2003 Fiscal Year Final Research Report Summary
Recognition mechanism between insect midgut epithelial cell membrane and insecticidal cry toxins
| Project/Area Number |
13306006
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
蚕糸・昆虫利用学
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| Research Institution | NIIGATA UNIVERSITY |
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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)
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| 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)
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| Project Period (FY) |
2001 – 2003
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| Keywords | Insect midgut epithelial cells / silkworm Bacillus thuringiensis / insecticidal toxin / novel binding protein / dissociation constant / liposome / aminopeptidase / アミノペプチデース |
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| 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
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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
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[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)
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