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Recognition mechanism between insect midgut epithelial cell membrane and insecticidal cry toxins

Research Project

Project/Area Number 13306006
Research Category

Grant-in-Aid for Scientific Research (A)

Allocation TypeSingle-year Grants
Section一般
Research Field 蚕糸・昆虫利用学
Research InstitutionNIIGATA UNIVERSITY

Principal Investigator

HORI Hidetaka  NIIGATA UNIVERSITY, Graduate School Science and Technology, Professor, 大学院・自然科学研究科, 教授 (00293241)

Co-Investigator(Kenkyū-buntansha) MITUI Toshiaki  NIIGATA UNIVERSITY, Department of Agriculture, Professor, 農学部, 教授 (70183960)
SATO Ryoichi  Tokyo University of Agriculture and Technology, Graduate School of Bio-Application and Systems Engineering, Associate Professor, 大学院・生物システム応用科学研究科, 助教授 (30235428)
HAYAKAWA Tohru  NIIGATA UNIVERSITY, Graduate School Science and Technology, Assistant, 大学院・自然科学研究科, 助手 (30313555)
Project Period (FY) 2001 – 2003
Project Status Completed (Fiscal Year 2003)
Budget Amount *help
¥35,360,000 (Direct Cost: ¥27,200,000、Indirect Cost: ¥8,160,000)
Fiscal Year 2003: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000)
Fiscal Year 2002: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000)
Fiscal Year 2001: ¥23,530,000 (Direct Cost: ¥18,100,000、Indirect Cost: ¥5,430,000)
KeywordsInsect midgut epithelial cells / silkworm Bacillus thuringiensis / insecticidal toxin / novel binding protein / dissociation constant / liposome / aminopeptidase / アミノペプチデース / Bombyx mori / 中腸上皮細胞 / アピカル膜 / アミノペブチデース / カドヘリン様蛋白 / 230kDa蛋白 / 90kDa蛋白 / バチラスチューリンゲンシス / 殺虫タンパク / Cry1Aa
Research Abstract

Interaction between insecticidal Cry toxins and apical cell membrane from the silkworm, Bombyx mori, midgut was investigated.
Four main results were obtained. 1)Novel P252 which bound Cry1Aa, Cry1Ab and Cry1Ac was discovered from brush border membrane. 2)Novel aminopeptidase, AP, which bound only Cry1Ac was discovered. This novel AP was thought to belong to group 1. 3)Liposomes containing BBM proteins were constructed and shown to have twice higher affinity with Cry1Aa, Cry1Ab and Cry1Ac compared to the liposome without the proteins. 4)Cry1A toxins have been thought to insert their α-helices in domain 1. But nothing is clear about which helices inserted into the membrane. We determined the portion of domain 1 which was inserted into apical cell membrane. About 24 kDa peptides has been thought to be inserted and it was shown to correspond to the stretch from α-helix 2 to α-helix 7. This was first concrete suggestion about the insertion region of domain 1.
P252 was solubilized with Triton … More X-100 or CHAPS and thus it was suggested to be localized in non raft region. P252 was thought to be a glycoprotein with homo tetramer. The sugar side chain was suggested to be tri antennal N-linked chain containing bisecting GlcNAc and O-linked sugar side chain with GalNAc. Value of kd of P252 with Cry1Aa, Cry1Ab and Cry1Ac were 10nM, 70nM and 20nM, respectively and which were thought to be one of the strongest bin ding in references. Heterologous binding assay suggested that the site of P252 for Cry1Aa shared by 50% with Cry1Ac and it for Cry1Ac shared by 80% with Cry1Aa. The value of kd of P252 with Cry1Ab was 70 nM and the value still low enough to thought that the binding was physiological event. But interestingly, the site for Cry1Ab was shared with Cry1Aa and Cry1Ab by 50% each and BSA also was shown to inhibit the binding.
P96 was recognized with antibody of APN 1. P96 was shown to bind only Cry1Ac among three Cry1A toxins and the binding was inhibited with GalNAc. Substrate specificity was surveyed and P96 was suggested to be APN. The reaction required divalent metal cations and optimum pH was 6-8. Reaction speed was enhanced at higher substrate concentration. Less

Report

(4 results)
  • 2003 Annual Research Report   Final Research Report Summary
  • 2002 Annual Research Report
  • 2001 Annual Research Report
  • Research Products

    (31 results)

All Other

All Publications (31 results)

