Chewn-Lang Ho

Purification and characterization of a lethal protein with phospholipase A1 activity from the hornet (Vespa basalis) venom

The hornet, Vespa basalis, is one of the most dangerous species of wasps found in Taiwan. The insect is aggressive and its venom is highly toxic. By gel filtration on a Fractogel TSK HW 50 column followed by cation-exchange chromatography on CM-Trisacryl M, a lethal protein was purified from the venom. It has a molecular mass of about 32 kDa and an i.v. LD50 value of 0.32 micrograms/g mouse. The toxin is capable of catalyzing the hydrolysis of emulsified phospholipids but not sphingomyelin. Analysis of the 1H-NMR spectra of the substrates and its hydrolytic products revealed that the toxin liberates fatty acid from the 1-position of sn-3-phosphoacylglycerols. This result indicates that the toxin possesses phospholipase A1 activity. The toxin exhibits an extremely potent hemolytic activity in washed red cells and diluted whole blood (HC50 = 0.09 micrograms/ml in mouse). The potency of direct hemolysis is about 100-times that of a basic phospholipase A2 from Naja nigricollis venom and about 1000-times that of a cardiotoxin from Naja naja atra venom. A positive correlation between the hemolytic activity and lethality of the toxin was found in three species of animals (mouse, rat and guinea pig). In the in vivo study, the toxin caused a marked increase in the plasma K+ concentration and a hyperkalemic change in the ECG of the treated rat. Hyperkalemia resulting from the hemolytic action of the toxin appears to be the main cause of death in the animal.

Effects of equinatoxin on the guinea-pig atrium

Equinatoxin is a lethal protein isolated from a sea anemone, Actinia equina. The toxin (0.1-3 micrograms/ml) caused an initial inhibition followed by an augmentation of contractions and beating rates in the isolated guinea-pig atrium. The inhibitory phase was transient (30-60 sec), while the stimulant phase lasted for about 30 min. The treated atrium showed tachyphylaxis to the toxin. The inhibitory effect of the toxin was diminished by tetrodotoxin and atropine and abolished by 8-phenyltheophylline or mepacrine. Dipyridamole, which blocks the uptake of adenosine in the heart, enhanced the inhibitory effect. The stimulant effect of the toxin was inhibited by indomethacin or mepacrine and abolished by a combination of both, but was not inhibited by propranolol. Bioluminescent assay performed during the inhibitory phase showed an increased release of ATP and radioimmunoassay during the stimulant phase revealed an increased release of prostaglandin E2 from the treated atrium. These results suggest that the cardiac inhibitory effect of equinatoxin is mainly due to release of adenyl compounds, while the cardiac stimulant effect of the toxin may result from the liberation of arachidonic acid and subsequent formation of prostaglandins in the guinea-pig atrium.

Site of action of caudoxin, a neurotoxic phospholipase A2 from the horned puff adder (Bitis caudalis) venom

Caudoxin, a single-chain phospholipase A2 isolated from the venom of Bitis caudalis is a toxic polypeptide with a formula weight of 13,332 dalton. The LD50 in mice (i.p.) was 0.18 (0.15-0.22) mg/kg. In the chick biventer cervicis muscle preparation the toxin (1-10 micrograms per ml) caused complete neuromuscular blockade without affecting the response of the muscle to acetylcholine. In the mouse phrenic nerve-diaphragm preparation, the toxin abolished the indirectly elicited contraction without inhibiting that evoked directly. When this preparation was bathed in a low calcium (0.6 mM) medium, the toxin induced a triphasic change in the indirectly evoked contractions: an immediate initial inhibition followed by augmentation and then a second phase of inhibition leading to irreversible neuromuscular blockade. Electrophysiological studies in the same preparation showed a similar triphasic change in the quantal content of endplate potentials. The frequency of miniature endplate potentials first increased and then decreased, while the resting membrane potential was not significantly decreased by the toxin. Histological study showed that the toxin caused local myonecrosis only at a higher dose (2 mg/kg mouse). It is concluded that caudoxin produced a neuromuscular block by acting selectively on a presynaptic site. However, the site of binding appears to be different from that of beta-bungarotoxin since combination of the toxin with beta-bungarotoxin caused potentiation of its neuromuscular blocking action rather than addition.

