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Tomohisa Ogawa

Publications and source records attributed to Tomohisa Ogawa.

At least 19 recordsLinked to original sources
PubMed2006

Isolation, characterization and molecular evolution of a novel pearl shell lectin from a marine bivalve, Pteria penguin.

A novel lectin, PPL, was isolated from the mantle of penguin wing oyster (Pteria penguin) by affinity chromatography on mucin-Sepharose 4B and cation exchange chromatography on HiTrap SP. This lectin was estimated to be a 21-kDa monomer by gel filtration, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted time of flight (MALDI-TOF) mass spectrometry. However, dynamic light scattering experiments revealed that a non-covalently linked dimer formed under high salt conditions (500 mM NaCl). Interestingly, PPL showed an increasing hemagglutinating activity with increasing salt concentration. The amino acid sequence of PPL was determined by direct protein sequence analysis and cDNA cloning. The 167-amino acid sequence included 24 lysine residues and had two tandemly repeated homologous domains (residues 20-78 and 107-165) with 44% internal homology. PPL showed sequence homology to L-rhamnose-binding lectins from fish eggs and a D-galactose-binding lectin from sea urchin eggs, with sequence identities in the range 37-48%. PPL agglutinated various animal erythrocytes independently of calcium ions. The minimum concentration of PPL needed to agglutinate rabbit erythrocytes was 0.5 micro g/ml, and the most effective saccharides to inhibit the hemagglutination were D-galactose, methyl-D-galactopyranoside and N-acetyl-D-lactosamine. Lactose also inhibited hemagglutination, but L-rhamnose did so only weakly despite the sequence homology with trout egg L-rhamnose-binding lectins. The carbohydrate-binding specificity of PPL was further examined by frontal affinity chromatography using 37 different pyridylaminated oligosaccharides. PPL was found to have strong binding affinity for various oligosaccharides that have Galbeta1-4Glu/GlcNAc, Galbeta1-3GalNAc/GlcNAc and Galalpha 1-4Gal moieties in their structure. PPL had a high thermal stability and retained 50% of its hemagglutinating activity after incubation at 70 degrees C for 100 min. It agglutinated some Gram-negative bacteria by recognizing lipopolysaccharides. Together, these results suggest that PPL is a new member of the trout egg lectin family which participates in the self-defense mechanism against bacteria and pathogens with a distinct carbohydrate-binding specificity. We conclude that the trout egg lectin family proteins, in particular their carbohydrate recognition domains, have acquired diverse carbohydrate-binding specificities during molecular evolution.

Amino Acid Sequence↗
PubMed2006

Structural characterization of a rhamnose-binding glycoprotein (lectin) from Spanish mackerel (Scomberomorous niphonius) eggs.

A rhamnose-binding glycoprotein (lectin), named SML, was isolated from the eggs of Spanish mackerel (Scomberomorous niphonius) by affinity and ion-exchange chromatographies. SML was composed of a non-covalently linked homodimer. The SML subunit was composed of 201 amino acid residues with two tandemly repeated domains, and contained 8 half-Cys residues in each domain, which is highly homologous to the N-terminal lectin domain of calcium-independent alpha-latrotoxin receptor in mammalian brains. Each domain has the same disulfide bonding pattern; Cys10-Cys40, Cys20-Cys99, Cys54-Cys86 and Cys67-Cys73 were located in the N-terminal domain, and Cys108-Cys138, Cys117-Cys195, Cys152-Cys182 and Cys163-Cys169 were in the C-terminal domain. SML was N-glycosylated at Asn168 in the C-terminal domain. The structure of the sugar chain was determined to be NeuAc-Galbeta1-4GlcNAcbeta1-2Manalpha1-6-(NeuAc-Galbeta1-4GlcNAcbeta1-2Manalpha1-3)Manbeta1-4GlcNAcbeta1-4GlcNAc-Asn.

Amino Acid Sequence↗
PubMed2006

Discovery of novel [Arg49]phospholipase A2 isozymes from Protobothrops elegans venom and regional evolution of Crotalinae snake venom phospholipase A2 isozymes in the southwestern islands of Japan and Taiwan.

