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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.

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%&ndash, 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.

The cyanelle is a primitive chloroplast that contains a peptidoglycan layer between its inner and outer membranes. Despite the fact that the envelope structure of the cyanelle is reminiscent of Gram-negative bacteria, the Cyanophora paradoxa genome appears to lack genes encoding homologs of putative peptidoglycan-associated outer membrane proteins and outer membrane channels. These are key components of Gram-negative bacterial membranes, maintaining structural stability and regulating permeability of outer membrane, respectively. Here, we discovered and characterized two dominant peptidoglycan-associated outer membrane proteins of the cyanelle (∼2 × 10(6) molecules per cyanelle). We named these proteins CppF and CppS (cyanelle peptidoglycan-associated proteins). They are homologous to each other and function as a diffusion channel that allows the permeation of compounds with Mr

Enhancement of sugar content and sweetness is desirable in some vegetables and in almost all fruits; however, biotechnological methods to increase sugar content are limited. Here, a completely novel methodological approach is presented that produces sweeter tomato fruits but does not have any negative effects on plant growth. Sucrose-induced repression of translation (SIRT), which is mediated by upstream open reading frames (uORFs), was initially reported in Arabidopsis AtbZIP11, a class S basic region leucine zipper (bZIP) transcription factor gene. Here, two AtbZIP11 orthologous genes, SlbZIP1 and SlbZIP2, were identified in tomato (Solanum lycopersicum). SlbZIP1 and SlbZIP2 contained four and three uORFs, respectively, in the cDNA 5'-leader regions. The second uORFs from the 5' cDNA end were conserved and involved in SIRT. Tomato plants were transformed with binary vectors in which only the main open reading frames (ORFs) of SlbZIP1 and SlbZIP2, without the SIRT-responsive uORFs, were placed under the control of the fruit-specific E8 promoter. Growth and morphology of the resulting transgenic tomato plants were comparable to those of wild-type plants. Transgenic fruits were approximately 1.5-fold higher in sugar content (sucrose/glucose/fructose) than nontransgenic tomato fruits. In addition, the levels of several amino acids, such as asparagine and glutamine, were higher in transgenic fruits than in wild-type fruits. This was expected because SlbZIP transactivates the asparagine synthase and proline dehydrogenase genes. This 'sweetening' technology is broadly applicable to other plants that utilize sucrose as a major translocation sugar.

Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.

We evaluated the impact of increased temperature and sunlight exposure, features of global warming, on the skin coloration of ‘Kyoho’ table grape berries. Skin coloration, anthocyanin accumulation, abscisic acid (ABA) content, and transcript levels of anthocyanin biosynthetic genes in the skins were examined during the ripening season under continuous 24 °C, 27 °C and 30 °C temperatures together with a sun or shade treatment. Skin coloration and anthocyanin accumulation were sufficient in the berries of plants ripened at 24 °C with a sun-exposure treatment. They also had higher ABA contents and anthocyanin biosynthetic gene transcript levels than grapes under higher temperatures. The skins of grapes ripened at 27 °C or 30 °C showed insufficient coloration and low levels of anthocyanin accumulation, ABA content, and anthocyanin biosynthetic gene transcripts levels, irrespective of the light conditions. These results suggest that temperatures exceeding 27 °C during the ripening season lead to insufficient berry coloration as a result of low levels of ABA and anthocyanin biosynthetic gene expression levels.

Venomous animals have specialized venom delivery apparatus such as nematocysts, stings, and fangs in addition to the poisonous organs consisting venom gland or sac, which produce and stock the venom. Snake is one of the major venomous animals, of which fangs are connected to the venom gland to inject the venom into prey. Snake’s venomous fangs showed the unique characteristics including mechanical strength and chemical stability. Especially, Protobothrops flavoviridis (habu) snake fangs showed the resistance against its venom digestive proteases, whereas the bones and teeth of mouse were completely digested in the gastrointestinal tract, although habu fangs were also drawn into the body with the prey. These observations suggest that structural differences exist between venomous fangs and mammalian bones and teeth.

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.

