Tamotsu Omori-Satoh

The antihemorrhagic factor, erinacin, from the European hedgehog (Erinaceus europaeus), a metalloprotease inhibitor of large molecular size possessing ficolin/opsonin P35 lectin domains

From muscle extracts of the European hedgehog, Erinaceus europaeus, an antihemorrhagic factor, erinacin, was purified by ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, hydroxylapatite and gel filtration columns. A purification of approx. 1400-fold was achieved with an overall yield of 21% in antihemorrhagic activity. The molecular weight of erinacin determined by gel filtration was approx. 1000 kDa. SDS-PAGE of erinacin under reducing conditions indicates that it consists of two types of subunits, alpha and beta, with molecular weights of 37 and 35 kDa, respectively, in a ratio of 1:2. In the presence of 6 M guanidine-HCl, erinacin dissociates into alpha-subunits and beta-subunit decamers. From these results the subunit assembling of erinacin has been formulated as alpha(10).2beta(10). The molecular weight of the subunits and of the beta-subunit decamer was confirmed by MALDI-TOF mass spectrometry. Erinacin inhibits the hemorrhagic and proteolytic activity of the major hemorrhagic metalloprotease from the venom of Bothrops jararaca. Complete inhibition was achieved in an equimolar mixture of inhibitor and enzyme suggesting an equimolar complex. Erinacin is not inhibiting serine proteases such as trypsin and chymotrypsin, it was characterized to be a metalloprotease inhibitor. In electronmicroscopy, flower bouquet-like structures characteristic for some animal lectins were observed. Amino acid sequence analysis indicated that both subunits are almost identical and are composed of common amino terminal, collagen- and fibrinogen-like domains homologous to proteins of the ficolin/opsonin P35 lectin family.

Identification of alpha-bungarotoxin (A31) as the major postsynaptic neurotoxin, and complete nucleotide identity of a genomic DNA of Bungarus candidus from Java with exons of the Bungarus multicinctus alpha-bungarotoxin (A31) gene.

The Malayan krait (Bungarus candidus) is one of the most medically significant snake species in Southeast Asia. No specific antivenom exists to treat envenoming by this species. Death within 30 min after its bite has been reported from Java, suggesting the presence of highly lethal postsynaptic neurotoxins in the venom of these snakes. We purified and identified the major postsynaptic toxin in the venom of B. candidus from Java. The toxin was indistinguishable from alpha-bungarotoxin (A31), a toxin originally isolated from Bungarus multicinctus, in its mass (7983.75 Da), LD50 (0.23 microg/g in mice i.p.), affinity to nicotinic acetylcholine receptors, and by its 40 N-terminal amino acid residues as determined by Edman degradation. Identity with alpha-bungarotoxin was confirmed by cloning and sequencing a genomic DNA from B. candidus which encodes the 74 amino acid sequence of alpha-bungarotoxin (A31) and part of its signal peptide, revealing complete identity to the alpha-bungarotoxin (A31) gene in exon and 98.9% identity in intron sequences. The entire mitochondrial cytochrome b gene of the krait species B. candidus from Java and B. multicinctus from Taiwan was sequenced for comparison, suggesting that these snakes are phylogenetically closely related. alpha-Bungarotoxin appears to be widely present and conserved in Southeast and East Asian black-and-white kraits across populations and taxa.

Cross-neutralization of Thai cobra (Naja kaouthia) and spitting cobra (Naja siamensis) venoms by Thai cobra antivenom

The neutralizing capacity of antivenom prepared against Thai cobra (Naja kaouthia) venom was compared in mice using the homologous venom and that of spitting cobra (Naja siamensis). The amounts of antivenoms neutralizing a dose of 4 LD50 of the test venom were determined. Four antivenom preparations were used: three purified antivenoms and a crude antivenom, which were made using N. kaouthia venom only. Almost the same neutralizing capacity was obtained with the purified antivenoms, whereas a slightly lower capacity was seen with the crude antivenom. However, ratios of the amounts of four antivenoms neutralizing the homologous and heterologous test venoms were almost constant. These results indicated that Thai cobra antivenom possesses neutralizing capacity against spitting cobra venom.