Edema-inducing activity of a lethal protein with phospholipase A1 activity isolated from the black-bellied hornet (Vespa basalis) venom

The lethal protein of the hornet (Vespa basalis) venom is a phospholipase A1 toxin (mol. wt approximately 32,000) with a potent hemolytic activity. Subplantar injection of the toxin caused a dose-dependent swelling in the rat hind paw. Its potency was higher than those of phospholipases A2 and cardiotoxin from cobra venoms. Hind-paw edema induced by the toxin was inhibited by antiserotonin drugs (cyproheptadine and methysergide), indomethacin and betamethasone. Antihistamine (chlorpheniramine) showed a relatively weak inhibition. Intradermal injection of the toxin into back skin of the rat also induced local edema which was inhibited by chlorpheniramine and methysergide. Rats pretreated with multiple doses of compound 48/80 showed a moderate decrease in the histamine and serotonin content of rat skin, and a slight decrease in paw edema induced by the toxin, while a single dose of reserpine markedly diminished the toxin-induced edema in association with depletion of serotonin in rat skin. The edema-inhibitory action of amine-depleting agents appeared to correlate with their potencies to deplete serotonin in the skin. It is suggested that serotonin, prostaglandin E2, and to a lesser extent of histamine are involved in producing the local effect of the toxin. However, serotonin released by the toxin appears to be the major factor mediating the toxin-induced edema in the rat.

Structural requirements for the edema-inducing and hemolytic activities of mastoparan B isolated from the hornet (Vespa basalis) venom

Mastoparan B (MP-B) is a cationic tetradecapeptide isolated from the black-bellied hornet (Vespa basalis) venom. It has a primary structure (LKLKSIVSWAKKVL-CONH2) distinct from other vespine mastoparans. The peptide caused a dose-dependent swelling in rat hind paw and showed a potent hemolytic activity in guinea pig red blood cells. Studies on the structure activity relationship of the peptide showed that replacing lysine at position 2 (Lys2) by asparagine (Asn) in the MP-B sequence caused about 40% decrease in its edema-inducing activity at 50 micrograms/paw and 90% decrease in hemolytic activity at 30 microM of the peptide, while the same substitution at Lys4 did not cause a significant change in either activity. Replacing either Lys11 or Lys12 by leucine (Leu) caused little or no decrease in the edema-inducing and hemolytic activities. Decreases in both activities were observed when both Lys11 and Lys12 were replaced by Leu. On the other hand, replacing tryptophan at position 9 (Trp9) by tyrosine or phenylalanine in MP-B sequence almost abolished its hemolytic activity, while the edema-inducing activity was only partially inhibited. Circular dichroism spectra of the peptides measured in 20% trifluoro-ethanol revealed that substitution of Lys and Trp did not cause a significant change in the conformation of MP-B. it appears that Lys2 is crucial for both hemolytic and edema-inducing activities of MP-B, while Trp9 is of special importance to the hemolytic activity of MP-B. Lys11 and Lys12 in MP-B probably play a lesser role in both activities.

Cardiovascular effects of mastoparan B and its structural requirements

Mastoparan B is a cationic, amphiphilic tetradecaeptide (LKLKSIVSWAKKVL-CONH2) isolated from the venom of the hornet Vespa basalis. Intravenous injection of the peptide into rats caused a profound depression of blood pressure and cardiac function, which was inhibited by cyproheptadine, reserpine and multiple doses of compound 48/80, but not by diphenhydramine and cromolyn. Mastoparan from Paravespula lewisii showed little cardiovascular inhibitory activity. A synthetic mastoparan B analog in which lysine at position 2 was replaced by asparagine showed a marked decrease in the cardiovascular depressor effects, while replacing lysine at position 4, 11 or 12 with leucine did not cause a significant reduction in these effects. Replacing lysine at position 12 with leucine even caused a more sustained depressor effect. However, the analog in which lysines at positions 11 and 12 were replaced by leucine lost its cardiovascular inhibitory activity. Replacing tryptophan at position 9 with phenylalanine in mastoparan B did not affect its activity. It is concluded that mastoparan B is involved in the cardiovascular disturbances induced by the hornet venom. Lysine at position 2 is a critical residue for the cardiovascular effects of mastoparan B. A peptide molecule with two lysine residues, one located close to the amino terminus and the other near the carboxyl end of the peptide, appears to be the optimal structure for eliciting the cardiovascular depressor effects.

Three toxins with phospholipase activity isolated from the yellow-legged hornet (Vespa verutina) venom

