Staphylococcus aureus expresses several hemolytic pore-forming toxins (PFTs), which are all commonly composed of three domains: cap, rim and stem. PFTs are expressed as soluble monomers and assemble to form a transmembrane beta-barrel pore in the erythrocyte cell membrane. The stem domain undergoes dramatic conformational changes to form a pore. Staphylococcal PFTs are classified into two groups: one-component alpha-hemolysin (alpha-HL) and two-component gamma-hemolysin (gamma-HL). The alpha-HL forms a homo-heptamer, whereas gamma-HL is an octamer composed of F-component (LukF) and S-component (Hlg2). Because PFTs are used as materials for nanopore-based sensors, knowledge of the functional properties of PFTs is used to develop new, engineered PFTs. However, it remains challenging to design PFTs with a beta-barrel pore because their formation as transmembrane protein assemblies requires large conformational changes. In the present study, aiming to investigate the design principles of the beta-barrel formed as a consequence of the conformational change, chimeric mutants composed of the cap/rim domains of alpha-HL and the stem of LukF or Hlg2 were prepared. Biochemical characterization and electron microscopy showed that one of them assembles as a heptameric one-component PFT, whereas another participates as both a heptameric one- and heptameric/octameric two-component PFT. All chimeric mutants intrinsically assemble into SDS-resistant oligomers. Based on these observations, the role of the stem domain of these PFTs is discussed. These findings provide clues for the engineering of staphylococcal PFT beta-barrels for use in further promising applications.

Amyloid beta peptides (A beta s) are generated from amyloid precursor protein (APP) through multiple cleavage steps mediated by gamma-secretase, including endoproteolysis and carboxypeptidase-like trimming. The generation of neurotoxic A beta 42/43 species is enhanced by familial Alzheimer's disease (FAD) mutations within the catalytic subunit of gamma-secretase, presenilin 1 (PS1). FAD mutations of PS1 cause partial loss-of-function and decrease the cleavage activity. Activating mutations, which have the opposite effect of FAD mutations, are important for studying A beta production. Aph1 is a regulatory subunit of gamma-secretase; it is presumed to function as a scaffold of the complex. In this study, we identified Aph1 mutations that are active in the absence of nicastrin (NCT) using a yeast gamma-secretase assay. We analyzed these Aph1 mutations in the presence of NCT; we found that the L30F/T164A mutation is activating. When introduced in mouse embryonic fibroblasts, the mutation enhanced cleavage. The Aph1 mutants produced more short and long A beta s than did the wild-type Aph1, without an apparent modulatory function. The mutants did not change the amount of gamma-secretase complex, suggesting that L30F/T164A enhances catalytic activity. Our results provide insights into the regulatory function of Aph1 in gamma-secretase activity.

Snakebites are one of the major causes of death and long-term disability in the developing countries due to the presence of various bioactive peptides and proteins in snake venom. In Japan, the venom of the habu snake (Protobothrops flavoviridis) causes severe permanent damage due to its myonecrotic toxins. Antivenom immunoglobulins are an effective therapy for snakebites, and antivenom was recently developed with effective suppressive activity against myonecrosis induced by snake venom. To compare the properties of an antivenom having anti-myonecrotic activity with those of conventional antivenom with no anti-myonecrotic activity, this study applied focused proteomics analysis of habu venom proteins using 2D gel electrophoresis. As a target protein for antivenom immunoglobulins with anti-myonecrotic activity, we identified a thrombin-like serine protease, TLSP2 (TLf2), which was an inactive proteolytic isoform due to the replacement of the active site, His43 with Arg. Additionally, we identified the unique properties and a novel synergistic function of pseudoenzyme TLf2 as a myonecrosis-enhancing factor. To our knowledge, this is the first report of a function of a catalytically inactive snake serine protease.

ABSTRACT N-glycan-mediated activation of the thrombopoietin receptor (MPL) under pathological conditions has been implicated in myeloproliferative neoplasms induced by mutant calreticulin, which forms an endogenous receptor-agonist complex that constitutively activates the receptor. However, the molecular basis for this mechanism has not been studied because no external agonists existed. We describe the structure and function of a marine sponge-derived MPL agonist, thrombocorticin (ThC), a homodimerized lectin with calcium-dependent fucose-binding properties. ThC-induced activation persists due to limited receptor internalization. The strong synergy between ThC and thrombopoietin suggests that ThC catalyzes the formation of receptor dimers on the cell surface. MPL is subject to sugar-mediated activation, where the kinetics differ from those of cytokines. This result suggests the presence of diverse receptor activation pathways in human thrombopoiesis. One-sentence summary A marine sponge lectin catalyzes thrombopoietin receptor dimerization and activation, exhibiting strong synergy with thrombopoietin, and modulates internalization of the receptor.

Multicopper oxidases have a wide range of substrate specificity to be involved in various physiological reactions. Pseudomonas syringae, a plant pathogenic bacterium, has a multicopper oxidase, CumA. Multicopper oxidases have ability to degrade plant cell wall component, lignin. Once P. syringae enter apoplast and colonize, they start to disrupt plant immunity. Therefore, deeper understanding of multicopper oxidases from plant pathogens helps to invent measures to prevent invasion into plant cell, which brings agricultural benefits. Several biochemical studies have reported lower activity of CumA compared with other multicopper oxidase called CotA. However, the mechanisms underlying the difference in activity have not yet been revealed. In order to acquire insight into them, we conducted a biophysical characterization of PsCumA. Our results show that PsCumA has weak type I copper EPR signal, which is essential for oxidation activity. We propose that difference in the coordination of copper ions may decrease reaction frequency.

Venomics projects have been conducted to disclose the divergent profiles and evolution of various venomous animals. Here, we describe the venomics project including genome and transcriptome of habu snake, leading to drug discovery. Venomics project including the decoding of their whole genomes revealed partly a producing mechanism of various venom proteins including accelerated evolution and alternative splicing and how the toxic organisms have evolved from the nontoxic ones. In addition, the venomics analysis of transcriptomes and proteomes beyond species reveals the relationship between the geographical distribution and evolution of toxic organisms. The abundance of different gene products within a gene family caused by accelerated evolution and alternative splicing may contribute to expand the repertoire of effective weapons to prey capture accompanied with neofunctionalization.

Previously, we isolated jacalin-related lectins termed PPL2, PPL3 (PPL3A, 3B and 3C) and PPL4 from the mantle secretory fluid of Pteria penguin (Mabe) pearl shell. They showed the sequence homology with the plant lectin family, jacalin-related beta-prism fold lectins (JRLs). While PPL3s and PPL4 shared only 35%-50% homology to PPL2A, respectively, they exhibited unique carbohydrate binding properties based on the multiple glycan-binding profiling data sets from frontal affinity chromatography analysis. In this paper, we investigated biomineralization properties of these lectins and compared their biomineral functions. It was found that these lectins showed different effects on CaCO3 crystalization, respectively, although PPL3 and PPL2A showed similar carbohydrate binding specificities. PPL3 suppressed the crystal growth of CaCO3 calcite, while PPL2A increased the number of contact polycrystalline calcite composed of more than one crystal with various orientations. Furthermore, PPL4 alone showed no effect on CaCO3 crystalization; however, PPL4 regulated the size of crystals collaborated with N-acetyl-D-glucosamine and chitin oligomer, which are specific in recognizing carbohydrates for PPL4. These observations highlight the unique functions and molecular evolution of this lectin family involved in the mollusk shell formation.

Snake venoms are complex mixtures of toxic proteins encoded by various gene families that function synergistically to incapacitate prey. A huge repertoire of snake venom genes and proteins have been reported, and alternative splicing is suggested to be involved in the production of divergent gene transcripts. However, a genome-wide survey of the transcript repertoire and the extent of alternative splicing still remains to be determined. In this study, the comprehensive analysis of transcriptomes in the venom gland was achieved by using PacBio sequencing. Extensive alternative splicing was observed in three venom protein gene families, metalloproteinase (MP), serine protease (SP), and vascular endothelial growth factors (VEGF). Eleven MP and SP genes and a VEGF gene are expressed as a total of 81, 61, and 8 transcript variants, respectively. In the MP gene family, individual genes are transcribed into different classes of MPs by alternative splicing. We also observed trans-splicing among the clustered SP genes. No other venom genes as well as non-venom counterpart genes exhibited alternative splicing. Our results thus indicate a potential contribution of mRNA alternative and trans-splicing in the production of highly variable transcripts of venom genes in the habu snake.

We determined the primary structures of jacalin-related lectins termed PPL3s (PPL3A, 3B, and 3C, which are dimers consisting of sequence variants α + α, α + β, β + β, respectively) and PPL4, which is heterodimer consisting of α + β subunits, isolated from mantle secretory fluid of Pteria penguin (Mabe) pearl shell. Their carbohydrate-binding properties were analyzed, in addition to that of PPL2A, which was previously reported as a matrix protein. PPL3s and PPL4 shared only 35-50% homology to PPL2A, respectively; they exhibited significantly different carbohydrate-binding specificities based on the multiple glycan binding profiling data sets from frontal affinity chromatography analysis. The carbohydrate-binding specificity of PPL3s was similar to that of PPL2A, except only for Man3Fuc1Xyl1GlcNAc2 oligosaccharide, while PPL4 showed different carbohydrate-binding specificity compared with PPL2A and PPL3s. PPL2A and PPL3s mainly recognize agalactosylated- and galactosylated-type glycans. On the other hand, PPL4 binds to high-mannose-and hybrid-type N-linked glycans but not agalactosylated- and galactosylated-type glycans.

Encapsulation of guest molecules into the vacant space of biomacromolecular crystals has been utilized for various purposes including functioning as a protein container to protect against physical stress and structural determination of the guest. Todarodes pacificus hemocyanin (TpHc) is a hollow cylindrical decameric protein complex with an inner space 110 Å in diameter and 160 Å in height. In the crystal, TpHc forms a straw-like bundle and contains one reactive Cys (Cys3246) in the inner domain of each protomer. Here, we conjugated biotin onto Cys3246 of TpHc followed by incubation with streptavidin. The streptavidin was immobilized into the inner space of TpHc due to its interaction with biotin. Moreover, the complex containing TpHc and streptavidin was crystallized under the same conditions used for unmodified TpHc. In order to expand this methodology for a variety of proteins, we conjugated the ligand nitrilotriacetic acid (NTA) chelated to a Ni2+ ion (Ni2+-NTA) to TpHc. We found that His-tagged green fluorescent protein (GFP) was encapsulated into the Ni2+-NTA-conjugated TpHc via the interaction between the His-tag and the Ni2+-NTA group. X-ray crystallography demonstrated that the crystal packing of the complex containing TpHc and GFP was identical to that of the unmodified TpHc. Our guest immobilization method is distinct from previous approaches that are dependent on diffusion of the guest into the host crystal. Thus, our findings may accelerate the development of proteinaceous crystal engineering.

Phospholipase A2 (PLA2) is one of the representative toxic components of snake venom. PLA2s are categorized into several subgroups according to the amino acid at position 49, which comprises either Asp49, Lys49, Arg49 or Ser49. Previous studies suggested that the Lys49-PLA2 assembles into an extremely stable dimer. Although the behavior on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions suggested the presence of intermolecular disulfide bonds, these bonds were not observed in the crystal structure of Lys49-PLA2. The reason for this discrepancy between the crystal structure and SDS-PAGE of Lys49-PLA2 remains unknown. In this study, we analyzed a Lys49-PLA2 homologue from Protobothrops flavoviridis (PflLys49-PLA2 BPII), by biophysical analyses including X-ray crystallography, SDS-PAGE, native-mass spectrometry, and analytical ultracentrifugation. The results demonstrated that PflLys49-PLA2 BPII spontaneously oligomerized in the presence of SDS, which is one of the strongest protein denaturants.

Encapsulation of guest molecules into the hollow spaces of crystals has been applied for a variety of purposes such as structure determination, separation, and catalysis of the guest. Although host-guest studies have been developed mainly in crystals of small molecules, those of biomacromolecules have recently been applied. In those reports, a huge hollow space in the protein crystal is commonly used for encapsulation of the guest. Our previous study revealed that cylindrical hemocyanins stack inside the crystal as a linear hollow structure. The diameter of the linear hollow is approximately 110 Å, which is large enough for most proteins to pass through. In the present study, we evaluated the potential of hemocyanin crystals as a host to encapsulate biomacromolecules. Confocal microscopy revealed that hemocyanin crystals encapsulate proteins of molecular mass up to 250 kDa, i.e., 27 kDa green fluorescence protein, 105 kDa allophycocyanin, 220 kDa C-phycocyanin, and 250 kDa phycoerythrin, and DNAs up to 200-bp long, whereas 440 kDa ferritin not. Further analysis revealed that hemocyanin crystals prefer a negatively charged guest rather than a positive charge to encapsulate. Moreover, a photobleaching experiment showed that the guest does not move once entrapped. This knowledge of the host-guest study using the hollow hemocyanin crystal should be of significance for further application of hollow proteinaceous crystals as a host.

Evolution of novel traits is a challenging subject in biological research. Several snake lineages developed elaborate venom systems to deliver complex protein mixtures for prey capture. To understand mechanisms involved in snake venom evolution, we decoded here the ~1.4-Gb genome of a habu, Protobothrops flavoviridis. We identified 60 snake venom protein genes (SV) and 224 non-venom paralogs (NV), belonging to 18 gene families. Molecular phylogeny reveals early divergence of SV and NV genes, suggesting that one of the four copies generated through two rounds of whole-genome duplication was modified for use as a toxin. Among them, both SV and NV genes in four major components were extensively duplicated after their diversification, but accelerated evolution is evident exclusively in the SV genes. Both venom-related SV and NV genes are significantly enriched in microchromosomes. The present study thus provides a genetic background for evolution of snake venom composition.

The nacreous layer of pearl oysters is one of the major biominerals of commercial and industrial interest. Jacalin-related lectins, including PPL3 isoforms, are known to regulate biomineralization of the Pteria penguin pearl shell, although the molecular mechanisms are largely unknown. The PPL3 crystal structures were determined partly by utilizing microgravity environments for 3 isoforms, namely, PPL3A, PPL3B, and PPL3C. The structures revealed a tail-to-tail dimer structure established by forming a unique inter-subunit disulfide bond at C-termini. The N-terminal residues were found in pyroglutamate form, and this was partly explained by the post-translational modification of PPL3 isoforms implied from the discrepancy between amino acid and gene sequences. The complex structures with trehalose and isomaltose indicated that the novel specificity originated from the unique α-helix of PPL3 isoforms. Docking simulations of PPL3B to various calcite crystal faces suggested the edge of a β-sheet and the carbohydrate-binding site rich in charged residues were the interface to the biomineral, and implied that the isoforms differed in calcite interactions.

Rice bran lectins, named as RBA1 and RBA2, were isolated from Oryza sativa in two chromatography steps: affinity chromatography and cation-exchange chromatography. RBA1 was found to be composed of a covalently linked heterodimer of 20- and 12-kDa subunits, and RBA2 was a noncovalently linked dimer of 12-kDa subunits. Both RBA1 and RBA2 bound to desialylated complex glycoproteins such as fetuin, α1-acid glycoprotein, and transferrin, and agalactosylated complex glycoproteins such as agalacto fetuin, agalacto-α1-acid glycoprotein, and agalacto-transferrin, in addition to chitooligosacchrides. RBAs were heat stable up to 80 °C and stable at pH 4-10. RBA1 increased the transport of the fluorescent marker, rhodamine 123, which is known to be transported via the P-glycoprotein-mediated efflux pathway across human intestinal Caco-2 cell monolayers. Furthermore, RBA1 itself was transported to the basolateral side of the monolayers via an endocytotic pathway.

