Koji Muramoto

Tohoku University, Sendai-shi, Miyagi, Japan

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Publications (197)409.87 Total impact

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    ABSTRACT: 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.
    No preview · Article · Sep 2015 · The Protein Journal
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    ABSTRACT: 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).
    No preview · Article · Sep 2015 · Food Science and Technology Research
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    ABSTRACT: 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.
    No preview · Article · May 2015 · Molecules
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    ABSTRACT: Nacreous layers of pearl oyster are one of the major functional biominerals. By participating in organic compound-crystal interactions, they assemble into consecutive mineral lamellae-like photonic crystals. Their biomineralization mechanisms are controlled by macromolecules; however, they are largely unknown. Here, we report two novel lectins termed PPL2A and PPL2B, which were isolated from the mantle and the secreted fluid of Pteria penguin oyster. PPL2A is a hetero-dimer composed of α and γ subunits, and PPL2B is a homo-dimer of β subunit, all of which surprisingly shared sequence homology with the jacalin-related plant lectin. On the basis of knockdown experiments at the larval stage, the identification of PPLs in the shell matrix, and in vitro CaCO3 crystallization analysis, we conclude that two novel jacalin-related lectins participate in the biomineralization of P. penguin nacre as matrix proteins. Furthermore, it was found that trehalose, which is specific recognizing carbohydrates for PPL2A and is abundant in the secreted fluid of P. penguin mantle, functions as a regulatory factor for biomineralization via PPL2A. These observations highlight the unique functions, diversity and molecular evolution of this lectin family involved in the mollusk shell formation.
    Full-text · Article · Nov 2014 · PLoS ONE
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    ABSTRACT: 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. © 2013 Springer Science+Business Media Dordrecht. All rights are reserved.
    No preview · Article · Nov 2013
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    ABSTRACT: Abstract 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.
    No preview · Article · Oct 2013 · Journal of Enzyme Inhibition and Medicinal Chemistry
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    ABSTRACT: 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 µg/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.
    No preview · Article · Sep 2013 · Bioscience Biotechnology and Biochemistry
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    ABSTRACT: 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.
    No preview · Article · May 2013 · Fisheries Science
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    ABSTRACT: 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.
    No preview · Article · Jul 2012 · Journal of Biological Chemistry
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    ABSTRACT: 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.
    No preview · Article · Jul 2012 · Fish & Shellfish Immunology
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    ABSTRACT: 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. © 2012 The Japanese Society for Plant Cell and Molecular Biology.
    No preview · Article · Jan 2012 · Plant Biotechnology
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    ABSTRACT: 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.
    Full-text · Article · Nov 2011
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    Full-text · Article · Sep 2011 · Journal of Molecular Biology
  • J. Yamashita · H.-M. Chen · T. Naganuma · T. Ogawa · K. Muramoto
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    ABSTRACT: 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, Fe2+ chelating, superoxide dismutase-like activity, and β-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.
    No preview · Article · Dec 2010 · ACS Symposium Series
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    ABSTRACT: 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).
    No preview · Article · Oct 2010 · Meat Science
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    ABSTRACT: 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 9weeks. 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. KeywordsAngiotensin-I-converting enzyme-Hypotensive-Oyster-Peptide-Spontaneously hypertensive rat
    No preview · Article · Sep 2010 · Fisheries Science
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    Shunji Sugawara · Koji Muramoto

    Preview · Article · Apr 2010 · The Journal of Immunology
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    Koji Muramoto · Shunji Sugawara
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    ABSTRACT: 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. The Journal of Immunology, 2003, 170: 5690 -5696.
    Preview · Article · Apr 2010 · The Journal of Immunology
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    ABSTRACT: 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. 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. 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.
    Full-text · Article · Feb 2010 · BMC Evolutionary Biology
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    ABSTRACT: 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.
    No preview · Article · Jan 2010 · Plant Biotechnology

Publication Stats

5k Citations
409.87 Total Impact Points


  • 1976-2015
    • Tohoku University
      • • Department of Biomolecular Sciences
      • • Graduate School of Life Sciences
      • • Division of Biological Resource Sciences
      • • Graduate School of Agricultural Science
      • • Department of Applied Chemistry
      Sendai-shi, Miyagi, Japan
  • 2009
    • National Institute of Advanced Industrial Science and Technology
      • Research Center for Medical Glycoscience
      Tsukuba, Ibaraki, Japan
  • 1979-2009
    • Kitasato University
      • Graduate School of Fisheries Sciences
      Edo, Tōkyō, Japan
  • 2002
    • Port Elizabeth Museum
      Port Elizabeth, Eastern Cape, South Africa
  • 1989
    • Teikyo University
      • Faculty of Pharmaceutical Sciences
      Edo, Tōkyō, Japan
  • 1985
    • Archbold Biological Station
      Florida, United States
  • 1980-1984
    • University of California, San Francisco
      • Department of Biochemistry and Biophysics
      San Francisco, California, United States