Researcher Profile and Settings

Master

Affiliation (Master)

• Faculty of Fisheries Sciences　Marine Life Science　Marine Biotechnology and Microbiology

Affiliation (Master)

• Faculty of Fisheries Sciences　Marine Life Science　Marine Biotechnology and Microbiology

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Profile and Settings

Profile and Settings

• Name (Japanese)

  Tanaka

• Name (Kana)

  Hiroyuki

• Name

  201301038147868829

Achievement

Research Interests

• invertebrate   molecular biology   水産化学   タンパク質の構造・機能相関   生体高分子   Marine biochemistry(6302)   Structure-function relationships of protein(5802)   Biopolymer(4706)   

Research Areas

• Life sciences / Marine/Aquatic life sciences
• Life sciences / Functional biochemistry
• Nanotechnology/Materials / Biochemistry

Research Experience

• 2007 - 大学院水産科学研究院 助教

Published Papers

• Nonmuscular Troponin-I is required for gastrulation in sea urchin embryos.

  Mai Kamata, Yuri Taniguchi, Junko Yaguchi, Hiroyuki Tanaka, Shunsuke Yaguchi

  Developmental dynamics : an official publication of the American Association of Anatomists 2023/12/10 

  BACKGROUND: Gastrulation is one of the most important events in our lives (Barresi and Gilbert, 2020, Developmental Biology, 12th ed.). The molecular mechanisms of gastrulation in multicellular organisms are not yet fully understood, since many molecular, physical, and chemical factors are involved in the event. RESULTS: Here, we found that one of muscle components, Troponin-I (TnI), is expressed in future gut cells, which are not muscular cells at all, and regulates gastrulation in embryos of a sea urchin, Hemicentrotus pulcherrimus. When we block the function of TnI, the invagination was inhibited in spite that the gut-cell specifier gene is normally expressed. In addition, blocking myosin activity also induced incomplete gastrulation. CONCLUSION: These results strongly suggested that TnI regulates nonmuscular actin-myosin interactions during sea urchin gastrulation. So far, Troponin system is treated as specific only for muscle components, especially for striated muscle, but our data clearly show that TnI is involved in nonmuscular event. It is also reported that recent sensitive gene expression analysis revealed that Troponin genes are expressed in nonmuscular tissues in mammals (Ono et al., Sci Data, 2017;4:170105). These evidences propose the new evolutionary and functional scenario of the involvement of Troponin system in nonmuscular cell behaviors using actin-myosin system in bilaterians including human being.
• Effects of omecamtiv mecarbil on the contractile properties of skinned porcine left atrial and ventricular muscles

  Tomohiro Nakanishi, Kotaro Oyama, Hiroyuki Tanaka, Fuyu Kobirumaki-Shimozawa, Shuya Ishii, Takako Terui, Shin’ichi Ishiwata, Norio Fukuda

  Frontiers in Physiology 13 2022/08/23 [Refereed][Not invited]
   

  Omecamtiv mecarbil (OM) is a novel inotropic agent for heart failure with systolic dysfunction. OM prolongs the actomyosin attachment duration, which enhances thin filament cooperative activation and accordingly promotes the binding of neighboring myosin to actin. In the present study, we investigated the effects of OM on the steady-state contractile properties in skinned porcine left ventricular (PLV) and atrial (PLA) muscles. OM increased Ca²⁺ sensitivity in a concentration-dependent manner in PLV, by left shifting the mid-point (pCa₅₀) of the force-pCa curve (ΔpCa₅₀) by ∼0.16 and ∼0.33 pCa units at 0.5 and 1.0 μM, respectively. The Ca²⁺-sensitizing effect was likewise observed in PLA, but less pronounced with ΔpCa₅₀ values of ∼0.08 and ∼0.22 pCa units at 0.5 and 1.0 μM, respectively. The Ca²⁺-sensitizing effect of OM (1.0 μM) was attenuated under enhanced thin filament cooperative activation in both PLV and PLA; this attenuation occurred directly via treatment with fast skeletal troponin (ΔpCa₅₀: ∼0.16 and ∼0.10 pCa units in PLV and PLA, respectively) and indirectly by increasing the number of strongly bound cross-bridges in the presence of 3 mM MgADP (ΔpCa₅₀: ∼0.21 and ∼0.08 pCa units in PLV and PLA, respectively). It is likely that this attenuation of the Ca²⁺-sensitizing effect of OM is due to a decrease in the number of “recruitable” cross-bridges that can potentially produce active force. When cross-bridge detachment was accelerated in the presence of 20 mM inorganic phosphate, the Ca²⁺-sensitizing effect of OM (1.0 μM) was markedly decreased in both types of preparations (ΔpCa₅₀: ∼0.09 and ∼0.03 pCa units in PLV and PLA, respectively). The present findings suggest that the positive inotropy of OM is more markedly exerted in the ventricle than in the atrium, which results from the strongly bound cross-bridge-dependent allosteric activation of thin filaments.
• The effect of alkaline pretreatment on the biochemical characteristics and fibril-forming abilities of types I and II collagen extracted from bester sturgeon by-products.

  Dawei Meng, Hiroyuki Tanaka, Taishi Kobayashi, Hirosuke Hatayama, Xi Zhang, Kazuhiro Ura, Shunji Yunoki, Yasuaki Takagi

  International journal of biological macromolecules 131 572 - 580 2019/06/15 [Refereed][Not invited]
   

  Non-mammalian collagens have attracted increasing attention for industrial and biomedical use. We have therefore evaluated extraction conditions and the biochemical properties of collagens from aquacultured sturgeon. Pepsin-soluble type I and type II collagen were respectively extracted from the skin and notochord of bester sturgeon by-products, with yields of 63.9 ± 0.19% and 35.5 ± 0.68%. Collagen extraction efficiency was improved by an alkaline pretreatment of the skin and notochord (fewer extraction cycles were required), but the final yields decreased to 56.2 ± 0.84% for type I and 31.8 ± 1.13% for type II. Alkaline pretreatment did not affect the thermal stability or triple-helical structure of both types of collagen. Types I and II collagen formed re-assembled fibril structures in vitro, under different conditions. Alkaline pretreatment slowed down the formation of type I collagen fibrils and specifically inhibited the formation of thick fibril-bundle structures. In contrast, alkaline pretreatment did not change type II collagen fibril formation. In conclusion, alkaline pretreatment of sturgeon skin and notochord is an effective method to accelerate collagen extraction process of types I and II collagen without changing their biochemical properties. However, it decreases the yield of both collagens and specifically changes the fibril-forming ability of type I collagen.
• Smooth muscle-like Ca2+-regulation of actin–myosin interaction in adult jellyfish striated muscle

  Hiroyuki Tanaka, Shiori Ishimaru, Yasuhiro Nagatsuka, Keisuke Ohashi

  Scientific Reports 8 (1) 7776  2018/12 [Refereed][Not invited]
   

  Cnidaria is an animal phylum, whose members probably have the most ancestral musculature. We prepared and characterized, for the first time to our knowledge, native actomyosin from the striated myoepithelium of the adult moon jelly Aurelia sp. The actomyosin contained myosin, paramyosin-like protein, Ser/Thr-kinase, actin, and two isoforms of tropomyosin, but not troponin, which is known to activate contraction dependent on intracellular Ca2+ signaling in almost all striated muscles of bilaterians. Notably, the myosin comprised striated muscle-type heavy chain and smooth muscle-type regulatory light chains. In the presence of Ca2+, the Mg-ATPase activity of actomyosin was stimulated and Ser21 of the regulatory light chain was concomitantly phosphorylated by the addition of calmodulin and myosin light chain kinase prepared from chicken smooth muscle. Collectively, these results suggest that, similar to smooth muscle, the contraction of jellyfish striated muscle is regulated by Ca2+-dependent phosphorylation of the myosin light chain.
• Troponin-I is present as an essential component of muscles in echinoderm larvae

