N49 phospholipase A2, a unique subgroup of snake venom group II phospholipase A2.

A novel phospholipase A2 (PLA2) with Asn at its site 49 was purified from the snake venom of Protobothrops mucrosquamatus by using SP-Sephadex C25, Superdex 75, Heparin-Sepharose (FF) and HPLC reverse-phage C18 chromatography and designated as TM-N49. It showed a molecular mass of 13.875 kDa on MALDI-TOF. TM-N49 does not possess enzymatic, hemolytic and hemorrhagic activities. It fails to induce platelet aggregation by itself, and does not inhibit the platelet aggregation induced by ADP. However, it exhibits potent myotoxic activity causing inflammatory cell infiltration, severe myoedema, myonecrosis and myolysis in the gastrocnemius muscles of BALB/c mice. Phylogenetic analysis found that that TM-N49 combined with two phospholipase A2s from Trimeresurus stejnegeri, TsR6 and CTs-R6 cluster into one group. Structural and functional analysis indicated that these phospholipase A2s are distinct from the other subgroups (D49 PLA2, S49 PLA2 and K49 PLA2) and represent a unique subgroup of snake venom group II PLA2, named N49 PLA2 subgroup.

Potent histamine-releasing activity of atrahagin, a novel snake venom metalloproteinase.

Poisonous snakebite wound is a popular disease worldwide. However, the pathogenesis remains unclear. In the present study, a novel metalloproteinase atrahagin in Chinese cobra (Naja atra) snake venom was purified, using heparin-sepharose followed by Superdex 75 gel filtration chromatography. Apart from its alpha-fibrinogenase activity, atrahagin potently activated human colon, lung and tonsil mast cells with the net histamine release being 25.9+/-4.4, 17.0+/-1.9, 13.2+/-3.6%, respectively. Time course studies revealed that the peak histamine release induced by atrahagin occurred at 12, 12 and 8 min following incubation of the enzyme with colon, lung and tonsil mast cells, respectively. The response of mast cells to atrahagin was abolished by preincubation of the cells with metabolic inhibitors or pertussis toxin, and by removal of Ca2+ and Mg2+ from the challenge buffer. In conclusion, activation of human mast cells by atrahagin indicated that the enzyme might contribute to the pathogenesis of snakebite wound.

Purification, characterization and cytokine release function of a novel Arg-49 phospholipase A(2) from the venom of Protobothrops mucrosquamatus.

Group IIA phospholipase A(2) (PLA(2)) are major components in Viperidae/Crotalidae venom. In the present study, a novel PLA(2) named promutoxin with Arg at the site 49 has been purified from the venom of Protobothrops mucrosquamatus by chromatography. It consists of 122 amino acid residues with a molecular mass of 13,656 Da assessed by MALDI-TOF. It has the structural features of snake venom group IIA PLA(2)s, but has no PLA(2) enzymatic activity. Promutoxin shows higher amino acid sequence identity to the K49 PLA(2)s (72-95%) than to D49 PLA(2)s (52-58%). Promutoxin exhibits potent myotoxicity in the animal model with as little as 1 microg of promutoxin causing myonecrosis and myoedema in the gastrocnemius muscle of mice. Promutoxin is also able to stimulate the release of IL-12, TNFalpha, IL-6 and IL-1beta from human monocytes, and induce IL-2, TNFalpha and IL-6 release from T cells, indicating that this snake venom group IIA PLA(2) is actively involved in the inflammatory process in man caused by snake venom poisoning.

Characterization and molecular cloning of dabocetin, a potent antiplatelet C-type lectin-like protein from Daboia russellii siamensis venom.