During a certain critical period in the development of the central and peripheral nervous systems, axonal branches and synapses are massively reorganized to form mature connections. In this process, neurons search their appropriate targets, expanding and/or retracting their axons. Recent work suggested that the caspase superfamily regulates the axon morphology. Here, we tested the hypothesis that caspase 3, which is one of the major executioners in apoptotic cell death, is involved in regulating the axon arborization. The embryonic chicken ciliary ganglion was used as a model system of synapse reorganization. A dominant negative mutant of caspase-3 precursor (C3DN) was made and overexpressed in presynaptic neurons in the midbrain to interfere with the intrinsic caspase-3 activity using an in ovo electroporation method. The axon arborization pattern was 3-dimensionally and quantitatively analyzed in the ciliary ganglion. The overexpression of C3DN significantly reduced the number of branching points, the branch order and the complexity index, whereas it significantly elongated the terminal branches at E6. It also increased the internodal distance significantly at E8. But, these effects were negligible at E10 or later. During E6-8, there appeared to be a dynamic balance in the axon arborization pattern between the "targeting" mode, which is accompanied by elongation of terminal branches and the pruning of collateral branches, and the "pathfinding" mode, which is accompanied by the retraction of terminal branches and the sprouting of new collateral branches. The local and transient activation of caspase 3 could direct the balance towards the pathfinding mode.

Background: Nickel (Ni) is the most frequent metal allergen and induces a TH1-dependent type-IV allergy. Although Ni2+ is considered to bind to endogenous proteins, it currently remains unclear whether these Ni-binding proteins are involved in Ni allergy in vivo. We previously reported the adjuvant effects of lipopolysaccharide (LPS) in a Ni allergy mouse model. As LPS induces a number of inflammatory mediators, we hypothesized that Ni-binding protein(s) are also induced by LPS. Objective: The objective of this study was to purify and identify Ni-binding protein(s) from serum taken from LPS-injected mice (referred as LPS serum) and examined the augmenting effects of these Ni-binding protein(s) on Ni allergy in an in vivo model. Methods: BALB/cA mice were sensitized with an i.p. injection of NiCl2 and LPS. Ten days after sensitization, mice were challenged with NiCl2 by an i.d. injection into ear pinnae. Ni-binding protein(s) were purified by Ni-affinity column chromatography and gel filtration. Results: Lipopolysaccharide serum, but not serum taken from saline-injected mice, augmented ear swelling induced by Ni-allergic inflammation. Ni-binding, but not non-binding fraction, purified from LPS serum augmented Ni-allergic inflammation. Mass spectrometry and Western blotting detected CXCL4 in the active fraction. A batch analysis with Ni-sepharose and a surface plasmon resonance analysis revealed direct binding between CXCL4 and Ni2+. Recombinant CXCL4 augmented Ni-allergic inflammation and exerted adjuvant effects at the sensitization phase. Conclusions: These results indicate that CXCL4 is a novel Ni-binding protein that augments Ni allergy at the elicitation and sensitization phases. This is the first study to demonstrate that the Ni-binding protein augments Ni allergy in vivo.

The water solubility of rice bran protein (RBP) was improved by deamidation under alkaline conditions. The degree of deamidation was found to be a major factor in improving the solubility of RBP. The decrease in molecular mass or the degradation of peptide bonds was not detected in deamidated REP under the conditions used. The thermal property and secondary structure of deamidated RBP measured by differential scanning calorimetry and Fourier transform infrared spectroscopy indicated that the secondary structure of RBP was well preserved during alkaline deamidation. By raising pH and temperature for deamidation, the generation rates of constituent amino acid racemization and lysinoalanine increased. The highest solubility (similar to 90%) of RBP was achieved by treatment at pH 12 and 120 degrees C for 15 - 30 min by enduring side reactions. Moderate solubility (similar to 40%) could be achieved by deamidation at pH 8 and 100 degrees C for 30 min to minimize side reactions.

Proteins contained in the green leaf mucus of Japanese bunching onion (Allium fistulosum L.) were isolated by ammonium sulfate precipitation and separated by hydrophobic interaction chromatography. The mucus proteins had an enhanced effect on tumor necrosis factor (TNF)-alpha production in the RAW 264 murine macrophage cell line. Protein composition was analyzed by SDS-polyacrylamide gel electrophoresis and N-terminal amino acid sequencing. The effect on RAW 264 cells was mainly attributed to mannose-binding lectin and thaumatin-like proteins, which occupied 34% of the mucus proteins. Other major proteins contained in the mucus showed homologous amino acid sequences to those of lachrymatory factor synthase, beta-1,3-glucanase, 2S albumin precursor, and pathogenesis-related protein 4. Protein composition ratios in the mucus of 13 cultivars of Japanese bunching onion were also examined. Taken together, the green leaf mucus of Japanese bunching onion may be a possible source of various bioactive proteins.

Free radical scavenging activities of two series of tripeptide libraries were investigated using ABTS, DPPH, and ORAC assays. Two Tyr-containing tripeptides showed higher scavenging activities against the hydrophilic ABTS and AAPH radicals than two His-containing tripeptides, showing that Tyr residues play important roles in free radical scavenging activity. Higher concentrations of tripeptides were required to reveal apparent DPPH radical scavenging activity. The antioxidant activities of the tripeptide libraries obtained from this study were compared with previous results obtained using several assays, including antioxidant activity against the peroxidation of linoleic acid, FRAP assay, and peroxynitrite (PN) scavenging activity. The antioxidant activity of the tripeptides against the peroxidation of linoleic acid showed high correlations with the ABTS and ORAC assays (correlation coefficients (R) = 0.725 and 0.731, respectively), and low correlations with FRAP, PN scavenging, and DPPH assays. The highest correlation was found between the ABTS and ORAC assays (R = 0.905).

The effect of a chum salmon egg lectin (CSL3) on tight junction (TJ) of Caco-2 cell monolayers was investigated. The lectin opened TJ as indicated by the decrease of the transepithelial electrical resistance (TER) value and the increase of the permeation of lucifer yellow, which is transported via the TJ-mediated paracellular pathway. The effects of CSL3 were inhibited by the addition of 10 mM L-rhamnose or D-galactose which were specific sugars for CSL3. The lectin increased the intracellular Ca2+ of Caco-2 cell monolayers, that could be inhibited by the addition of L-rhamnose. The fluorescence immunostaining of -actin in Caco-2 cell monolayers revealed that the cytoskeleton was changed by the CSL3 treatment, suggesting that CSL3 depolymerized -actin to cause reversible TJ structural and functional disruption. Although Japanese jack bean lectin and wheat germ lectin showed similar effects in the decrease of the TER values and the increase of the intracellular Ca2+, they could not be inhibited by the same concentrations of simple sugars, such as D-glucose and N-acetyl-D-glucosamine.

Apios tuber lectin, named ATL, was isolated from Apios americana Medikus by two chromatography steps, hydrophobic chromatography and anion-exchange chromatography. The minimum concentration required for the hemagglutination activity toward rabbit erythrocytes of ATL was 4 μg/mL. ATL was composed of a homodimer of 28.4 kDa subunits. The amino acid sequence of ATL was similar to those of other legume lectins. The lectin showed moderate stability toward heating and acidic pH, and the binding affinity against several monosaccharides, such as D-glucosamine and D-galactosamine. ATL also bound to desialylated or agalactosylated glycoproteins such as asialo and agalacto transferrin. ATL decreased the transepithelial electrical resistance across human intestinal Caco-2 cell monolayers, suggesting the effect on the tight junction-mediated paracellular transport.

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10 kDa, respectively, and under non-reducing conditions, 26 kDa was observed. The inhibitor was shown to inhibit bovine trypsin (Ki of 3.45 nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.

Protein evolution is a process of molecular design leading to the diversity of functional proteins found in nature. Recent advances in bioinformatics and structural biology, in addition to recombinant protein expression techniques, enable us to analyze more directly the molecular evolution of proteins by a new method using ancestral sequence reconstruction (ASR), the so-called experimental molecular archaeology. ASR has been used to reveal molecular properties and structures correlating with changing geology, ecology, and physiology, and to identify the structure elements important to changing physiological functions to fill substantial gaps in the processes of protein evolution. In this chapter, we describe ASR as a new method of protein engineering studies, and their application to analyzing lectins, of which evolutionary processes and structural features contributing to molecular stability, specificity, and unique functions have been elucidated. Experimental molecular archeology using ASR and crystal structures of full-length ancestral proteins is useful in understanding the evolutionary process of the functional and structural diversified lectins by tracing ancestral specificities.

featured in Nature Chemical Biology’s News & Views, January Issue 2015

© 2013 Springer Science+Business Media Dordrecht. All rights are reserved. During the past several decades, intensive investigations have been conducted to clarify the biochemical and physiological properties of lectins from marine organisms, including cyanobacteria, algae, and invertebrates and fish. These investigations have revealed that lectins are highly diversified in terms of not only structural aspects but also functional aspects, including unique carbohydrate-binding specificities. Lectins are still being intensively investigated to understand their biological roles in cell recognition and biodefense as well as to employ them as valuable tools for studying complex carbohydrates in solution and on cell surfaces. Here, we review the structures and activities of lectins from marine organisms and their applications as carbohydrate recognition molecules and medicinal agents with antitumor and antiviral activities.

The effects of 16 lectins isolated from foodstuff on the transport system across human intestinal Caco-2 cell monolayers were investigated by using four fluorescent markers: lucifer yellow (LY) for the paracellular pathway, fluorescein (FL) for the monocarboxylic acid transporter-mediated pathway, rhodamine 123 for the P-glycoprotein-mediated efflux pathway, and calcein for the multidrug resistance associated protein-related efflux pathway. The transepithelial electrical resistance (TER) values for the monolayers were also measured. WGA from wheat germ, ABA from white mushroom, AOL from Aspergillus oryzae, and CSL3 from chum salmon eggs (each at 100 mu g/mL) decreased the TER value by 20-40% which resulted in increased LY transport. These lectins, as well as such other lectins as SBA from soybean, RBA from rice bran, and Con A from jack bean, affected other transport pathways too. These results indicate that the lectins modulated the transepithelial transport system in different ways, probably because of their specific binding characteristics toward Caco-2 cell monolayers.

Rhamnose-binding lectins (RBLs) have been isolated and characterized from various kinds of fish. RBLs recognize the structures of lipopolysaccharides and lipoteichoic acid on microbes, and have an opsonic effect, suggesting that RBLs play an important role as pattern recognition proteins in innate immunity. However, the function of RBLs in the removal of pathogens from the body has not yet been elucidated. In this study, we investigated the effect of three RBLs-named CSL1, 2, and 3-that were isolated from chum salmon Oncorhynchus keta eggs on the respiratory burst activity in a macrophage cell line. The CSLs induced reactive oxygen species in the peritoneal macrophage cell line from rainbow trout Oncorhynchus mykiss (RTM5). However, this activity was not affected by l-rhamnose or dl-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, an inhibitor for glucosylceramide synthesis, indicating that the interaction between CSLs and Gb3 on cell surfaces was not involved in the induction of respiratory burst activity.

Congerin is a proto-type galectin distributed on the skin and mucosal epithelia of the upper digestive tract of the Japanese conger eel Conger myriaster. It has at least 2 isotypes, namely, congerin I and II, and plays a role in bio-defense at the body surface. In the current study, we identified both isotypes in the peritoneal fluid and peritoneal cells of C. myriaster by western blot and mass spectrometry (MS)/MS analysis. Cucullanus nematodes parasitize the abdominal cavity of C. myriaster, and immunohistochemical analyses demonstrated that congerins can bind to both the body surface of the encapsulated nematodes and the encapsulating cells. Furthermore, adhesion of the peritoneal cells to Sepharose particles was greatly accelerated when the microspheres were coated with congerin. Indeed, this effect was significantly hampered by the addition of lactose. These results indicate that congerin participates in the cellular encapsulation of the Cucullanus nematode via the induction of cellular adhesion to the parasites depending on lectin-glycoside recognition.

Conger eel has two galectins, termed congerins I and II (Con I and II), that function in mucus as biodefense molecules. Con I and II have acquired a novel protein fold via domain swapping and a new ligand-binding site by accelerated evolution, which enables recognition of some marine bacteria. In this study, we identified a new congerin isotype, congerin P (Con-P), from the peritoneal cells of conger eel. Although Con-P displayed obvious homology with galectins, we observed substitution of 7 out of 8 amino acid residues in the carbohydrate recognition domain that are conserved in all other known galectins. To understand the structure-function relationships of this unique galectin, recombinant Con-P was successfully expressed in Escherichia coli by using a Con II-tagged fusion protein system and subsequently characterized. In the presence of D-mannose, Con-P displayed 30-fold greater hemagglutinating activity than Con I; however, no activity was observed without mannose, indicating that D-mannoside can act as a modulator of Con- P. Frontal affinity chromatography analysis showed that activated Con-P, allosterically induced by mannose, displayed affinity for oligomannose-type sugars as well as N-acetyllactosamine-type beta-galactosides. Thus, Con-P represents a new member of the galectin family with unique properties.

Dioscorea batatas tuber lectin 1 (DB1) is a storage protein isolated from yam tuber and has been shown to be a mannose-binding lectin. Here we produced transgenic rice plants expressing cDNA of DB1 under the control of phloem-specific promoter of rice sucrose synthase-1 gene. DB1 accumulated at a level of 0.07% of total soluble protein. We then evaluated its efficacy for brown planthopper (BPH). After releasing the first instar of BPH on the transgenic rice plants, the number of survived BPH adults was reduced up to 30% compared to that of wild-type rice. The number of the next generation BPH was suppressed to 22% on average in the seven most-resistant plants compared to that of wild-type rice plants when female adult BPH was inoculated. These results demonstrated that DB1 is effective to confer BPH resistance in terms of decreased survival and fecundity.

Ancestral structures of fish galectins (congerins) were determined. The extant isoforms I and II of congerin are the components of a fish biological defense system and have rapidly differentiated under natural selection pressure, by which congerin I has experienced a protein-fold evolution. The dimer structure of the ancestral congerin demonstrated intermediate features of the extant isoforms. The protein-fold evolution was not observed in the ancestral structure, indicating it specifically occurred in congerin I lineage. Details of hydrogen bonding pattern at the dimer interface and the carbohydrate-binding site of the ancestor were different from the current proteins. The differences implied these proteins were under selection pressure for stabilizing dimer structure and differentiation in carbohydrate specificity. The ancestor had rather low cytotoxic activity than offspring, indicating selection was made to enhance this activity of congerins. Combined with functional analyses, the structure revealed atomic details of the differentiation process of the proteins.

Cathepsin D was purified from ostrich (Struthio camelus) skeletal muscle using pepstatin-A chromatography. The enzyme was comprised of two subunits (29.1 and 14 kDa). The N-terminal amino acid sequence of both subunits were determined and showed high amino acid sequence identity to other cathepsin D homologs. Ostrich cathepsin D was optimally active at pH 4 and at a temperature of 45°C, and was strongly inhibited by pepstatin-A (K(i)=3.07×10(-9)M) and dithiothreitol. Cathepsin D activities from five ostriches were monitored over a 30-day period. Cathepsin D remained substantially active throughout the 30-day storage period with an average remaining activity of 112±8.57% at day 30 (mean value from 5 ostriches).