  Shunsuke Yaguchi, Junko Yaguchi, Hiroyuki Tanaka

  SCIENTIFIC REPORTS 7 43563  2045-2322 2017/03 [Refereed][Not invited]
   

  The troponin complex, composed of Troponin-I, Troponin-T and Troponin-C, is an essential mediator of the contraction of striated muscle downstream of calcium signaling in almost all bilaterians. However, in echinoderms and hemichordates, collectively termed Ambulacraria, the components of the troponin complex have never been isolated, thus suggesting that these organisms lost the troponin system during evolution. Here, by analyzing genomic information from sea urchins, we identify the troponin-I gene and isolate its complete mRNA sequence. Using this information, we reveal that the larval muscles express this gene and its translated product and that the protein is definitely a functional molecule expressed in sea urchin larvae by showing that Troponin-I morphants are unable to swallow algae. We conclude that muscular contraction in all bilaterians universally depends on a regulatory system mediated by Troponin-I, which emerged in the common ancestor of bilaterians.
• Ca2+-binding properties and regulatory roles of lobster troponin C sites II and IV

  Hiroyuki Tanaka, Hiroki Takahashi, Takao Ojima

  FEBS LETTERS 587 (16) 2612 - 2616 0014-5793 2013/08 [Refereed][Not invited]
   

  Invertebrate troponin C typically contains Ca2+-specific binding sites, sites II and IV, in the N- and C-terminal domains, respectively. To investigate the roles of these sites for Ca2+-dependent regulation of muscle contraction, we generated lobster troponin C mutants, and analyzed their Ca2+-binding properties and regulatory effects on actomyosin-tropomyosin Mg-ATPase activity. The results suggest that Ca2+ binding to site IV is responsible for regulation at relatively low Ca2+ concentrations, while site II has an essential role in full activation at higher Ca2+ concentrations. (C) 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
• Structure of the Ca 2 +-saturated C-terminal domain of scallop troponin C in complex with a troponin i fragment

  Yusuke S Kato, Fumiaki Yumoto, Hiroyuki Tanaka, Takuya Miyakawa, Yumiko Miyauchi, Daijiro Takeshita, Yoriko Sawano, Takao Ojima, Iwao Ohtsuki, Masaru Tanokura

  Biological Chemistry 394 (1) 55 - 68 1431-6730 2013/01 [Refereed][Not invited]
   

  Troponin C (TnC) is the Ca 2 +-sensing subunit of troponin that triggers the contraction of striated muscles. In scallops, the striated muscles consume little ATP energy in sustaining strong contractile forces. The N-terminal domain of TnC works as the Ca 2 + sensor in vertebrates, whereas scallop TnC uses the C-terminal domain as the Ca 2 + sensor, suggesting that there are differences in the mechanism of the Ca 2 +-dependent regulation of muscles between invertebrates and vertebrates. Here, we report the crystal structure of Akazara scallop ( Chlamys nipponensis akazara ) adductor muscle TnC C-terminal domain (asTnC C ) complexed with a short troponin I fragment (asTnI S ) and Ca 2 +. The electron density of a Ca 2 + ion is observed in only one of the two EF-hands. The EF-hands of asTnC C can only be in the fully open conformation with the assistance of asTnI S. The number of hydrogen bonds between asTnC C and asTnI S is markedly lower than the number in the vertebrate counterparts. The Ca 2 + modulation on the binding between asTnC C and asTnI S is weaker, but structural change of the complex depending on Ca 2 + concentration was observed. Together, these findings provide a detailed description of the distinct molecular mechanism of contractile regulation in the scallop adductor muscle from that of vertebrates.
• Different Ca2+-sensitivities between the EF-hands of T- and L-plastins

  Takuya Miyakawa, Hiroto Shinomiya, Fumiaki Yumoto, Yumiko Miyauchi, Hiroyuki Tanaka, Takao Ojima, Yusuke S. Kato, Masaru Tanokura

  Biochemical and Biophysical Research Communications 429 (3-4) 137 - 141 0006-291X 2012/12/14 [Refereed][Not invited]
   

  Plastins are Ca2+-regulated actin-bundling proteins, and essential for developing and stabilizing actin cytoskeletons. T-plastin is expressed in epithelial and mesenchymal cells of solid tissues, whereas L-plastin is expressed in mobile cells such as hemopoietic cell lineages and cancer cells.Using various spectroscopic methods, gel-filtration chromatography, and isothermal titration calorimetry, we here demonstrate that the EF-hand motifs of both T- and L-plastin change their structures in response to Ca2+, but the sensitivity to Ca2+ is lower in T-plastin than in L-plastin. These results suggest that T-plastin is suitable for maintaining static cytoskeletons, whereas L-plastin is suitable for dynamic rearrangement of cytoskeletons. © 2012 Elsevier Inc.
• Purification and biochemical characterization of a cellulase from the digestive organs of the short-spined sea urchin Strongylocentrotus intermedius

  Syuto Hasegawa, Kazuhiro Ura, Hiroyuki Tanaka, Takao Ojima, Yasuaki Takagi

  FISHERIES SCIENCE 78 (5) 1107 - 1115 0919-9268 2012/09 [Refereed][Not invited]
   

  We isolated a cellulase from the digestive organs of the short-spined sea urchin Strogylocentrotus intermedius using a combination of ion-exchange chromatography and gel filtration together with an assay for carboxymethylcellulase activity. The isolated cellulase was stained as a single band by Congo red. The molecular weight of the isolated cellulase, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, was 59 kDa. The isolated cellulase exhibited hydrolytic activity toward carboxymethyl cellulose, with an optimum temperature and pH of 30 A degrees C and pH 8.0, respectively. The thermal stability of the enzyme was characterized by determining the temperature at which activity decreased by 50 % with treatment for 30 min at pH 7.0 and found to be 32 A degrees C. Cellulase activity remained at a high level at 5-20 A degrees C, which is the growth temperature of the short-spined sea urchin. These results confirm that the short-spined sea urchin should preferably be reared at a water temperature of < 20 A degrees C.
• cDNA cloning of an alginate lyase from a marine gastropod Aplysia kurodai and assessment of catalytically important residues of this enzyme

  Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima

  BIOCHIMIE 93 (10) 1720 - 1730 0300-9084 2011/10 [Refereed][Not invited]
   

  Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5'- and 3'-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed similar to 40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg: however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1. (C) 2011 Elsevier Masson SAS. All rights reserved.
• cDNA cloning and bacterial expression of an endo-beta-1,4-mannanase, AkMan, from Aplysia kurodai

  Umme Afsari Zahura, Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 159 (4) 227 - 235 1096-4959 2011/08 [Refereed][Not invited]
   