A novel C-type lectin-like protein, dabocetin, was purified from Daboia russellii siamensis venom. On SDS-polyacrylamide gel electrophoresis, it showed a single band with an apparent molecular weight of 28 kDa and two distinct bands with the apparent molecular weights of 15.0 kDa and 14.5 kDa under non-reducing and reducing conditions, respectively. cDNA clones containing the coding sequences for dabocetin alpha and beta subunits were isolated and sequenced. The deduced protein sequences of both subunits were confirmed by N-terminal amino acid sequencing and trypsin-digested peptide mass fingerprinting. Dabocetin did not induce platelet aggregation in platelet-rich plasma. It also had little effect on the platelet aggregation induced by ADP, TMVA or stejnulxin. Whereas, dabocetin inhibited ristocetin-induced platelet agglutination in platelet-rich plasma in a dose-dependent manner with an IC50 value of 0.35 microM. Flow cytometry analysis showed that dabocetin significantly inhibited mAb SZ2 binding to platelet membrane glycoprotein Ib alpha, indicating that platelet membrane glycoprotein Ib is involved in the inhibitory effect of dabocetin on ristocetin-induced platelet agglutination.

[Effect of Yunnan-cobra venom factor in overcoming acute humoral rejection after allograft cardiac transplantation in presensitized recipients: experiment with rats].

OBJECTIVE: To investigate the effect of Yunnan-cobra venom factor (Y-CVF) in overcoming acute humoral rejection after allograft cardiac transplantation in presensitized recipients. METHODS: Fifteen Lewis rats received the transplantation of full-thickness skin graft of BN rats three times so as to be presensitized. Fifteen pairs of Lewis rat, as recipients of heart, and BN rat, as heart donors, were randomly divided into 2 groups: experimental group (n = 8), and control group (n = 7). The Lewis rats in the experimental group received heart transplantation of the heart of the BN rat 7 approximately 10 days after the third skin transplantation, and were injected with Y-CVF 80 microg/kg 24 hours before the heart transplantation. The Lewis rat in the control group received only the heart transplantation without Y-CVF injection. Blood samples were collected from all rats before pre-sensitization and 7 days after the third skin transplantation so as to determine the titer of anti-BN rat lymphocyte antibody. 0 and 24 hours, and 6 and 8 days after Y-CVF injection blood samples were collected from the Lewis rats to determine the total complement activity with the complement activity before Y-CVF injection defined as 100%. The survival time of the transplanted heart was observed. After the transplanted hearts stopped to beat, they were resected and underwent HE staining and microscopy. Immunohistochemistry was used to examine the deposition of IgG and complement 3 (C3). RESULTS: The titer of anti-BN rat lymphocyte antibody was 0 before the pre-sensitization, and increased to 1:1028 - 1:2056 7 days after the third skin pre-sensitization. The serum total complement activity of the Lewis rats decreased to 0 twenty-four hours after the Y-CVF injection, recovered to 2.01% - 15.41% 6 days after, and returned to the normal level (89.61% - 109.46%) 8 days after. The mean survival time of the transplanted hearts of the control group was 12.71 +/- 13.94 hours (with a range of 1.5 - 15 hours), significantly shorter than that of the experimental group (99.50 +/- 38.72 hours, with a range of 23 - 153 hours, P < 0.01). Pathological examination showed that the acute humoral rejection in the control group was mainly complement-dependent antibody-mediated humoral rejection characterized by intravascular thrombosis, and that in the experimental group was mainly cellular rejection, characterized by extensive infiltration of mononuclear cells. Immunohistochemistry showed that remarkable IgG deposition was seen in the cardiac muscle cells and vascular endothelial cells in both groups; however, C3 deposition in vascular endothelial cells could be seen only in the control group. CONCLUSION: Administration of CVF is effective in overcoming the acute humoral rejection after allograft cardiac transplantation in presensitized recipients.

Molecular characterization of a weak fibrinogen-clotting enzyme from Trimeresurus jerdonii venom.