Protobothrops (formerly Trimeresurus) elegans, a Crotalinae snake, inhabits Ishigaki and Iriomote islands of the Sakishima Islands of Japan which are located between Okinawa island of Japan and Taiwan. Two phospholipase A(2) (PLA(2)) isozymes were purified to homogeneity from P. elegans venom and sequenced. This led to a discovery of novel PLA(2) isozymes with Arg at position 49, that is, [Arg(49)]PLA(2) forms, named PeBP(R)-I and PeBP(R)-II. They are polymorphic at position 3, Val for PeBP(R)-I and Ile for PeBP(R)-II. The cDNAs encoding PeBP(R)-I and PeBP(R)-II were cloned. The cDNA encoding an [Asp(49)]PLA(2) named PePLA(2) was also obtained. In contrast to PLA(2) isozymes from Protobothrops genus with 122 amino acid residues, PeBP(R)-I and PeBP(R)-II are composed of 121 amino acid residues due to lack of Pro at position 90. They exhibited necrotic and edema-inducing activities but no hemorrhagic activity was detected. A phylogenetic tree constructed for venom PLA(2) isozymes of Protobothrops genus and of related genera in the southwestern islands of Japan and Taiwan revealed that PeBP(R)-I and PeBP(R)-II of P. elegans are evolutionarily much closer to PmK49PLA(2), a [Lys(49)]PLA(2), from P. mucrosquamatus (Taiwan) than BPI and BPII, both [Lys(49)]PLA(2) forms, from P. flavoviridis (Amami-Oshima and Tokunoshima islands of Japan). Such evolutionary relationships are also seen in neutral [Asp(49)]PLA(2) isozymes from the three Protobothrops species. Thus, P. elegans is the species much closer to P. mucrosquamatus than P. flavoviridis. Their evolutionary distances seem to be well related to geological history of the islands where they have lived. In addition, it was clearly noted that Ovophis okinavensis (Amami-Oshima), which had formerly belonged to the Trimeresurus genus, and Trimeresurus stejnegeri (Taiwan) are the species fairly distant from Protobothrops genus.

Amino Acid Sequence↗
PubMed2006

Effect of lectins on the transport of food factors in caco-2 cell monolayers.

The effect of three plant lectins, soybean lectin (SBA), Japanese jack bean lectin (CGA), and wheat germ lectin (WGA), on the transport of various food factors, such as isoflavones, quercetin, dipeptides, and calcium ions, were investigated by use of an intestinal tract model, Caco-2 cell monolayers. The lectins increased the isoflavone transport but had no effect on aglycon transport. SBA increased the transport of quercetin glycosides, whereas CGA and WGA had no effect. The lectins increased the transport of calcium ions but showed no effect on the transport of dipeptides, carnosine, and anserine. Although SBA did not change the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayers, CGA and WGA decreased the TER value. These results indicate that plant lectins affect the transport of food factors in different manners, presumably due to their specific sugar binding activity.

Biological Transport↗
PubMed2006

Structure based studies of the adaptive diversification process of congerins.

The isoforms of a fish galectin, congerins I and II, have several features that make them suitable for a study of accelerated process of molecular diversification based on 3D structures: They have been generated by a gene duplication, and still maintain 47% amino acid sequence identity to each other. Their genes show very high K A: /K S: ratio, and are though to be components of fish defense system. The crystal systems for a high-resolution analysis are known for both proteins. A series of works with biochemistry, molecular biology, and X-ray crystallography techniques have suggested that the two proteins might have evolved under differential selection pressures. Congerin I appeared to be a stabilized version of galectin-1. Congerin II was shown to be adapted to a new carbohydrate-ligand. The 3D structures of the wild type and mutant proteins have revealed the probable cause and consequence of the selection pressure responsible for the diversification of congerins.

Adaptation, Physiological↗
PubMed2006

The efficacy of povidone-iodine products against periodontopathic bacteria.

A total of 8 strains of 6 bacterial species, Porphyromonas gingivalis ATCC33277 and TDC286, Actinobacillus actinomycetemcomitans ATCC29523 and JP2, Fusobacterium nucleatum No. 2, Tannerella forsythensis ATCC43937, Prevotella intermedia ATCC25611 and Streptococcus anginosus ATCC33397, were treated with povidone-iodine (PVP-I) gargle (PVP-I: 0.47 and 0.23% w/v) or chlorhexidine gluconate (CHG) gargle (CHG: 0.002% w/v) for 15, 30 or 60 s, after which they were inoculated into various media, cultured and counted for residual bacteria. At both concentrations, PVP-I gargle reduced the viable cell count of all 8 bacterial strains to below the measurable limit within 15 s. By contrast, there were more than 1,000 viable colonies 60 s following treatment with the CHG gargle. The results demonstrate that povidone-iodine gargle has rapid bactericidal activity against the causative bacteria of periodontal disease.