  Previously we isolated an endo-beta-1,4-mannanase (EC 3.2.1.78), AkMan, from the digestive fluid of a common sea hare Aplysia kurodai and demonstrated that this enzyme had a broad pH optimum spanning 4.0 to 7.5 and an appreciably high heat stability in this pH range (Zahura et al., Comp. Biochem. Physiol., B157, 137-148 (2010)). In the present study, we cloned the cDNA encoding AkMan and constructed a bacterial expression system for this enzyme to enrich information about the primary structure and the characteristic properties of this enzyme. cDNA fragments encoding AkMan were amplified by PCR followed by 5'- and 3'-RACE PCRs from the A. kurodai hepatopancreas cDNA using degenerated primers designed on the basis of partial amino-acid sequences of AkMan. The cDNA including entire translational region of AkMan consisted of 1392 bp and encoded 369 amino-acid residues. The N-terminal region of 17 residues of the deduced sequence except for the initiation Met was regarded as the signal peptide of AkMan and the mature enzyme region was considered to comprise 351 residues with a calculated molecular mass of 39961.96 Da. Comparison of the primary structure of AkMan with other beta-1,4-mannanases indicated that AkMan belongs to the subfamily 10 of glycosyl-hydrolase-family-5 (GHF5). Phylogenetic analysis for the GHF5 beta-1,4-mannanases indicated that AkMan together with other molluscan beta-1,4-mannanases formed an independent clade of the subfamily 10 in the phylogenetic tree. The recombinant AkMan (recAkMan) was expressed with an Escherichia coli BL21 (DE3)-pCold1 expression system as an N-terminal hexahistidine-tagged protein and purified by Ni-NTA affinity chromatography. The recAkMan showed the broad pH optimum in acidic pH range as did native AkMan; however, heat stability of recAkMan was considerably lower than that of native enzyme. This may indicate that the stability of AkMan is derived from an appropriate folding and/or some posttranslational modifications in Aplysia cells. (C) 2011 Elsevier Inc. All rights reserved.
• Proteomic analysis of inviable salmonid hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss during early embryogenesis

  L. Zheng, H. Tanaka, S. Abe

  JOURNAL OF FISH BIOLOGY 78 (5) 1508 - 1528 0022-1112 2011/05 [Refereed][Not invited]
   

  Early embryos of inviable hybrids between female masu salmon Oncorhynchus masou masou and male rainbow trout Oncorhynchus mykiss at 9, 12, 15 and 20 days after fertilization were examined for protein expression profiles. A total of 44 proteins, mostly down-regulated products of house-keeping genes and those involved in nucleic acid metabolism or chromatin replication, were identified in hybrid embryos by mass spectrometry analysis and protein database searching. The identified down-regulated proteins may be responsible for the inviability in the hybrids. (C) 2011 The Authors Journal of Fish Biology (C) 2011 The Fisheries Society of the British Isles
• Isolation and characterization of two alginate lyase isozymes, AkAly28 and AkAly33, from the common sea hare Aplysia kurodai

  Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 157 (4) 317 - 325 1096-4959 2010/12 [Refereed][Not invited]
   

  Two alginate lyase isozymes, AkAly28 and AkAly33, with approximate molecular masses of 28 and 33 kDa, respectively, were isolated from the digestive fluid of the common sea hare, Aplysia kurodai. Both of AkAly28 and AkAly33 were regarded as the endolytic polymannuronate (poly(M)) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate producing unsaturated tri- and disaccharides and rapidly decreased the viscosity of sodium alginate solution in the initial phase of degradation. Optimal pH and temperature of the two enzymes were similarly observed at pH 6.7 and 40 degrees C, respectively. Temperature that caused a half inactivation of the two enzymes during 20-min incubation was also similar to each other, i.e., 38 degrees C. However, NaCl requirement and activity toward oligosaccharide substrates of the two enzymes were significantly different from each other. Namely, AkAly28 showed practically no activity in the absence of NaCl and the maximal activity at NaCl concentrations higher than 0.2 M, whereas AkAly33 showed similar to 20% of maximal activity despite the absence of NaCl and the maximal activity at around 0.1 M NaCl. AkAly28 hardly degraded oligosaccharides smaller than tetrasaccharide, while AkAly33 could degrade oligosaccharides larger than disaccharide producing disaccharide and 2-keto-3-deoxy-gluconaldehyde (an open chain form of unsaturated monosaccharide). Analysis of the N-terminal and internal amino-acid sequences of AkAly28 and AkAly33 indicated that both of the two enzymes belong to polysaccharide lyase family 14. (C) 2010 Elsevier Inc. All rights reserved.
• An endo-beta-1,4-mannanase, AkMan, from the common sea hare Aplysia kurodai

  Umme Afsari Zahura, Mohammad Matiur Rahman, Akira Inoue, Hiroyuki Tanaka, Takao Ojima

  COMPARATIVE BIOCHEMISTRY AND PHYSIOLOGY B-BIOCHEMISTRY & MOLECULAR BIOLOGY 157 (1) 137 - 143 1096-4959 2010/09 [Refereed][Not invited]
   

  A mannan-degrading enzyme was isolated from the digestive fluid of the common sea hare Aplysia kurodai by ammonium sulfate fractionation followed by conventional column chromatography. The purified enzyme, named AkMan in the present paper, showed a single band with an approximate molecular mass of 40,000 Da on SDS-PAGE and preferably degraded a linear beta-1,4-mannan from green algae Codium fragile producing tri-and disaccharides. The optimal temperature of AkMan was 55 degrees C at pH 7.0 and temperature that caused 50% inactivation of AkMan during a 20-min incubation was 52 degrees C. AkMan retained high activity at pH 4.0-7.5 and was not inactivated in such acidic pH range by the incubation at 40 degrees C for 20 min. AkMan could degrade glucomannan from konjak root and galactomannan (tara gum and guar gum) as well as the linear beta-1,4-mannan, while not carboxymethyl cellulose, agarose, dextran and xylan. These results indicate that AkMan is a typical endo-beta-1,4-mannanase (EC 3.2.1.78) splitting internal beta-1,4-mannosyl linkages of mannan. The N-terminal and internal amino-acid sequences of AkMan shared similar to 55% amino-acid identity to the corresponding sequences of an abalone beta-1,4-mannanase, HdMan, which belongs to glycosyl hydrolase family 5 (GHF5). Thus, AkMan was also regarded as a member of GHF5 beta-1,4-mannanases. (C) 2010 Elsevier Inc. All rights reserved.
• Protein kinase A-dependent modulation of Ca2+ sensitivity in cardiac and fast skeletal muscles after reconstitution with cardiac troponin

  Douchi Matsuba, Takako Terui, Jin O-Uchi, Hiroyuki Tanaka, Takao Ojima, Iwao Ohtsuki, Shin&apos, ichi Ishiwata, Satoshi Kurihara, Norio Fukuda

  JOURNAL OF GENERAL PHYSIOLOGY 133 (6) 571 - 581 0022-1295 2009/06 [Refereed][Not invited]
   

  Protein kinase A (PKA)-dependent phosphorylation of troponin (Tn)I represents a major physiological mechanism during beta-adrenergic stimulation in myocardium for the reduction of myofibrillar Ca2+ sensitivity via weakening of the interaction with TnC. By taking advantage of thin filament reconstitution, we directly investigated whether or not PKA-dependent phosphorylation of cardiac TnI (cTnI) decreases Ca2+ sensitivity in different types of muscle: cardiac (porcine ventricular) and fast skeletal (rabbit psoas) muscles. PKA enhanced phosphorylation of cTnI at Ser23/24 in skinned cardiac muscle and decreased Ca2+ sensitivity, of which the effects were confirmed after reconstitution with the cardiac Tn complex (cTn) or the hybrid Tn complex (designated as PCRF; fast skeletal TnT with cTnI and cTnC). Reconstitution of cardiac muscle with the fast skeletal Tn complex (sTn) not only increased Ca2+ sensitivity, but also abolished the Ca2+-desensitizing effect of PKA, supporting the view that the phosphorylation of cTnI, but not that of other myofibrillar proteins, such as myosin-binding protein C, primarily underlies the PKA-induced Ca2+ desensitization in cardiac muscle. Reconstitution of fast skeletal muscle with cTn decreased Ca2+ sensitivity, and PKA further decreased Ca2+ sensitivity, which was almost completely restored to the original level upon subsequent reconstitution with sTn. The essentially same result was obtained when fast skeletal muscle was reconstituted with PCRF. It is therefore suggested that the PKA-dependent phosphorylation or dephosphorylation of cTnI universally modulates Ca2+ sensitivity associated with cTnC in the striated muscle sarcomere, independent of the TnT isoform.
• Comparative study on general properties of alginate lyases from some marine gastropod mollusks

