protein^?1 hr^?1. The enzyme with a molecular weight of 126, 000 has an excess of acidic amino acids over basic amino acids. The N-terminal amino acid analysis gave leucine as the only N-terminal amino acid with a free amino group. There are no common antigenic sites between the B. fasciatus venom acetylcholinesterase and acetylcholinesterases from bovine erythrocytes and electric eel.     

A novel serine protease inhibitor from Bungarus fasciatus venom

By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.     

Predicted structure model of Bungarotoxin from Bungarus fasciatus snake

Snake venoms are cocktails comprising combinations of different proteins, peptides, enzymes and toxins. Snake toxins have diverse characteristics having different molecular configuration, structure and mode of action. Many toxins derived from snake venom have distinct pharmacological activities. Venom from Bungarus fasciatus (commonly known as banded krait) is a species of elapid snake found on the South East Asia and Indian sub-continent, mainly contains neurotoxins. Beta bungartotoxin is the major fraction of Bungarus venom and particularly act pre-synaptically by obstructing neurotransmitter release. This toxin in other snake species functionally forms a heterodimer containing two different subunits (A and B). Dimerization of these two chains is a pre-requisite for the proper functionality of this protein. However, B. fasciatus bungartotoxin contains only B chain and their structural orientation in yet to be resolved. Therefore, it is of interest to describe the predicted structure model of the toxin for functional insights. In this work we analyzed the neurotoxic nature, their alignments, secondary and three dimensional structures, functions, active sites and stability with the help of different bioinformatical tools. A comprehensive analysis of the predicted model provides approaching to the functional interpretation of its molecular action.     

A bactericidal homodimeric phospholipases A2 from Bungarus fasciatus venom

Group IIA secretory phospholipases A(2) (sPLA(2)-II) is generally known to display potent gram-positive bactericidal activity, while group IA sPLA(2) (sPLA(2)-I) reportedly is not. In this work, a novel sPLA(2)-I named BFPA was identified from Bungarus fasciatus venom, and its antimicrobial activity was studied as well. The amino acid sequence of the venomous protein precursor was 145-amino acid in length, and contained a predicted 27-amino acid signal peptide and a 118-amino acid mature protein. Unlike the well-known sPLA(2)-Is, which have 14 half-cysteines forming 7 intramolecular disulfide bridges, BFPA possesses 15 half-cysteines. The additional cysteine might contribute to the formation of an intermolecular disulfide bridge of the homodimeric protein. In the biological activities assays, BFPA displayed the activities of anticoagulation and bactericidal against Escherichia coli and Staphylococcus aureus. This study is the first report about gram-positive bactericidal activity of sPLA(2)-I.     

Solution structure of a Kunitz-type chymotrypsin inhibitor isolated from the elapid snake Bungarus fasciatus

Bungarus fasciatus fraction IX (BF9), a chymotrypsin inhibitor, consists of 65 amino acid residues with three disulfide bridges. It was isolated from the snake venom of B. fasciatus by ion-exchange chromatography and belongs to the bovine pancreatic trypsin inhibitor (BPTI)-like superfamily. It showed a dissociation constant of 5.8 x 10(-8) m with alpha-chymotrypsin as measured by a BIAcore binding assay system. The isothermal titration calorimetry revealed a 1:1 binding stoichiometry between this inhibitor and chymotrypsin and apparently no binding with trypsin. We further used CD and NMR to determine the solution structure of this venom-derived chymotrypsin inhibitor. The three-dimensional NMR solution structures of BF9 were determined on the basis of 582 restraints by simulated annealing and energy minimization calculations. The final set of 10 NMR structures was well defined, with average root mean square deviations of 0.47 A for the backbone atoms in the secondary structure regions and 0.86 A for residues The side chains of Phe(23), Tyr(24), Tyr(25), Phe(35), and Phe(47) exhibited many long-range nuclear Overhauser effects and were the principal components of the hydrophobic core in BF9. To gain insight into the structure-function relationships among proteins in the BPTI-like superfamily, we compared the three-dimensional structure of BF9 with three BPTI-like proteins that possess distinct biological functions. These proteins possessed similar secondary structure elements, but the loop regions and beta-turn were different from one another. Based on residues at the functional site of each protein, we suggest that the flexibility, rigidity, and variations of the amino acid residues in both the loop and beta-turn regions are related to their biological functions.     