  • [Publications] M.D.Hossain, et al.: "Characterization of a novel plasma membrane protein expressed in the midgut epithelia of Bombyx mori that binds to Cry1A toxins."Appl Environ Microbiol.(米国微生物学会誌). 印刷中(In press). (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] D.Takano, et al.: "Differential extraction of the proteins localizing on cell membrane of midgut from Bombyx mori with Triton X-114."Bulletin of the Facultuy of Agriculture, Niigata University.. 55(2). 133-141 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] D.Takano, et al.: "Activity of phophatidylinositol/UDP-GlcNAc : GlcNAc transferase in midgut tissue of Bombyx mori."Bulletin of the Facultuy of Agriculture, Niigata University.. 55(2). 123-131 (2003)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] N.S.Kumaraswami, et al.: "Lipid of brush border membrane vesicle (BBMV) from Plutella xylostella resistant and susceptible to Cry1Ac δ-endotoxin of Bacillus thuringiensis"Comp.Biochem.Physiol.. 129(1). 173-183 (2001)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] D.M.Hossain et al.: "A novel 252kDa protein from BBMV of Bombyx mori, which bound with Cry1A toxins but did not bind with anti-APN or BtR175 antibodies."Proceeding of 5th Pacific rim conference on the biotechnology of Bacillus thuringiensis and its environmental impact. In press. (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Egawa et al.: "Allergenic assessment of Cry1Aa using rat digestive juices and mast cells."Proceeding of 5th Pacific rim conference on the biotechnology of Bacillus thuringiensis and its environmental impact. In press. (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Hayakawa, et al.: "Novel binding proteins for Cry1A of Bacillus thuringiensis toxins and complicated killing mechanism."Proceeding of 5th Pacific rim conference on the biotechnology of Bacillus thuringiensis and its environmental impact. In press. (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Shitomi, et al.: "Characterization of Bombyx moriAP96 which binds with Cry1Ac."Proceeding of 5th Pacific rim conference on the biotechnology of Bacillus thuringiensis and its environmental impact. In press. (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] K.Tomimoto, et al.: "Detection of membrane-inserted region of Cry1Aa of Bacillus thuringiensis."Proceeding of 5th Pacific rim conference on the biotechnology of Bacillus thuringiensis and its environmental impact. In press. (2004)

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] M.D.Hossain, Y.Shitomi, K.Moriyama, M.Higuchi, T.Hayakawa, T.Mitsui, R.Sato, H.Hori: "Characterization of a novel plasma membrane protein expressed in the midgut epithelia of Bombyx mori that binds to Cry1A toxins."Appl.Environ.Microbiol.. (in press). (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] D.Takano, T.Kishimoto, T.Hayakawa, M.Higuchi, Y.Shitomi, T.Mitsui, H.Hori: "Differential extraction of the proteins localizing on cell membrane of midgut from Bombyx mori with Triton X-114."Bulletin of the Faculty of Agriculture, Niigata University.. 55(2). 133-141 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] D.Takano, T.Kishimoto, T.Hayakawa, T.Mitsui, H.Hori: "Activity of phophatidylinositol/UDP-GlcNAc:GlcNAc transferase in midgut tissue of Bombyx mori."Bulletin of the Faculty of Agriculture, Niigata University.. 55(2). 123-131 (2003)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] N.S.Kumarasami, T.Maruyama, S.Kurabe, T.Kishimoto, T.Mitsui, H.Hori: "Lipid of brush border membrane vesicle (BBMV) from Plutella xylostella resistant and susceptible to Cry1Ac δ-endotoxin of Bacillus thuringiensis."Comp.Biochem.Physiol.. 129(1). 173-183 (2001)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] D.M.Hossain, Y.Shitomi, K.Moriyama, M.Higuchi, T.Hayakawa, R.Sato, H.Hori: "A niovel 252kDa protein from BBMV of Bombyx mori, which Bound with Cry1A Toxins but did not bind with anti-APN or BTR175 antibodies"Proceeding of 5th Pacific rim conference on the biotechnology of Bacillus thuringiensis on the biotechnology of Bacillus thuringiensis and its environmental impact, Hanoi, Vietnam.. (in press). (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Egawa, K.Okuyama, T.Hara, K.Tomimoto, T.Hayakawa, H.Hori: "Allergenic assessment of Cry1Aa using rat digestive juices and mast cells"Proceeding of 5th Pacific rim conference on the biotechnology of Bacillus thuringiensis on the biotechnology of Bacillus thuringiensis and its environmental impact, Hanoi, Vietnam.. (in press). (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] T.Hayakawa, Y.Shitomi, D.M.Hossain, M.Higuchi, R.Sato, T.Maruyama, K.Miyamaoto, H.Hori: "Novel binding proteins for Cry1A of Bacillus thuringiensis toxins and complicated Killing mechanism"Proceeding of 5th Pacific rim conference on the biotechnology of Bacillus thuringiensis on the biotechnology of Bacillus thuringiensis and its environmental impact, Hanoi, Vietnam.. (in press). (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] Y.Shitomi, K.Moriyama, D.M.Hossain, M.Higuchi, T.Hayakawa, K.Miyamaoto, T.Mitsui, K.Nakanishi, R.Sato, H.Hori: "Characterization of Bombyx mori AP96 which binds with Cry1Ac"Proceeding of 5th Pacific rim conference on the biotechnology of Bacillus thuringiensis on the biotechnology of Bacillus thuringiensis and its environmental impact, Hanoi, Vietnam.. (in press). (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] K.Tomimoto, T.Hayakawa, T.Mitsui, H.Hori: "Detection of membrane-inserted region of Cry1Aa of Bacillus thuringiensis."Proceeding of 5th Pacific rim conference on the biotechnology of Bacillus thuringiensis on the biotechnology of Bacillus thuringiensis and its environmental impact, Hanoi, Vietnam.. (in press). (2004)