  Mami Hata, Yuya Kumagai, Mohammad Matiur Rahman, Satoru Chiba, Hiroyuki Tanaka, Akira Inoue, Takao Ojima

  FISHERIES SCIENCE 75 (3) 755 - 763 0919-9268 2009/05 [Refereed][Not invited]
   

  Alginate lyase (EC 4.2.2.3) is an enzyme that splits glycosyl linkages of alginate chain via beta-elimination, producing unsaturated oligoalginates. This enzyme is widely distributed in herbivorous marine mollusks, brown algae, and marine and soil bacteria. In the present study, we determined the general properties and partial amino acid sequences of alginate lyases from three Archeogastropoda, i.e., Haliotis discus hannai, H. iris, and Omphalius rusticus, and one Mesogastropoda, i.e., Littorina brevicula, in order to enrich the information about functional and structural diversity in gastropod alginate lyases. The alginate lyases were extracted from hepatopancreas of these animals and purified by ammonium sulfate fractionation followed by conventional column chromatography. Single alginate lyases with molecular masses of approximately 28, 34, and 34 kDa were isolated from H. discus, H. iris, and O. rusticus, respectively. While three alginate lyases with molecular masses of 35, 32, and 28 kDa were isolated from L. brevicula. These enzymes were identified as poly(M) lyase (EC 4.2.2.3) since they preferably degraded poly(M)-rich substrate. Western blot analysis using an antiserum raised against H. discus enzyme suggested that H. iris, and O. rusticus enzymes shared similar primary/higher-order structure with H. discus enzyme, but the L. brevicula enzymes did not. H. discus, H. iris, and O. rusticus enzymes were classified to polysaccharide-lyase family-14 by the analysis of partial amino acid sequences, while the L. brevicula enzymes were not.
• Preparation of beta-1,3-glucanase from scallop mid-gut gland drips and its use for production of novel heterooligosaccharides

  Yuya Kumagai, Akira Inoue, Hiroyuki Tanaka, Takao Ojima

  FISHERIES SCIENCE 74 (5) 1127 - 1136 0919-9268 2008/10 [Refereed][Not invited]
   

  The mid-gut gland of scallop Patinopecten yessoensis has been discarded in scallop processing factories as a fishery waste and various attempts have been made to turn the waste into valuable resources. In the present study, we tried to use mid-gut gland drips from scallop as a source of beta-1,3-glucanase. The mid-gut gland drips were collected in a local fishery factory in Yubetsu-cho, Hokkaido Prefecture. beta-1,3-Glucanase was purified from the mid-gut gland drips by ammonium sulfate fractionation followed by successive chromatography on Toyopearl Phenyl-650M and Toyopearl DEAE-650M. The scallop beta-1,3-glucanase, named PyLam38 in the present study, showed a molecular mass of approximately 38 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and hydrolyzed laminarin, a beta-1,3-glucan from Laminaria sp., producing laminaribiose and glucose with an optimal pH and temperature of 6.0 and 45 degrees C, respectively. PyLam38 exhibited high transglycosylation activity toward various accepter substrates such as monosaccharides, alcohols and xylooligosaccharides. Thus, PyLam38 was found to be useful for the production of various novel heterooligosaccharides consisting of laminarioligosaccharides and various accepters.
• Structure-function relationships of molluscan troponin T revealed by limited proteolysis

  Hiroyuki Tanaka, Hisoka Suzuki, Iwao Ohtsuki, Takao Ojima

  BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 1784 (7-8) 1037 - 1042 1570-9639 2008/07 [Refereed][Not invited]
   

  Molluscan troponin regulates muscle contraction through a novel Ca2+-dependent activating mechanism associated with Ca2+-binding to the C-terminal domain of troponin C. To elucidate the further details of this regulation, we performed limited chymotryptic digestion of the troponin complex from akazara scallop striated muscle. The results indicated that troponin T is very susceptible to the protease, compared to troponin C or troponin 1. The cleavage occurred at the C-terminal extension, producing an N-terminal 33-kDa fragment and a C-terminal 6-kDa fragment. This extension is conserved in various invertebrate troponin T proteins, but not in vertebrate troponin T. A ternary complex composed of the 33-kDa fragment of troponin T, troponin 1, and troponin C could be separated from the 6-kDa troponin T fragment by gel filtration. This complex did not show any Ca2+-dependent activation of the Mg-ATPase activity of rabbit-actomyosin-scallop-tropomyosin. In addition, the actin-tropomyosin-binding affinity of this complex was significantly decreased with increasing Ca2+ concentration. These results indicate that the C-terminal extension of molluscan troponin T plays a role in anchoring the troponin complex to actin-tropomyosin filaments and is essential for regulation. (C) 2008 Elsevier B.V. All rights reserved.
• Spectroscopic and ITC study of the conformational change upon Ca2+-binding in TnC C-lobe and TnI peptide complex from Akazara scallop striated muscle

  Fumiaki Yumoto, Hiroyuki Tanaka, Koji Nagata, Yumiko Miyauchi, Takuya Miyakawa, Takao Ojima, Masaru Tanokura

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 369 (1) 109 - 114 0006-291X 2008/04 [Refereed][Not invited]
   

  Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca2+ ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca2+-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC Globe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify the Ca2+-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca2+ -binding properties of the complex of TnC Globe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca2+-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the Globe of scallop TnC. We therefore assumed that the effect of Ca2+-binding must lead to a change in the interaction mode between the Globe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca2+ signal to TnI in Tit ternary complex. (c) 2007 Elsevier Inc. All rights reserved.
• Isolation and primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus

  Yukiko Nishida, Ken-ichi Suzuki, Yuya Kumagai, Hiroyuki Tanaka, Akira Inoue, Takao Ojima

  BIOCHIMIE 89 (8) 1002 - 1011 0300-9084 2007/08 [Refereed][Not invited]
   

  Glycoside-hydrolase-family 9 (GHF9) cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54 kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal cellulases. (c) 2007 Elsevier Masson SAS. All rights reserved.
• Crystallization and preliminary X-ray analysis of the Ca2+-bound C-terminal lobe of troponin C in complex with a troponin I-derived peptide fragment from Akazara scallop

  Fumiaki Yumoto, Koji Nagata, Yumiko Miyauchi, Takao Ojima, Hiroyuki Tanaka, Kiyoyoshi Nishita, Iwao Ohtsuki, Masaru Tanokura

  ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY AND CRYSTALLIZATION COMMUNICATIONS 63 535 - 537 1744-3091 2007/06 [Refereed][Not invited]
   

  Troponin C (TnC) is the Ca2+-binding component of troponin and triggers muscle contraction. TnC of the invertebrate Akazara scallop can bind only one Ca2+ at the C-terminal EF-hand motif. Recombinant TnC was expressed in Escherichia coli, purified, complexed with a 24-residue synthetic peptide derived from scallop troponin I (TnI) and crystallized. The crystals diffracted X-rays to 1.80 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 32.1, b = 42.2, c = 60.0 angstrom. The asymmetric unit was assumed to contain one molecular complex of the Akazara scallop TnC C-lobe and TnI fragment, with a Matthews coefficient of 1.83 angstrom(3) Da(-1) and a solvent content of 33.0%.
• Drastic Ca2+ sensitization of myofilament associated with a small structural change in troponin I in inherited restrictive cardiomyopathy

  F Yumoto, QW Lu, S Morimoto, H Tanaka, N Kono, K Nagata, T Qjima, F Takahashi-Yanaga, Y Miwa, T Sasaguri, K Nishita, M Tanokura, Ohtsuki, I