Purification and properties of the soluble NAD glycohydrolase from Bungarus fasciatus venom

The NAD glycohydrolase (NADase) (EC 3.2.2.5) from Bungarus fasciatus (banded krait) venom was purified (1000-fold) to electrophoretic homogeneity through a 3-step purification procedure, the last step being affinity chromatography on Cibacron blue agarose. The purified NADase is a glycoprotein containing two subunits of Mr = 62,000 each. Nicotinamide and adenosine diphosphoribose were produced in a 1:1 stoichiometry and were the only products formed when the purified NADase was incubated with NAD. These results were confirmed by high performance liquid chromatography. The enzyme exhibited a brod pH profile with optimum pH for hydrolysis at 7.5 with very little change in Km from pH 6.0 to pH 8.5. The NADase is only slightly affected by changes in ionic strength. The enzyme studied titrimetrically at pH 7.5 and 38 degrees C exhibited a Km of 14 microM and a Vmax of 1380 mumol of NAD cleaved/min/mg of protein. The activation energy for the enzyme-catalyzed hydrolysis of NAD was 15.7 kcal/mol. In addition to NAD and NADP, a number of NAD analogs were shown to function as substrates for the enzyme. Product inhibition studies demonstrated nicotinamide to be a noncompetitive inhibitor with a KI of 1.5 mM and adenosine diphosphoribose a competitive inhibitor with a KI of 0.36 mM. Procion blue HB (Cibacron blue F3GA) was shown to be a competitive inhibitor with a KI of 33 nmol. The purified NADase catalyzed the pyridine base exchange reaction between 3-acetylpyridine and the nicotinamide moiety of NAD.     

Thermal stability of acetylcholinesterase from Bungarus fasciatus venom as investigated by capillary electrophoresis

Previous studies on the conformation of the monomeric acetylcholinesterase (AChE) from the krait (Bungarus fasciatus) venom showed that the protein possesses a large permanent dipole moment. These studies predicted that thermal irreversible denaturation must occur via partially unfolded states. The thermal stability of Bungarus AChE was determined using capillary electrophoresis (CE) with optimized conditions. Runs performed at convenient temperature scanning rates provided evidence for an irreversible denaturation process according to the Lumry and Eyring model. The mid-transition temperature, T(m), and the effective enthalpy change, DeltaH(m) were determined at different pH. The temperature dependence of the free energy, DeltaG, of Bungarus AChE unfolding was drawn using values of T(m), DeltaH(m) and DeltaC(p) determined by CE. The thermodynamic parameters for the thermal denaturation of the monomeric snake enzyme were compared with those of different dimeric and tetrameric ChEs. It was shown that the changes in the ratio of DeltaH(cal/)DeltaH(vH) and DeltaC(p) reflect the oligomerization state of these proteins. All these results indicate that wild-type monomeric Bungarus AChE is a stable enzyme under standard conditions. However, designed mutants of this enzyme capable of degrading organophosphates have to be engineered to enhance their thermostability.     

两个金环蛇蛇毒磷脂酶A2基因的克隆与序列报道

从广西产金环蛇（Bungarus fasciatus)毒腺中抽提总RNA,经mRNA纯化后构建了金环蛇毒腺cDNA文库,根据已发表的眼镜蛇科蛇毒磷脂酶A2基因序列中的保守区设计探针筛选克隆,得到2个磷脂酶A2基因,测定两者序列,其cDNA的阅读框均为435bp,编码145个氨基酸的磷脂酶A2前体,其中包括27个氨基酸组成的信号肽,118个氨基酸组成的成熟蛋白质,两者均属于第一类磷脂酶A2,其等电点经计算机软件推算分别为7．96和7．95。根据序列比较分析,这2个磷脂酶A2基因所编码的蛋白质序列结构均区别于已报道的金环蛇蛇毒磷脂酶A2,是2个新的金环蛇蛇毒磷酯酶A2,分别命名为金环蛇蛇毒磷脂酶A2Ⅰ（Bf-PLA2I)和金环蛇磷脂酶A2Ⅱ（Bf-PLA2Ⅱ)。     

Sepharose 4B一步法对金环蛇蛇毒磷脂酶A2的分离纯化

利用经酸处理的Sepharose 4B为层析介质,以含0.2mol/L半乳糖,pH7.4台氏液作为洗脱液,从广西产金环蛇(Bungarus fasciatus)蛇毒中一步分离得到一种磷脂酶A2.用SDS-聚丙烯酰胺凝胶电泳测定其分子量为14 kDa.N端部分序列测定表明,所分离得到的磷脂酶A2其N端16个氨基酸残基序列与已报道的金环蛇蛇毒磷脂酶A2同功酶Ⅵ(Lu & Lo,1978)一致.该酶糖含量较高,为13.4%;具有弱的磷脂酶A2活性,无毒,也无溶血和出血毒活性.     

Some actions of purified toxins from Bungarus fasciatus venom on isolated pig-heart mitochondria.