A fibrinogen-clotting enzyme designed as jerdonobin-II was isolated from the venom of Trimeresurus jerdonii. It differed in molecular weight and N-terminal sequence with the previously isolated jerdonobin, a thrombin-like enzyme from the same venom. The enzyme consists of a single polypeptide chain with molecular weights of 30,000 and 32,000 under non-reducing and reducing conditions, respectively. Jerdonobin-II showed weak fibrinogen clotting activity and its activity unit on fibrinogen was calculated to be less than one unit using human thrombin as standard. The precursor protein sequence of jerodonobin-II was deduced from cloned cDNA sequence. The sequence shows high similarity (identity=89%) to TSV-PA, a specific plasminogen activator from venom of T. stejnegeri. Despite of the sequence similarity, jerdonobin-II was found devoid of plasminogen activating effect. Sequence alignment analysis suggested that the replacement of Lys239 in TSV-PA to Gln239 in jerdonobin-II might play an important role on their plasminogen activating activity difference.

Prolonged cardiac xenograft survival in guinea pig-to-rat model by a highly active cobra venom factor.

A highly active cobra venom factor (CVF) was isolated from the venom of Naja kaouthia by sequential column chromatography. It displays strong anticomplementary activity, and has 1515 U of anticomplementary activity per mg protein. A single dose of 0.1 mg/kg CVF given i.v. to rats completely abrogated complement activity for nearly 5 days. Given 0.02 mg/kg of CVF, the complement activity of rats was reduced by more than 96.5% in 6 h. In guinea pig-to-rat heart transplant model, rats treated with a single dose of 0.05 mg/kg CVF had significantly prolonged xenograft survival (56.12+/-6.27 h in CVF-treated rats vs. 0.19+/-0.07 h in control rats, P<0.001).

TMVA, a snake C-type lectin-like protein from Trimeresurus mucrosquamatus venom, activates platelet via GPIb.

TMVA is a C-type lectin-like protein with potent platelet activating activity from Trimeresurus mucrosquamatus venom. In the absence of von Willebrand factor (vWF), TMVA dose-dependently induced aggregation of washed platelets. Anti-GP Ib monoclonal antibodies (mAbs), HIP1, specifically inhibited TMVA-induced aggregation in a dose-dependent manner. The aggregation was also inhibited by mAb P2 (an anti-GP IIb mAb). Flow cytometric analysis revealed that FITC-TMVA bound to human formalin-fixed platelets in a saturable manner, and its binding was specifically blocked by HIP1 in a dose-dependent manner. Flow cytometric analysis showed that TMVA did not bind to platelet GPIX, GPIIb, GPIIIa, GPIa, GPIIa and GPIV. Moreover, the platelet aggregation induced by TMVA was partially inhibited when platelet was pretreated with mocarhagin, a snake venom protease that specifically cleaves human GPIb. These results suggest that TMVA is a strong platelet agonist via GPIb and it might have multiple functional binding-sites on GPIb molecule or on other unknown receptor.

Purification, cloning and biological characterization of a novel disintegrin from Trimeresurus jerdonii venom.

A novel disintegrin, jerdonin, was purified from the Trimeresurus jerdonii venom by means of gel filtration and reverse phase high pressure liquid chromatography. Its coding cDNA was also isolated from the venom gland. The jerdonin coding cDNA is part of a precursor composed of proprotein, metalloproteinase, and disintegrin domains. From the deduced amino acid sequence, jerdonin is composed of 71 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonin was determined to be 7483Da by matrix-assisted laser desorption ionization time of flight mass spectrometry. Jerdonin inhibited ADP- and collagen-induced human platelet aggregation with IC(50) of 220 and 240 nM, respectively. In vivo, jerdonin inhibited the growth of subcutaneously inoculated B16 solid tumor in C57BL/6 mice and improved the survival time of the tumor-bearing mice.

A novel high molecular weight metalloproteinase cleaves fragment F1 of activated human prothrombin.

A hemorrhagic proteinase, jerdohagin, was purified from Trimeresurus jerdonii venom by gel filtration and ion-exchange chromatographies. It was a single chain polypeptide with an apparent molecular weight of 96 kDa as estimated by SDS-PAGE under the non-reducing and reducing conditions. Internal peptide sequencing indicated that it consisted of metalloproteinase, disintegrin-like and cysteine-rich domains and belonged to the class III snake venom metalloproteinases (class P-III SVMPs). Like other typical metalloproteinases, hemorrhagic activities of jerdohagin were completely inhibited by EDTA, but not by PMSF. Jerdohagin preferentially degraded alpha-chain of human fibrinogen. Interestingly, jerdohagin did not activate human prothrombin, whereas it cleaved human prothrombin and fragment F1 of activated human prothrombin.