Marine bioresources produce a great variety of specific and potent bioactive molecules including natural organic compounds such as fatty acids, polysaccharides, polyether, peptides, proteins, and enzymes. Lectins are also one of the promising candidates for useful therapeutic agents because they can recognize the specific carbohydrate structures such as proteoglycans, glycoproteins, and glycolipids, resulting in the regulation of various cells via glycoconjugates and their physiological and pathological phenomenon through the host-pathogen interactions and cell-cell communications. Here, we review the multiple lectins from marine resources including fishes and sea invertebrate in terms of their structure-activity relationships and molecular evolution. Especially, we focus on the unique structural properties and molecular evolution of C-type lectins, galectin, F-type lectin, and rhamnose-binding lectin families.

BACKGROUND: Conger eel galectins, congerin I (ConI) and congerin II (ConII), show the different molecular characteristics resulting from accelerating evolution. We recently reconstructed a probable ancestral form of congerins, Con-anc. It showed properties similar to those of ConII in terms of thermostability and carbohydrate recognition specificity, although it shares a higher sequence similarity with ConI than ConII. RESULTS: In this study, we have focused on the different amino acid residues between Con-anc and ConI, and have performed the protein engineering of Con-anc through site-directed mutagenesis, followed by the molecular evolution analysis of the mutants. This approach revealed the functional importance of loop structures of congerins: (1) N- and C-terminal and loop 5 regions that are involved in conferring a high thermostability to ConI; (2) loops 3, 5, and 6 that are responsible for stronger binding of ConI to most sugars; and (3) loops 5 and 6, and Thr38 residue in loop 3 contribute the specificity of ConI toward lacto-N-fucopentaose-containing sugars. CONCLUSIONS: Thus, this methodology, with tracing of the molecular evolution using ancestral mutants, is a powerful tool for the analysis of not only the molecular evolutionary process, but also the structural elements of a protein responsible for its various functions.

The extent of current research in ancient biology is not restricted by the presence of fossil records, owing to the developments in computational analysis and engineering techniques of proteins. Ancestral sequences of extant genes/proteins can be inferred with the molecular phylogeny reconstruction methods, which are mainly based on maximum-likelihood estimation. Once an ancestral sequence is determined, a reconstruction of the ancestral protein is not difficult by using the current protein engineering techniques, and reproduced protein can be experimentally tested either biochemically or biophysically. To date, such 'experimental molecular archeology' has been examined on several enzymes, elongation factor, photo-reactive proteins, nuclear receptor, and lectin, and largely helped to elucidate evolutionary processes of stability, specificity, and structure of the proteins. Although this method would promise a new field in protein engineering, several problems still remain to be solved, the largest among which should be accuracy of inferred ancestral sequence as a whole. In this chapter, the reported studies of ancestral protein reconstruction are reviewed with a focus on our recent results on the ancestral congerin, which is the ß-galactoside specific lectin of fish involved in the biological defense system. The reconstruction of ancestral congerin is important because two extant isoforms of this protein, congerins I and II, have been rapidly evolving, and their molecular properties and structures have been differentiating due to the natural selection pressures operating on the genes. © 2010 Nova Science Publishers, Inc. All rights reserved.

We examined changes of functional components and antioxidative activity of soybeans during the processes for producing a fermented soybean food, natto. Proteolytic hydrolysis of proteins was followed along with the changes in the contents of isoflavones, saponins, and phytic acid. The antioxidative activities were measured by radical scavenging, Fe(2+) chelating, superoxide dismutase-like activity, and beta-carotene-linoleic acid assay. Radical scavenging activity under hydrophilic conditions increased with protein hydrolysis during fermentation. At the same time, total isoflavones decreased. Total saponins and phytic acid were kept at similar levels. The analyses of functional components and antioxidative activities showed that antioxidative peptides generated during fermentation contributed to the total antioxidative capacity of natto.

Dioscorea batatas tuber lectin 1 (DB1) is a storage protein isolated from yam tuber, and is shown to be a mannose-binding lectin. It has 58% amino-acid identity to insecticidal snowdrop bulb lectin GNA. In this study, we demonstrated that ≥ 1 mg ml-1 DB1 in an artificial diet significantly decreased the survival and fecundity of green peach aphid, Myzus persicaeca. We produced transgenic tobacco plants expressing cDNA of DB1 under the control of Cauliflower mosaic virus 35S promoter (35S-DB1) or phloem-specific promoter of rice sucrose synthase-1 gene (RSs1-DB1), and evaluate the degree of aphid resistance in whole plant bioassays. The number of survival aphids was reduced to 60% in transgenic lines with 35S-DB1 and RSs1-DB1, which accumulated DB1 at a level of 1.8% and 0.25%, respectively, of total soluble protein. Our results indicate that DB1 can be used to enhance resistance to sap-sucking insects in transgenic crops. © 2010 The Japanese Society for Plant Cell and Molecular Biology.

Columns of phosphorylcholine (PC) immobilized on silica gel were shown to be useful for size exclusion chromatography (SEC) of proteins. The columns provided good separation of proteins in 50 mM sodium phosphate buffer (pH 6.9) containing 0.25 M NaCl, and there was a linear relationship between the retention times and the logarithmic values of the molecular weights with a correlation coefficient (R2) of 0.978-0.992. The columns were used in analyzing the subunit structures of the rhamnose-binding lectins CSL1, CSL2, and CSL3, isolated from chum salmon (Oncorhynchus keta) eggs. Although the lectins, which are a group of carbohydrate-binding and hydrophobic proteins, behaved anomalously in SEC with conventional matrices, they could be eluted from the immobilized PC columns without non-size-related retention, thereby allowing their molecular weights to be reliably estimated. © 2009 Elsevier B.V. All rights reserved.

Yeasts of the genus Lipomyces are known as fat yeasts, and they store large amounts of lipids. Because the major lipid produced by Lipomyces is triglyceride, which can be used as a food and energy resource, the control of lipid production by Lipomyces sp. is an important issue. Here we report the effects of UV irradiation on lipid production in Lipomyces lipofer cells. UV irradiation (315-400 nm) led to a 4-fold increase in the amount of triglyceride per cell. We discovered a novel phenomenon, that UV irradiation promotes triglyceride accumulation in L. lipofer.

The crystal structure of the L-rhamnose-binding lectin CSL3 was determined to 1.8 A resolution. This protein is a component of the germline-encoded pattern recognition proteins in innate immunity. CSL3 is a homodimer of two 20 kDa subunits with a dumbbell-like shape overall, in which the N- and C-terminal domains of different subunits form lobe structures connected with flexible linker peptides. The complex structures of the protein with specific carbohydrates demonstrated the importance of the most variable loop region among homologues for the specificity toward oligosaccharides. CSL3 and Shiga-like toxin both use Gb(3) as a cellular receptor to evoke apoptosis. They have very different overall architecture but share the separation distance between carbohydrate-binding sites. An inspection of the structure database suggested that the pseudo-tetrameric structure of CSL3 was unique among the known lectins. This architecture implies this protein might provide a unique tool for further investigations into the relationships between architecture and function of pattern recognition proteins.

The amino acid sequence of mannose-binding lectin, named DB1, from the yam (Dioscorea batatas, synonym Dioscorea polystachya) tubers was determined. The lectin was composed of two isoforms DB1 (Cys86) and DB1 (Leu86) consisting of 108 amino acid residues with 90% sequence homology between them. DB1 showed a high sequence similarity to snowdrop (Galanthus nivalis) bulb lectin, GNA; especially, the carbohydrate-binding sites of GNA were highly conserved in DB1. DB1 interacted with D-mannose residues of oligosaccharides, and the oligosaccharides carrying two mannose-alpha-1,3-D-mannose units showed high binding affinity. DB1 was examined for insecticidal activity against Helicoverpa armigera (Lepidoptera: Noctuidae) larvae at different stages of development. The rate of adults successfully emerging from pupae fed on DB1 was 33%, when incorporated into an artificial diet at a level of 0.01% (w/w). Although DB1 had no or marginal inhibitory effects on gut proteolytic and glycolic enzymes, the lectin strongly bound to larval brush border and peritrophic membrane detected by immunostaining. The results show that DB1 may fulfill a defense role against insect pests.

Rice bran protein fraction (RBPF)-albumin, globulin, glutelin and prolamin were hydrolyzed with proteases M, N, P, S and pepsin under their optimal conditions for 24 h. Hydrolysates of various hydrolysis periods were collected and subjected to peptide mapping and the antioxidative activity measured by the 2,2-Azino-bis-3-ethylbenzothiazoline-6-sulfonic Acid (ABTS) method. Protease M hydrolysates showed high degree of hydrolysis (DH), but low antioxidative activity. On the contrary, pepsin hydrolysates showed low DH with high activity. Albumin and globulin hydrolysates had higher DH values, but lower values for glutelin and prolamin. The globulin hydrolysate (Opep2) from 2 h-pepsin hydrolysis was separated by using three consecutive purification steps with RP-HPLC. Nineteen antioxidative peptides were isolated and their amino acid sequences were determined by a gas-phase protein sequencer and MALDI-TOF mass spectrometry. These peptides were composed of 6-30 amino acid residues with molecular masses ranging from 670-3,611 Da. Tyr-Leu-Ala-Gly-Met-Asn had the highest antioxidative activity among them.

Rice bran contains underutilised protein materials. Sequential extraction of rice bran protein (RBP) from defatted rice bran was conducted based on the differences in their solubility. Three extraction methods were investigated. Method 1 involved the isoelectric and acetone precipitation using water, 50 g kg-1 NaCl, 0.02 mol L-1 NaOH and 70% ethanol as extracting solvents for albumin (pH 4.1), globulin (pH 4.3), glutelin (pH 4.8) and prolamin, respectively. Method 2 adopted dialysis and sequential extraction was carried out with 20 g kg-1 NaCl, 70% ethanol, 0.1 mol L-1 acetic acid and 0.1 mol L-1 NaOH solution as extracting solvents. Method 3 combined dialysis, isoelectric and acetone precipitation for the extraction. Based on the yields and data obtained from sodium dodecyl sulphate polyacrylamide gel electrophoresis, size-exclusion chromatography and differential scanning calorimetry, method 3 was chosen for the isolation and characterization of RBPs. Rice bran protein fraction (RBPF)-albumin, globulin, glutelin and prolamin were obtained in good yields. Denaturation temperature and enthalpy values of denaturation of RBPF vary. Highest phytate content was found in albumin and lowest in prolamin. The highest antioxidative and hemagglutinating activities were observed in albumin.

L-Rhamnose-binding lectins (RBLs) have been isolated from various kinds of fish and invertebrates and interact with various kinds of bacteria, suggesting RBLs are involved in various inflammatory reactions. We investigated the effect of RBLs from chum salmon (Oncorhynchus keta), named CSL1, 2 and 3. on the peritoneal macrophage cell line from rainbow trout (Oncorhynchus mykiss) (RTM5) and an established fibroblastic-like cell line derived from gonadal tissue of rainbow trout (RTG-2). CSLs were bound to the surface of RTM5 and RTG-2 cells and induced proinflammatory cytokines, including IL-1 beta 1, IL-1 beta 2, TNF-alpha 1, TNF-alpha 2 and IL-8 in both cells by recognizing globotriaosylceramide (Gb3). In addition, CSLs had an opsonic effect on RTM5 cells and this effect was significantly inhibited by L-rhamnose, indicating that CSLs enhanced their phagocytosis by binding to Gb3 on cell surfaces. This is the first finding that Gb3 plays a role in innate immunity by cooperating with natural ligands, RBLs. (C) 2008 Elsevier Ltd. All rights reserved.

A novel recombinant expression system in Escherichia coli was developed using conger eel galectin, namely, congerin II, as an affinity tag. This system was applied for the functional expression of myotoxic lysine-49-phospholipase A2 ([Lys49]PLA2), termed BPII and obtained from Protobothrops flavoviridis (Pf) venom. Recombinant Pf BPII fused with a congerin tag has been successfully expressed as a soluble fraction and showed better quantitative yield when folded correctly. The solubility of the recombinant congerin II-tagged BPII increased up to >90% in E. coli strain JM109 when coexpressed with the molecular chaperones GroEL, GroES, and trigger factor (Tf). The tag protein was cleaved by digestion with restriction protease, such as α-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for α-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity. © 2007 Elsevier Inc. All rights reserved.

The modulating effect of invertebrate C-type lectins on the crystallization of calcium carbonate was investigated. The multiple C-type lectins, named BRA-1, -2 and -3, isolated from the acorn barnacle Megabalanus rosa inhibited the nucleation and growth of calcium carbonate crystals. Among BRA, BRA-2 most efficiently inhibited the crystal nucleation of aragonite and calcite at the concentrations of >3.3 and >26 μg/mL, respectively. The inhibitory activities of BRA were enhanced by the addition of various biomolecules such as D-glucosamine, D-galactosamine, chitosan oligosaccharides, L-arginine and L-aspartic acid. Although the crystals of aragonite and calcite were formed in the presence of lower concentrations of BRA, the shape and size of the crystals were changed. These results further indicate the participation of the lectins in biomineralization. © 2008 Japanese Society of Fisheries Science.

The development of degradable and edible films from protein sources has drawn significant attention for the utilisation of natural resources as well as for the alleviation of the environmental burden. Rice bran protein (RBP) was applied to protein film preparation in this study. The protein solutions were casted on plastic tissue culture dishes with glycerol as a plasticiser after heat treatment. Functional properties of the films were then measured. The puncture strength (PS) of RBP films increased up to pH 8.0 and then decreased. PS of protein films depends on the degree of protein purity, quality and composition. Higher concentration of glycerol weakened the films. The pH affected the water solubility of RBP films and the films showed least solubility at pH 3.0. RBP could be utilised in the preparation of degradable protein-based films. The RBP-based film had functional properties comparable to those of the soy protein-based ones. © 2007 Institute of Food Science and Technology Trust Fund.

Because sugar-binding proteins, so-called lectins, play important roles in many biological phenomena, the lectin-selective labeling should be useful for investigating biological processes involving lectins as well as providing molecular tools for analysis of saccharides and these derivatives. We describe herein a new strategy for lectin-selective labeling based on an acyl transfer reaction directed by ligand-tethered DMAP (4-dimethylaminopyridine). DMAP is an effective acyl transfer catalyst, which can activate an acyl ester for its transfer to a nucleophilic residue. To direct the acyl transfer reaction to a lectin of interest, we attached the DMAP to a saccharide ligand specific for the target lectin. It was clearly demonstrated by biochemical analyses that the target-selective labeling of Congerin II, an animal lectin having selective affinity for Lactose/LacNAc (N-acetyllactosamine), was achieved in the presence of Lactethered DMAPs and acyl donors containing probes such as fluorescent molecules or biotin. Conventional peptide mapping experiments using HPLC and tandem mass-mass analysis revealed that the acyl transfer reaction site-specifically occurred at Tyr 51 of Cong II. This strategy was successfully extended to other lectins by changing the ligand part of the ligand-tethered DMAP. We also demonstrated that this labeling method is applicable not only to purified lectin in test tubes, but also to crude mixtures such as E. coli lysates or homogenized animal tissue samples expressing Congerin. © 2008 American Chemical Society.

