Venom Of Agkistrodon Halys Pallas Research Articles

Purification and characterization of AHPM, a novel non-hemorrhagic P-IIIc metalloproteinase with α-fibrinogenolytic and platelet aggregation-inhibition activities, from Agkistrodon halys pallas venom

A novel non-hemorrhagic metalloproteinase, AHPM, was purified from the venom of Agkistrodon halys pallas by a combination of ion-exchange and gel filtration chromatography. AHPM is a dimeric glycoprotein with multiple pIs around pH 7.9 and has a molecular mass of 110kDa with two blocked N-terminuses. Partial sequence of AHPM obtained by LC-MS/MS analysis together with its dimeric nature reveals that it is a P-IIIc snake venom metalloproteinase composed of metalloproteinase, disintegrin-like and cysteine-rich domains. AHPM has a conserved DECD sequence in the disintegrin-like domain. AHPM hydrolyzes casein and fibrinogen and also dissolves fibrin clots and the proteolytic activity is abolished by EDTA, but not by PMSF, suggesting that it is a metalloproteinase. The protease hydrolyzes rapidly the Aα-chain of fibrinogen followed by the Bβ-chain and does not cleave the γ-chain. AHPM contains endogenous Zn(2+) and Ca(2+) ions at a molar ratio of 1:1.9 and 1:4.2, respectively, and Zn(2+) ions are essential for its proteolytic activity. AHPM inhibits collagen-and ADP-induced platelet aggregation with half maximal inhibitory concentrations of 200±8nM and 280±10nM, respectively. EDTA markedly attenuates the inhibition of ADP-induced platelet aggregation by AHPM, indicating that the fibrinogenolytic activity of AHPM is involved in its inhibition of ADP-induced platelet aggregation. AHPM is devoid of hemorrhagic activity when injected (up to 30μg) subcutaneously into mice. AHPM is so far identified as first non-hemorrhagic P-IIIc SVMP which has both fibrinolytic and platelet aggregation-inhibition activities. The bifunctional enzyme may have a potential clinical application as a thrombolytic agent.

Cloning of cDNAs and molecular characterisation of C-type lectin-like proteins from snake venoms

C-type lectin-like proteins (CTLPs) isolated from snake venoms are the largest and most complex non-mammalian vertebrate C-type lectin-like domain family. In the present study, we simultaneously amplified four cDNAs encoding different types of CTLP subunits from the venoms of two different species of snakes by RT-PCR with a single sense primer and a nested universal primer – two CTLP subunit-encoding cDNAs were cloned from Deinagkistrodon acutus venom and two from Agkistrodon halys Pallas venom. All four cloned CTLP subunits exhibited typical motifs in their corresponding domain regions but with relatively-low sequence similarities to each other. Compared with previously-published CTLPs, the four cloned CTLPs subunits showed slight variations in the calcium-binding sites and the disulphide bonding patterns. To our knowledge, these data constitute the first example of co-expression of CTLP platelet glycoprotein Ib-binding subunits and coagulation factors in Agkistrodon halys Pallas venom.

Acute liver injury and expression of P-selectin in serum of mice induced by agkistrodon halys pallas venom

Objective To explore the relationship between pathological changes of liver,serum ALT,expression of P-selectin in serum of mice and different dose of venom,the poisoning time induced by Agkistrodon Halys Pallas venom.Methods Using two -factor factorial design,72 male Kunming mice of clean grade were randomly ( random number) divided into 9 groups (n =8).As per the different doses of snake venom injected and different lengths of time after venom injected,the 9 groups were 0-dose (0.0 mg/kg) 3 hours after saline given group,0-dose 8 hours after saline given group,0-dose 24 hours after saline given group,low-dose (0.5 mg/kg) 3 hours after venom given group,low-dose 8 hours after venom given group,low-dose 24 hours after venom given,high-dose ( 1.0 mg/kg) 3 hours after venom given group,high-dose 8 hours after venom given group and high-dose 24 hours after venom given group.The pathological change of liver tissue was determined,and ALT and expression of P-selectin in serum were detected.Results In the venom given groups,the liver histopathologic scores,serum ALT value and expression of Pselectin in serum were significantly higher than those in control group ( P ＜ 0.05 ),and those in high-dose group were higher than those in low-dose group (P ＜ 0.05 ),and those in 8h groups were higher than those in 3h groups (P＜0.05),and those in 24 h groups were higher than those in 8 h groups (P ＜0.05).As the dose of venom increased and the length of time after venom given extended,the liver histopathologic scores,serum ALT and expression of P-selectin in serum were higher.A positive correlation between Pselectin expression and liver histopathologic scores was found after correlation analysis ( r =0.98,P ＜0.05 ).Conclusions Agkistrodon snake venom can cause acute liver injury with elevated ALT and Pselectin in mice.As higher dose of venom given and longer length of time after venom given extended,more serum ALT and P-selectin are produced.Between acute liver injury and high expression of P-selectin exists a correlation,suggesting overexpression of P-selectin is one of the mechanisms of acute liver injury induced by Agkistrodon Halys Pallas venom. Key words: Agkistrodon Halys Pallas venom; Acute liver injury; P-selectin; Alanine aminotransferase; Mouse