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 338 (3) 1519 - 1526 0006-291X 2005/12 [Refereed][Not invited]
   

  bacterially expressed and purified these human cTnI mutants and examined their functional and structural consequences. Inserting the human cTnI into skinned cardiac muscle fibers showed that these mutations had much greater Ca2+ sensitizing effects on force generation than the cTnI mutations in hypertrophic cardiomyopathy (HCM). The mutation K178E in the second actin-tropomyosin (Tm) binding region showed a particularly potent Ca2+-sensitizing effect among the six RCM-causing mutations nuclear magnetic resonance spectroscopy revealed that this Mutation does not extensively affect the Structure of the whole cTnI molecule, but induces all unexpectedly subtle change in the structure of a region around the Mutated residue. The results indicate that the K178E mutation has a localized effect on a structure that is critical to the regulatory function of the second actin-Tin binding region of cTnI. The present study also Suggests that both HCM and RCM involving cTnI Mutations share a common feature of increased Ca2+ sensitivity of cardiac myofilament, but more severe change in Ca2+ sensitivity is associated with the clinical phenotype of RCM. (c) 2005 Elsevier Inc. All rights reserved.
• Comparative studies on the functional roles of N- and C-terminal regions of molluskan and vertebrate troponin-I

  Hiroyuki Tanaka, Yuhei Takeya, Teppei Doi, Fumiaki Yumoto, Masaru Tanokura, Iwao Ohtsuki, Kiyoyoshi Nishita, Takao Ojima

  FEBS Journal 272 (17) 4475 - 4486 1742-464X 2005/09 [Refereed][Not invited]
   

  Vertebrate troponin regulates muscle contraction through alternative binding of the C-terminal region of the inhibitory subunit, troponin-I (TnI), to actin or troponin-C (TnC) in a Ca(2+)-dependent manner. To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI. The C-terminal fragment of Akazara scallop TnI (ATnI(232-292)), which contains the inhibitory region (residues 104-115 of rabbit TnI) and the regulatory TnC-binding site (residues 116-131), bound actin-tropomyosin and inhibited actomyosin-tropomyosin Mg-ATPase. However, it did not interact with TnC, even in the presence of Ca(2+). These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On the other hand, ATnI(130-252), which contains the structural TnC-binding site (residues 1-30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC. Moreover, the ternary complex consisting of this fragment, troponin-T, and TnC activated the ATPase in a Ca(2+)-dependent manner almost as effectively as intact Akazara scallop troponin. Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC-binding site to the inhibitory region of TnI. Together with the observation that corresponding rabbit TnI-fragment (RTnI(1-116)) shows similar activating effects, these findings suggest the importance of the TnI N-terminal region not only for maintaining the structural integrity of troponin complex but also for Ca(2+)-dependent activation.
• Functional importance of Ca2+-deficient N-terminal lobe of molluscan troponin C in troponin regulation

  T Doi, A Satoh, H Tanaka, A Inoue, F Yumoto, M Tanokura, Obtsuki, I, K Nishita, T Ojima

  ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 436 (1) 83 - 90 0003-9861 2005/04 [Refereed][Not invited]
   

  Ca2+-binding sites I and II in the N-terminal lobe of Molluscan troponin C (TnC) have lost the ability to bind Ca2+ due to substitutions of the amino acid residues responsible for Ca2+ liganding. To evaluate the functional importance of the Ca2+-deficient N-terminal lobe in the Ca2+-regulatory function of Molluscan troponin, we constructed chimeric TnCs comprising the N-terminal lobes from rabbit fast muscle and squid mantle muscle TnCs and the C-terminal lobe from akazara scallop TnC, TnC(RA), and TnC(SA), respectively. We characterized their biochemical properties as compared with those of akazara scallop wild-type TnC (TnC(AA)). According to equilibrium dialysis using Ca-45(2+), TnC(RA), and TnC(SA) bound stoichiometrically 3 mol Ca2+/mol and 1 mol Ca2+/Mol, respectively, as expected from their primary structures. All the chimeric TnCs exhibited difference-UV-absorption spectra at around 280-290 nm upon Ca2+ binding and formed stable complexes with akazara scallop troponin I, even in the presence of 6 M urea, if Ca2+ was present. However, when the troponin complexes were constructed from chimeric TnCs and akazara scallop troponin T and troponin 1, they showed different Ca2+-regulation abilities from each other depending on the TnC species. Thus, the troponin containing TnC(SA) conferred as high a Ca2+ sensitivity to Mg-ATPase activity of rabbit actomyosin-akazara scallop tropomyosin as did the troponin containing TnC(AA), whereas the troponin containing TnC(RA) conferred virtually no Ca2+ sensitivity. Our findings indicate that the N-terminal lobe of Molluscan TnC plays important roles in molluscan troponin regulation, despite its inability to bind Ca2+. (c) 2005 Elsevier Inc. All rights reserved.
• Cloning and sequencing of cDNAs encoding walleye pollack alpha-skeletal actin isoforms

  Hiroyuki TANAKA, Yuichi MAEZAWA, Takao OJIMA, Kiyoyoshi NISHITA

  Fisheries Science Japanese Society of Fisheries Science 70 (1) 198 - 200 0919-9268 2004/02 [Refereed][Not invited]
  
• Amino acid sequence of phospholipase A(2) from the pyloric ceca of starfish Asterina pectinifera

  H Kishimura, T Ojima, H Tanaka, K Hayashi, K Nishita

  FISHERIES SCIENCE 66 (1) 104 - 109 0919-9268 2000/02 [Refereed][Not invited]
   

  The complete amino acid sequence of phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera was determined by automated Edman degradation. The A. pectinifera PLA(2) (APLA(2)) consists of 137 amino acids with an unblocked N-terminus and its molecular weight is calculated to be 15 300.1. The enzyme contains 14 cysteine (Cys) residues at the corresponding positions of the same residues which have been shown to be involved in intramolecular disulfide bonds in mammalian pancreatic PLA(2). The region involving an active site and a Ca2+-binding loop shows fairly high sequence homology (75%) between the APLA(2) and porcine pancreatic PLA(2). The APLA(2) conserved the amino acid sequence of the loop portion of the porcine pancreatic PLA(2) except for the deletion of two amino acids. These features indicate that the APLA(2) can be classified into the group 1 type PLA(2). In contrast, the homology between the APLA(2) and porcine pancreatic PLA(2) was calculated to be 47% in the whole region. Further, the insertion of sixteen residues and the deletion of three residues were required in the sequence of the APLA(2) to align the corresponding region to the beta-wing of porcine pancreatic PLA(2). These differences in amino acid sequence of the APLA(2) may account for its specific properties such as the higher activity and the characteristic substrate specificity.
• Amino Acid Sequence of Troponin-I from Akazara Scallop Striated Adductor Muscle

  H. Tanaka, T. Ojima, K. Nishita

  Journal of Biochemistry Japanese Biochemical Society 124 (2) 304 - 310 0021-924X 1998/08/01 [Refereed][Not invited]
   

  The complete amino acid sequence of Akazara scallop, Chlamys nipponensis akazara, troponin-I was determined by automated Edman degradation. It is composed of 292 amino acid residues with a blocked N-terminus. The Mr is calculated to be 34,678, about 14,000 larger than that of vertebrate skeletal troponin-I but significantly smaller than the 52,000 that had been estimated by SDS-polyacrylamide gel electrophoresis. The homologous sequence to vertebrate and arthropoda troponin-Is is found in the C-terminal region. In particular, the sequence of the regions essential for binding to actin and troponin-C is highly conserved. On the other hand, Akazara scallop troponin-I has 100-133 extra residues at the N-terminus compared with vertebrate troponin-I. This extra region is rich in Glu and Arg and has a unique sequence, that shows in part a high sequence homology with the tropomyosin-binding site of troponin-T and caldesmon.
• Amino Acid Sequence of C-Terminal 17 kDa CNBr-Fragment of Akazara Scallop Troponin-I

  Takao Ojima, Hiroyuki Tanaka, Kiyoyoshi Nishita

  The Journal of Biochemistry 117 (1) 158 - 162 0021-924X 1995/01 [Refereed]
  
• Cloning and Sequence of a cDNA Encoding Akazara Scallop Troponin C

  T. Ojima, H. Tanaka, K. Nishita

  Archives of Biochemistry and Biophysics 311 (2) 272 - 276 0003-9861 1994/06 [Refereed][Not invited]
  
• Amino acid sequence of troponin C from scallop striated adductor muscle.