Crude rice bran protein (CRBP) was prepared by alkaline extraction and then treated with 0.6 m HCl to remove phytic acid. The phytate-free rice bran protein (PFRBP) was hydrolysed with proteases M, N, S, P and pepsin under optimal conditions. Hydrolysates obtained from various hydrolysis periods were subjected to analysis for their degree of hydrolysis (DH) and functional properties. The hydrolysates were fractionated by reversed-phase column chromatography on Kaseigel ODS resin (120-140 μm) using a stepwise gradient of aqueous ethanol, and their activities were measured. The 40% ethanol fraction of protease P 4 h-hydrolysate was separated by successive reversed-phase high-performance liquid chromatography and the amino acid sequences of isolated antioxidative peptides were determined by a protein sequencer and matrix-assisted laser desorption ionisation-time of flight mass spectrometry. Crude rice bran protein had higher antioxidative activity than PFRBP, due to the presence of phytic acid. Phytate contents of rice bran, CRBP and PFRBP were 2.5%, 1.42% and 0%, respectively. The activity of PFRBP increased upon protease digestion. Protease M hydrolysates showed the highest DH, but the lowest antioxidative activity. Hydrolysates with DH below 10% had higher antioxidative activity than those above 20%. This result indicates that the antioxidative activity of the hydrolysates is inherent to their characteristics amino acid sequences of peptides depending on the protease specificities. © 2007 Institute of Food Science and Technology Trust Fund.

A rhamnose-binding lectin, named SFL, was isolated from the eggs of ayu (sweet fish, Plecoglossus altivelis) by affinity and ion-exchange chromatographies. SFL revealed 287 amino acid residues with 3 tandemly repeated domains, and contained 8 half-Cys residues in each domain. The lectin was shown to have a highly specific binding affinity to globotriaosylceramide (Gb3) by frontal affinity chromatography using 100 oligosaccharides. SFL was localized in several tissues and serum of both mate and female ayu, such as gill, liver, ovary, testis, intestine, stomach, brain, kidney and serum. The lectin agglutinated the spores of the microsporidian Glugea plecoglossi, which is a pathogen of ayu. Although SFL bound to glycoproteins and glycolipids of G. plecoglossi spores, Gb3 could not be detected in either of them. The results suggest that SFL could interact with various glycoconjugates of pathogens to play a role in the adhesion of microorganisms invading in the body. (c) 2007 Elsevier Ltd. All rights reserved.

Recently, many cases of rapid adaptive evolution, which is characterized by the higher substitution rates of nonsynonymous substitutions to synonymous ones, have been identified in the various genes of venomous and biodefense proteins, including the conger eel galectins, congerins I and II (ConI and ConII). To understand the evolutionary process of congerins, we prepared a probable ancestral form, Con-anc, corresponding to the putative amino acid sequence at the divergence of ConI and ConII in phylogenetic tree with 76% and 61% sequence identities to the current proteins, respectively. Con-anc and ConII had comparable thermostability and similar carbohydrate specificities in general, whereas ConI was more thermostable and showed different carbohydrate specificities. Con-anc showed decreased specificity to oligosaccharides with alpha 2,3-sialyl galactose moieties. These suggest that ConI and ConII have evolved via accelerated evolution under significant selective pressure to increase the thermostability and to acquire the activity to bind to α2,3-sialyl galactose present in pathogenic bacteria, respectively. Furthermore, comparative mutagenesis analyses of Con-anc and congerins revealed the structural basis for specific recognition of ConII to α2,3-sialyl galactose moiety, and strong binding ability of ConI to oligosaccharides including lacto-N-biosyl (Galβ1-3GlcNAc) or lacto-N-neobiosyl (Galβ1-4GlcNAc) residues, respectively. Thus, protein engineering using a probable ancestral form presented here is a powerful approach not only to determine the evolutionary process but also to investigate the structure-activity relationships of proteins. © The Author 2007. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved.

Rice bran contains 120-200 g kg-1 protein in addition to a large amount of fat, carbohydrate, and phytic acid. Rice bran protein (RBP) fractions were refined by a two-step preparation to eliminate residual carbohydrate. The first step involved the sequential extraction of defatted rice bran into RBP fractions using their distinct solubility to give 37 g kg-1 of albumin, 31 g kg-1 of globulin, 27 g kg-1 of glutelin, and 2 g kg-1 of prolamin. In the second step, carried out by dissolving in respective solvent and isoelectric precipitation, the protein content of each fraction increased from 69% to 97% for albumin, from 71% to 90% for globulin, from 74% to 83% for glutelin, and from 18% to 20% for prolamin. The low protein content in the prolamin fraction might be due to its low solubility in the protein assay. Emulsifying stability index and surface hydrophobicity increased in the second-step preparation of albumin and globulin, but not of glutelin. Emulsifying properties of RBPs were lower than that of a soybean protein isolate. Denaturation temperatures and enthalpy values of denaturation for albumin, globulin, glutelin, and prolamin were 50.1°C/1.2 J g-1, 79.0°C/1.8 J g-1, 74.5°C/3.0 J g-1, and 78.5°C/8.1 J g-1, respectively. No significant differences in the denaturation temperatures and enthalpy values of denaturation of RBP fractions were obtained with these two-step preparations (P < 0.05). © 2007 Society of Chemical Industry.

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-Gal beta 1-4GlcNAc beta 1-2Man alpha 1-6-(NeuAc-Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man beta 1-4GlcNAc beta 1-4GlcNAc-Asn. (c) 2006 Elsevier B.V All rights reserved.

The sugar-binding specificities of C-type lectins isolated from marine invertebrates were investigated by frontal affinity chromatography (FAC) using 100 oligosaccharides. The lectins included BRA-2 and BRA-3, multiple lectins from the hemolymph of the acorn barnacle, Megabalanus rosa, and BRL from the acorn barnacle, Balanus rostatus. The diverse sugar-binding specificities of the C-type lectins were determined by FAC analysis. BRA-2 recognized α2-6 sialylation but not α2-3 sialylation on glycans. On the other hand, BRA-3 showed high affinity for oligosaccharides with α-linked non-reducing terminal galactose, but not for sialylated forms, and BRL showed enhanced recognition activity towards Lewisx and Lewisa epitopes.

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 A2 (PLA2) isozymes were purified to homogeneity from P. elegans venom and sequenced. This led to a discovery of novel PLA2 isozymes with Arg at position 49, that is, [Arg49]PLA2 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 [Asp49]PLA2 named PePLA2 was also obtained. In contrast to PLA2 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 PLA2 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 PmK49PLA2, a [Lys49]PLA2, from P. mucrosquamatus (Taiwan) than BPI and BPII, both [Lys49]PLA2 forms, from P. flavoviridis (Amami-Oshima and Tokunoshima islands of Japan). Such evolutionary relationships are also seen in neutral [Asp49]PLA2 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. © 2006 Elsevier Ltd. All rights reserved.

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 μg/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 Galβ1-4Glu/GlcNAc, Galβ1-3GalNAc/GlcNAc and Galα 1-4Gal moieties in their structure. PPL had a high thermal stability and retained 50% of its hemagglutinating activity after incubation at 70°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. © 2006 Springer Science+Business Media, Inc.

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 KA/KS 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.

Lectins distributing in all animal phyla form a diverse group of protein families that have in common the ability to recognize and bind certain carbohydrates. Although at least 13 animal lectin families are known to exist, many of marine invertebrate lectins are categorized in C-type lectin family, which was named from the Ca^<2+>-dependency for their carbohydrate binding activities. In contrast to a growing list of C-type lectins in marine invertebrates, their physiological roles are not fully understood. This review summarizes the structures and functions of marine invertebrate C-type lectins with our new findings.

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. © 2006 American Chemical Society.

Carnivorous plants usually grow in nutrient-deficient habitats, and thus they partly depend on insects for nitrogen and phosphate needed for amino acid and nucleotide synthesis. We report that a sticky digestive liquid from a sundew, Drosera adelae, contains an abundant amount of an S-like ribonuclease (RNase) that shows high amino acid-sequence similarity to S-like RNases induced by phosphate starvation or wounding in normal plants. By giving leaves an RNase "coat", D. adelae seems to achieve two requirements simultaneously to adapt itself to its specific surroundings: it obtains phosphates from insects, and defends itself against pathogen attack. © 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima and Okinawa. A phospholipase A2 (PLA 2) 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]PLA 2s, 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 [Asp 49]PLA2) type, basic [Asp49]PLA2 type, neurotoxic [Asp49]PLA2 type and [Lys 49]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. © 2005 Elsevier Ltd. All rights reserved.

A C-type lectin (BRA-2) isolated from the acorn barnacle Megabalanus rosa, which was a glycoprotein having an N-linked sugar chain, was deglycosylated by N-glycopeptidase F. The structure of the released sugar chains was determined by a 2-D mapping method after derivatization with a fluorescent reagent, 2-aminopyridine, to be Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1- 4(Fucα1-6)GlcNAc and Manα1-6(GlcNAcβ1-2Manα1-3) Manβ1-4GlcNAcβ1-4(Fucα1-6)GlcNAc. The structures were confirmed by matrix-assisted laser desorption ionization mass spectrometry and a comparison with authentic sugar chains by high-pressure liquid chromatography. Various properties of BRA-2 were examined before and after deglycosylation. The susceptibility of BRA-2 to protease digestion was increased by deglycosylation. However, the inhibitory activity toward calcium carbonate crystallization as well as the hemagglutinating activity of deglycosylated BRA-2 was significantly decreased. These results suggest that the sugar chains of BRA-2 are important to both its structural stability and its function.

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 Å, 1.8 Å, and 1.6 Å 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 KA/KS 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. © 2005 Elsevier Ltd. All rights reserved.

The acid hydrolysis of proteins was miniaturized and simplified by employing microcapillary tubes (100 μl 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°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.

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. © 2004 Elsevier Ltd. All rights reserved.

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. © 2004 Springer Science+Business Media, Inc.

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. © 2004 Elsevier Inc. All rights reserved.

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 Cys28-Cys187, whereas DB3L had one interchain disulfide bond (Cys40-Cys 40′) in addition to the intrachain disulfide bond (Cys 28-Cys188) to form a 66-kDa subunit. DB1 and DB3 agglutinated rabbit erythrocytes at 2.7 and 3.9 μg/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 Ca2+ 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.

The microstructure and orientation distribution of aragonite crystals in the nacreous layer of cultured Pteria penguin are investigated in this paper. Helical patterns formed by growth forefronts of the nacreous layer for good-quality shells are observed using a laser microscope, whereas no clear pattern is observed on the nacreous layer surfaces of bad-quality shells. The observed aragonite crystals are plate-shaped and hexagonal for both the good and bad-quality shells, in which the top and bottom faces are parallel to the (001) plane and the side faces are parallel to the {110} and (010) planes. The aragonite crystals in the nacreous layer of good-quality shells seem to be harmonically oriented along a crystallographic direction. These orientation distributions basically indicate that the (001) basal planes are parallel to the inner shell surface, and the [100] and [010] axes are oriented in almost the same direction, respectively. Some of the aragonite crystals are rotated about the c axis by approximately ±60° from the basic orientation distribution. On the other hand, the aragonite crystals of bad-quality shells seem to be randomly oriented in the nacreous layer. These orientation distributions indicate that the (001) basal planes are parallel to the inner shell surface in a similar manner as those of good-quality shells, but their [1001 and [010] axes are randomly oriented about the c axis. Therefore, it is considered that such different orientation distributions result in different qualities of pearls that are developed in the shells.

特集１：STS-107追悼ー我が国の多様な取り組みと成果

We investigated the effect of several lectins, such as soy bean lectin (SBA), concanavalin A (Con A), and wheat germ agglutinin (WGA), on the transport of some food ingredients (isoflavones, quercetin glycosides, carnosine/anserine) across Caco-2 cell monolayers. After incubation of food ingredients (0.03∼2 mmol/L) in the presence or absence of lectins (1∼180 μg/ml) on the apical side, aliquots were taken from the apical and basolateral solution, and were subjected to HPLC analysis. We also examined the effect of lectins on the permeability of the tight junction by measuring the transepithelial electrical resistance (TER) value of the Caco-2 cell monolayer. Isoflavones, which was not transported to the basolateral solution without lectins, could be transported in the presence of lectins, whereas their aglycones were detected at the same levels with or without the lectin treatment. The transport of quercetin glycosides also increased in the presence of lectins, however, that of peptides was not affected by the lectins. Con A and WGA, but SBA, decreased the TER value, indicating that Con A and WGA increased the transport via paracellular pathway, whereas SBA did via a different pathway. © 2004 - IOS Press and the authors. All rights reserved.

Ed. by Scientific Program Committee of ISBC 2003, Maruzen Co. Ltd.

After two decades of study, we draw the conclusion that venom-gland phospholipase A2 (PLA2) isozymes, including PLA 2 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. © 2003 Elsevier Ltd. All rights reserved.

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of AmamiOshima 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 LD 50 for mice was 1.34 μg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the AmamiOshima and the Tokunoshima T. flavoviridis venomgland 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.

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.

Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A2 (PLA2), 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 PLA2 from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA2, named PLA-B′, which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp49]PLA2 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 β1-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 PLA2, 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 PLA2 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 PLA2 isozymes are clearly separated into three groups, PLA2 type, basic [Asp49]PLA2 type, and [Lys49]PLA2 type. Basic [Asp49]PLA2-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys49]PLA2 type.

In B2-type FeAl, supersaturated vacancies retained upon rapidly quenching are absorbed near surface during an aging treatment, being agglomerated into nano- to meso-clusters through the absorption process. Eventually, surface morphology is self-patterned in nano-order by the vacancy clustering. If FeAl was not quenched from high temperature or plastic strain remained near surface, the surface self-patterning never occurs, indicating that the change in surface morphology is caused by the clustering of supersaturated vacancies. The clusters have specific shape with cluster surfaces faceted toward {100} planes. Thus, the shape of the clusters formed near surface is controllable by changing surface orientation. Vacancy cluster size and its distribution density can be also controlled by varying the concentration of supersaturated vacancies and/or the clustering condition. These indicate that the vacancy clustering is a unique process to efficiently pattern the surfaces of metals, alloys and intermetallics in nano-scale.

Three L-rhamnose-binding lectins, named CSL1, CSL2, and CSL3, were isolated from eggs of chum salmon (Oncorhynchus keta) by affinity chromatography and reversed-phase high-performance liquid chromatography. The yields of CSL1, CSL2, and CSL3 from 2 kg eggs were 2.7, 1.7 and 29.6 mg, respectively. The subunit of CSL1 was composed of 286 amino acid residues with three tandemly repeated domains, while the subunits of both CSL2 and CSL3 were composed of 195 amino acid residues with two tandemly repeated domains. The amino acid sequence homologies among CSL showed 42-52% identities. CSL showed 94-97% sequence identities to three corresponding L-rhamnose-binding lectins, named STL1, STL2 and STL3, from steelhead trout (Oncorhynchus mykiss) eggs. Each CSL showed different hemagglutinating activities against rabbit and human erythrocytes. The lectins also showed different inhibition patterns in the hemagglutination towards rabbit erythrocytes by lipopolysaccharides from Gram-negative bacteria including a fish pathogen, Aeromonas salmonicida. CSL agglutinated Escherichia coli and Bacillus subtilis. These results indicate that CSL could be useful biochemical tools for recognizing specific molecules either in soluble forms or on cell surfaces.