Cholangiocarcinoma is an aggressive malignant tumor originating from intrahepatic or extrahepatic bile ducts. Its malignant phenotypes may be assumed by cancer stem cells (CSC). Here, we demonstrate that CD274 (PD-L1), known as an immunomodulatory ligand, has suppressive effects on CSC-related phenotypes of cholangiocarcinoma. Using two human cholangiocarcinoma cell lines, RBE and HuCCT1, we attempted to isolate the CD274(low) and CD274(high) cells from each cell line, and xenografted them into immunodeficient NOD⁄scid⁄γcnull (NOG) mice. We found that the CD274(low) cells isolated from both RBE and HuCCT1 are highly tumorigenic in NOG mice compared with CD274(high) cells. Furthermore, the CD274(low) cells possess several CSC-related characteristics, such as high aldehyde dehydrogenase (ALDH) activity, reduced reactive oxygen species production and a dormant state in the cell cycle. Furthermore, depletion of CD274 expression by shRNA in RBE cells enhances their tumorigenicity and increases ALDH activity. These findings are compatible with our observation that clinical cholangiocarcinoma specimens are classified into low and high groups for CD274 expression, and the CD274 low group shows poorer prognosis when compared with the CD274 high group. These results strongly suggest that CD274 has a novel function in the negative regulation of CSC-related phenotypes in human cholangiocarcinoma, which is distinct from its immunomodulatory actions.

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 β-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 phloemspecific 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.

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.

We estimated genetic and environmental variance components for fruit quality traits (cluster weight, berry weight, soluble solids content, total sugar content, sugar composition, free acid content, total amino acid content, amino acid composition), and a functional trait (gamma-aminobutyric acid (GABA) content) of three commercial tetraploid table grape cultivars (Kyoho, Pione, Suiho) grown in Japan over three successive years. ANOVA showed that the effect of genotype was significant (P

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).

Angiotensin-I-converting enzyme (ACE) plays a crucial role in the crisis of hypertension. Some peptides that originate from protease hydrolysates are known to suppress ACE activity in vitro and in vivo. Here, we investigated whether trypsin hydrolysate of oyster Crassostrea gigas showed hypotensive activity and ACE inhibition. The hydrolysate significantly suppressed systolic blood pressure and ACE activity in spontaneously hypertensive rats following a one-shot oral administration and a long-term feeding experiment lasting 9 weeks. Each hydrolysate from oyster tissue showed ACE inhibitory activity, indicating the hypotensive effect was due to synergism. One potent ACE inhibitory peptide, Asp-Leu-Thr-Asp-Tyr, was identified from the hydrolysate of the striate muscle, and the peptide exhibited hypotensive activity in vivo. Protease digestion analysis suggested that Asp-Tyr could be the real effector of this penta-peptide in vivo.

The ability of grape cultivars to produce resveratrol in response to UV radiation is associated with their resistance to fungal pathogens. In this study, we evaluated the UV-induced resveratrol production potential of grape flowers and green berries of 72 grape genotypes. Their ability to produce resveratrol in response to UV radiation was used to establish a selection criterion for screening genotypes for resistance to gray mold and powdery mildew. We quantified resveratrol in grape flower extracts from pre-bloom and early bloom flowers after exposure to UV radiation. There was a strong negative correlation between UV-induced resveratrol production and susceptibility to Botrytis infection. The grape genotype was considered to be susceptible to gray mold when the resveratrol content of whole flower clusters was less than 10 μg g−1 FW after the UV treatment. We determined resveratrol production in response to UV radiation in whole grape berries from 0 to 30 days after full bloom. The ability to produce resveratrol in response to UV increased by 8- to 20-times during this period, depending on the genotype. At 30 days after full bloom, Vitisvinifera genotypes generally had low levels of resveratrol ( 50 μg g−1 FW). There were clear varietal differences in resistance to Erysiphe (powdery mildew) infection. Susceptibility to Erysiphe infection was strongly negatively correlated with UV-induced resveratrol production.

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.

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-1beta1, IL-1beta2, TNF-alpha1, TNF-alpha2 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.

The primary structure of prolactin from fin whale pituitary has been elucidated. It consists of 199 amino acid residues with three disulfide bridges formed by residues 6–11, 58–174 and 191–199. The fin whale prolactin is highly homologous with other mammalian prolactins, especially with the porcine hormone.

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.

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 cyrstals. Among BRA, BRA-2 most efficiently inhibited the crystal nucleation of aragoite and calcite at the concentrations of >3.3 and >26 ⧎g/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.