  K. Nishita, H. Tanaka, T. Ojima

  Journal of Biological Chemistry 269 (5) 3464 - 3468 0021-9258 1994/02 [Refereed][Not invited]
   

  Troponin and its Ca(2+)-binding subunit troponin C (TnC) of the Ezo-giant scallop, Patinopecten yessoensis, have been revealed to bind only 1 mol of Ca2+/mol irrespective of the presence and absence of Mg2+. The amino acid sequence of the TnC has been determined by the automated Edman degradation. TnC is composed of 152 residues including 3 tryptophans at positions 53, 70, and 109, 4 cysteines at positions 19, 31, 67, and 145, and no proline. The molecular weight is calculated as 17,410. The NH2 terminus of TnC is blocked by an acetyl group. The sequence of scallop TnC required deletion of three residues in the D/E linker region to maximize sequence homology to other TnCs and shows considerably lower homology to vertebrate skeletal TnCs (27-30%), ascidian TnC (26%), arthropoda TnCs (30-37%), and chicken calmodulin (37%). Further, we conclude that Ezo-giant scallop TnC binds Ca2+ at site IV, a site specific for Ca2+. The other sites I, II, and III appeared to lose the Ca2+ binding ability due to substitutions of some important residues.
• Purification and Characterization of a Novel 160 kDa Actin-Binding Protein from Surf Clam Foot Muscle.

  Satoru Chiba, Takao Ojima, Hiroyuki Tanaka, Kiyoyoshi Nishita

  NIPPON SUISAN GAKKAISHI 日本水産學會 59 (10) 1783 - 1791 0021-5392 1993 [Refereed]
   

  A 160kDa actin-binding protein was isolated from surf clam foot muscle by DEAE-Toyopearl column chromatography. The protein was found to complex readily with F-actin to form turbidity. The turbidity reached a maximum when bound in a ratio of approx. 0.3:1 (w/w). The binding ratio as well as turbidity decreased steeply as the KCl increased concentration up to 80-100mM. In these events, F-actin filaments were aggregated side by side and formed complicated networks, whose morphological features revealed close similarity to those reported for F-actin bundles formed by caldesmon. However, the binding features of the 160kDa protein to F-actin are different from those with caldesmon in the presence of Ca²⁺-calmodulin. Besides, further comparison of molecular weights, amino acid compositions, and α-helix contents revealed that the 160kDa protein is different from caldesmon, α-actinin, and several other actin-binding proteins. Thus, we consider that the 160kDa protein is a novel actin-binding protein.
• Cyanogen Bromide Fragments of Akazara Scallop Mr 52,000 Troponin-I1

  Takao Ojima, Hiroyuki Tanaka, Kiyoyoshi Nishita

  The Journal of Biochemistry The Japanese Biochemical Society 108 (4) 519 - 521 0021-924X 1990/10 [Refereed][Not invited]
   

  Akazara scallop troponin-I of Mr 52,000 (52K) was cleaved into two fragments of 17K and 35K with cyanogen bromide. The 17K fragment, along with tropomyosin, inhibited weakly the rabbit actomyosin Mg-ATPase activity, however, the 35K fragment did not affect it at all. In the presence of Akazara scallop TnT (40K component), the 17K fragment, in turn, strongly inhibited the activity, while the 35K fragment did not. The amlno acid composition and partial amino acid sequence suggested that the 17K and 35K fragments were derived from C- and N-terminal regions of the TnI, respectively, and that structural similarity to TnIs from other animals is present in the 17K region.

MISC

• Comparative study on general properties of alginate lyases from some marine gastropod mollusks

  RAHMAN Mohammad Matiur  日本水産學會誌 = Bulletin of the Japanese Society of Scientific Fisheries  76-  (4)  580  -580  2010/07/15  [Not refereed][Not invited]
  
• エゾアワビ肝膵臓におけるアルギン酸代謝に関する研究

  木本雄太, 熊谷祐也, 澤辺智雄, 田中啓之, 井上晶, 尾島孝男  日本水産学会大会講演要旨集  2010-  2010
• 1P149 Regulatory roles of inhibitory region of Akazara scallop troponin-I

  Tanaka H, Ohtsuki I, Ojima T  Biophysics  45-  (1)  S69  2005/10/19  [Not refereed][Not invited]
  
• 2P149 Functional analysis of NH_2- and COOH- terminal regions of Akazara scallop troponin-T

  Suzuki H, Tanaka H, Ohtsuki I, Ojima T  Biophysics  45-  (1)  S157  2005/10/19  [Not refereed][Not invited]
  

Association Memberships

• THE ZOOLOGICAL SOCIETY OF JAPAN   日本生化学会   日本水産学会   日本付着生物学会   

Works

• Fish of the month -Jellyfish-

  田中啓之 2022/07 - Today
• 水圏生化学実験（タンパク質）

  田中啓之 2022/06 

  学生実験用教材（全27本の動画）
• クラゲ

  田中啓之 LASBOS Moodle 2020/07

Research Projects

• Investigation for regulatory mechanisms of cnidarian muscle contraction

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2019/04 -2024/03 

  Author : 田中 啓之

   

  ミズクラゲ横紋筋から精製したミオシンの画分に少量含まれていることが確認された新規カルシウムイオン結合タンパク質、AaCBPを認識する抗体を精製した。また、ミズクラゲ横紋筋アクトミオシンの構成成分であるものの、機能が不明なパラミオシン様タンパク質について、C末端断片を大腸菌発現させ、ウサギに免疫して抗血清を作製した。これらの抗体を用いて、ウェスタンブロッティングや免疫染色を行い、これらのタンパク質の発現解析を行った。また、ミズクラゲのポリプや成体の下傘、上傘、口腕など各種の組織・部位からのRT-PCRによる発現解析も行った。それらの結果、これらのタンパク質はいずれも横紋筋を含む組織に特異的に発現しており、横紋筋に特有の形態形成、収縮や収縮の調節等に関与している可能性が示された。特に、AaCBPに関しては、サルコメア構造の中でもミオシンフィラメントの部位に存在することが明らかとなった。また、横紋筋から抽出・精製した天然アクトミオシンには、AaCBPが結合していること、そしてそれは、天然アクトミオシンにキモトリプシンを作用させてミオシンの重鎖が切断されると遊離することも示された。以上の結果から、AaCBPはミオシンに結合して機能するタンパク質であると考えられ、筋細胞への刺激伝達時に細胞内で濃度が上昇するカルシウムイオンを結合して、情報をミオシンに伝達し、アクチン-ミオシン相互作用を促進して筋収縮を引き起こすという、筋収縮調節機構の存在が示唆された。
• Regulatory mechanisms for echinoderm muscle contraction

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2012/04 -2016/03 

  Author : Tanaka Hiroyuki, HADANO Junji, MORITA Takahiro, NOTO Kouki, ZUSHI Hideki, HATTORI Kazuki

