Orientotoxin--a new presynaptic neurotoxin from the venom of the giant hornet Vespa orientalis

Orientotoxin, a novel presynaptically acting neurotoxin from the venom of giant hornet Vespa orientalis, has been isolated by gel filtration and ion exchange chromatography and characterized. The toxin has a molecular mass of 18,000. Highly purified preparations of orientotoxin possessed clearly manifested lysophospholipase activity and can block both induced and spontaneous release of neurotransmitter from the presynaptic nerve membrane.     

Low molecular weight peptides from the venom of the giant hornet Vespa orientalis. Structure and function

Three 14-member linear peptides (HR-1, HR-2 and HR-3) capable of degranulating mast cells and thus initiating histamine release were isolated from the venom of the giant hornet Vespa orientalis, using reverse phase high performance liquid chromatography. The complete amino acid sequence of the peptides HR-1 and HR-2 molecules and partial structure of peptide HR-3 were determined, using automatic degradation by the Edman method. It was shown that peptide HR-1 at relatively low concentrations (2-20 micrograms/ml) selectively liberated histamine from rat mast cells and, when taken at higher doses (50-100 micrograms/ml), exerted a non-selective cytotoxic action. Besides, this peptide caused erythrocyte hemolysis, inhibited Ca2+-ATPase with concomitant uncoupling of Ca2+ transport and ATP hydrolysis as well as induced the conductance of lipid bilayer membranes, predominantly for monovalent cations due to the formation of nonspecific single permeability channels.     

Properties of phospholipase A2 from the venom of the large hornet Vespa orientalis

Some properties (catalytic and hemolytic activity, pH and temperature optima, stability, substrate specificity, effects of detergents and metal ions, N-terminal sequence, chemical modification of histidine in the enzyme active center, etc.) of phospholipase A2 from hornet (Vespa orientalis) venom were studied. It was shown that phospholipase A2 from hornet venom differs essentially from other enzymes of this species in terms of stability, catalytic properties and structural features. The active center of the enzyme contains an essential histidine residue, similar to other phospholipases A2 from various sources. Unlike other known forms of phospholipase A2, the enzyme under study exerts a pronounced hemolytic action. The hemolysis is inhibited by Ca2+ at concentrations capable of inducing the activation of the hydrolytic activity of the enzyme.     

Amino acid sequence of orientotoxins I and II from the venom of the hornet Vespa orientalis

Orientotoxin I, a neurotoxin of presynaptic effect having a lysophospholipase activity, and orientotoxin II, a highly toxic phospholipase A2, were isolated from the hornet Vespa orientalis venom, and their primary structures were determined. Despite their different functional activity, orientotoxin I and II proved to be structural homologues, differing significantly in the amino acid sequence from well-known toxic phospholipase from other sources.     

Hemolytic effect of phospholipase A2 and orientotoxin from venom of the great hornet, Vespa orientalis

The effect of toxic phospholipase A2 and orientotoxin from the venom of the giant hornet Vespa orientalis on human erythrocytes was studied. It was shown that these venom components are potent hemolytic agents, the efficiency of the latter being by about two orders of magnitude as high as that of phospholipase A2. The hemolytic function of the both components is enhanced in the presence of low concentrations of Ca2+, whereas high concentrations of this cation exert an inhibiting action. Polyvalent cations, in particular, ruthenium red, peptide HR-1, mellitin and cytotoxins Us-1 and Us-5 synergetically increase the hemolytic effect of phospholipase A2. During erythrocyte hemolysis the synergistic effect is manifested upon a combined action of phospholipase A2 and orientotoxin. The combination of these toxins increases the total hemolytic activity and produces a far greater effect than in could be expected in the case of each of these compounds taken separately.     

Purification and characterization of a trypsin-like proteinase from the midgut of the larva of the hornet, Vespa orientalis

A proteinase from the larval midgut of Vespa orientalis was purified by exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Sephadex G-75. This purified enzyme was proved to be homogeneous by electrophoresis on a cellulose acetate membrane. The molecular weight was calculated to be 27,000 by gel filtration. Optimum pH for the hydrolysis of N-benzoyl-arginine-ethyl ester (BAEE) was 7·5 to 8·5, and optimum temperature with casein as a substrate was 60°C at pH 8·0 for 20 min. According to studies with synthetic inhibitors the hornet protease belongs to the ‘serine proteases’, being inhibited by phenylmethyl sulphonylfluoride (PMSF) and tosyl-lysyl chloromethane (TLCK). The hydrolysis of different amino acid ester bonds and the cleavage specificity on the B chain of oxidized insulin allow us to speak of a trypsin-like protease.     