Biotin, a water-soluble vitamin of the B complex, functions as a cofactor of carboxylases that catalyze an indispensable cellular metabolism. Although significant decreases in serum biotin levels have been reported in patients with chronic inflammatory diseases, the biological roles of biotin in inflammatory responses are unclear. In this study, we investigated the effects of biotin deficiency on TNF-α production. Mice were fed a basal diet or a biotin-deficient diet for 8 weeks. Serum biotin levels were significantly lower in biotin-deficient mice than biotin-sufficient mice. After i.v. administration of LPS, serum TNF-α levels were significantly higher in biotin-deficient mice than biotin-sufficient mice. A murine macrophage-like cell line, J774.1, was cultured in a biotin-sufficient or -deficient medium for 4 weeks. Cell proliferation and biotinylation of intracellular proteins were decreased significantly in biotin-deficient cells compared with biotin-sufficient cells. Significantly higher production and mRNA expression of TNF-α were detected in biotin-deficient J774.1 cells than biotin-sufficient cells in response to LPS and even without LPS stimulation. Intracellular TNF-α expression was inhibited by actinomycin D, indicating that biotin deficiency up-regulates TNF-α production at the transcriptional level. However, the expression levels of TNF receptors, CD14, and TLR4/myeloid differentiation protein 2 complex were similar between biotin-sufficient and -deficient cells. No differences were detected in the activities of the NF-κB family or AP-1. The TNF-α induction by biotin deficiency was down-regulated by biotin supplementation in vitro and in vivo. These results indicate that biotin deficiency may up-regulate TNF-α production or that biotin excess down-regulates TNF-α production, suggesting that biotin status may influence inflammatory diseases.

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 male 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.

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 alpha2,3-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 alpha2,3-sialyl galactose moiety, and strong binding ability of ConI to oligosaccharides including lacto-N-biosyl (Galbeta1-3GlcNAc) or lacto-N-neobiosyl (Galbeta1-4GlcNAc) 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.

Congerin, a mucosal galectin of the Japanese conger eel, provides chemical fortification through its agglutinating and opsonizing activity. Congerin is produced in the epidermis, and the epithelia of the oral cavity to the esophagus, but not in the stomach or intestine. We hypothesized that congerin secreted from the upper digestive tract can reach and function in the intestinal lumen. We found that congerin possessed marked resistance against digestion by gastric and enteric enzymes of conger eel. It was not degraded until 6h of incubation with stomach extract or intestinal digestion juice. Western blotting demonstrated that congerin essentially remained in the intestinal mucus. The mucus agglutinated rabbit erythrocytes, and the agglutination was hampered by anti-congerin antibody. Furthermore, congerin could bind to some enteric bacteria. These results support the above hypothesis.

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 alpha2-6 sialylation but not alpha2-3 sialylation on glycans. On the other hand, BRA-3 showed high affinity for oligosaccharides with alpha-linked non-reducing terminal galactose, but not for sialylated forms, and BRL showed enhanced recognition activity towards Lewis(x) and Lewis(a) epitopes.

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). Copyright © 2007 Society of Chemical Industry

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 β-actin in Caco-2 cell monolayers revealed that the cytoskeleton was changed by the CSL3 treatment, suggesting that CSL3 depolymerized β-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.

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

We recently demonstrated that the conger eel galectins, Congerin I and II, have evolved under significant selective pressure, with the emergence of a new strand-swap structure between subunits in Congerin I. To understand the structure-thermostability relationship of Congerins, and seek clues to the mechanism of their evolution, we improved the thermostability of Congerin II by rational design and in vitro evolutionary protein engineering. A resulting Tyr16/Thr88 double mutant exhibited structural features and thermostability similar to the naturally thermostable isoform, Congerin I. Thus, under artificial selective pressure, Congerin II partially reproduced the natural evolution of Congerin I.

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.