   

  Most of the musculature in echinoderms are smooth muscle and the contraction of which is believed to be regulated by the phosphorylation of myosin light chain depending on intracellular Ca2+ concentration as in the case of other smooth muscles in higher animals. In this study, troponin which is known as an actin-linked regulatory protein of striated muscles was revealed to be present in the various smooth muscles of sea urchin. The subunits of sea urchin troponin were obtained by the expression system constructed in Escherichia coli and were confirmed to be functional in vitro, even though they are lacking some regions which are known to be essential for the Ca2+-dependent activation of vertebrate striated muscle contraction. Therefore, the sea urchin troponin should activate the contraction by the acting mechanisms different from those of vertebrate troponin.
• Temperature adaptation and regulatory mechanisms of molluscan muscle contraction

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2008 -2011 

  Author : TANAKA Hiroyuki, OJIMA Takao

   

  We revealed that molluscan troponin-tropomyosin, the regulatory protein of muscle contraction, functions through the novel molecular mechanism that is different from those known for vertebrate troponin-tropomyosin. In addition, we suggested that the novel mechanism is present in arthropods and echinoderms and ubiquitous for invertebrate animals. Furthermore, we revealed that molluscs express various isoforms of the regulatory proteins, the biochemical properties of which are significantly different from each other. We proposed that the presence of these isoforms should be associated with the temperature adaptation of the molluscs and the difference of the locomotor habits of molluscan species.
• Study on the metabolic pathways for seaweed polysaccharides in abalone

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2007 -2010 

  Author : OJIMA Takao, INOUE Akira, TANAKA Hiroyuki, SAWABE Tomoo

   

  Herbivorous marine gastropods like abalone and sea hare possess various polysaccharide-degrading enzymes which can degrade seaweeds' polysaccharides such as alginate, laminaran, mannan, xylane, and cellulose to oligo- and monosaccharides. These saccharides are considered to be metabolized via glycolytic pathway and TCA cycle. In the present study, we isolated several polysaccharide-degrading enzymes, e.g., alginate lyase, laminarinase and mannanase, from abalone and sea hare and investigated their biological roles in the metabolism of seaweeds' polysaccharides in gastropods.
• イガイ類の付着機構に関する生化学的研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2003 -2005 

  Author : 田中 啓之

   

  イガイ類の付着は足から分泌されるタンパク質が硬化して形成される足糸によって行われる。この足糸タンパク質は、生合成から分泌に至るまでの間に細胞内で様々な翻訳後修飾を受けることが知られている。本研究では、翻訳後修飾に関わる酵素や分泌に関与するタンパク質を明らかにするため、足糸タンパク質と相互作用するタンパク質を、酵母Two-Hybrid systemを用いてスクリーニングすることにした。 函館湾においてイガイ類を採取し、まず、既報のPCR法によって種の同定を行った。その結果、採取した10個体はいずれもムラサキイガイであることを確認した。次に、そのうち1個体の足よりmRNAを分離してcDNAの合成を行った後、PCRによって2種類の足糸タンパク質(MGFP1およびMGFP2)をコードするcDNAの増幅を行った。MGFP1-cDNAの増幅産物については、様々なサイズを示したが、5'端側から分析した部分塩基配列はいずれも相同で、既報の配列にほぼ一致した。一方、MGFP2-cDNAの増幅産物についても、様々なサイズのものが得られ、同様に5'端側の相同性は高かったが、3'端側の繰り返し領域は、繰り返し単位の欠損や重複により、少なくとも3つの配列パターンに分類されることがわかった。さらに、これらのアイソフォームは異なる複数の遺伝子にコードされていることが示唆された。これらの結果は、MGFP2の基本構造が種を超えて保存されているとする既報の知見とは異なっていた。また、配列も既報のものとはアミノ酸配列レベルで約85%の相同性を示すに留まり、異なっていることがわかった。さらに、MGFP1およびMGFP2をコードするこれらのcDNAをBaitベクターにサブクローニングする一方、cDNAライブラリーをPreyベクターに構築して酵母Two-Hybrid systemによるスクリーニングの準備をした。
• Studies on primary structures and genes of cellulases from marine invertebrates

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 2003 -2005 

  Author : OJIMA Takao, KISHIMURA Hideki, TANAKA Hiroyuki

   

  Distribution of cellulases in marine invertebrates was investigated by measuring the cellulase activity (CMCase activity) of digestive fluids from various invertebrates. Accordingly, herbivorous invertebrates such as abalone, sea urchin, and scallop were found to possess cellulases, whereas carnivorous invertebrates like starfish and Neptune snail were considered to possess no cellulases. Subsequently, cellulases (endo-1,4-β-glucanases) were isolated from abalone, sea urchin, and scallop by conventional column chromatographies and their cDNA and structural genes were cloned to deduce their primary structure. According to the deduced structures, the abalone, sea urchin, and scallop enzymes were classified to glycoside hydrolase family 9 (GHF9) as in case of termite and crayfish cellulases previously reported. Phylogenetic analysis of cellulase genes indicated that abalone, sea urchin, and scallop cellulases were derived from a common ancestral gene along with arthropod enzymes. In the abalone digestive fluid, an isozyme possessing a family-II carbohydrate-binding module in the N-terminus was found. This enzyme is the first animal cellulase possessing the family-II CBM in the N-terminus of the GHF9-type catalytic domain. Degrading abilities of the invertebrate cellulases toward cello-oligosaccharides were investigated. The abalone cellulase could degrade trisaccharide to disaccharide and glucose ; however, the sea urchin and scallop enzymes could not. Thus smallest substrate was trisaccharide for the abalone enzyme but tetrasaccharide for sea urchin and scallop enzymes. Further, when tetrasaccharide was allowed to react, the abalone enzyme split it into two moles of disaccharide, while the sea urchin and scallop enzymes split into trisaccharide and glucose as well as 2 moles of disaccharide. Therefore, the catalytic mechanism of invertebrate cellulases was considered to vary depending on the animal species although the enzymes all belong to GHF9.
• 軟体動物アルギン酸リアーゼの起源と遺伝子構造に関する研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 2003 -2004 

  Author : 尾島 孝男, 岸村 栄毅, 田中 啓之

   

  本年度は、アワビ・エンド型アルギン酸リアーゼ(HdAly)の構造遺伝子を染色体DNAライブラリーからクローニングし、そのイントロンとエキソンの配置を解析した。構造遺伝子の塩基配列とcDNAの塩基配列とを比較した結果、HdAlyの構造遺伝子は2784bpから成り、2つのイントロンで分断された3つのエキソンから構成されることが明らかになった。イントロンの位置は5'-非翻訳領域内部および分泌シグナルと触媒部位の連結部位にそれぞれ存在していた。なお、HdAlyの触媒部位のコード領域は1つのエキソンから構成されていたが、これはHdAlyの構造遺伝子の起源が比較的新しいことを示すものと考えられる。一方、本年度はエゾアワビ消化液から新たにエキソ型のアルギン酸リアーゼHdAlexを単離した。HdAlexはSDS-PAGEで分子量約36,000の単一バンドを示し、poly(M)-blockを特異的に分解した。また、ポリマンヌロン酸分子の還元末端より2番目のグリコシド結合を切断することが明らかになった。次いでアワビ肝膵臓cDNAライブラリーからHdAlexのcDNAをクローン化し、その塩基配列を分析した。その結果、cDNAの翻訳領域822bpからHdAlex前駆体の273残基のアミノ酸配列が演繹された。さらに、HdAlexは翻訳後修飾により256アミノ酸残基から成る成熟体に変換されることが明らかになった。HdAlexのアミノ酸配列は、アワビのエンド型リアーゼHdAlyと67.2%、サザエのエンド型リアーゼと61.8%、およびクロレラウィルス・アルギン酸リアーゼ(CL2)と34.1%の相同性を示した。これらの結果より、エンド型とエキソ型のアルギン酸リアーゼ遺伝子が共通の祖先遺伝子から分化したことが強く示唆された。
• cDNA Cloning and Amino Acid Sequence of Myosin and Actin of valuable Fishes for Food Industry