I. In the presence of succinate as an oxidation substrate, neurotoxins alpha, beta and gamma induce the following. Firstly, an increasing stimulation of oxygen uptake, which in potentiated by 25 muM Ca2+, Mg2+ 1.3 mM completely inhibits the effect of toxin alpha but not of toxins beta and gamma. Secondly, a depletion of the Mg2+, Ca2+ and K+ content of the water-soluble and membrane-bound mitochondrial compartments, following complex kinetics, which suggest a multistep interaction mechanism of the toxins with the mitochondria. 25 muM Ca2+ also potentiates the effect of the toxins on these ionic flows. Thirdly, no decrease of turbidity with toxin alpha, and a limited decrease with toxins beta and gamma. 2. In the absence of respiration, the neurotoxins induce a cationic depletion, the kinetics of which are different than with succinate, suggesting an instantaneous maximal effect on the inner membrane. Toxins beta and gamma (but not alpha) induce, under these conditions, a turbidity decrease of large amplitude, which is proportional to the amount of toxin added and tends to reach a maximum. With gamma toxin this turbidity decrease is faster than the rate of water uptake (which never exceeds 18%) indicating that it is due rather to structural modifications than to swelling. The same is observed with beta toxin, provided the mitochondrial protein concentration to be lower than 0.7 mg/ml. For higher concentrations, a continuous decrease of turbidity with a considerable uptake of water probably reflects the onset of phospholipasic activities. 3. It is postulated that structural modifications of the mitochondrial membranes are initiated which lead to the loss of their selective impermeability. The simultaneous loss of respiratory control with succinate may be due to the direct (though Ca2+-potentiated) displacement of the fraction of the membrane-bound Mg2+ ions which controls its energy-transducing properties. 4. In addition, correlations between the effects of the toxins on mitochondria and their neurotoxicity in vivo are discussed.     

Amino acid sequence of a short chain neurotoxin from the venom of banded krait (Bungarus fasciatus)

The amino acid sequence of a short chain neurotoxin obtained from Bungarus fasciatus venom consists of 64 amino acid residues: Arg-Ile-Cys-Leu-Asn-Gln-Gln-Gln-Ser- Thr-Pro-Glu-Asp-Gln-Pro-Thr-Asn-Gly-Gln-Cys-Tyr-Ile-Lys-Thr-Asp-Cys-Gln- Asn-Lys - Thr-Trp-Asn-Thr-His-Arg-Gly-Ser-Arg-Thr-Asp-Arg-Gly-Cys-Gly-Cys-Pro-Lys- Val-Lys - Pro-Gly-Ile-Asn-Leu-Arg-Cys-Cys-Lys-Thr-Asp-Lys-Cys-Asn-Glu. The above result was obtained primarily from the amino acid analyses and sequencing of tryptic peptides accompanied with the necessary analyses and sequencing of the chymotryptic and lysyl endopeptidic peptides for alignment.     

The synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met is a potent chemotactic agonist for mouse formyl peptide receptor

Formyl peptides are potent neutrophil chemoattractants. In humans and rabbits, the formyl peptide receptor (FPR) binds N-formyl-Met-Leu-Phe (fMLF) with high affinity (K(d) approximately 1 nM). The mouse FPR (mFPR) is a low-affinity receptor for fMLF (K(d) approximately 100 nM); therefore, other agonists for this receptor may exist. Using mFPR-transfected rat basophilic leukemia cells, we found that a recently identified synthetic peptide Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) is a potent agonist for mFPR. WKYMVm induced calcium mobilization with an EC(50) of 1.2-1.5 nM. Optimal chemotaxis was achieved with 1 nM of WKYMVm, but it required 100 nM of fMLF. WKYMVm stimulated rapid and potent phosphorylation of the mitogen-activated protein kinases extracellular signal-related kinases 1 and 2 when used at 50 nM. Pertussis toxin only partially blocked calcium mobilization and production of inositol 1,4,5-trisphosphate in the stimulated mFPR cells, suggesting the possibility that this receptor couples to Galpha proteins other than Gi and Go. Competitive binding and desensitization data suggest that both peptides interact with the same receptor but may use nonoverlapping binding sites because WKYMVm was unable to effectively displace [(3)H]fMLF bound to mFPR. These results provide evidence for the presence of an alternative potent agonist for mFPR, and suggest a potential usage of WKYMVm for probing the ligand-receptor interactions with the murine formyl peptide receptor homologs.     

The complete amino-acid sequence of a basic phospholipase A2 in the venom of Bungarus fasciatus

A basic phospholipase A2 (PLA2) was isolated and purified from the venom of Bungarus fasciatus. Four kinds of enzymes, lysyl endopeptidase, endoproteinase Asp-N, endoproteinase Glu-C and trypsin, were employed to elucidate the complete primary structure by means of gas-phase sequencing. The amino-acid sequence reveals 118 amino-acid residues containing seven pairs of half-cystine. It has 78% and 61% structural identities with PLA2 from Bungarus multicinctus and Naja melanoleuca DE-II, respectively.     

C-type natriuretic peptide is a potent activator of guanylate cyclase in endothelial cells from brain microvessels.