Characterization and cloning of a novel phospholipase A(2) from the venom of Trimeresurus jerdonii snake.

A phospholipase A(2) (PLA(2)), called jerdoxin, was isolated from Trimeresurus jerdonni snake venom and partially characterized. The protein was purified by three chromatographic steps. SDS-polyacrylamide gel electrophoresis in the presence or absence of dithiothreitol showed that it had a molecular mass of 15 kDa. Jerdoxin had an enzymatic activity of 39.4 micro mol/min/mg towards egg yolk phosphatidyl choline (PC). It induced edema in the footpads of mice. In addition, jerdoxin exhibited indirect hemolytic activity. About 97% hemolysis was observed when 2 micro g/ml enzyme was incubated for 90 min in the presence of PC and Ca(2+). No detectable hemolysis was noticed when PC was not added. Ca(2+) was necessary for jerdoxin to exert its hemolytic activity, since only 52% hemolysis was seen when Ca(2+) was absent in the reaction mixture. Furthermore, jerdoxin inhibited ADP induced rabbit platelet aggregation and the inhibition was dose dependent with an IC(50) of 1.0 micro M. The complete amino acid sequence of jerdoxin deduced from cDNA sequence shared high homology with other snake venom PLA(2)s, especially the D 49 PLA(2)s. Also, the residues concerned to Ca(2+) binding were conserved. This is the first report of cDNA sequence of T. jerdonii venom PLA(2).

Purification and cloning of a novel C-type lectin-like protein with platelet aggregation activity from Trimeresurus mucrosquamatus venom.

TMVA, a novel C-type lectin-like protein that induces platelet aggregation in a dose-dependent manner, was purified from the venom of Trimeresurus mucrosquamatus. It consists of two subunits, alpha (15536 Da) and beta (14873 Da). The mature amino acid sequences of the alpha (135 amino acids) and beta subunits (123 amino acids) were deduced from cloned cDNAs. Both of the sequences show great similarity to C-type lectin-like venom proteins, including a carbohydrate recognition domain. The cysteine residues of TMVA are conserved at positions corresponding to those of flavocetin-A and convulxin, including the additional Cys135 in the alpha subunit and Cys3 in the beta subunit. SDS-PAGE, mass spectrometry analysis and amino acid sequence showed that native TMVA exists as two convertible multimers of (alpha beta)(2) and (alpha beta)(4) with molecular weights of 63680 and 128518 Da, respectively. The (alpha beta)(2) complex is stabilized by an interchain disulfide bridge between the two alpha beta-heterodimers, whereas the stabilization of the (alpha beta)(4) complex seems to involve non-covalent interactions between the (alpha beta)(2) complexes.

Actions of two serine proteases from Trimeresurus jerdonii venom on chromogenic substrates and fibrinogen.

Jerdonobin and jerdofibrase are two serine proteases purified from the venom of Trimeresurus jerdonii. The Michaelis constant K(m) and the catalytic rate constant K(cat) of jerdonobin or jerdofibrase on three chromogenic substrates, H-D-Pro-Phe-Arg-pNA (S2302), H-D-Phe-pipecolyl-Arg-pNA (S2238), and H-D-Val-Leu-Lys-pNA (S2251) were obtained from lineweaver-Burk plots. Jerdofibrase could hydrolyze all three substrates, but jerdonobin had no detectable activity on S2251, suggesting a relatively broader substrate specificity for jerdofibrase than jerdonobin. By SDS-PAGE, jerdofibrase preferentially degraded Bbeta-chain of fibrinogen. It also degraded Aalpha-chain of fibrinogen with relatively slow activity, but did not act on the gamma-chain. In contrast, jerdonobin did not degrade fibrinogen within 12 h. Fibrinopeptides liberation test, identified by HPLC, showed jerdonobin released fibrinopeptide A and a small amount of fibrinopeptide B. Unlike jerdonobin, jerdofibrase mainly released fibrinopeptide B. These results indicate that the two enzymes differ in their ability to hydrolyze chromogenic substrates and in their actions on fibrinogen.