Interfacial activation of snake venom phospholipases A 2 ( svPLA 2) probed by molecular dynamics simulations

Asp49 plays a key role coordinating calcium in the catalytic site of phospholipases A 2 (PLA 2’s), thus assisting the stabilization of the ES intermediary transition state, which induces the enzyme activation. In this work, molecular dynamics simulations of an acidic PLA 2 from the venom of Agkistrodon halys pallas were performed in water, methanol and octanol. Because of the low affinity of svPLA 2’s for calcium ion in aqueous systems, different positional restraint forces were applied and the coordinates describing svPLA 2–Ca 2+ ligand properties were simulated and evaluated. The results of the simulations were used to propose an interfacial activation model for svPLA 2’s. In this model, events related with enzyme activation involve: (a) the reorganization of calcium binding loop at membrane proximity followed by the Ca 2+ uptake; (b) side chain reorientation of Trp31, which defines a new specificity pocket for the phospholipid chain; (c) reduction of the distance between His48 and Asp49, increasing the nucleophilicity of Nε−His48; (d) side chain reorientation of Lys69 concomitant with projection of the 69-loop to solvent. In addition to existing biochemical and crystallographic data, our results allow us to describe a more detailed model for the interfacial activation of phospholipases A 2.

Three-step Column Chromatographic Method for Separation and Purification of Thrombin-like Enzyme from Venom of Agkistrodon halys pallas

A three-step column chromatographic method, utilizing CM Sepharose CL-6B, DEAE Sepharose Fast Flow and Sephadex G-75 in sequence, for the separation and purification of thrombin-like enzyme from crude venom of Agkistrodon halys pallas was developed. Based on the separation results on lab-scale chromatographic column, this isolation and purification process was amplified according to the diameter of the chromatographic columns used in labscale. It was found that when the amount of the sample loaded on the large-scale columns was 25 times as that loaded on the lab-scale columns, the quality level of the thrombin-like enzyme obtained with the large-scale columns was almost the same as that obtained with lab-scale columns. The relative molecular mass of thrombin-like enzyme obtained was about 33500, and its purity was about 96%. When using this method to isolate thrombin-like enzyme from crude venom of Agkistrodon halys pallas, the total protein yield and total activity yield of thrombin-like enzyme were about 0.3% and 64% respectively, and specific activity was more than 2000 U/mg.

Purification, crystallization and preliminary crystallographic analysis of AHP IX-bp, a zinc ion and pH-dependent coagulation factor IX binding protein from Agkistrodon halys Pallas venom.

A new coagulation factor IX binding protein, AHP IX-bp, has been purified from Agkistrodon halys Pallas venom and estimated to be an AB heterodimer of about 25 kDa consisting of two chains (an A chain of 15.5 kDa and a B chain of 15 kDa) linked by one or more disulfide bonds. The N-terminal sequence of AHP IX-bp has been determined and aligned with C-type lectin-like proteins. The protein has a high sequence similarity to some snake-venom C-type lectin-like proteins. AHP IX-bp binds to coagulation factor IX but not to coagulation factor X. Moreover, AHP IX-bp shows binding to coagulation factor IX in both zinc ion-dependent and pH-dependent manners. The affinity between AHP IX-bp and coagulation factor IX is higher under neutral or weakly alkaline conditions than under weakly acidic conditions. Single crystals of AHP IX-bp grown at pH 6.5 and 7.5 diffract X-rays to 2.0 and 1.8 A resolution, respectively. Both crystals are isomorphous and belong to the space group P1; only one AB heterodimer is present in the unit cell.