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2003 Final Research Report Summary
  • [Publications] D.Takano, et al.: "Differential extraction of the proteins localizing on cell membrane of midgut from Bombyx mori with Triton X-114"Bulletin of the Facultuy of Agriculture, Niigata University. 55(2). 133-141 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] D.Takano, et al.: "Activity of phophatidylinositol/UDP-GlcNAc:GlcNAc transferase in midgut tissue of Bombyx mori"Bulletin of the Facultuy of Agriculture, Niigata University. 55(2). 123-131 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] N.S.Kumaraswami, et al.: "Lipid of brush border membrane vesicle (BBMV) from Plutella xylostella resistant and susceptible to Cry1Acδ-endotoxin of Bacillus thuringiensis"Comp.Biochem.Physiol. 129(1). 173-183 (2001)

    • Related Report
      2003 Annual Research Report
  • [Publications] D.M.Hossain et al.: "A novel 252kDa protein from BBMV of Bombyx mori, which bound with Cry1A toxins but did not bind with anti-APN or BtR175 antibodies"5th Pacific rim conference on the biotechnology of Bacillus thuringiensis and its environmental impact, Proceeding. (In press). (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Y.Egawa et al.: "Allergenic assessment of Cry1Aa using rat digestive juices and mast cells"5th Pacific rim conference on the biotechnology of Bacillus thuringiensis and its environmental impact, Proceeding. (In press). (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] T.Hayakawa, et al.: "Novel binding proteins for Cry1A of Bacillus thuringiensis toxins and complicated killing mechanism"5th Pacific rim conference on the biotechnology of Bacillus thuringiensis and its environmental impact, Proceeding. (In press). (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] Y.Shitomi, et al.: "Characterization of Bombyx mori AP96 which binds with Cry1Ac"5th Pacific rim conference on the biotechnology of Bacillus thuringiensis and its environmental impact, Proceeding. (In press). (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] K.Tomimoto, et al.: "Detection of membrane-inserted region of Cry1Aa of Bacillus thuringiensis"5th Pacific rim conference on the biotechnology of Bacillus thuringiensis and its environmental impact, Proceeding. (In press). (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 堀秀隆: "「次世代の農薬開発-ニューナノテクノロジーによる探索と創製」害虫防除 第II編「新規ターゲットの探索と新しいタイプの防除剤」4章Bacillus thuringiensis殺虫剤(農薬学会編)"ソフトサイエンス社. 134-144 (2003)

    • Related Report
      2003 Annual Research Report
  • [Publications] 福田雅夫, 大山卓爾, 堀秀隆, 小島誠, 早川徹: "微生物からのメッセージ-21世紀に活かす道-第3章昆虫を殺す細菌 担当"エンタープライズ社. 129-207 (2001)

    • Related Report
      2003 Annual Research Report
  • [Publications] 堀秀隆: "農薬学会編「次世代の農薬開発-ニューナノテクノロジーによる探索と創製」害虫防除4章Bacillus thuringiensis殺虫剤、134-144ページ"フラトサィエンス社. 316 (2003)

    • Related Report
      2002 Annual Research Report
  • [Publications] Hidetaka HORI et al.: "Comparative Study of Proteins and Neutral Glyusylceramides of Nidgut Eputelial Cell Membrine in Cry1Ac Susceptible and highly Resistant Diamondbook Moth"Proceeding of 4th Pacific Rim Conference on the Biotechnology of Bacillus thuringiensis and its Environmental Impact. (印刷中).

    • Related Report
      2001 Annual Research Report
  • [Publications] Yasuyuki SHITOMI et al.: "Partial Purification of Animopeptidase from BBM of B.mori and their Binding Speciticitig with Cry1A Toxins"Proceeding of 4th Pacific Rim Conference on the Biotechnology of Bacillus thuringiensis and its Environmental Impact. (印刷中).

    • Related Report
      2001 Annual Research Report

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Published: 2001-04-01   Modified: 2016-04-21  

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