Cystein proteinases (gingipains) from Porphyromonas gingivalis cleave a broad range of in-host proteins and are considered to be key virulence factors in the onset and development of adult periodontitis and host defense evasion. In periodontitis, an inflammatory disease triggered by bacterial infection, the production of hepatocyte growth factor (HGF) is induced not only by various factors derived from the host, such as inflammatory cytokines, but also by bacterial components. In this study we examined the possible enhanced production of HGF produced by human gingival fibroblasts upon stimulation with gingipains. Arginine-specific gingipain (Rgp) caused a marked production of HGF into the supernatant, the induction of HGF expression on the cell surface, and the up-regulation of HGF mRNA expression in a dose-dependent and an enzymatic activity-dependent manner. Because it has been reported that Rgp activated protease-activated receptors (PARs), we examined whether the induction of HGF triggered by Rgps on human gingival fibroblasts occurred through PARs. An RNA interference assay targeted to PAR-1 and PAR-2 mRNA revealed that gingipains-induced secretion of HGF was significantly inhibited by RNA interference targeted to PAR-1 and PAR-2. In addition, the Rgps-mediated HGF induction was completely inhibited by the inhibition of phospholipase C and was clearly inhibited by RNA interference targeted to p65, which is an NF-κB component. These results suggest that Rgps activated human gingival fibroblasts to secrete HGF in the inflamed sites and the mechanism(s) involved may actively participate in both inflammatory and reparative processes in periodontal diseases.

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 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.

Symbioses between zooxanthellae (Symbiodinium spp.) and marine invertebrates, including corals, are common in shallow marine environments. The zooxanthellae contribute to host nutrition by translocating photosynthetic products and enabling them to effloresce in oligotrophic conditions. Coral mainly acquire Symbiodinium spp. by capturing freeswimming cells from the environment (1). Cultured Symbiodinium cells show a diel growth cycle with alternation between motile and non-motile cell stages once a day (2, 3), and the cell divides only during the latter stage (2). When associated with a host, however, cells are arrested in a non-motile stage while healthy cell division is maintained (4). We deduced that host-directed and chemical-based mechanisms are responsible for this phenomenon since SLL-2, a lectin that binds to carbohydrate chains with a D-galactosyl moiety, produced by the octocoral Sinularia lochmodes, is localized on the cell surface of the Symbiodinium harbored in the host (5). Here we describe SLL-2 as the key chemical factor for arresting Symbiodinium in the cell-dividing, non-motile stage, while some nonsymbiotic microalgae were even destroyed by SLL-2. Symbiotic associations between photosynthetic dinoflagellates (Symbiodinium spp.) and invertebrate hosts such as corals are crucial for the survival of the host animals since Symbiodinium supplies organic compounds to them and enables them to prosper in the oligotrophic environment

L-amino acid oxidase (LAAO) activity, as well as mechanisms of antimicrobial action of aplysianin A, a sea hare-derived 340 kDa homotetrameric protein, was determined. Spectrophotometric and High-performance liquid chromatography analyses of aplysianin A indicated that one flavin adenine dinucleotide, a cofactor of LAAO, bound to each subunit of the homotetramer. Aplysianin A can specifically catalyze oxidation of basic amino acids (L-arginine and L-lysine), and is the first protein from marine invertebrate animals with LAAO activity. Substrate specificity of aplysianin A is markedly different from that of commonly known LAAO, such as snake venom LAAO, which prefer hydrophobic amino acids. Km value of aplysianin A was the smallest of those for all known LAAO reported. In the presence of catalase, the antibacterial activity of aplysianin A was inhibited as expected, indicating that the antibacterial action of aplysianin A results from hydrogen peroxide production during the reaction with substrates. Interestingly, aplysianin A acted as an antibacterial agent even in the presence of excess catalase. Antibacterial assays in various media suggested that this phenomenon was simply attributed to the consumption of amino acids required for bacterial growth in the media by aplysianin A.

Protease-activated receptors (PARs) compose a family of G protein-coupled receptors activated by proteolysis with exposure of their tethered ligand. Recently, we reported that a neutrophil-derived serine proteinase, proteinase 3 (PR3), activated human oral epithelial cells through PAR-2. The present study examined whether other neutrophil serine proteinases, human leukocyte elastase (HLE), and cathepsin G (Cat G) activate nonepithelial cells, human gingival fibroblasts (HGF). HLE and Cat G as well as PR3 activated HGF to produce IL-8 and monocyte chemoattractant protein 1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor, an inhibitor of HLE and Cat G, and recombinant secretory leukocyte protease inhibitor clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HGF express PAR-1 and PAR-2 mRNA in the cells and the proteins on the cell surface. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca2+ mobilization and rendered cells refractory to subsequent stimulation with HLE and Cat G. The production of cytokine induced by HLE and Cat G and the PAR-2 agonist peptide was completely abolished by inhibition of phospholipase C. These findings suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines and may control a number of inflammatory processes such as periodontitis.