Cardioactive effects of hornet venom, Vespa mandarinia

In the experiment of electrocardiogram, the crude venom of giant hornet (Vespa mandarinia) showed cardioactive effects on rat heart. The heart rate was accelerated within 5 min after injection of the venom intraperitoneally, then the heart beat was blocked, resulting in conduction delay. The cardioactive constituent was separated into two components by gel filtration. One which was high molecular species such as protein showed complete atrioventricular block. Another component, having a low molecular weight, was fractionated in 5 peaks, which accelerated heart rate.     

Structural-functional characteristics of vasoactive peptides from the Vespa orientalis venom

Two vasoactive peptides are isolated from the Vespa orientalis venom by gel filtration and ion-exchange chromatography. The physicochemical and functional properties of peptides are studied. Vasoactive peptides show the myotropic activity and hypotensive action which is of prolonged character as compared with bradykinin. Their complete amino acidic sequences are determined. One of the peptides is a similar structural analogue of bradykinin.     

Building activity and longevity of queenless groups of the Oriental hornet,Vespa orientalis

The effect of group size on behavioral parameters of the Oriental hornet,Vespa orientalis, was assessed experimentally under laboratory conditions. Hornet groups of various sizes (ranging from 1 to 100 individuals per group) comprised of young individuals (0–24 hr of age) devoid of a queen were placed in artificial breeding boxes (ABBs). The following three quantitative parameters were evaluated: the amount and rate of building as a function of the number of hornets in the group, the rate of oviposition as, related to group size and the longevity of hornets as a function of their group size. The probability for the occurrence of these events was similarly considered and additional behavioral parameters were only assessed qualitatively. Results of this investigation revealed a relation between the three mentioned quantitative behavioral parameters and the number of hornets per group. The number of hornets per group was positively related to the extent of building, the number of cells built by a group is , but negatively related to the rate of building. As for the delay of building, a non-monotone relation was found. The relation between number of hornets per group and the oviposition delay was found to be non-monotone; the number of hornets per group and their longevity were found to be inversely related. Discrepanices were recorded on the very small (1–2 individuals) or very large (100 individuals) hornet groups.     

Purification and properties of a presynaptically acting neurotoxin, mandaratoxin, from hornet (Vespa mandarinia)

A hornet (Vespa mandarinia) neurotoxin, mandaratoxin (MDTX), was purified by simple procedures with column chromatography made on Sephadex G-50 and CM-Sephadex by using an acetate buffer. The molecular weight of homogeneous MDTX was calculated to be approximately 20000 by gel filtration, NaDodSO4 disc gel electrophoresis, and amino acid analysis. MDTX is a single-chain polypeptide. MDTX did not migrate electrophoretically in a basic buffer at pH 8.3 but did so when the buffer was acidic, at pH 4.3. The isoelectric point of the toxin was determined at 9.1 by isoelectric focusing. A relatively high amount of lysine was found in the amino acid analysis. A280nm1% was 15.1. Glucosamine and galactosamine were not detectable by amino acid analysis. MDTX had neither hemolytic nor enzymatic activity. The toxin was heat labile. By use of neuromuscular junctions of a lobster walking leg, it was found that the nanomole range of MDTX irreversibly blocked the excitatory postsynaptic potential without appreciable change in the resting conductance of the postsynaptic membrane. Intracellular recording from the presynaptic nerve fiber showed that MDTX blocked the action potential mainly by reducing the sodium current.     

Characterization of some membrane-active components of Vespa orientalis venom

The molecular weight distribution of the components of giant hornet (Vespa orientalis) venom was studied, using gel-filtration on a column with Sephadex G-50. The effects of the venom and its constituent fractions on the permeability and stability of artificial bilayer phospholipid membranes, potassium ions release from the erythrocytes and mitochondrial oxidative phosphorylation parameters, as well as on the activity and stability of polyenzymic systems of the mitochondrial respiratroy chain, were studied. The data obtained suggest that the high molecular weight fractions contain phospholipases, whose activities are much higher than those of presently known venoms. Despite the fact that the hemolytic effect is typical for two low molecular weight fractions, no fractions possessing high activity of bee venom of the melitin type were found.     