The crystal structure of congerin II, a galectin family lectin from conger eel, was determined at 1.45 Angstrom resolution. The previously determined structure of its isoform, congerin I, had revealed a fold evolution via strand swap; however, the structure of congerin II described here resembles other prototype galectins. A comparison of the two congerin genes with that of several other galectins suggests acceralated evolution of both congerin genes following gene duplication. The presence of a Mes (2-[N-morpholino]ethanesulfonic acid) molecule near the carbohydrate-binding site in the crystal structure points to the possibility of an additional binding site in congerin II. The binding site consists of a group of residues that had been replaced following gene duplication suggesting that the binding site was built under selective pressure. Congerin II may be a protein specialized for biological defense with an affinity for target carbohydrates on parasites' cell surface. (C) 2002 Elsevier Science Ltd. All rights reserved.

An expression system for recombinant conger eel galectins, congerins I and II, were constructed using the pTV 118N plasmid vector and Escherichia coli.Recombinant congerins I and II could be obtained in the soluble active form with high quantitative yield. Mutation of codons for Val andLeu located in the N-terminal region of Con I increased the expression efficiency. Purification of recombinant proteins were done by only two chromatographical steps from E. coli extract. The purified recombinant congerins were found to be almost the same as the native ones except for the acetyl group at the N-terminus; that is, they showed the same structures and carbohydrate binding activities, suggesting that N-terminal acetyl groups of congerins were not significant for activity. © 2002, Taylor & Francis Group, LLC. All rights reserved.

The interaction between bacteria and three L-rhamnose-binding lectins, named STL1, STL2, and STL3, from steelhead trout (Oncorhynchus mykiss) eggs was investigated. Although STLs bound to most Gram-negative and Gram-positive bacteria, they agglutinated only Escherichia coli K-12 and Bacillus subtilis among the bacteria tested. The binding was inhibited by L-rhamnose. STLs bound to distinct serotypes of lipopolysaccharides (LPSs), and showed much higher binding activities to smooth-type LPSs of Escherichia coli K-12 and Shigella flexneri 1A than to their corresponding rough-type LPSs. STLs also bound to lipoteichoic acid (LTA) of Bacillus subtilis. These results indicate that STLs bound to bacteria by recognizing LPSs or LTA on the cell surfaces. © 2002 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.

An anticoagulant protein named AaACP was isolated from Agkistrodon actus (hundred-pace snake of Taiwan, Viperidae) venom. AaACP inhibited the factor Xa-induced plasma coagulation in a concentration-dependent manner. Thus, AaACP seems to bind to factor Xa in prothrombinase complex. AaACP was composed of A and B chains linked by disulphide bond(s). The amino acid sequences of A and B chains of AaACP were analysed with a few residues unidentified which were complemented from the nucleotide sequences of their cDNAs. The A chain consisted of 129 amino acid residues and the B chain 123 amino acid residues. Their amino acid sequences were highly similar to those of A and B chains of a series of anticoagulant proteins which had been purified from the venoms of some Viperidae snakes. The A and B chains structurally belong to C-type lectin-like protein family of snake venom origin. Construction of phylogenetic tree of C-type lectins and C-type lectin-like proteins based on their amino acid sequences indicated that their A and B chains diverged before speciation of snake species. The comparison of the nucleotide sequences of the cDNAs encoding A and B chains of AaACP and of Trimeresurus flavoviridis (Viperidae) venom-gland factors IX/X-binding protein and factor IX-binding protein showed that the mature protein-coding region is much more variable than the signal peptide-coding domain and the 5′- and 3′-untranslated regions, being in contrast to the case of the ordinary isoprotein genes. The ratios of the numbers of nucleotide substitutions per nonsynonymous site (KA) and per synonymous site (KS) in the mature protein-coding region in the cDNA pairs were about three times greater than those for the ordinary isoprotein genes, suggesting that these genes have been evolving in an accelerated manner. Taking account of the functional diversities of venom-gland C-type lectins and C-type lectin-like proteins including factors IX and/or X-binding proteins, it can be said that their functional diversities have been acquired by accelerated evolution. © 2002 Elsevier Science Ltd. All rights reserved.

The localization of three L-rhamnose-binding lectins named STL1, STL2, and STL3 from eggs of steelhead trout (Oncorhynchus mykiss) was analyzed by indirect immunohistochemical staining with specific antisera against individual lectins. In early previtellogenic oocyte, STL1 was localized not only in the cortical vesicles, but also in the plasma membrane and germinal vesicle. On the other hand, STL2 and STL3 were localized only in the cortical vesicles. In pre-fertilization mature egg, STLs were localized in a thin layer of cortical granules at the cytoplasmic side of the plasma membrane. STLs were accumulated on the surface of cytoplasm and inner membrane 30 min after fertilization. The strong staining with anti-STL1 antiserum was observed in several tissues and cells of the steelhead trout, such as spleen, thrombocytes, and blood leukocytes, but not erythrocytes. STL1 was also identified in exocrine cells, such as goblet cells of intestine and mucous cells of gill. These results indicate that the multiple lectins found in eggs of the steelhead trout play physiological roles not only in eggs, but also in various cells related to the innate immunity. © 2002 Elsevier Science Ltd. All rights reserved.

Many cases of accelerated evolution driven by positive Darwinian selection are identified in the genes of venomous and reproductive proteins. This evolutional phenomenon might have important consequences in their gene-products' functions, such as multiple specific toxins for quick immobilization of the prey and the establishment of barriers to fertilization that might lead to speciation, and in the molecular evolution of novel genes. Recently, we analyzed the molecular evolution of two galectins isolated from the skin mucus of conger eel (Conger myriaster), named congerins I and II, by cDNA cloning and X-ray structural analysis, and we found that they have evolved in the rapid adaptive manner to emergence of a new structure including strand-swapping and a unique new ligand-binding site. In this review article we summarize and discuss the molecular evolution, especially the rapid adaptive evolution, and the structure-function relationships of conger eel galectins.

L-Rhamnose-binding lectins were isolated from white-spotted charr (Salvelinus leucomaenis) eggs to understand the distribution and molecular evolution of the lectins in Salmonidae. Only two L-rhamnose-binding lectins, named WCL1 and WCL3, were isolated from white-spotted charr eggs, though three lectins, named STL1, STL2, and STL3, had been obtained from steelhead trout (Oncorhynchus mykiss) eggs. The cDNAs of WCL1 and WCL3 included 1,245 and 838 bp nucleotides with open reading frames of 933 and 651 nucleotides, respectively, and encoded for the complete amino acid sequences of mature proteins consisted of 288 (WCL1) and 195 (WCL3) residues, and signal sequences of 23 and 22 residues, respectively. WCLs were composed of three (for WCL1) or two (for WCL3) tandemly repeated homologous domains, which consisted of about 95 amino acid residues, and showed 91 and 93% sequence identities to STL1 and STL3, respectively. The mRNAs of WCL1 and WCL3 were detected exclusively in liver and ovary, respectively, however, neither a protein nor mRNA corresponding to STL2 could be identified in white-spotted charr. The phylogenetic tree of the 16 sequences encoding carbohydrate recognition domains of 7 lectins from 4 species shows 5 functional clusters and their evolutional process. These results indicate that multiple L-rhamnose-binding isolectins have diverged by gene duplication and exon shuffling to play various biological roles in each species. © 2002 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.

Tissue-specific expression of three L-rhamnose-binding lectins, named STL1, STL2, and STL3, in the steelhead trout (Oncorhynchus mykiss) was investigated. STL2 and STL3 mRNAs were restricted in the oocytes. In contrast, STL1 mRNA was detected only in the liver. The transcription of STL2 and STL3 started in previtellogenic oocytes. These results showed distinct expression profiles of rhamnose-binding lectins in the fish. © 2002 by Japan Society for Bioscience, Biotechnology, and Agrochemistry.

The crystal structure of congerin II, a galectin family lectin from conger eel, was determined at 1.45 Å resolution. The previously determined structure of its isoform, congerin I, had revealed a fold evolution via strand swap; however, the structure of congerin II described here resembles other prototype galectins. A comparison of the two congerin genes with that of several other galectins suggests acceralated evolution of both congerin genes following gene duplication. The presence of a Mes (2-[N-morpholino]ethanesulfonic acid) molecule near the carbohydrate-binding site in the crystal structure points to the possibility of an additional binding site in congerin II. The binding site consists of a group of residues that had been replaced following gene duplication suggesting that the binding site was built under selective pressure. Congerin II may be a protein specialized for biological defense with an affinity for target carbohydrates on parasites' cell surface. © 2002 Elsevier Science Ltd. All rights reserved.

Two phospholipases A2 (PLA2s) were purified from the venom of Trimeresurus flavoviridis (Crotalinae) inhabiting Tokunoshima island, Japan, and named PLA-A and PLA-B in the order of elution on a cation-exchange column. Lipolytic activities of PLA-A and PLA-B toward mixed micelles and liposomes were substantially lower than that of PLA2 (an [Asp49]PLA2) which had been isolated from the same venom. Both PLA-A and PLA-B consisted of 122 amino acids and contained aspartate at position 49 (the numbering according to the aligned sequences of PLA2s in Fig. 8), thus belonging to an [Asp49]PLA2 subgroup. PLA-A and PLA-B were identical in sequence with an exception at position 79. PLA-B contained Asn-Gly at positions 79 and 80 which are located in the β-sheet region. On the other hand, PLA-A had β-Asp-Gly and α-Asp-Gly in high and low proportion, respectively, at the corresponding positions which were produced from Asn-Gly through the base-catalyzed formation and hydrolysis of the succinimide type intermediate. Thus, PLA-A is derived from PLA-B. PLA-B is similar in sequence to PL-X, which had been purified from the venom of T. flavoviridis inhabiting Amami-Oshima island, Japan, and to PL-X′, whose cDNA had been cloned from Tokunoshima T. flavoviridis venom gland, rather than PLA2. PLA-B showed strong edema-inducing activity, while PLA-A exhibited rather lower activity. The sequence around position 79 which constitutes a β-turn segment seems to be crucial for edema-inducing activity. Phylogenetic tree of Tokunoshima T. flavoviridis venom PLA2 isozymes indicated that PLA-B and PL-X′ diverged from PLA2 after branching of [Asp49]PLA2 forms and [Lys49]PLA2 forms. © 2001 Elsevier Science Ltd.

An L-rhamnose-binding isolectin named STL3 (subunit Mr, 21.5 k) was isolated from eggs of the steelhead trout (Oncorhynchus mykiss) in addition to STL1 (subunit Mr, 31.4 k) and STL2 (subunit Mr, 21.3 k) that had been already isolated. STLs were composed of non-covalently linked subunits. The primary structures of STL1 and STL3 were analyzed by the combined use of protein sequencing and cDNA sequencing. A cDNA encoding STL2, of which the protein sequence had been previously studied, was also analyzed. The STL1 subunit (289 amino acid residues) had different structural properties compared to those of the STL2 subunit (195 amino acid residues) and the STL3 subunit (195 amino acid residues); e.g., the number of repeated domain (three for STL1, and two for STL2 and STL3), although all of them were composed of tandemly repeated homologous domains (40 to 53% identities). The lectin levels in various tissues and during the embryonic development showed that STL1 had different distribution and expression profiles from those of STL2 and STL3. Although STL1 could be detected in several tissues and serum of both male and female steelhead trout, STL2 and STL3 were only abundant in the ovary. STL2 and STL3 levels dramatically decreased just after hatching, however, the STL1 level increased temporarily. These results indicate that the multiple lectins from eggs of the steelhead trout form a novel rhamnose-binding lectin family with different structures and tissue distribution to share distinct functions in eggs.

The amino acid sequence of a D-galactose-binding lectin isolated from the hemolymph of the acorn barnacle Balanus rostratus was determined. The lectin (BRL) (Mr 120K) is a multimeric protein whose subunit consists of 182 amino acids. The amino acid sequence was compared with those of multiple lectins (BRA-2, BRA-3) from Megabalanus rosa to explore the relationship between the structures and the inhibitory activity toward the crystal growth of calcium carbonate. Although BRL was 46% identical to BRA-2 and 15% identical to BRA-3, the lectin had no inhibitory activity, unlike BRA-2 and BRA-3. Both the number and the localization of acidic amino acid residues and their amide forms were different among them. Observations by scanning electron microscopy revealed modifications in the size and morphology of the calcium carbonate crystals grown in the presence of the lectins.

Conventional chromatographic analysis showed that phospholipase A2 (PLA2) isoenzymes of the venom of Trimeresurus flavoviridis (Habu snake) of Okinawa island are profoundly different in composition from those of T. flavoviridis of Amami-Oshima and Tokunoshima islands. The most striking feature was that myotoxic [Lys49]PLA2 isoenzymes, called BPI and BPII, which are expressed abundantly in the venoms of Amami-Oshima and Tokunoshima T. flavoviridis, are missing from the venom of Okinawa T. flavoviridis. Northern blot analysis of Okinawa T. flavoviridis venom-gland mRNA species showed the absence of BPI and BPII mRNA species. Analysis by single-stranded conformational polymorphism-PCR of venom-gland mRNA species of T. flavoviridis from three islands, with reference to five DNA species each encoding different PLA2 isoenzymes from Tokunoshima T. flavoviridis venom gland, also suggested that BPI and BPII mRNA species are not expressed in Okinawa T. flavoviridis venom gland. In contrast, genomic Southern blot analysis with a variety of probes showed that only the bands corresponding to the upstream and downstream regions of the genes for BPI and/or BPII can be detected in Okinawa T. flavoviridis. These results suggested that the genes for BPI and BPII in Okinawa T. flavoviridis genome had been inactivated to form pseudogenes. Differently from Amami-Oshima and Tokunoshima T. flavoviridis genomic DNAs, PCR amplification of the segments of BPI and BPII genes between the 5' moiety of second exon and the middle portion of second intron failed for Okinawa T. flavoviridis genomic DNAs. In sequence analysis of the two segments involving polymorphism between BPI and BPII genes, which are located in first exon and third exon, respectively, only one base was detected at the polymorphic positions for pseudogene in Okinawa T. flavoviridis genome. Based on these facts, it became evident for pseudogene that the upstream region of BPI gene down to the 5' moiety of second exon and the downstream region of BPII gene starting from the middle portion of second intron are in a linked form with a possible insertion. Such observations suggest that venom-gland genes for PLA2 isoenzymes in T. flavoviridis snakes isolated for one to two million years have evolved independently. Their evolution is regional and seems, from several lines of consideration and observation, to be adaptive to the environment.

In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5'- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions. Copyright (C) 1999 Elsevier Science Ltd.

Effects of culture conditions on the lectin level by a unicellular laboratory culture of Microcystis aeruginosa M228 (freshwater cyanobacterium) were investigated. The lectin level per cell at stationary phase increased two- or fourfold by lowering the light intensity from 45 or 90μE/m2 per s to 12μE/m2 per s, respectively, at 25°C as measured by the enzyme-linked immunosorbent assay method. The hemagglutinating activity against rabbit erythrocytes increased in response to the lectin level. The lectin level was threefold greater at 15°C than at 25°C, and nearly ninefold greater than at 30°C. Thus, although the specific growth rate of the cyanobacterium decreased with lowering light intensity and temperature, the lectin level per cell increased significantly. When the cyanobacterium was cultured at 15°C with a light intensity of 12μE/m2 per s, the lectin level reached the maximum near the end of exponential phase, and then decreased toward stationary phase. The level near the end of exponential phase was nearly threefold greater than that of the culture incubated at 25°C with a light intensity of 45μE/m2 per s. These findings show that the lectin production in M. aeruginosa M228 was induced by unfavorable conditions for the cell growth.