Aggregatibacter actinomycetemcomitans↗
PubMed2005

In vitro evolutionary thermostabilization of congerin II: a limited reproduction of natural protein evolution by artificial selection pressure.

The thermostability of the conger eel galectin, congerin II, was improved by in vitro evolutionary protein engineering. Two rounds of random PCR mutagenesis and selection experiments increased the congerin II thermostability to a level comparative to its naturally thermostable isoform, congerin I. The crystal structures of the most thermostable double mutant, Y16S/T88I, and the related single mutants, Y16S and T88I, were determined at 2.0 angstroms, 1.8 angstroms, and 1.6 angstroms resolution, respectively. The exclusion of two interior water molecules by the Thr88Ile mutation, and the relief of adjacent conformational stress by the Tyr16Ser mutation were the major contributions to the thermostability. These features in the congerin II mutants are similar to those observed in congerin I. The natural evolution of congerin genes, with the K(A)/K(S) ratio of 2.6, was accelerated under natural selection pressures. The thermostabilizing selection pressure artificially applied to congerin II mimicked the implied natural pressure on congerin I. The results showed that the artificial pressure made congerin II partially reproduce the natural evolution of congerin I.

Amino Acid Sequence↗
PubMed2005

Molecular diversity and accelerated evolution of C-type lectin-like proteins from snake venom.

A number of C-type lectin-like proteins that affect thrombosis and hemostasis by inhibiting or activating specific platelet membrane receptors or blood coagulation factors have been isolated from the venom of various snake species and characterized and more than 80 have been sequenced. Recent data on the primary sequences and 3D structures of C-type lectins and C-type lectin-like proteins from snake venoms have enabled us to analyze their molecular evolution. Statistical analysis of their cDNA sequences shows that C-type lectin-like proteins, with some exceptions, have evolved in an accelerated manner to acquire their diverse functions. Phylogenetic analysis shows that the A and B chains of C-type lectin-like proteins are clearly separated from C-type lectins and that the A and B chains are further divided into a group of platelet receptor-binding proteins and a group of coagulation factor-binding proteins. Elucidation of the tertiary structures of several C-type lectin-like proteins led to the discovery of a unique domain-swapping interaction between heterodimeric subunits, which creates a concave surface for ligand binding.

Amino Acid Sequence↗
PubMed2005

Amino acid sequence of a basic aspartate-49-phospholipase A2 from Trimeresurus flavoviridis venom and phylogenetic analysis of Crotalinae venom phospholipases A2.

Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima and Okinawa. A phospholipase A2 (PLA2) of basic nature (pI 8.5) was isolated from the venom of Amami-Oshima T. flavoviridis. Its amino acid sequence determined by the ordinary procedures was completely in accord with that predicted from the nucleotide sequence of the cDNA previously cloned from Amami-Oshima T. flavoviridis venom gland, which was named PLA-B'. It consists of 122 amino acid residues and has aspartate at position 49. It induced edema in a mouse footpad assay and caused necrosis in mouse skeletal muscles. PLA-B' is similar in sequence to PLA-B (Tokunoshima) and PL-Y (Okinawa), both basic [Asp49]PLA2s, with a few amino acid substitutions, indicating occurrence of interisland mutation. Although PLA2s of Crotalinae subfamily were phylogenetically classified into four types, PLA2 (acidic or neutral [Asp49]PLA2) type, basic [Asp49]PLA2 type, neurotoxic [Asp49]PLA2 type and [Lys49]PLA2 type, it was ascertained that PLA2s of PLA2 type and [Lys49]PLA2 type are most essential as toxic components for Crotalinae snake venoms and that basic [Asp49]PLA2-type PLA2s are uniquely contained only in the venoms of T. flavoviridis species. Prediction of physiological activities of some PLA2s was made based on their location in the phylogenetic tree. Relationship of divergence of PLA2s via accelerated evolution followed by less rapid mutation and physiological activities was discussed.

Amino Acid Sequence↗
PubMed2005

Acid hydrolysis of protein in a microcapillary tube for the recovery of tryptophan.