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.

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.

Proteinase 3 (PR3), a 29-kDa serine proteinase secreted from activated neutrophils, also exists in a membrane-bound form, and is suggested to actively contribute to inflammatory processes. The present study focused on the mechanism by which PR3 activates human oral epithelial cells. PR3 activated the epithelial cells in culture to produce IL-8 and monocyte chemoattractant protein-1 and to express ICAM-1 in a dose- and time-dependent manner. Incubation of the epithelial cells for 24 h with PR3 resulted in a significant increase in the adhesion to neutrophils, which was reduced to baseline levels in the presence of anti-ICAM-1 mAb. Activation of the epithelial cells by PR3 was inhibited by serine proteinase inhibitors and serum. The epithelial cells strongly express protease-activated receptor (PAR)-1 and PAR-2 mRNA and weakly express PAR-3 mRNA. The expression of PAR-2 on the cell surface was promoted by PR3, and inhibited by cytochalasin B, but not by cycloheximide. PR3 cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. Treatment with trypsin, an agonist for PAR-2, and a synthetic PAR-2 agonist peptide induced intracellular Ca2+ mobilization, and rendered cells refractory to subsequent stimulation with PR3 and vice versa. The production of cytokine induced by PR3 and the PAR-2 agonist peptide was completely abolished by a phospholipase C inhibitor. These findings suggest that neutrophil PR3 activates oral epithelial cells through G protein-coupled PAR-2 and actively participates in the process of inflammation such as periodontitis.

Seven kinds of angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated from the hydrolysates of wakame (Undaria pinnatifida) by Protease S "Amano" (from Bacillus stearothermophilus) by using three-step high-performance liquid chromatography (HPLC) on a reverse-phase column. These peptides were identified by amino acid composition analysis, sequence analysis, and liquid chromatography-mass spectrometry (LC-MS), as Val-Tyr (IC(50) = 35.2 microM), Ile-Tyr (6.1 microM), Ala-Trp (18.8 microM), Phe-Tyr (42.3 microM), Val-Trp (3.3 microM), Ile-Trp (1.5 microM), and Leu-Trp (23.6 microM). These peptides have resistance against gastrointestinal proteases in vitro. Each peptide was determined to have an antihypertensive effect after a single oral administration in spontaneously hypertensive rats (SHR). Among them, the blood pressure significantly decreased by Val-Tyr, Ile-Tyr, Phe-Tyr, and Ile-Trp in a dose of 1 mg/kg of body weight (BW). The present study showed that antihypertensive effect in the hydrolysates of wakame by Protease S "Amano" was attributed to these peptides.

The epidermis of premetamorphic leptocephali of Japanese conger Conger myriaster consisted of only a few cell layers including congerin- (a galectin) containing club cells. The epidermis increased in thickness after metamorphosis, mainly due to a preponderant development of club cells. This suggested that the biological significance of congerins increased at this stage, which may be related to a change in life style. Epidermal mucous cells showed only limited distribution. Immuno-positive cells in the mucosal epithelia of the buccal cavity wall and oesophagus were not found in the youngest individuals. Though these cells were observed at the commencement of metamorphosis, the development of the epithelia and club cells was apparently retarded compared to that of the epidermis. No immuno-staining was found in the gills at any stages.

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.

BRA-2, a major C-type lectin in the hemolymph of the acorn barnacle Megabalanusrosa was detected in shell-associated proteins. Immobilized BRA-2 promoted CaCO3 crystallization as determined by an in vitro pH-drift mineralization assay. In nature, calcification occurs as wound repair in M. rosa after breakage of the shell plate. To observe this phenomenon, we inserted a rubber cube inside the mantle cavity of M. rosa through the basal plate, and kept the organisms in a tank for several weeks. The rubber cube was covered first with brownish-colored material, probably a melanin, and then with CaCO3 5 weeks after the insertion. In the CaCO3-associated proteins, BRA-3, a minor component of M. rosa hemolymph lectins, was present in addition to BRA-2. The presence of both the lectins and their ligands on the hemocyte was also observed. These results suggest that the C-type lectins of the M. rosa hemolymph participate in mineralization as well as defense in these organisms.