Koji Muramoto

Tohoku University, Sendai-shi, Miyagi-ken, Japan

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Publications (154)368.14 Total impact

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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.
    Journal of Enzyme Inhibition and Medicinal Chemistry 10/2013; · 1.50 Impact Factor
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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.
    Bioscience Biotechnology and Biochemistry 09/2013; · 1.27 Impact Factor
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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.
    Journal of Biological Chemistry 07/2012; 287(37):31061-72. · 4.65 Impact Factor
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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.
    Fish &amp Shellfish Immunology 07/2012; 33(4):780-7. · 2.96 Impact Factor
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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.
    Journal of amino acids. 01/2011; 2011:838914.
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    Journal of Molecular Biology - J MOL BIOL. 01/2011; 412(4):751-751.
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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).
    Meat Science 10/2010; 87(3):196-201. · 2.75 Impact Factor
  • Shunji Sugawara, Koji Muramoto
    The Journal of Immunology 04/2010; 184(7):4042. · 5.52 Impact Factor
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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.
    The Journal of Immunology 04/2010; 184(7):4043. · 5.52 Impact Factor
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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.
    BMC Evolutionary Biology 02/2010; 10:43. · 3.29 Impact Factor
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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
    Fisheries Science 01/2010; 76(5):865-872. · 0.90 Impact Factor
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    ABSTRACT: 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 50mM 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 (R(2)) 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.
    Journal of Chromatography A 11/2009; 1216(48):8563-6. · 4.61 Impact Factor
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    ABSTRACT: 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.
    Journal of Molecular Biology 07/2009; 391(2):390-403. · 3.91 Impact Factor
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    ABSTRACT: A myofibril-bound serine protease (MBSP) was partially purified from ostrich (Struthio camelus) skeletal muscle. MBSP was dissociated from the myofibrillar fraction by ethylene glycol treatment at pH 8.5, followed by partial purification via Toyopearl Super Q 650 S and p-aminobenzamidine column chromatographies. Ostrich MBSP revealed a major protein band of approximately 21 kDa on SDS-PAGE, showing proteolytic activity after casein zymography. Optima pH and temperature of ostrich MBSP were 8 and 40 degrees C, respectively. Substrate specificity analysis revealed that the enzyme cleaved synthetic fluorogenic substrates at the carboxyl side of arginine residues. Kinetic parameters (K(m) and V(max) values) were calculated from Lineweaver-Burk plots. The kinetic characteristics of ostrich MBSP were compared to values obtained for commercial bovine trypsin in this study, as well as those obtained for MBSP from mouse and various fish species. The results suggest that ostrich MBSP is a tryptic-like serine protease. Ostrich MBSP exhibited low sequence identity to commercial bovine trypsin (44%), MBSP from lizard fish skeletal muscle (33%) and trypsinogen from ostrich pancreas (22%).
    Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 07/2009; 154(2):229-34. · 1.61 Impact Factor
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    ABSTRACT: 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.
    Journal of Agricultural and Food Chemistry 05/2009; 57(7):2896-902. · 3.11 Impact Factor
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    ABSTRACT: 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.
    European Journal of Allergy and Clinical Immunology 01/2009; 25(4):442 - 448. · 1.30 Impact Factor
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    ABSTRACT: 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 24h. 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 2h-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 670–3,611Da. Tyr-Leu-Ala-Gly-Met-Asn had the highest antioxidative activity among them.
    European Food Research and Technology 01/2009; 228(4):553-563. · 1.39 Impact Factor
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    ABSTRACT: 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, 50gkg−1 NaCl, 0.02molL−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 20gkg−1 NaCl, 70% ethanol, 0.1molL−1 acetic acid and 0.1molL−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.
    European Food Research and Technology 12/2008; 228(3):391-401. · 1.39 Impact Factor
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    ABSTRACT: 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.
    Developmental & Comparative Immunology 10/2008; 33(2):187-97. · 3.24 Impact Factor
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    ABSTRACT: 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 A(2) ([Lys(49)]PLA(2)), 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 alpha-thrombin or Microbacterium liquefaciens protease (MLP), to obtain completely active recombinant BPII. Thus, the congerin-tagged fusion systems containing the cleavage recognition site for alpha-thrombin or MLP were demonstrated to be highly efficient and useful for producing proteins of desired solubility and activity.
    Protein Expression and Purification 05/2008; 58(2):194-202. · 1.43 Impact Factor

Publication Stats

2k Citations
368.14 Total Impact Points

Institutions

  • 1994–2012
    • Tohoku University
      • • Graduate School of Life Sciences
      • • Graduate School of Agricultural Science
      Sendai-shi, Miyagi-ken, Japan
  • 1984–2012
    • Kitasato University
      • • Graduate School of Fisheries Sciences
      • • Department of Marine Biosciences
      • • Department of Biosciences
      Edo, Tōkyō, Japan
  • 2007–2010
    • Nelson Mandela Metropolitan University
      • Department of Biochemistry and Microbiology
      Port Elizabeth, Province of Eastern Cape, South Africa
  • 2006–2009
    • Nagahama Institute of Bio-Science and Technology
      • Department of Bioscience
      Нагахама, Shiga, Japan
  • 2004
    • National Institutes of Health
      • Laboratory of Bioorganic Chemistry (LBC)
      Bethesda, MD, United States
  • 1999–2002
    • Nagoya University
      • Graduate School of Engineering
      Nagoya-shi, Aichi-ken, Japan
  • 1997–2002
    • Port Elizabeth Museum
      Port Elizabeth, Eastern Cape, South Africa
  • 2001
    • Kanazawa University
      • School of Electrical and Computer Engineering
      Kanazawa, Ishikawa, Japan
  • 1998
    • Sendai University
      Sendai, Kagoshima, Japan
  • 1993
    • University of São Paulo
      • Instituto de Biociências (IB) (São Paulo)
      São Paulo, Estado de Sao Paulo, Brazil
  • 1989
    • Teikyo University
      • Faculty of Pharmaceutical Sciences
      Tokyo, Tokyo-to, Japan