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1998 -2001 

  Author : NISHITA Kiyoyoshi, TANAKA Hiroyuki, OJIMA Takao

   

  The cDNA libraries were constructed from dorsal muscle of four species of fishes. In case of salmon, the libraries were also constructed from dark and cardiac muscles. Then, the cDNAs for the myosin heavy chains and actins were cloned and amplified by PCR. Nucleotide sequences were determined by thermal cycle sequencing. Results obtained were as follows; 1. Two species of myosin heavy chain cDNA clones were found in the salmon ordinary muscle. The nucleotide sequences of the both clones were determined and deduced the complete amino acid sequences of 1,937 residues. The sequences show low homology as 71 % and dispersed amino acid replacements. 2. The myosin cDNA clone was prepared from salmon dark muscle and cardiac muscle. From their nucleotide sequences, amino acid sequences of 246 residues were deduced According to the sequence comparison and database search, one of the clones found in the salmon ordinary muscle was concluded to be identical with the dark muscle myosin clone. 3. From the cDNA library of the ordinary muscle of arabesque greenling, two species of cDNA clones encoding ordinary muscle myosin and dark muscle myosin were obtained. Thereby, complete amino acid sequence of 1931 residues of the "dark muscle type" myosin was determined. The sequence of 832 residues of the "dark muscle type" was also determined. 4. As for the mackerel, partial amino acid sequences of 245 residues were determined of the both ordinary and dark muscle type myosins. 5. Three species of actin cDNA clones of salmon, walleye pollack, and arabesque greenling were obtained and determined the nucleotide sequences deducing the complete amino acid sequences of 375 residues. 6. The sequence comparison and discussion on the fish myosins and actins were made from various viewpoints. Two manuscripts were made and submitted.
• 軟体動物トロポニンIのN端伸長領域の存在意義の研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1999 -2000 

  Author : 田中 啓之

   

  1.ホタテガイ類TnIの一次構造と機能の相関の検討 アカザラガイTnIのcDNAを鋳型として種々の領域をPCR増幅し、それらを発現ベクターに組み込んで大腸菌によるTnI断片発現系を構築した。現在、N端伸長領域を含む断片の発現には至っておらず、脊椎動物TnIと相同性を示すC端側の領域に含まれる残基130-292、残基130-252および残基231-292の領域について発現を行った。さらにこれらの領域に相当するウサギTnIの領域についても同様にして発現系を構築した。これらの断片のTnCとの結合能を比較した結果、残基231-292について、ウサギTnIではCa依存的な強い結合が認められるのに対し、アカザラガイTnIでは全く結合しないなどの大きな相異が認められた。従って、アカザラガイTnIの収縮調節における作用機構は脊椎動物TnIのそれとは異なっていることが示唆された。 2.N端伸長領域と相互作用する蛋白質の酵母Two-Hybrid systemによる検索 アカザラガイTnIのN端伸長領域にあたる残基1-129、脊椎動物TnIと相同性を示す残基130-292およびTnC結合部位である残基130-183の領域をコードするDNAを調製し、それらをpHybLex/Zeoベクターに組み込んで酵母EGY48(pSH18-34)を形質転換した。さらにそれらをpYESTrp2ベクターに構築したアカザラガイ横紋筋cDNAライブラリーで形質転換し、Leu非要求性およびLacZの発現を指標として、上述の領域と相互作用する蛋白質を検索した。その結果、残基130-292に関してはTnCおよびTnTが、残基130-183に関してはTnCが相互作用蛋白質として検出されるなど予想通りの結果が得られる一方で、N端伸長領域である残基1-129に関しては相互作用蛋白質を検出するには至らなかった。
• Amino Acid Sequence Analysis of Fish Myosin

  Japan Society for the Promotion of Science：Grants-in-Aid for Scientific Research

  Date (from‐to) : 1995 -1997 

  Author : NISHITA Kiyoyoshi, TANAKA Hiroyuki, OJIMA Takao

   

  We have determined the sequence of 1-1287 amino acids of walleye pollack myosin by cDNA cloning. In this year, the homology between the sequences determined with cDNA and myosin protein, and modified amino acid were investigated. Heavy meromyosin (HMM) was prepared by alpha-chymotryptic digestion of walleye pollack myofibrils and its light chains were removed with urea-acetone. By digestion of HMM with CNBr, lysylendopeptidase, arginylendopeptidase, alpha-chymotrypsin, and trypsin, 6,37,14,6, and 5 peptides, respectively, were obtained. From these peptides, 725 amino acid residues were sequenced by automated Edman method. Accordingly, 355 and 370 residues in S-1 and S-2 regions, respectively, were sequenced. Walleye pollack myosin was found to possess unmodified lysine at the position 35 where the monomethyl lysine exists in chicken myosin and the sequence homology around it is 52-57% to vertebrate myosins. Further, trimethyl lysine was detected at 550 and the homology around it is 71-81%. Moreover, the sequence ^<834>Val-Tyr-Tyr-Lys-Ile-Lys^<839> in the S-2 region waspredicted to be formed beta-sheet structure instead of alpha-helix of carp and rabbit myosins. Existence of two myosin protein isoforms were suggested in adding to two other isoforms of the cDNA clones.
• ホタテガイ類トロポニンの一次構造決定と一次構造・機能相関の研究

  日本学術振興会：科学研究費助成事業

  Date (from‐to) : 1996 -1996 

  Author : 田中 啓之

   

  アカザラガイ横紋閉殻筋トロポニンの一次構造を決定することができた。このトロポニンIはアミノ酸292個からなり、端はアセチル化によってブロックされていた。分子量は34,678と計算され、従来SDS電気泳動法で見積もられてきた52,000に比べ小さいことがわかったが、これはこのトロポニンIがGluに非常に富んだ領域を持ち、電気泳動において異常な移動度を示すためであると推測された。アカザラガイトロポニンIの一次構造を既に一次構造が報告されている他種のトロポニンIのそれと比較すると、C端側の領域(残基134-292)が脊椎動物トロポニンIに対し26-30%、また節足動物トロポニンIに対し39%の相同性を示すことがわかった。特にこのC端側領域の中でも脊椎動物トロポニンIにおいてアクチンやトロポニンCとの結合部位と考えられている部位については他種トロポニンIとの相同性が高かった。一方、N端部分は脊椎動物骨格筋トロポニンIに比べて約130残基、また節足動物トロポニンIに比べて約50から110残基も突出しており、この突出したN端領域(残基1-133)はGluとArgに富んだ特異な配列からなっていた。また、配列データベースの検索により、このN端領域の中でも残基76-115の領域がトロポニンTやカルデスモンのトロポミオシン結合部位と相同性を示すことが検出され、トロポミオシンとの相互作用の機能を担っている可能性が示唆された。そこで、アカザラガイトロポニンIをCNBr消化しN端断片(残基1-129)とC端断片(残基158-292)を調製し、これらのトロポミオシン・アクチン複合体ととの結合性を超遠心共沈法によって検討したが、C端断片がトロポミオシン・アクチン複合体と結合する一方で、N端断片は結合せず、N端領域にトロポミオシンとの相互作用機能が存在することを証明するには至らなかった。
• 水産生物タンパク質の構造、機能および発現の解析
• Structural, functional, and expressional analysis of protein in marine organisms