An exposure of endothelial cells from rat brain microvessels to C-type natriuretic peptide (CNP) resulted in a rapid and large increase in cGMP formation. The action of CNP did not require inhibitors of phosphodiesterases to be observed and occurred at nanomolar concentrations. Other natriuretic peptides (ANP and BNP) also stimulated cGMP formation in endothelial cells from brain microvessels but with a potency that was at least 100 times less than that of CNP. In contrast, endothelial cells from the aorta showed large cGMP responses to low concentrations of ANP and BNP but were unresponsive to CNP up to concentrations as large as 100 nM. It is concluded that endothelial cells from brain microvessels and from aorta express different receptors subtypes for natriuretic peptides. Endothelial cells from brain microvessels express CNP specific ANPB receptors; aortic endothelial cells express ANP (and BNP) specific ANPA receptors. CNP may play an important role in the regulation of water and electrolyte movements across the blood brain barrier.     

Cutting edge: mast cell antimicrobial activity is mediated by expression of cathelicidin antimicrobial peptide

Cathelicidins (caths) are peptides that are expressed at high levels in neutrophils and some epithelia and can act as natural antibiotics by directly killing a wide range of microorganisms. We hypothesized that caths are expressed in mast cells (MCs), because these cells have been previously associated with inherent antimicrobial activity. Cultured murine MCs contained abundant amounts of cathelin-related antimicrobial peptide (AMP), the murine cath, and this expression was inducible by LPS or lipoteichoic acid. Human skin MCs also expressed cath as detected by immunohistochemical analysis for the human cath LL-37. The functional significance of this expression was shown by comparing MCs cultured from normal mice to MCs from littermates deficient in the cathelin-related AMP gene (Cnlp(-)). MCs derived from Cnlp(-/-) animals had a 50% reduction in their ability to kill group A STREPTOCOCCUS: These MCs expressed equivalent amounts of mRNA for murine beta-defensin-4, a beta-defensin AMP. Thus, different antimicrobials can be identified in MCs, and the presence of cath is necessary for efficient bacterial killing. These observations suggest that the presence of cath is vital to the ability of mammalian MCs to participate in antimicrobial defense.     

Production of an immunoenzymatic tracer combining a scFv and the acetylcholinesterase of Bungarus fasciatus by genetic recombination.

We constructed a plasmid containing a chimeric gene composed of the gene encoding acetylcholinesterase (AChE) from Bungarus fasciatus venom and a gene encoding a single chain antibody fragment (scFv) directed against one of the two subunits of a presynaptic neurotoxin from rattlesnake. Large quantities of the fusion protein were produced in the culture medium of transfected COS cells. Fusion to AChE did not affect the ability of the scFv to recognise its antigen. Similarly, the AChE activity was not impaired in the fusion. The fusion protein was purified from the culture medium in a single step by affinity chromatography. The immunoconjugate obtained consisted of a soluble monomeric form of AChE fused to scFv. It was monovalent and had a molecular weight of 94 kDa. The properties of this scFv-AChE fusion show that the simple, reproducible preparation of various recombinant monovalent immunoenzymatic tracers with low molecular weight is possible. In addition, in the construct presented, the scFv domain can be easily changed to another one taking advantage of the SfiI-NotI restriction sites surrounding this domain.     

Structure-activity relationships of fowlicidin-1, a cathelicidin antimicrobial peptide in chicken

Cationic antimicrobial peptides are naturally occurring antibiotics that are actively being explored as a new class of anti-infective agents. We recently identified three cathelicidin antimicrobial peptides from chicken, which have potent and broad-spectrum antibacterial activities in vitro (Xiao Y, Cai Y, Bommineni YR, Fernando SC, Prakash O, Gilliland SE & Zhang G (2006) J Biol Chem281, 2858-2867). Here we report that fowlicidin-1 mainly adopts an alpha-helical conformation with a slight kink induced by glycine close to the center, in addition to a short flexible unstructured region near the N terminus. To gain further insight into the structural requirements for function, a series of truncation and substitution mutants of fowlicidin-1 were synthesized and tested separately for their antibacterial, cytolytic and lipopolysaccharide (LPS)-binding activities. The short C-terminal helical segment after the kink, consisting of a stretch of eight amino acids (residues 16-23), was shown to be critically involved in all three functions, suggesting that this region may be required for the peptide to interact with LPS and lipid membranes and to permeabilize both prokaryotic and eukaryotic cells. We also identified a second segment, comprising three amino acids (residues 5-7) in the N-terminal flexible region, that participates in LPS binding and cytotoxicity but is less important in bacterial killing. The fowlicidin-1 analog, with deletion of the second N-terminal segment (residues 5-7), was found to retain substantial antibacterial potency with a significant reduction in cytotoxicity. Such a peptide analog may have considerable potential for development as an anti-infective agent.