Yuji Samejima

Oral Therapeutic Agents with Highly Clustered Globotriose for Treatment of Shiga Toxigenic Escherichia coli Infections

Shiga toxin (Stx) is a major virulence factor in infection with Stx-producing Escherichia coli (STEC). We developed a series of linear polymers of acrylamide, each with a different density of trisaccharide of globotriaosylceramide (Gb3), which is a receptor for Stx, and identified Gb3 polymers with highly clustered trisaccharides as Stx adsorbents functioning in the gut. The Gb3 polymers specifically bound to both Stx1 and Stx2 with high affinity and markedly inhibited the cytotoxic activities of these toxins. Oral administration of the Gb3 polymers protected mice after administration of a fatal dose of E. coli O157:H7, even when the polymers were administered after the infection had been established. In these mice, the serum level of Stx was markedly reduced and fatal brain damage was substantially suppressed, which suggests that the Gb3 polymers entrap Stx in the gut and prevent its entrance into the circulation. These results indicate that the Gb3 polymers can be used as oral therapeutic agents that function in the gut against STEC infections.

Specificity of two types of phospholipase A2 inhibitors from the plasma of venomous snakes

Specificity of two different types of phospholipase A2 (PLA2) inhibitory proteins from the blood plasma of venomous snakes was investigated. Two Crotalidae inhibitors, having a carbohydrate recognition domain (CRD) in their sequences, inhibited specifically the group‐II acidic PLA2s of their own snake venom. On the other hand, Elapidae inhibitor, having two tandem patterns of cysteine residues found in proteins of the Ly‐6 superfamily, inhibited not only the group‐I PLA2 from its own snake venom but also the group‐I, ‐II, and ‐III PLA2s from other snake venom. Amino acid sequences of PLA2s that were specifically inhibited by the inhibitors were compared with those of the other PLA2s. A unique aromatic patch structure appeared on the group‐II acidic PLA2s was suggested to be involved in the binding to the Crotalidae inhibitors; and residues located in or close to the Ca2+ binding loop of PLA2, in the binding to the Elapidae inhibitor.

Structural Analysis of the Interaction between Shiga Toxin B Subunits and Linear Polymers Bearing Clustered Globotriose Residues

ABSTRACT We previously developed linear polymers bearing clustered trisaccharides of globotriaosylceramide (Gb3) as orally applicable Shiga toxin (Stx) neutralizers. Here, using a Gb3 polymer with a short spacer tethering the trisaccharide to the core, we found that shortening the spacer length markedly reduced the binding affinity for Stx2 but not Stx1. Moreover, mutational analysis revealed that the essential binding sites of the terminal trisaccharides were completely different between Stx1 and Stx2. These results provide the molecular basis for the interaction between Stx B subunits and Gb3 polymers.

Structure of acidic phospholipase A2 for the venom of Agkistrodon halys blomhoffii at 2.8Åresolution

The crystal structure of acidic phospholipase A2 from the venom of Agkistrodon halys blomhoffii has been determined by molecular replacement methods based on the known structure of Crotalus atrox PLA2, a same group II enzyme. The overall structures, except the calcium-binding regions, are very similar to each other. A calcium ion is pentagonally ligated to two carboxylate oxygen atoms of Asp-49 and each carbonyl oxygen atoms of Tyr-28, Gly-30 and Ala-31. A reason why the former enzyme functions as monomeric form, while the latter one does as dimer, could be presumed by the structural comparison of these calcium-binding regions. Although Gly-32 is usually participated as a ligand in the coordination with calcium ion in group I PLA2, it is characteristically replaced to Ala-31 in the present structure, and thus the coordination geometry of calcium ion is rather different from the usually observed one.

Amino acid sequence of a myotoxin from venom of the eastern diamondback rattlesnake (Crotalus adamanteus).

The complete amino acid sequence of a myotoxin isolated from Crotalus adamanteus venom (CAM-toxin) was determined. The total number of amino acid residues was 45, giving a mol. wt of 5202. The amino acid sequence was compared with those of previously determined Crotalus myotoxins. The CAM-toxin shows a surprisingly close relationship to those of peptide c (C. v. helleri), myotoxin a (C. v. viridis), crotamine (C. d. terrificus), myotoxin I and II (C. v. concolor) with homologies of approximately 98, 83, 90, 95 and 91%, respectively. The hydropathy profile of the CAM-toxin is almost identical to peptide c and myotoxin I, as can be assumed from the sequence homology. Despite the absence of any close sequence homology with the myotoxic phospholipase A2 enzymes, CAM-toxin contains a similar cationic myotoxic region at residues 2-10 as found in the other five myotoxic peptides.

Amino acid sequence studies on cytolytic toxins from sea anemone Heteractis magnifica, Entacmaea quadricolor and Stichodactyla mertensii (Anthozoa)

The complete amino acid sequence of a cytolytic toxin, HmT, isolated from sea anemone Heteractis magnifica was determined. It is composed of 177 amino acid residues and lacks half-cystines. Partial N-terminal sequences of three other cytolysins from Entacmaea quadricolor (EnT) and Stichodactyla mertensii (SmT-1 and SmT-2) were also determined. Comparing these sequences with those of other sea anemone cytolysins, a high degree of homology was observed.

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.

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.

Blomhotin: a novel peptide with smooth muscle contractile activity identified in the venom of Agkistrodon halys blomhoffii

A novel peptide has been isolated from the venom of Agkistrodon halys blomhoffii using a bioassay that monitors the stimulant effect on rat stomach fundus. The 11-amino acid peptide, named blomhotin, was purified to homogeneity by gel-filtration column chromatography and reverse-phase HPLC. The amino acid sequence of blomhotin was determined to be pGlu-Gly-Arg-Pro-Pro-Gly-Pro-Pro-Ile-Pro-Arg, which is similar to that of bradykinin-potentiating peptides which themselves cause no contraction of smooth muscle. The contraction induced by blomhotin showed homologous desensitization, implicating the involvement of a blomhotin-specific site in the response.

Amino acid sequence of two neurotoxins from the venom of the Egyptian black snake (Walterinnesia aegyptia).

The venom of the Egyptian black snake Walterinnesia aegyptia contains at least three toxins, which act postsynaptically to block the neuromuscular transmission of isolated rat phrenic nerve-diaphragm and chicken biventer cervicis muscle. The complete amino acid sequence of the two toxins, W-III and W-IV, consisting of 62 amino acid residues, was elucidated by Edman degradation of fragments obtained after Staphylococcus aureus protease and prolylpeptidase digestion. Although the toxins exhibit close structural homology to other short-chain postsynaptic neurotoxins from Elapidae venoms, toxin IV is unique by having a free SH-group (cysteine) at position 16. In position 35 of W-III, which is located at the tip of the central loop, threonine is replaced by lysine, which may alter the interaction of the toxin with the acetylcholine receptor, since the toxin is seven times less lethal than toxin W-IV.