The acid hydrolysis of proteins was miniaturized and simplified by employing microcapillary tubes (100 microl in volume) with 6 M HCl containing 1% 2-mercaptoethanol and 3% phenol for an amino acid compositional analysis. The method not only eliminated the laborious evacuation step for the hydrolysis tube but also decreased the destruction of tryptophan during hydrolysis. The recovery of tryptophan was 79% by acid hydrolysis at 145 degrees C for 4 h. Since the acid mixture could be removed under vacuum, the hydrolysate was subjected to an amino acid analysis without neutralization or dilution.

Amino Acids↗
PubMed2004

Complementary DNA cloning and molecular evolution of opine dehydrogenases in some marine invertebrates.

The complete complementary DNA sequences of genes presumably coding for opine dehydrogenases from Arabella iricolor (sandworm), Haliotis discus hannai (abalone), and Patinopecten yessoensis (scallop) were determined, and partial cDNA sequences were derived for Meretrix lusoria (Japanese hard clam) and Spisula sachalinensis (Sakhalin surf clam). The primers ODH-9F and ODH-11R proved useful for amplifying the sequences for opine dehydrogenases from the 4 mollusk species investigated in this study. The sequence of the sandworm was obtained using primers constructed from the amino acid sequence of tauropine dehydrogenase, the main opine dehydrogenase in A. iricolor. The complete cDNA sequence of A. iricolor, H. discus hannai, and P. yessoensis encode 397, 400, and 405 amino acids, respectively. All sequences were aligned and compared with published databank sequences of Loligo opalescens, Loligo vulgaris (squid), Sepia officinalis (cuttlefish), and Pecten maximus (scallop). As expected, a high level of homology was observed for the cDNA from closely related species, such as for cephalopods or scallops, whereas cDNA from the other species showed lower-level homologies. A similar trend was observed when the deduced amino acid sequences were compared. Furthermore, alignment of these sequences revealed some structural motifs that are possibly related to the binding sites of the substrates. The phylogenetic trees derived from the nucleotide and amino acid sequences were consistent with the classification of species resulting from classical taxonomic analyses.

Amino Acid Oxidoreductases↗
PubMed2004

Characterization of the yam tuber storage proteins from Dioscorea batatas exhibiting unique lectin activities.

Four major proteins designated DB1, DB2, DB3, and DB4 were isolated and characterized from the yam tuber Dioscorea batatas. The ratios of their yields were 20:50:20:10. DB1 was a mannose-binding lectin (20 kDa) consisting of 10-kDa subunits and was classified as the monocot mannose-binding lectin family. DB2, accounting for 50% of the total protein, was the storage protein, commonly called dioscorins consisting of a 31-kDa subunit. On the basis of amino acid sequence, DB2 was classified to be dioscorin A. DB3 was a maltose-binding lectin, having an apparent molecular mass of 120 kDa and composed of a 66-kDa subunit and two 31-kDa subunits (DB3S). The 66-kDa subunit was further composed of two 31-kDa subunits (DB3L) cross-linked by disulfide bonds. DB3L and DB3S (242 and 241 amino acid residues, respectively) were homologous with each other with 72% sequence identity. They showed a sequence homology to dioscorin B and dioscorin A from Dioscorea alata, with 90 and 93% identity, respectively, and to carbonic anhydrase from Arabidopsis thaliana with about 45% identity. DB3S had one intrachain disulfide bond located at Cys(28)-Cys(187), whereas DB3L had one interchain disulfide bond (Cys(40)-Cys(40)') in addition to the intrachain disulfide bond (Cys(28)-Cys(188)) to form a 66-kDa subunit. DB1 and DB3 agglutinated rabbit erythrocytes at 2.7 and 3.9 microg/ml, respectively. Despite the structural homology between DB2 and DB3, DB2 had no lectin activity. The 66-kDa subunit itself revealed the full hemagglutinating activity of DB3, indicating that DB3L but not DB3S was responsible for the activity. The hemagglutinating activity of DB3 required Ca(2+) ions and was exclusively inhibited by maltose and oligomaltoses (e.g. maltopentaose and maltohexaose) but not by d-glucose. DB3 could not be classified into any known plant lectin family. DB4 was a chitinase, homologous to an acidic chitinase from Dioscorea japonica. DB1, DB2, and DB3 did not show any activity of carbonic anhydrase, amylase, or trypsin inhibitor activity. These results show that two of the four major proteins isolated from the yam tubers D. batatas have unique lectin activities.

Amino Acid Sequence↗
PubMed2004

Long-sarafotoxins: characterization of a new family of endothelin-like peptides.