A novel disintegrin, jerdonatin, inhibits platelet aggregation and sperm-egg binding.

A novel disintegrin, jerdonatin, was purified to homogeneity from Trimeresurus jerdonii venom by gel filtration and reversed-phase high-pressure liquid chromatography. We isolated the cDNA encoding jerdonatin from the snake venom gland. Jerdonatin cDNA precursor encoded pre-peptide, metalloprotease and disintegrin domain. Jerdonatin is composed of 72 amino acid residues including 12 cysteines and the tripeptide sequence Arg-Gly-Asp (RGD), a well-known characteristic of the disintegrin family. Molecular mass of jerdonatin was determined to be 8011 Da by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). Jerdonatin inhibited ADP- and collagen-induced human platelet aggregation with IC50 of 123 and 135 nM, respectively. We also investigated the effect of jerdonatin on the binding of B6D2F1 hybrid mice spermatozoa to mice zona-free eggs and their subsequent fusion. Jerdonatin significantly inhibited sperm-egg binding in a concentration-dependent manner, but had no effect on the fusion of sperm-egg. These results indicate that integrins on the egg play a role in mammalian fertilization.

Purification, characterization and primary structure of a chymotrypsin inhibitor from Naja atra venom.

A chymotrypsin inhibitor, designated NA-CI, was isolated from the venom of the Chinese cobra Naja atra by three-step chromatography. It inhibited bovine alpha-chymotrypsin with a Ki of 25 nM. The molecular mass of NA-CI was determined to be 6403.8 Da by matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) analysis. The complete amino acid sequence was determined after digestion of S-carboxymethylated inhibitor with Staphylococcus aureus V8 protease and porcine trypsin. NA-CI was a single polypeptide chain composed of 57 amino acid residues. The main contact site with the protease (P1) has a Phe, showing the specificity of the inhibitor. NA-CI shared great similarity with the chymotrypsin inhibitor from Naja naja venom (identities=89.5%) and other snake venom protease inhibitors.

A Highly Active Anticomplement Factor from the Venom of Naja kaouthia.

A highly active anticomplement factor (cobra venom factor) from the venom of Naja kaouthia in South Yunnan, China was isolated by sequential column chromatography (SP-Sephadex-C-25, Q Sepharose HP and Sephadex G-150). It displays strong anticomplement activities in vitro and in vivo, and has anticomplement activity of 1 515 u per mg. The purified anticomplement factor was homogeneous on SDS-PAGE with a molecular weight of 149 kD. It is composed of three polypeptide chains which are connected by disulfide bonds. The molecular weights of the three chains were determined to be 65.4 kD (alpha-chain), 52.1 kD (beta-chain), and 35.5 kD (gamma-chain). The gamma-chain displayed size heterogeneity, and two bands could be clearly detected in gels. All polypeptide chains were stained with periodate-Schiff reagent, suggesting the presence of carbohydrate. Neutral sugar and sialicacid were determined to be 1.78% and 0.38%, respectively. The isoelectric point of this anticomplement factor was 6.2. Its amino acid composition was analyzed and N-terminal amino acid sequence of each polypeptide chain was determined.

Purification, characterization and biological activity of an L-amino acid oxidase from Trimeresurus mucrosquamatus venom.