Protein hydrolysates, prepared by enzymatic digestion of soybean protein and egg white albumin using several proteases, inhibited the crystal growth of calcium carbonate. Each hydrolysate showed different inhibitory activities, suggesting the key role of peptide structures in the inhibition. The deamidation of protein hydrolysates by glutaminase increased not only the inhibitory activity toward the crystal growth of calcium carbonate but also the resistance of the hydrolysates against peptic digestion. Furthermore, the addition of sodium chloride, citric acid, or lactose into the reaction mixture enhanced the inhibitory activity. The protein hydrolysates inhibited both nucleation and crystal growth of calcium carbonate and also affected the crystal morphology.

The amino-acid sequence of α-amylase isolated from the pancreas of the ostrich, Struthio camelus was determined. The α-amylase (OPA) consisted of 497 amino acid residues with pyroglutamic acid at the N-terminus and no oligosaccharide. Amino acid identity between OPA and chicken, porcine and human pancreatic α-amylases individually, was found to be 88, 82 and 86%, respectively. (C) 2000 Elsevier Science Inc.

The work focused on the penultimate enzyme, prothrombin, in the coagulation cascade. Prothrombin was purified and characterized from ostrich plasma. The results obtained contribute to a better understanding of blood coagulation in the ostrich and the evolution of prothrombin and the coagulation cascade. Prothrombin was purified from ostrich plasma by barium chloride precipitation, ammonium sulfate fractionation, and DEAE-cellulose and Cu2+-chelate Sepharose chromatography. Ostrich prothrombin exhibited a Mr of 72 800 and a pI of 6.9 using SDS-PAGE and PAG-isoelectrofocusing, respectively. The N-terminal sequence of ostrich prothrombin showed 78 and 87% identity with human and bovine, respectively. The cDNA was isolated from ostrich liver and the predicted amino acid sequence compared with those from other species. Ostrich prothrombin shares sequence identity with chicken (84%), human (60%), bovine (59%), rat (60%), mouse (59%) and hagfish (50%) prothrombin, suggesting a common function of prothrombin in these vertebrates. Amino acid sequence identities indicate that the thrombin β-chain (62%) and the propeptide-Gla (75%) domains are the regions most constrained for the common functions of vertebrate prothrombins. Ostrich prothrombin, therefore, shows similarity in structure to other vertebrate prothrombins. © 2000 Elsevier Science Ltd.

Microcystis viridis NIES-102 strain, a unicellular freshwater bloom-forming cyanobacterium, showed transient hemagglutinating activity in laboratory culture during stationary phase under nonaeration conditions. However, the hemagglutinating activity which was inhibited with yeast mannan could not be observed during culture with aeration. A mannan-binding lectin named MVL was isolated with the assay of the hemagglutinating activity against rabbit erythrocytes from the cyanobacterium by successive hydrophobic and gel filtration chromatography. MVL was composed of a single polypeptide of 13 kDa. The gene (mvl) for MVL was cloned from a genomic DNA of NIES-102 strain as a template, and its sequence was determined. The deduced amino acid sequence showed that MVL consisted of 113 amino acid residues and was composed of two tandemly repeated homologous domains of 54 amino acid residues. MVL showed no sequence homology to any other lectins or proteins.

Background: Congerin I is a member of the galectin (animal β- galactoside-binding lectin) family and is found in the skin mucus of conger eel. The galectin family proteins perform a variety of biological activities. Because of its histological localization and activity against marine bacteria and starfish embryos, congerin I is thought to take part in the eels' biological defense system against parasites. Results: The crystal structure of congerin I has been determined in both lactose-liganded and ligand-free forms to 1.5 Å and 1.6 Å resolution, respectively. The protein is a homodimer of 15 kDa subunits. Congerin I has a β-sheet topology that is markedly different from those of known relatives. One of the β-strands is exchanged between two identical subunits. This strand swap might increase the dimer stability. Of the known galectin complexes, congerin I forms the most extensive interaction with lactose molecules. Most of these interactions are substituted by similar interactions with water molecules, including a π- electron hydrogen bond, in the ligand-free form. This observation indicates an increased affinity of congerin I for the ligand. Conclusions: The genes for congerin I and an isoform, congerin II, are known to have evolved under positive selection pressure. The strand swap and the modification in the carbohydrate-binding site might enhance the cross-linking activity, and should be the most apparent consequence of positive selection. The protein has been adapted to functioning in skin mucus that is in direct contact with surrounding environments by an enhancement in cross-linking activity. The structure of congerin I demonstrates the emergence of a new structure class by accelerated evolution under selection pressure.

Cloning of cDNAs encoding bradykinin-potentiating peptides (BPPs)-C-type natriuretic peptide (CNP) precursor or its homologue was performed for cDNA libraries of Bothrops jararaca (South American snake), Trimeresurus flavoviridis, Trimeresurus gramineus and Agkistrodon halys blomhoffi (Asian snakes), all belonging to Crotalinae subfamily. Each cDNA library was constructed from the venom glands of a single snake to preclude ambiguity by intraspecies variation in venom components. Thirteen positive clones derived from B. jararaca were divided into two types depending on restriction sites. Differences in the nucleotide sequence arise at three locations and two of them accompanied amino acid conversions. Despite the differences, both types of cDNA clones encode the BPP-CNP precursor of 256 amino acid residues. Sequence analysis demonstrated that cDNA clones from three Asian snakes encode homologues of the BPP-CNP precursor from B. jararaca. In a precursor polypeptide, a signal sequence (~25 aa) at the N-terminus is followed by sequences of BPP or the analogue (5-13 aa) with flanking spacer sequences (indefinite number of aa), an intervening linker sequence (~100 aa) with unidentified function, and a CNP sequence (22 aa) with a preceding processing signal sequence (10 aa). cDNA clones from A. halys blomhoffi encode two distinct peptides in place of BPP, and T. flavoviridis and T. gramineus were shown to have considerably different sequences in the BPP domain from those known as BPP sequences. The present results provide evidence for a wide distribution of the orthologous gene expressing a series of bioactive peptides among Crotalinae subfamily. Copyright (C) 1999 Elsevier Science B.V.

The complete amino acid sequence of an isogalectin, named congerin II, isolated from the skin mucus of conger eel, was determined by sequencing of the protein and its peptides generated by enzymatic and chemical cleavages. Congerin II consisted of 135 amino acids residues containing an acetylated N-terminus. Congerin II was found to be only 46% homologous in sequence to congerin I which was previously determined (Muramoto K., Kamiya H., Biochem. Biophys. Acta, 1992;1116:129-136), suggesting that the galectins with diverse molecular properties are present in the skill mucus of conger eel. However, it was confirmed by analysis of the secondary structures using circular dichroism that both congerins I and II shared similar folds characterized by beta structures. Congerins I and II showed different molecular properties such as thermostability, pH dependency for hemagglutinating activity and for binding specificity against the pyridylamino derivative of lactose. Congerin I showed more strict recognition specificity for lactose than did congerin II. Furthermore, the effects of chemical modification on congerins I and II were investigated in order to identify the type of amino acids involved in their different lectin activities. Modification of tyrosine and lysine residues did not affect the carbohydrate-binding activities of congerins. However, modification of tryptophan, arginine, histidine, glutamic acid and aspartic acid residues led to considerable loss of their activities, and a different mode of binding activity was observed between modified congerins I and II. These results suggest that multiple galectins from conger eel with the same scaffold have different biological functions and properties. (C) 1999 Elsevier Science Inc. All rights reserved.

The complete amino acid sequence of an isogalectin, named congerin II, isolated from the skin mucus of conger eel, was determined by sequencing of the protein and its peptides generated by enzymatic and chemical cleavages. Congerin II consisted of 135 amino acids residues containing an acetylated N-terminus. Congerin II was found to be only 46% homologous in sequence to congerin I which was previously determined (Muramoto K., Kamiya H., Biochem. Biophys. Acta, 1992;1116:129-136), suggesting that the galectins with diverse molecular properties are present in the skin mucus of conger eel. However, it was confirmed by analysis of the secondary structures using circular dichroism that both congerins I and II shared similar folds characterized by β structures. Congerins I and II showed different molecular properties such as thermostability, pH dependency for hemagglutinating activity and for binding specificity against the pyridylamino derivative of lactose. Congerin I showed more strict recognition specificity for lactose than did congerin II. Furthermore, the effects of chemical modification on congerins I and II were investigated in order to identify the type of amino acids involved in their different lectin activities. Modification of tyrosine and lysine residues did not affect the carbohydrate-binding activities of congerins. However, modification of tryptophan, arginine, histidine, glutamic acid and aspartic acid residues led to considerable loss of their activities, and a different mode of binding activity was observed between modified congerins I and II. These results suggest that multiple galectins from conger eel with the same scaffold have different biological functions and properties. Copyright (C) 1999 Elsevier Science Inc.

Two cDNAs encoding galectins named congerins I and II from the skin mucus of conger eel (Conger myriaster) were isolated and sequenced. Comparison of the nucleotide sequences of congerins I and II showed that the sequence similarities of the 5′ and 3′ untranslated regions (86 and 88%, respectively) were much higher than those of the protein-coding region (73%). The numbers of nucleotide substitutions per site (KN) for the untranslated regions are smaller than the numbers of nucleotide substitutions per synonymous site (KS) for the protein coding region. Furthermore, nonsynonymous nucleotide substitutions have accelerated more frequently than synonymous nucleotide substitutions in the protein coding region (KA/KS=2.57). These results suggest that accelerated substitutions have occurred in the protein-coding regions of galectin genes to generate diverse galectins with different molecular properties. Northern blot analysis showed that both congerins were expressed not only in the skin tissues but also in the stomach of conger eel. © 1999, Taylor & Francis Group, LLC. All rights reserved.

Two L-rhamnose-binding lectins named STL1 and STL2 were isolated from eggs of steelhead trout (Oncorhynchus mykiss) by affinity chromatography and ion exchange chromatography. The apparent molecular masses of purified STL1 and STL2 were estimated to be 84 and 68 kDa, respectively, by gel filtration chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry of these lectins revealed that STL1 was composed of noncovalently linked trimer of 31.4-kDa subunits, and STL2 was non-covalently linked trimer of 21.5-kDa subunits. The minimum concentrations of STL1, a major component, and STL2, a minor component, needed to agglutinate rabbit erythrocytes were 9 and 0.2 μg/ml, respectively. The most effective saccharide in the hemagglutination inhibition assay for both STL1 and STL2 was L-rhamnose. Saccharides possessing the same configuration of hydroxyl groups at C2 and C4 as that in L-rhamnose, such as L-arabinose and D-galactose, also inhibited. The amino acid sequence of STL2 was determined by analysis of peptides generated by digestion of the S-carboxamidomethylated protein with Achromobacter protease I or Staphylococcus aureus V8 protease. The STL2 subunit of 195 amino acid residues proved to have a unique polypeptide architecture; that is, it was composed of two tandemly repeated homologous domains (STL2-N and STL2-C) with 52% internal homology. These two domains showed a sequence homology to the subunit (105 amino acid residues) of D- galactoside-specific sea urchin (Anthocidaris crassispina) egg lectin (37% for STL2-N and 46% for STL2-C, respectively). The N terminus of the STL1 subunit was blocked with an acetyl group. However, a partial amino acid sequence of the subunit showed a sequence similarity to STL2. Moreover, STL2 also showed a sequence homology to the ligand binding domain of the vitellogenin receptor. We have also employed surface plasmon resonance biosensor methodology to investigate the interactions between STL2 and major egg yolk proteins from steelhead trout, lipovitellin, and β'-component, which are known as vitellogenin digests. Interestingly, STL2 showed distinct interactions with both egg yolk proteins. The estimated values for the affinity constant (Kα) of STL2 to lipovitellin and β' component were 3.44 x 106 and 4.99 x 106, respectively. These results suggest that the fish egg lectins belong to a new family of animal lectin structurally related to the low density lipoprotein receptor superfamily.

A part of the 3'-flanking region of BP-II gene, which is one of Trimeresurus flavoviridis venom gland phospholopase A2 (PLA2) isozyme genes, has a region homologous to avian chicken repeat 1 (CR1)-element. In the present study, ten CR1-like elements were further identified in T. gramineus venom gland PLA2 isozyme genes, T. flavoviridis PLA2 inhibitor (PLI) genes, and T. flavoviridis and T. gramineus TATA-box binding protein (TBP) genes. Southern blot analysis using a probe for CR1 showed that Crotalinae snake genomes contain a number of CR1-like elements.

Site-directed mutagenesis studies of recombinant Trimeresurus flavoviridis venom gland phospholipase A2 (PLA2) showed that the Asn residue at position 67 takes part in recognition of the substrate 2-arachidonoyl sn-glycero-3-phosphocholine in both monomeric and micellar states. The amount of arachidonate released from phosphatidylcholine mixed micelles was reduced to 30% for N67D and N67K mutants, and to 70% for N67G mutant, but remained unchanged for N67S mutant. In contrast, for monomeric substrate, the activity was decreased to 40% for N67D and N67G and to 60% for N67K but remained unchanged for N67S. These results suggest that the properties of the side chain of residue 67 exert a significant influence on recognition of 2-arachidonoyl sn-glycero-3-phosphocholine.

Inhibitors (PLIs) against snake venom gland phospholipases A2 (PLA2s) have been found in their sera. A cDNA encoding a PLI from Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) serum, cPLI-A, was isolated from the Tf liver cDNA library and sequenced. Northern blot analysis with cPLI-A showed that PLIs are expressed only in liver. Genes for PLIs, gPLI-A and gPLI-B, were isolated from the Tf genomic DNA library and their nucleotide (nt) sequences were determined. The genes consisted of four exons and three introns, and exon 4 encoded the carbohydrate recognition domain (CRD)-like motif. Comparison of the nt sequences between gPLI-A and gPLI-B showed that these genes are highly homologous, including introns, except that exon 3 is rich in nonsynonymous nt substitutions which are almost four times as frequent as synonymous nt substitutions. This evolutionary feature of PLI genes is different from that of venom gland PLA2 isozyme genes in which nonsynonymous nt substitutions are spread over the entire mature protein-coding region.

A cDNA encoding a subunit of the verrucotoxin (VTX) has been identified from a cDNA library derived from stonefish venom glands. It encodes a polypeptide of 708 amino-acid residues, followed by a 3'-untranslated region of 895 bp long. The ORF contains the complete mature sequence of the β-subunit of the VTX, as inferred from both the presence of an identical N-terminus sequence and 96% homology among the 506 amino terminus residues found in the partial sequence of the β-subunit of the stonustoxin from Synanceia horrida (Ghadessy, F.J., Jeyaseelan, K., Chung, M.C.M., Khoo, H.E. and Yuen, R. (1994) Toxicon 32, 1684-1688). Upstream the mature sequence, we noticed the presence of an incomplete peptide of a 13 amino acids, whose unusual primary structure supports the idea of the existence of a propeptide and/or of a new secretion signal.