Hornetin: the lethal protein of the hornet (Vespa flavitarsus) venom

By gel permeation on a Fractogel TSK HW 50 column followed by ion-exchange chromatography on carboxymethylcellulose CM 52, a lethal protein, designated hornetin, was purified from the venom of Vespa flavitarsus. Hornetin is a highly basic protein (pI 10.2) with a molecular mass of about 32 kDa. Its amino acid composition is characterized by a high content of lysine, aspartic and glutamic acid, and is devoid of tryptophan and cysteine. The lack of cysteine in the molecule is distinct from other known vespid venom proteins of comparable size. The i.v. LD50 of the toxin is 0.42 microgram per g mouse. Assayed on the red blood cells of the mouse and guinea-pig as well as isolated nerve muscle preparations of the chick and mouse, hornetin showed direct hemolytic activity and presynaptic neurotoxicity at microgram level and displayed musculotropic effect at higher concentrations.     

Purification and characterization of two new allergens from the venom of Vespa magnifica

Due to poor diagnostic facilities and a lack of medical alertness, allergy to Vespa wasps may be underestimated. Few allergens have been identified from Vespa wasps.Possible native allergen proteins were purified from the wasp venoms (WV) (Vespa magnifica Smith) by gel filtration, ion exchange chromatography, respectively. Their sequences were determined by Edman degradation and cDNA cloning. Their allergenicities were assayed by enzyme-linked immunosorbent assay inhibition tests (ELISA-IT), immunoblots, and skin prick tests (SPTs). Their cross allergencities with Tab y 2 and Tab y 5 purified from the horsefly (Tabanus yao Macquart) were also determined. Two native allergens were identified from the WV, respectively. They are a 25-KDa antigen 5 protein (Ag5) (Vesp ma 5) and a 35-KDa hyaluronidase (Vesp ma 2). They represented major allergens in Vespa magnifica by immunoblots and SPTs. ELISA inhibition of pooled sera IgE reactivity to both the WV and the horsefly salivary gland extracts (HSGE) using four purified allergens (Vesp ma 2, Vesp ma 5 and previously purified Tab y 2 and Tab y 5) was significant. Their cross allergenicities were confirmed by ELISA-IT, immunoblots, and SPTs. They represented the cross reactive allergens from wasp and horsefly and proved the so called wasp-horsefly syndrome.     

Enzymatic and chemical properties of an endopeptidase from the larva of the hornet Vespa crabro.

An endopeptidase from the larvae of the hornet Vespa crabro has been purified to homogeneity. The enzyme has been characterized with respect to molecular weight, amino acid compositon, and amino- and carboxyl-terminal sequences. The catalytic properties of the hornet protease are similar to those of bovine chymotrypsin with respect to inactivation by phenylmethanesulfonyl fluoride and carbobenzoxyphenylalanine chloro ketone and preferential peptide bond cleavage at aromatic amino acid residues. In contrast to bovine chymotrypsin, the hornet protease is not inhibited by the basic pancreatic Kunitz inhibitor, soybean inhibitor, or chicken ovomucoid. The molecular weight, as determined by several independent methods, was found to be 14 500. The protease is a single-chain protein containing two disulfide bonds. The terminal sequences are: NH2-Ile-Val-Gly-Gly-Ile-Asp.....Gly-Lys-Tyr-Pro-Tyr-Gln-Val-Ser-Leu-Arg-COOH.     

Hornet (Vespa orientalis) venom sac extract causes hepatic injury in cats

1. Injection of sublethal doses of hornet venom to cats was followed by increases in serum aminotransferases and alkaline phosphatase, together with hyperglycemia, uremia and hyperkalemia. 2. The blood pH and PO2 fell significantly. 3. All changes occurred within 30 min of injection and were found to be partially reversible. 4. These results may be due to specific liver cell injury. 5. The possibility of a generalized metabolic effect, such as shock, cannot be discounted, although it is quite unlikely.