An L-amino acid oxidase (TM-LAO) from the venom of Hunan Trimeresurus mucrosquamatus was purified to homogeneity by three steps including DEAE Sephadex A-50 ion-exchange chromatography, Sephadex G-75 gel filtration and Resource Q ion-exchange chromatography. TM-LAO is composed of two identical subunits with a molecular weight of 55 kD by SDS-polyacrylamide gel electrophoresis. The molecular weight was different with that of LAO purified from the same species distributed in Taiwan that was 70 kD. The 24 N-terminal amino acid sequence of TM-LAO is ADNKNPLEECFRETNYEEFLEIAR, which shares high similarity with other Viperid snake venom LAOs and has moderate similarity with Elapid snake venom LAOs. Further studies found that TM-LAO inhibited the growth of E. coli, S. aurues and B. dysenteriae. TM-LAO also showed cytotoxicity and platelet aggregation activity. All the biological activities were eliminated by catalase, a H(2)O(2) scavenger. It was shown that these biological effects were possibly due to the formation of H(2)O(2) produced by TM-LAO.

A Novel Myotoxin from the Venom of Trimeresurus mucrosquamatus.

A novel myotoxin, designated TMPB, was purified from the venom of Trimeresurus mucrosquamatus by Sephadex G-100 superfine gel chromatography and fast protein liquid chromatography (FPLC). The N-terminal sequence of 24 amino acid residues was determined by protein sequencer. The sequence similarities between TMPB and other two phospholipase A(2) (PLA2s) previously purified from the same venom were 41.7% and 54.2%, respectively, but TMPB showed no detectable PLA(2) hydrolytic activity. Its molecular weight was estimated to be 16 000 by reducing SDS-PAGE and isoelectric point was determined to be 9.2 by isoelectric focusing electrophoresis. TMPB exhibited strong myotoxicity and platelet aggregation inhibiting activity, and the two activities could all be inhibited by heparin.

Characterization of a new bradykinin-potentiating peptide (TmF) from Trimeresurus mucrosquamatus.

A novel bradykinin-potentiating peptide (BPP), designated as TmF, has been purified to homogeneity from the venom of Trimeresurus mucrosquamatus by 70% cold methanol extraction, Sephadex G-15 gel filtration and reverse-phase high performance liquid chromatography (RP-HPLC). The amino acid sequence of TmF was determined to be pGlu-Gly-Arg-Pro-Leu-Gly-Pro-Pro-Ile-Pro-Pro (pGlu denotes pyroglutamic acid), which shared high homology with other BPPs. The molecular mass of TmF was 1.1107 kD as determinated by electrospray ionization-mass spectrometry (ESI-MS), which was in accordance with the calculated value of 1.1106 kD. The potentiating unit of TmF to bradykinin-induced (BK-induced) contraction on the guinea-pig ileum in vitro was (1.13 +/-0.3) unit (mg/L), and TmF (5.0 x10(-4) mg/kg) increased the pressure-lowering-effect of bradykinin (5.0 x10(-5 )mg/kg) with approximate descent value of (14 +/-2) mmHg. In addition, TmF inhibited the conversion of angiotensin I to angiotensin II, 2 x10(-3) mg of TmF caused 50% inhibition (IC(50)) of angiotensin- converting enzyme (ACE) hydrolyzing activity to bradykinin.

Alpha-neurotoxins of Naja atra and Naja kaouthia snakes in different regions.

Recent studies have shown that there are geographic variation of alpha-neurotoxins in Naja kaouthia, but the cause is not clear yet. In this work, venoms were collected from adult Naja atra in Zhejiang Province and Naja kaouthia in Yunnan Province, well identified by morphological characters and cytochrome b gene analysis in summer season to avoid age and seasonal variation in the venom composition. Then alpha-neurotoxins were purified and cloned from these two kinds of snakes. Three alpha-neurotoxins from Naja kaouthia (Yunnan) and two from Naja atra (Zhejiang) were identified. Together with previously reported alpha-neurotoxins in Naja kaouthia (Thailand) and Naja atra (Taiwan Province), it was found that the alpha-neurotoxins of Naja kaouthia in Yunnan Province were similar to those of Naja atra in Zhejiang and Taiwan Provinces, but different from those of Naja kaouthia in Thailand. This result can hardly be explained by population phylogeny or geographic distance. It might be due to the different climate, habitat and prey in Thailand in comparison with those in Yunnan, Zhejiang and Taiwan Provinces.