The relationship between biological activity and the electronic structure and transfer of the whole acidic PLA 2 molecule in ab initio level

The electronic structure of the whole molecule of acidic phospholipase A 2 (PLA 2) from the venom of Agkistrodon halys pallas ( A. halys pallas) has been calculated using the extended negative factor counting (ENFC) method in which dimers were calculated at the ab initio level using a minimal basis set, with simulation of the aqueous environment. Hopping conductivities were determined by the use of random walk theory. The results show that the frontier orbitals are mainly localized to residues which are involved in the biological activity of acidic PLA 2. The C-terminal region might play some important role in biological activity because of its active electrons. The aromatic patch on the surface of the enzyme, together with two neighbouring acidic residues, has very active electrons that may be responsible for the inhibition of platelet aggregation. Trp30, which is involved in the interfacial recognition region, may transfer its electrons to the aggregated substrate. It is also concluded that the conductivity of the protein is caused mainly by holes transported through the valence band rather than electrons transferred in the conductive band. The a.c. conductivity of acidic PLA 2 confirms that proteins, if doped, are amorphous conductors. Moreover, the a.c. conductivities of acidic PLA 2 are approximately one order of magnitude higher than those of some other proteins. This suggests that the toxicity of acidic PLA 2 may be related to its high a.c. conductivity.

Diversity of cDNAs encoding phospholipase A 2 from Agkistrodon halys Pallas venom, and its expression in E. coli

As a step toward understanding the structure and function of phospholipase A 2(PLA 2), we isolated several novel cDNAs encoding Agkistrodon halys Pallas PLA 2 isoenzymes including B-PLA 2, Asn 49-PLA 2, A-PLA 2, A′-PLA 2 and BA 1-PLA 2 by polymerase chain reaction with oligonucleotide primers corresponding to the N- and C-terminus of these enzymes. The amino acid sequences of A-PLA 2 deduced from cDNA are consistent with that isolated from venom except for four residues. Asn 49–PLA 2 and B-PLA 2 are highly similar (>95%), but the critical residue Asp 49 in the active centre of B-PLA 2 is replaced by Asn 49 in Asn 49–PLA 2. The N-terminal residues (1–24) of BA1-PLA 2 shows high similarity to that of B-PLA 2 which has strong ability to hemolyze erythrocytes, while its C-terminal residues (72–125) are the same as that of A-PLA 2 which can inhibit platelet aggregation. The successful cloning of these isoenzymes not only provide excellent native material to study the structure-function relationship of PLA 2s, but also to disclose the genesis of structural diversity of PLA 2s, namely DNA modification and gene rearrangement. The cloned cDNA for A-PLA 2 has been expressed in E. coli. By Q-Sepharose column chromatography, denaturation–renaturation and FPLC, we obtained the active recombinant protein with the initiator Met. This is the first report of the production of an active recombinant PLA 2 with the initiator Met.

Molecular Cloning of a Cdna Encoding a Nerve Growth Factor Precursor from the Agkistrodon Halys Pallas

NGF cDNAs from many sources have been cloned. Here we report a new practical method for cloning a nearly full-length NGF cDNA. Due to the high conservation in the 5’ and 3’ untranslated regions, oligonucleotides complementary to all sequences coding for them were synthesized. Total RNA was extracted from the Agkistrodon halys Pallas venom gland. Accordingly, the first strand of cDNA was synthesized and used as template. The RT-PCR amplified DNA manifested as a single band of predicted size after electrophoresis.Nucleotide sequence analysis revealed an open reading frame of 726 bases encoding a 242 amino acid polypeptide, which includes Agkistrodon halys Pallas prepro-NGF, and whose sequence shows a high similarity to the prepro-NGF of cobra and krait Bungarus multicinctus. Comparison with known sequences shows that the 5’ and 3’ untranslated sequences show slightly higher conservation than the translated region. The prepro part of the precursor is less conserved. The Agkistrodon cDNA possesses the presumptive 18 residue amino terminal signal sequence. It also has two long highly conserved stretches, corresponding to functional domains, alternating with large divergent regions. Comparison of the mature NGF amino acid sequence revealed a high degree of sequence conservation of the structurally important residues. It suggested that the same overall three-dimension structure is adopted. The presence of aspartate introduces a potential N-glycosylation site, which doesn't exist in cobra NGF.Additionally, because the major region of prepro NGF is encoded in one exon, human and mouse genomic DNA, used as a template for amplificating NGF cDNA, also proved to be competent. It revealed the feasibility of isolating and amplifying unknown genes using genomic DNA as a template for PCR.