Sarafotoxins (SRTXs) constitute a family of vasoactive peptides that were initially isolated from the venom of Atractaspis engaddensis, and that are structurally and functionally related to endothelins (ETs). Analysis of the venom of Atractaspis microlepidota microlepidota revealed several new SRTX molecules manifesting some new structural and functional characteristics. These novel SRTXs are longer by three amino acids than the previously described SRTXs, and are designated here "long-SRTXs". Six isoforms, derived from new poly-cistronic precursors, have been identified so far in the venom of this snake. One of these isoforms, designated SRTX-m, was chemically synthesized and its biological properties were studied. Our results show that SRTX-m induces toxicity in mice, mostly due to vasoconstriction, and also that it has a lower toxicity and potency than the more potent SRTX described up to now: sarafotoxin-b (SRTX-b) from A. engaddensis.

Amino Acid Sequence↗
PubMed2003

Molecular evolution of myotoxic phospholipases A2 from snake venom.

After two decades of study, we draw the conclusion that venom-gland phospholipase A2 (PLA2) isozymes, including PLA2 myotoxins of Crotalinae snakes, have evolved in an accelerated manner to acquire their diverse physiological activities. In this review, we describe how accelerated evolution of venom PLA2 isozymes was discovered. This type of evolution is fundamental for other venom isozyme systems. Accelerated evolution of venom PLA2 isozyme genes is due to rapid change in exons, but not in introns and the flanking regions, being completely opposite to the case of the ordinary isozyme genes. The molecular mechanism by which proper base substitutions had occurred in the particular sites of venom isozyme genes is a puzzle to be solved in future studies. It should be noted that accelerated evolution occurred until the isozymes had acquired their particular function and, since then, they have evolved with less frequent mutation, possibly for functional conservation. We also found that interisland mutations occurred in venom PLA2 isozymes. The relationships between mutation and its driving force are speculative and the real mechanism remains a mystery.

Amino Acid Sequence↗
PubMed2003

Antioxidative properties of tripeptide libraries prepared by the combinatorial chemistry.

Two series of combinatorial tripeptide libraries were constructed, based on an antioxidative peptide isolated from a soybean protein hydrolysate. One was a library of 108 peptides containing either His or Tyr residues. Another was a library of 114 peptides related to Pro-His-His, which had been identified as an active core of the antioxidative peptide. The antioxidative properties of these libraries were examined by several methods, such as the antioxidative activity against the peroxidation of linoleic acid, the reducing activity, the radical scavenging activity, and the peroxynitrite scavenging activity. Two Tyr-containg tripeptides showed higher activities than those of two His-containing tripeptides in the peroxidation of linoleic acid. Tyr-His-Tyr showed a strong synergistic effects with phenolic antioxidants. However, the tripeptide had only marginal reducing activity and a moderate peroxynitrite scavenging activity. Cysteine-containing tripeptides showed the strong peroxynitrite scavenging activity. Change of either the N-terminus or C-terminus of Pro-His-His to other amino acid residues did not significantly alter their antioxidative activity. Tripeptides containing Trp or Tyr residues at the C-terminus had strong radical scavenging activities, but very weak peroxynitrite scavenging activity. The present results allow us to understand why protein digests have such a variety of antioxidative properties.

Antioxidants↗
PubMed2003

Interisland evolution of Trimeresurus flavoviridis venom phospholipase A(2) isozymes.

Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A(2) (PLA(2)), named PL-Y, was isolated from Okinawa T. flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA(2) from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA(2), named PLA-B', which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp(49)]PLA(2) isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima), and PLA-B' (Amami-Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the beta1-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different environments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA(2), named PLA2, ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA2 gene is a prototype among T. flavoviridis venom PLA(2) isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA(2) isozymes are clearly separated into three groups, PLA2 type, basic [Asp(49)]PLA(2) type, and [Lys(49)]PLA(2) type. Basic [Asp(49)]PLA(2)-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys(49)]PLA(2) type.

Amino Acid Sequence↗
PubMed2003

Interisland mutation of a novel phospholipase A2 from Trimeresurus flavoviridis venom and evolution of Crotalinae group II phospholipases A2.

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A2 (PLA2). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA2, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD50 for mice was 1.34 microg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA2's based on the amino acid sequences revealed that neurotoxic PLA2's including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA2 groups such as PLA2 type, basic [Asp49]PLA2 type, and [Lys49]PLA2 type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA2's showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA2's, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA2's evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation.

Amino Acid Sequence↗