The proteins that bind phospholipase A2 (PLA2) isozymes of Trimeresurus flavoviridis (habu snake, crotalinae) venom were fractionated from sera on four columns, each conjugated with one of four PLA2 isozymes. Five proteins, termed PLA2 inhibitors (PLI) I-V, were obtained as the binding components. The combinations of the binding components differed depending on the PLA2 isozymes. PLI-IV and PLI-V correspond to PLI-A and PLI-B, respectively, which were known to bind to a major [Asp49]PLA2, PLA2, and contained a segment similar to the carbohydrate-recognition domain of C-type lectins. PLI-I, which is a major component of inhibitory proteins against three basic PLA2 isozymes, PLA-B (a basic [Asp49]PLA2) and basic proteins I and II (both [Lys49]PLA2s), has been isolated, and its partial amino acid sequence has been determined. A cDNA encoding PLI-I was isolated from a T. flavoviridis liver cDNA library and sequenced. PLI-I cDNA encoded 200 amino acid residues, including a signal peptide of 19 amino acid residues. One sugar chain was predicted to occur at position 157. A gene coding for PLI-I was isolated. It is 9.6-kb long and consists of five exons and four introns. Comparison of the exon-intron structure of the PLI-I gene with those of genes encoding urokinase-type-plasminogen-activator receptor (uPAR), Ly-6, CD59 and neurotoxins showed that they have characteristic unit encoding approximately 90 amino acid residues, which is divided over two exons. This strongly suggests that the PLI-I gene belongs to the uPAR, Ly-6, CD59 and neurotoxin gene family. There are two types of structurally different inhibitors against PLA2 isozymes in T. flavoviridis serum with different evolutionary origins.

Recent studies revealed that animal toxins with unrelated biological functions often possess a similar architecture. To tentatively understand the evolutionary mechanisms that may govern this principle of functional prodigality associated with a structural economy, two complementary approaches were considered. One of them consisted of investigating the rates of mutations that occur in cDNAs and/or genes that encode a variety of toxins with the same fold. This approach was largely adopted with phospholipases A2 from Viperidae and to a lesser extent with three-fingered toxins from Elapidae and Hydrophiidae. Another approach consisted of investigating how a given fold can accommodate distinct functional topographies. Thus, a number of topologies by which three-fingered toxins exert distinct functions were investigated either by making chemical modifications and/or mutational analyses or by studying the three-dimensional structure of toxin-target complexes. This review shows that, although the two approaches are different, they commonly indicate that most if not all the surface of a snake toxin fold undergoes natural engineering, which may be associated with an accelerated rate of evolution. The biochemical process by which this phenomenon occurs remains unknown. © 1998 Academic Press

Eight cDNAs encoding serine proteases isolated from Trimeresurus flavoviridis (habu snake) and T. gramineus (green habu snake) venom gland cDNA libraries showed that nonsynonymous nucleotide substitutions have accumulated in the mature protein-coding regions to cause amino acid changes. Southern blot analysis of T. flavoviridis genomic DNAs using two proper probes indicated that venom gland serine protease genes form a multigene family in the genome. These observations suggest that venom gland serine proteases have diversified their amino acid sequences in an accelerating manner. Since a similar feature has been previously discovered in crotalinae snake venom gland phospholipase A2 (PLA2) isozyme genes, accelerated evolution appears to be universal in plural isozyme families of crotalinae snake venom gland.

The nucleotide sequences of two cDNAs and four genes encoding Trimeresurus gramineus venom gland phospholipase A2 (PLA2) isozymes were determined and compared internally and externally with those encoding Trimeresurus flavoviridis venom gland PLA2 isozymes. It was reveald that the protein-coding regions are much more diversified than the 5' and 3' untranslated regions (UTRs) and the introns except for the signal peptide domain. The numbers of nucleotide substitutions per site (K(N)) for the UTRs and the introns were approximately one-quarter of the numbers of nucleotide substitutions per synonymous site (K(s)) for the protein-coding regions and were at the same level as the K(N) value of T. gramineus and T. flavoviridis TATA box-binding protein (TBP) genes, indicating that the protein-coding regions of PLA2 isozyme genes are unusually variable and that the UTRs including the introns of venom gland PLA2 isozyme genes have evolved at similar rate to those of non-venomous genes. The numbers of nucleotide substitutions per non-synonymous genes. The numbers of nucleotide substitutions per non-synonymous site (K(A)) values were close to or larger than the K(s) values for the protein-coding regions in venom gland PLA2 isozyme genes, indicating that the protein-coding regions of snake venom gland PLA2 isozyme genes have evolved via accelerated evolution. Furthermore, the evolutionary trees derived from the combined sequences of the 5' and 3' UTRs and the signal peptide domain of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. Estimations of nucleotide divergence of genes and the phylogenetic analysis reveal that snake venom group II PLA2 isozyme genes have been evolving under adaptive pressure to acquire new physiological activities.

Three Trimeresurus okinavensis (To; himehabu snake, Crotalinae) venom gland phospholipase A2 (PLA2) isozyme-encoding genes, gPLA2-o1, gPLA2-o2 and gPLA2-o3, were isolated from its genomic DNA library. The nucleotide (nt) sequence analysis revealed that two of the three genes (gPLA2-o2 and gPLA2-o3) occasionally have been converted to inactivated genes by introduction of one base insertion or substitution. It was confirmed from Southern blot analysis that the To haploid genome contains only three venom gland PLA2 isozyme genes herein isolated. Comparison of these genes showed that nonsynonymous nt substitutions have occurred more frequently than synonymous nt substitutions in the protein-coding regions, except for the signal-peptide coding domain, implying that To venom gland PLA2 isozyme genes have evolved via accelerated evolution. Such an evolutionary feature of To venom gland PLA2 isozyme genes proves the general universality of accelerated evolution previously drawn for venom gland PLA2 isozyme genes of other crotalinae snakes. The variability in the mature protein-coding regions of three To venom gland PLA2 isozyme genes appears to have been brought about by natural selection for point mutations.

Trimeresurus flavoviridis (Habu snake) venom aspartate-49-phospholipase A2 (Asp-49-PLA2) was reacted at pH 9.0 with a 2-fold molar excess of 2,4,6-trinitrobenzenesulfonate in the absence of Ca2+ and two trinitrophenylated derivatives were isolated by HPLC. One was a derivative modified at Lys-11 and its activity was mostly retained. The other was a derivative modified at both Lys-11 and Lys-72 and its activity was 40% that of unmodified enzyme. Trinitrophenylation of Lys-72 appeared to bring about a conformational disorder at the lipid-water interface recognition site and thus a reduction of activity. When the enzyme was modified in the presence of Ca2+, activity decreased at a rate much faster than that in the absence of Ca2+ and Lys-69 came to be modified. These results suggested that conformational displacement of Asp-49-PLA2 of a local to global type occurs upon the binding of Ca2+. The derivative modified at Lys-69 had 28% activity and existed as a monomer. This supports a previous assumption that Lys-69 participates in dimerization of group II Asp-49-PLA2s [Brunie et al. (1985) J. Biol. Chem. 260, 9742-9749] and shows that dimerization is not necessarily essential for activity manifestation.

Basic proteins I and II (BP-I and BP-II) isolated from Trimeresurus flavoviridis venom, which are classified into a group of lysine-49-phospholipases A2 (Lys-49-PLA2), exhibited only limited lipolytic activity for the mixed micelles of various phospholipids. Based on the finding that BP-II elicits a strong contraction of guinea pig ileum due to the release of arachidonic acid, BP-II together with BP-I has been tested for their interaction with artificial phospholipid bilayer membranes. The dye leakage experiments indicated that BP-II interacts strongly with liposomes of β-arachidonoyl-γ-stearoyl-L-α-phosphatidylcholine. The perturbation of liposomes was observed only in the Ca2+-containing buffer, and as demonstrated by HPLC analyses, accompanied by the release of arachidonic acid. The concentration of Ca2+ which gave a half maximal activity of BP-II was 3.0×10-4M, suggesting that the affinity of BP-II for Ca2+ is more than 10 times stronger than that of BP-II without liposomes. These observations clearly show that Lys-49-PLA2 of BP-II is the enzyme responsible for the hydrolysis of membrane phospholipids and that Ca2+ is essential for such enzymatic activity. The interaction of BP-I with liposomes was much weaker than BP-II. BP-I and BP-II share a common sequence except for Asp-67 (BP-I) and Asn-67 (BP-II) in the aligned sequences. This implies that the amino acid at position 67 of Lys-49-PLA2s is the residue required for discriminatory recognition of phospholipid membranes.

The localization and expression profiles of phospholipases A2 (PLA2s) in Trimeresurus flavoviridis venom gland were studied by means of in situ hybridization and immunohistochemical techniques. Venom gland cells are tightly arrayed in a single layer along the inlet-like lumens in which venom proteins are stored. mRNAs for PLA2s were detected at the high level in cytoplasm. Using the immunohistochemical technique with polyclonal antiAsp-49-PLA2 antibody, Asp-49-PLA2 and, possibly, its isozymes were detected in intracellular granules and in venom lumens. The intracellular granules containing PLA2 proteins appear to be transferred from the nucleus towards the outer membrane facing the lumen, and then to be secreted. © 1995.

The nucleotide sequences of 13 cDNAs encoding group II phospholipases A2 (PLA2S), which are from viperidae snake venoms and from mammalian sources, were aligned and analyzed by phylogenetic trees constructed using various components of the sequences. The evolutionary trees derived from the combined sequences of the untranslated (5′ and 3′) region and the signal peptide region of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. These observations indicated that the mature protein-coding region has evolved through a process differently from the untranslated and signal peptide regions. The trees built from the nucleotide differences at each of three positions of codons in the mature protein-coding region suggested that snakevenom-gland PLA2 genes have evolved via a process different from mammalian PLA2 genes. The occurrence of accelerated evolution has been recently discovered in Trimeresurus flavoviridis venom-gland group II PLA2 isozyme genes (Nakashima et al. 1993, Proc Natl Acad Sci USA 90:5964–5968), so the present phylogenetic analysis together with the estimation of nucleotide divergence of cDNAs provides further evidence that snakevenom-group II PLA2 isozyme genes have evolved by accelerated evolution to gain diverse physiological activities. © 1995, Springer-Verlag New York Inc. All rights reserved.

Four acidic phospholipase A2 (PLA2) isozymes named PLA2-I, II, III and IV have previously been isolated from Trimeresurus gramineus (green habu snake) venom and sequenced [Oda et al. (1991) Toxicon29, 157; Fukagawa et al. (1992) Toxicon30, 1131; Fukagawa et al. (1993) Toxicon 31, 957]. They contain aspartate-49 which is known to bind Ca2+, essential for catalysis. In the present study, a basic PLA2 named PLA2-V containing lysine-49 was newly isolated from the same snake venom. Its isoelectric point was 9.4 and considerably higher than those (c. 4.5) of PLA2-I-IV. PLA2-V was 1.1% as active as PLA2-I toward egg-yolk emulsion but exhibited strong myotoxicity. The amino acid sequence of PLA2-V was determined by sequencing the S-carboxamidomethylated derivative and its peptide fragments produced by enzymatic (clostripain, chymotrypsin, Achromobacter protease I and Staphylococcus aureus V8 protease) cleavages. PLA2-V consists of 122 amino acid residues and is highly homologous (72-78%) to Lys-49 PLA2s so far isolated from Viperidae snake venoms but less homologous (52%) to PLA2-I. The presence of Asn-28, which is characteristic of Lys-49 PLA2s, was confirmed. © 1995.

The nucleotide sequences of four genes encoding Trimeresurus gramineus (green habu snake, crotalinae) venom gland phospholipase A2 (PLA2; phosphatidylcholine 2-acylhydrolase, EC 3.1.1.4) isozymes were compared internally and externally with those of six genes encoding Trimeresurus flavoviridis (habu snake, crotalinae) venom gland PLA2 isozymes. The numbers of nucleotide substitutions per site (K(N)) for the noncoding regions including introns were one-third to one-eighth of the numbers of nucleotide substitutions per synonymous site (K(S)) for the protein-coding regions of exons, indicating that the noncoding regions are much more conserved than the protein-coding regions. The K(N) values for the introns were found to be nearly equivalent to those of introns of T. gramineus and T. flavoviridis TATA box-binding protein genes, which are assumed to be a general (nonvenomous) gene. Thus, it is evident that the introns of venom gland PLA2 isozyme genes have evolved at a similar rate to those of nonvenomous genes. The numbers of nucleotide substitutions per nonsynonymous site (K(A)) were close to or larger than the K(S) values for the protein-coding regions in venom gland PLA2 isozyme genes. All of the data combined reveal that Darwinian-type accelerated evolution has universally occurred only in the protein-coding regions of crotalinae snake venom PLA2 isozyme genes.

A cDNA encoding the Trimeresurus gramineus (Tg; green habu snake) TATA-box-binding protein (TgTBP) was cloned and sequenced. The cDNA encodes a 33-kDa protein with an extensive sequence similarity to those derived from other organisms, except for the N-terminal domain. Genes encoding TgTBP and Trimeresurus flavoviridis (Tf; habu snake) TBP (TgTBP) were isolated using a TgTBP cDNA and their nt sequences were determined. They are the first TBP genes entirely sequenced in higher animals. Both genes span over 15 kb and are constructed from eight exons and seven introns. Comparison of the loci of introns on the aligned amino-acid sequences of TBP from six organisms (Tg, Tf, mouse, Arabidopsis thaliana, Schizosaccharomyces pombe and Acanthamoeba castellanii) indicated that there are three highly conserved loci in the C-terminal domain. © 1995.

As a step towards understanding the structure and function of phospholipases A2 (PLA2s), five cDNAs encoding Trimeresurus flavoviridis venom gland PLA2 isozymes have been sequenced. They revealed that the 5′ and 3′ un translated regions are much more homologous than the protein‐coding regions and that mbase substitutions have occurred at similar rates for the three positions of codons in the protein‐coding regions. Such novel findings are of great interest from the viewpoint of molecular evolution. To gain a further insight into this evolutional phenomenon, we have isolated and sequenced six T. flavoviridis PLA2 isozyme genes. Each gene consisted of four exons and three introns and encoded protein of 138 amino‐acid residues, including the signal sequence of 16 amino‐acid residues. The introns were much more homologous than the protein‐coding regions of exons except for the signal peptide‐coding region of the first exon. The absence of apparent functional role in the introns suggested that the protein‐coding regions, expect for the signal peptide‐coding domains, have evolved at greater substitution rates than introns. The fact that the numbers of nucleotide substitutions per non‐synonymous site are close to or larger than the number of nucleotide substitutions per synonymous site for relevant pairs of genes revealed that Darwinian‐type accelerated substitutions have occurred in the protein‐coding regions of exons. This is compatible with the presence of PLA2 species with diverse physiological activities in the venom. Copyright © 1995 John Wiley & Sons Ltd.

N.H. Tan, S.L.Oo, V. Thambyrajah and N. Azila Ed.<br /> Proceedings of the third asia pacific congress on animal, plant and microbial toxins, Malaysia, 27 june-1 July, 1993

As a step toward understanding the structure and function relationships of phospholipases A2 (PLA2s), we isolated and sequenced five cDNAs encoding Trimeresurus flavoviridis (Habu snake) venom PLA2 isozymes. Comparison of the nucleotide sequences of these cDNAs revealed that the homologies of the 5′ and 3′ untranslated regions were much higher than that of the protein-coding regions and that the base substitution rates at the first, second, and third position of codons are similar in the coding region. To gain further insight into this novel findings, six T. flavoviridis venom gland PLA2 isozyme genes were isolated and sequenced. They were found to consist of four exons and three introns. The numbers of nucleotide substitutions per site (KN) for introns are approximately one-fourth of the numbers of nucleotide substituions per synonymous site (KS) for the protein-coding regions, indicating that the introns are unusually conserved. The fact that the numbers of nucleotide substitutions per nonsynonymous site (KA) are close to or larger than KS values for relevant pairs of genes revealed that Darwinian-type accelerated substitutions have occurred in the protein-coding regions of exons. This is compatible with the presence of PLA2 species with diverse physiological activities in the venom. © 1994, Walter de Gruyter. All rights reserved.