The yellow-legged hornet, Vespa verutina, is widely distributed in both the mountain area and the suburbs of Taiwan and possesses highly toxic venom (LD50=0.02 microl/g mouse). By gel filtration on Fractogel (TSK HW 50f) followed by cation-exchange chromatography on Mono S column, three toxins designated as verutoxin 1, 2a and 2b (VT-1, VT-2a and VT-2b) were isolated from the venom. The toxin VT-1 had a molecular mass of 34,982 Da and an LD50 value of 3.61 microg/g mouse. Toxin VT-2a and 2b were more basic and more toxic than VT-1. VT-2a and 2b were isotoxins with molecular masses differing in only 14 Da (33,360 and 33,374 Da, respectively) and had a similar toxicity in mice (LD50=0.87 microg/g mouse). All three toxins were capable of catalyzing the hydrolysis of emulsified phospholipids and lysophosphatide, but not sphingomyelin. Analysis of the hydrolyzed products (fatty acid and lyso-compound) by a liquid chromatography/mass spectrometer revealed that the toxins liberates fatty acid mainly from the 1-position of the synthetic phospholipid. This result indicates that verutoxins possess phospholipase A1 activity. Toxin VT-1 showed higher phospholipase activity than VT-2a and 2b. However, the latter toxins exhibited much higher direct hemolytic activity toward the mouse red blood cells. Vespid phospholipases are known as one of the three major venom allergens in many species of wasps. Our studies indicate that vespid phospholipases A1, in addition to acting as allergens, possess direct toxic actions that may also cause death in animals. Toxin VT-2a and 2b which possess potent hemolytic activity and high lethality in mice may act as the lethal factor of V. verutina venom.

Purification and pharmacological characterization of a cardiotoxin-like protein from Formosan banded krait (Bungarus multicinctus) venom

By ion exchange chromatography followed by gel filtration, a polypeptide toxin was purified from Formosan banded krait (Bungarus multicinctus) venom. The toxin had a molecular weight of 7000 +/- 300 and showed an amino acid composition characteristic of cardiotoxin from cobra venom. The i.p. LD50 value of the toxin was 2.5 (1.9-3.2) mg per kg mouse. Pharmacological studies showed that the toxin (10 micrograms/ml) induced contracture in chick and mouse skeletal muscles, depolarized the cell membrane of the mouse diaphragm, arrested the contraction of spontaneously beating atria and the electrically driven ventricle strip of the rat, and caused direct hemolysis of guinea-pig erythrocytes. From these chemical and pharmacological characterizations it was concluded that this toxin has characteristics similar to those of cobra venom cardiotoxins.

Enhancing the hypotensive effect and diminishing the cytolytic activity of hornet mastoparan B by D-amino acid substitution

Mastoparan B (MP-B) is a cationic tetradecapeptide (LKLKSIVSWAKKVL-CONH(2)) isolated from the venom of the Taiwan hornet Vespa basalis. Unlike other vespid mastoparans, the peptide is capable of inducing short-term hypotension and causes hemolysis in animals. This study was aimed to find out MP-B analogs that possess higher hypotensive potency with the least lytic action by D-amino acid substitution, especially at lysine (Lys) residues. The synthetic MP-B isomer in which Lys(2) was replaced by D-Lys showed a significant decrease in both hemolytic and hypotensive activities. Substitution of Lys(4) by D-Lys in MP-B also caused a marked reduction of hemolytic activity, but its hypotensive action was only slightly affected. However, when Lys(11,12) were replaced by D-Lys, the resulting isomer ([D-Lys(11,12)]MP-B) exhibited a higher hypotensive activity with negligible hemolytic activity as compared with the native peptide. The D-antipot of MP-B in which all amino acid residues were replaced by D-isomers showed the highest hypotensive activity with a hemolytic activity about 1/5 that of MP-B. The results reveal that D-Lys substitution at the N-terminus of MP-B (Lys(2,4)) causes decreases in both hypotensive and hemolytic activities, while D-Lys substitution at the C-terminus (Lys(11,12)) leads to a significant increase in hypotensive activity of MP-B with a remarkable decrease in hemolytic activity. The hypotensive effect of [D-Lys(11,12)]MP-B was more prominent on spontaneously hypertensive rats. At a proper dose (0.3mg/kg) the peptide could reduce the high blood pressure (approximately 180 mmHg) of the rat to a normal level (approximately 120 mmHg) for more than 3h. [D-Lys(11,12)]MP-B which possesses a potent hypotensive action with the least cytolytic side effect is the best MP-B analog for studying the mechanism of cardiovascular inhibition by MP-B and could be useful as a hypotensive agent in hypertension crisis.

Comparison of the immunogenicity of wasp venom peptides with or without carbohydrate moieties

Three synthetic vespulakinin analogues either with or without carbohydrate moieties and mastoparan B isolated from Vespa basalis venom were investigated for their immunogenic activity and solution conformation. Mice immunized with these wasp venom peptides, with the exception of (Gal alpha)Thr3, (Gal alpha)Thr4-vespulakinin 1, showed positive antibody responses. However, the response elicited by mastoparan B was much higher than those induced by vespulakinin analogues. The class of antibody induced by these peptides was identified as an IgG1 isotype with kappa-light chain, suggesting stimulation of a T-cell-dependent immune response by these peptides. According to the circular dichroism spectra of these peptides, the structures of the vespulakinin analogues in solution were largely unordered, while mastoparan B exhibited a conformation rich in alpha-helices. The presence of carbohydrate moieties and the rather random structure in vespulakinins may interfere with T-cell recognition of the peptides, leading to lower immune responses.