Isolation, characterization and crystallization of a phospholipase A 2 myotoxin from the venom of the prairie rattlesnake ( Crotalus viridis viridis)

A myotoxin with phospholipase A 2 (PLA 2) activity was isolated from the venom of the prairie rattlesnake ( Crotalus viridis viridis, CVV) by cation-exchange chromatography. The toxin contains 123 amino acids and has an estimated mol. wt of 14,000. It is basic, with a p I above 9. Comparison of the N-terminal 33 residues of this myotoxin with other PLA 2 proteins from snake venoms showed that CVV myotoxin has highest homology (91%) to one isoform of the B component of crotoxin from Crotalus durissus terrificus venom, and less homology (73–75%) to mojave toxin from Crotalus scutulatus scutulatus venom and agkistrotoxin from Agkistrodon halys Pallas venom. It has the least homology (40–43%) to PLA 2s from venom of two other snakes in the Crotalus genus which are neither neurotoxic nor myotoxic. CVV myotoxin induces the type of myonecrosis typical of snake venom myotoxins with the PLA 2 structure, i.e. rapid disruption of the plasma membrane as indicated by the presence of delta lesions, hypercontraction and clumping of the myofilaments, and necrosis of affected skeletal muscle cells. Inhibition of the phospholipase activity of the toxin with p-bromophenacyl bromide inhibits the myotoxic activity, indicating that for some myotoxins with the PLA 2 structure, the catalytic activity is important for myotoxic activity. This is the first report of the isolation of a non-neurotoxic, single-chain PLA 2 myotoxin from the venom of a snake from the Crotalus genus.

Characterization of the structure and function of three phospholipases A 2 from the venom of Agkistrodon halys pallas

Y.-C. Chen, J. M. Maraganore, I. Reardon and R. L. Heinrikson. Characterization of the structure and function of three phospholipases A 2 from the venom of Agkistrodon halys pallas. Toxicon 25, 401 – 409. 1987.—Three monomeric phospholipases A 2 with isoelectric points 4.5, 6.9 and 9.3 were purified from the venom of Agkistrodon halys pallas. The complete amino acid sequence of the acidic enzyme and partial amino acid sequences of the neutral and basic phospholipases were determined in order to relate differences in enzymatic reactivities, pharmacologic activities and cytotoxicities to aspects of structure. Studies reported here and elsewhere demonstrate that the three phospholipases A 2 exhibit pronounced differences relative to function. The acidic enzyme maintains the highest reactivity toward hydrolysis of monolayers at the air — water interface and may share a feature in common with the acidic enzyme from A. h. blomhoffii, namely the inhibition of platelet aggregation. The neutral phospholipase A 2 designated agkistrotoxin, is characterized by potent activity as a pre-synaptic neurotoxin. Agkistrotoxin is the first single polypeptide chain, neurotoxic phospholipase A 2 to be documented with a Group II disulfide pattern and, in several respects, may be considered functionally and structurally analogous to notexin from the Australian tiger snake venom. Finally, the basic enzyme exhibits the abilities to hemolyze erythrocytes and to hydrolyze phospholipids of E. coli membranes in the presence of a bactericidal-permeability-increasing protein from neutrophil sources.

Study on the thrombin-like enzyme preferentially releasing fibrinopeptide B from the snake venom of agkistrodon halys pallas

Three components with arginine esterase activities were found in the venom of the Chinese pit viper (Agkistrodon halys Pallas). They were identified as bradykinin releasing, thrombin-like and fibrinogenolytic enzymes respectively. The thrombin-like enzyme of A. halys Pallas was purified to a homogeneous state. It was a glycoprotein with molecular weight of about 43,000. Though this enzyme displayed a higher esterase activity on BAEE than trypsin and human thrombin, it showed very weak clotting ability on fibrinogen. The kinetic analysis of the release of fibrinopeptides A (FPA) and B (FPB) showed that the action mode of this enzyme on fibrinogen was just opposite to that of mammalian thrombin. FPB was first released, followed by FPA after a long lag period.