Trimeresurus flavoviridis venom gland phospholipase A2(PLA2) genes pgPLA la and pgPLA 2a encode Asp-49-PLA2and genes pgPLA lb and pgPLA 2b encode an isozyme of Asp-49-PLA2. Polymorphisms were found in pairs of pgPLA la and pgPLA 2a and of pgPLA lb and pgPLA 2b for individuals of T. flavoviridis. The occurrence of both homozygotes and heterozygotes was demonstrated. © 1994, Japan Society for Bioscience, Biotechnology, and Agrochemistry. All rights reserved.

T. Fukagawa, T. Nose, Y. Shimohigashi, T. Ogawa, N. Oda, K. Nakashima, C.-C. Chang and M. Ohno. Purification, sequencing and characterization of single amino acid-substituted phospholipase A2 isozymes from Trimeresurus gramineus (green habu snake) venom. Toxicon 31, 957-967, 1993.-Two phospholipases A2 named PLA2-III and IV were newly isolated from Trimeresurus gramineus (green habu snake) venom in addition to PLA2-I and II reported previously [Oda et al. (1991) Toxicon 29, 157; Fukagawa et al. (1992) Toxicon 30, 133]. Their isoelectric points were determined to be about 4.5. PLA2-III and IV exhibited almost unchanged lipolytic activity toward egg-yolk when compared with PLA2-I. The amino acid sequences were determined by sequencing the native proteins and the peptides produced by enzymatic (Achromobacter protease I and clostripain) and chemical (hydroxylamine) cleavages of the S-carboxamidomethylated derivative of the proteins. Both proteins consisted of 122 amino acid residues. When compared with PLA2-I, PLA2-III showed only a single amino acid substitution at the N-terminal position; namely from His to Asn. PLA2-IV also showed a single substitution from Ala to Asp at position 72. It was inferred that these amino acid substitutions between PLA2-I and PLA2-III or IV are due to the single base substitution at the corresponding codons of genes, which might be preserved independently. The unique presence of Phe at position 28, where Tyr is commonly located and assumed to be a part of the Ca2+-binding loop, was conserved in both PLA2-III and IV as in PLA2-I. There was no significant difference in the dissociation constants (4.3-5.2 × 10-4 M) for Ca2+ between these PLA2s and Tyr-28-containing PLA2s. These results suggested that the p-hydroxy group of Try-28 does not play a crucial role in binding of PLA2s to Ca2+. © 1993.

Six Trimeresurus flavoviridis (Habu snake) venom gland phospholipase A2 (PLA2) isozyme genes were found to consist of four exons and three introns and to encode proteins of 138 amino acid residues, including the signal sequence of 16 amino acid residues. Comparison of the nucleotide sequences showed that the introns are much more homologous than the protein-coding regions of exons except for the signal peptide-coding region of the first exon. The numbers of nucleotide substitutions per site (AN) for introns are approximately one-fourth of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions, indicating that the introns are unusually conserved. The absence of an apparent functional role for the introns suggests that the protein-coding regions, except for the signal peptide-coding domains, have evolved at greater substitution rates than introns. The fact that the numbers of nucleotide substitutions per nonsynonymous site (KA) are close to or larger than KS values for relevant pairs of genes revealed that Darwinian-type accelerated substitutions have occurred in the protein-coding regions or exons. This is compatible with the presence of PLA2 species with diverse physiological activities in the venom.

A complete series of configurationally isomers (L‐L, L‐D, D‐L AND D‐D) of a dipeptide Leu‐Phe benzyl ester have been synthesized and assayed for chymotrypsin. In the conformational analysis by 400 MMz 1H NMR, the L‐D and D‐L isomers, but not hte L‐L and D‐D isomers, showed fairly large up field shifts (0.2–0.4 ppm) of Leu‐βCH2 and γCH proton signals, indicating the presence of shielding effects from the benzene ring. In addition to distinct signal splitting of Phe‐βCH2, the NOE enhancement observed between Leu‐δCH3 and Phe‐phenyl groups revealed that these groups are in close proximity. These data indicated that L‐D and D‐L isomers from a hydrophobic core between side chains of adjacent Leu and Phe residues. When the dipeptides were examined for inhibition of chymotrypsin using Ac‐Try‐OEt as a substrate, the L‐L isomer showed no inhibition, itself becoming a substrate. However, the other three isomers inhibited chymotrypsin in a competitive manner, and the D‐L isomer was strongest with Ki of 2.2 × 10−5 M. It was found that the D‐L isomer was only slowly hydrolysed but the L(or D)‐D isomer was not. H‐D‐Phe‐L‐Leu‐OBzl with the inverse sequence of H‐D‐Leu‐L‐Pre‐OBzl inhibited chymotrypsin more strongly (Ki = 6.3 × 10−6 M). Since the free acid analogue of the D‐L isomer exhibited no inhibition, the benzyl ester moiety itself was thought to be involved in the enzyme inhibition. It is assumed that in the inhibitory conformation the ester‐benzyl group fits the S1 site of chymotrypsin, while the side chain‐side chain complexing hydrophobic core fits the S2 site. Copyright © 1993 John Wiley & Sons Ltd.

In addition to phospholipase A2-I (PLA2-I) reported previously (Oda et al., 1991, Toxicon 29, 157), a new PLA2 named PLA2-II was isolated from Trimeresurus gramineus (green habu snake) venom, and its amino acid sequence was determined by sequencing the native protein and the peptides produced by enzymatic (Achromobacter protease I and clostripain) cleavages of the carboxamidomethylated derivative of the protein. The protein consisted of 122 amino acid residues and His-47, Asp-48, and Asp-98 which have been assumed to be essential for PLA2 activity were conserved. Its sequence similarity to PLA2-I was 79%, with 26 residual differences. In contrast to the unique presence of Phe-28 in PLA2-I, PLA2-II contains Tyr-28 as seen in most of other PLA2s. There was no significant difference between the dissociation constants of PLA2-I and PLA2-II for Ca2+. Secondary structure compositions of PLA2-II were similar to those of PLA2-I and Crotalus atrox PLA2. A striking difference was found between these isozymes in contractile activity of isolated smooth muscle preparation of guinea-pig ileum. PLA2-II was over ten times more potent than PLA2-I, although its lipolytic activity toward egg-yolk was even slightly weaker (73%) than that of PLA2-I. The difference in contractile activities of PLA2-I and PLA2-II could be assumed to be due to discriminative lipid recognition brought about by different amino acid residues at the 58th position (Asp for PLA2-I and Asn for PLA2-II). © 1992.

© 1992 The Chemical Society of Japan. Trimeresurus flavoviridis (Habu snake) venom contains phospholipase A2 (PLA2) isozymes which are rich in disulfide bond and show diverse activities in spite of their highly homologous structures. When reduced form of Tyr(NO2)-67-PLA2, which is a mimic of Asp-49-PLA2 mutant, was oxidized at pH 8.0 in the presence of 5 mM L-cysteine and 5 mM Ca2+, native form of Tyr(NO2)-67-PLA2 was produced as being judged from both activity regeneration and adequate HPLC profile. Reduced forms of two Lys-49-PLA2 isozymes with extremely low lipolytic activity, which are called basic proteins I and II, also generated native forms of proteins when oxidized under the same conditions as above. Protein disulfide isomerase accelerated proper folding of reduced Tyr(NO2)-67-PLA2 at an initial phase of oxidation and was effective for rearrangement of incorrectly paired disulfide bonds of Asp-49-PLA2 to native construction. However, this isomerase failed to convert reduced form of partially active L-fragment, Asp-49-PLA2 lacking N-terminal octapeptide, to the native form, indicating that the entire sequence of protein in a nascent state is primarily required for proper folding. The present data together with the fact that reduced Asp-49-PLA2 folded properly (S. Tanaka et al. J. Biochem., 96, 1443 (1984)) afforded a strong basis for the structure and function study of T. flavoviridis PLA2S by means of in vitro mutagenesis technique.

P. Goparakrishnakone and C. K. Tan, eds

As a step toward understanding the structure and function of phospholipases A2 (PLA2s), we isolated and sequenced several cDNAs encoding Trimeresurus flavoviridis venom PLA2 isozymes including two [Lys49]PLA2s called basic proteins I and II, [Thr37]PLA2, and PLX'-PLA2. Comparison of the nucleotide sequences of these cDNAs with the previously isolated [Asp49]PLA2 cDNA revealed some interesting findings from the viewpoint of evolution. First, the homologies of the 5' and 3' untranslated regions (98% and 89%, respectively) were much higher than that of the protein-coding regions (67%). The predicted secondary structure showed the characteristic stemloop structures for both the untranslated regions of the mRNAs, suggesting that these regions play some functional role(s) in translation or stability of mRNAs. Second, base substitutions appeared to have occurred at similar rates for the three positions of codons among these PLA2s. The results are discussed in terms of evolution of PLA2s. Northern blot analysis showed that these PLA2s are specific to venom gland.

A series of dipeptides with the sequence of H-D-Leu-L-Phe-R, where R denotes 4-phenylpiperidide, 4-phenylpiperazide, and their derivatives, have been synthesized to examine their ability to inhibit chymotrypsin. Dipeptide phenylpiperidide and phenylpiperazide inhibited chymotrypsin fairly strongly (K(i) = 7.5 x 10(-4) M and 1.4 x 10(-3) M, respectively) (1 M = 1 mol dm-3), while their phenyl-substituted analogs were inactive. In high resolution H-1 NMR measurements, it was found that the proton signals of D-Leu gamma-CH and beta-CH2 shifted considerably to the upfield when compared with those of H-D-Leu-L-Ala-OH, suggesting that these upfield shifts are due to the magnetic anisotropy effect by the benzene ring of the Phe residue. This was confirmed by measuring NOE between D-Leu-isobutyl and L-Phe-phenyl groups. These NMR data indicate that the side chains of both D-Leu and L-Phe are in close proximity, resulting in formation of a hydrophobic complex. It was suggested that, in the inhibitory conformation of dipeptides against chymotrypsin, the phenyl group of phenylpiperidide or phenylpiperazide moiety fits the S1 site, while the side chain-side hydrophobic complexing core fits the S2 site.

Y. Shimonishi ed.

Y. Shimonishi ed.

A cDNA clone of Trimeresurus flavoviridis phospholipase A2 was isolated from the venom gland cDNA library using DNA amplified by polymerase chain reaction with oligonu-cleotide primers corresponding to the N- and C-terminal sequences of this enzyme. The amino acid sequence deduced from the cDNA nucleotide sequence determined by the dideoxy termination method was found to be inconsistent in the segment comprising the 69th to 81st positions from that reported previously [Tanaka, S. et al. (1986) J. Biochem. 99, 281-289]. Reinvestigation of the amino acid sequence of the peptide covering the sequence in question showed that the amino acid sequence predicted from the nucleotide sequence is correct. The sequence contains Asn-Asn-Gly (positions 69-71) and it was found that the Asn-Gly bond easily undergoes α-β transpeptidation when digested with Achromobacter protease I at pH 9.0 but not seriously at pH 6.8. It is likely that the transpeptidation reaction caused a failure in the previous sequence determination. The cDNA clone obtained was 597 base pairs long and contained an open reading frame of 414 base pairs coding for 138 amino acid residues. A typical signal peptide sequence (16 amino acid residues long), located at the N-terminal moiety of the deduced sequence, was immediately followed by a polypeptide which corresponds to the mature enzyme. Northern blot analysis showed a single transcript only in the poly(A)+ RNA fraction of the venom gland but not in those of many other organs tested. © 1990 Copyright, 1990 by the Journal of Biochemistry.

Based on the analyzed conformation of a chymotrypsin inhibitor H-{down triangle, open}Phe-Phe-OMe, we have designed a series of diastereomeric phenylalanylphenylalanine methyl esters and derivatives as possible inhibitors. Among the peptides synthesized, H-D-Phe-L-Phe-OMe was found to be very resistant to chymotrypsin in spite of its L-Phe-OMe structure at the C-terminus. It inhibited the enzyme fairly strongly and competitively with Ki = 9.0 × 10-5 M in the assay using Ac-Tyr-OEt as a substrate. The measurements of the NOEs in high-resolution 1H-NMR analyses indicated the presence of the hydrophobic core built by the intramolecular interaction between the D-Phe-phenyl and ester-methyl groups. It was suggested that this core interacts with the chymotrypsin S2 site (Trp215) and Phe2 with the S1 site. The backbone structure of this dipeptide was assumed to be in an inhibitory conformation that fits the active center of the enzyme. © 1990.

Dipeptides containing 2,3-methanophenylalanine, a sterically constrained amino acid with alpha,beta-cyclopropane ring, showed fairly strong inhibitory activity for the hydrolysis of Ac-L-Tyr-OEt by chymotrypsin. Kinetic analyses of the inhibition by dipeptides, H-inverted delta E Phe-Leu (or Phe)-OMe (or OH), indicated the mode of inhibition to be competitive. Comparative analyses of the inhibitory constants Ki have clarified several structural elements necessary to elicit an inhibitory activity. Those include the (2R,3S)-configuration of the inverted delta E Phe residue, the phenyl side chain at position 2 and the C-terminal methylesterification. These structural conditions suggested that the peptides are in specific inhibitory conformation. In the conformational analyses of these dipeptide inhibitors by a high resolution 1H-NMR (270 MHz), the measurement of enhancements of the nuclear Overhauser effect indicated that an intramolecular hydrophobic bonding exists between the inverted delta Phe-phenyl and ester-methyl to construct the hydrophobic core in the molecule. It was suggested that this hydrophobic core interacts with the chymotrypsin S2 site and prevents the hydrolysis of methyl ester. The Leu2 or Phe2 interacts with the enzyme S1 site. The structure of dipeptides containing 2,3-methanophenylalanine has been characterized as an enzyme-inhibitory conformation that interacts with the enzyme at its catalytic center.

N. Yanaihara ed.

N. Yanaihara ed.

N. Yanaihara ed.

Both isomers of (E)-2,3-methanophenylalanine (▽EPhe), a sterically restricted amino acid, were incorporated into peptides in order to examine their possible enzyme inhibitory activity. Both (2R,3S)- and (2S,3R)-▽EPhe-Phe(or Leu)-OMe were found to inhibit effectively the hydrolysis of Ac-Tyr-OEt by chymotrypsin in a competitive manner. The ester groups of these dipeptides were quite resistant to chymotrypsin hydrolysis, and the ▽EPhe-Phe peptide bond was also entirely stable. The inhibition constant (Ki) of the most potent dipeptide of H-(2R,3S)-▽EPhe-Phe-OMe was 0.16 mM at 25°C. The inhibitory action of ▽Phe-containing peptides was found to depend on the configuration of the ▽Phe residue. The electrophilic nature of the cyclopropane ring which is conjugated with both the phenyl ring and the ester carbonyl group appears to be relevant to the inhibitory activity. Fully irreversible inactivation of chymotrypsin was achieved by its incubation with H-(2R,3S)-▽EPhe-Leu-OMe. An enzyme carboxylate group is thought to be responsible for nucleophilic attack on the cyclopropane ring leading to irreversible inactivation. © 1989.

M. Ueki ed.

T. Shiba &amp; S. Sakakibara ed.