Subunit structure and inhibition specificity of α-type phospholipase A2 inhibitor from Protobothrops flavoviridis ☆

The alpha-type phospholipase A2 inhibitor (PLIalpha) in the plasma of the Habu snake, Protobothrop flavoviridis, was shown to be a trimer of two homologous subunits, PLIalpha-A and PLIalpha-B, each of which contains one C-type lectin-like domain (CTLD). Since one molecule of trimeric PLIalpha binds stoichiometrically to one molecule of P. flavoviridis acidic phospholipase A2 (PLA2), the trimeric structure is critical for its inhibitory activity. Hydrophobic chromatography separated the purified P. flavoviridis PLIalpha into four different trimeric subspecies, A3-PLIalpha, A2B-PLIalpha, AB2-PLIalpha, and B3-PLIalpha, with different combinations of the two subunits. The trimeric PLIalpha could be reconstituted from the purified subunits, and the four different trimeric subspecies were formed through random association of the two subunits. The inhibitory activity of the PLIalpha-A homotrimer (A3-PLIalpha) was more specific than that of the PLIalpha-B homotrimer (B3-PLIalpha). This difference in inhibitory properties between the two homotrimers was probably caused by the amino acid differences at residues 10-37.

Comparison of antihemorrhagic activities in skeletal muscle extracts from various animals against Bothrops jararaca snake venom

Antihemorrhagic activities of skeletal muscle extracts from various animals were compared in inhibiting the hemorrhagic activity of Bothrops jararaca venom. The muscle extracts of the European hedgehog (Erinaceus europaeus) exhibited the strongest activity, followed by those of other insectivores such as the shrew (Crocidura russula) and mole (Talpa europaea). The antihemorrhagic activities of muscle extracts from experimental animals such as mice, rats, guinea-pigs, hamsters and rabbits were negligible.

A PROTEASE FROM THE MARINE SPONGE Callyspongia schulzi

Aqueous extracts of 25 marine sponge species (from coral reefs of Papua New Guinea) were screened for proteolytic activity. Only one sponge, Callyspongia schulzi, showed remarkable activity. A protease hydrolyzing casein as well as the synthetic substrate α‐N‐benzoyl‐L‐arginine ethyl ester was isolated from the sponge extract by gel filtration, ion‐exchange and HPLC absorption chromatography. The enzyme was homogenous in SDS‐PAGE exhibiting an apparent molecular weight of 80 kDa. Its pH optimum was in the range of 9‐11, it was remarkably heat‐stable and was not inhibited by phenylmethane sulfonylfluoride, soybean trypsin inhibitor, aprotinin or α1‐antitrypsin, but by EDTA and 1,10‐phenanthroline suggesting properties of a metalloprotease. The protease hydrolyzed the oxidized insulin B‐chain between Arg22‐Gly23 and Lys29‐Ala30, similar to trypsin.

Antihemorrhagin in the blood serum of king cobra (Ophiophagus hannah): purification and characterization

King cobra (Ophiophagus hannah) serum was found to possess antihemorrhagic activity against king cobra hemorrhagin. The activity was stronger than that in commercial king cobra antivenom. An antihemorrhagin has been purified by ion exchange chromatography, affinity chromatography and gel filtration with a 22-fold purification and an overall yield of 12% of the total antihemorrhagic activity contained in crude serum. The purified antihemorrhagin was homogeneous in disc-PAGE and SDS-PAGE. Its apparent molecular weight determined by SDS-PAGE was 120 kDa. The antihemorrhagin was also active against other hemorrhagic snake venoms obtained in Thailand and Japan such as Calloselasma rhodostoma, Trimeresurus albolabris, Trimeresurus macrops and Trimeresurus flavoviridis (Japanese Habu). It inhibited the proteolytic activity of king cobra venom. It is an acid- and thermolabile protein and does not form precipitin lines against king cobra venom.