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

昆虫中肠上皮细胞膜与杀虫叫声毒素的识别机制

基本信息

批准号：
13306006
负责人：
HORI Hidetaka
金额：
$ 22.63万
依托单位：
NIIGATA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2003
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13306006/
关键词：
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 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

项目摘要

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

本文研究了杀虫毒素Cry与家蚕中肠顶端细胞膜的相互作用。1)从刷状缘膜中发现了与Cry 1Aa、Cry 1Ab和Cry 1Ac结合的新的P252。2)发现了一种仅与Cry 1Ac结合的新型氨肽酶AP。这种新型AP被认为属于第1组。3)构建了含有BBM蛋白的脂质体，并显示与不含蛋白的脂质体相比，其与Cry 1Aa、Cry 1Ab和Cry 1Ac的亲和力高两倍。4）Cry 1A毒素被认为在结构域1中插入它们的α-螺旋。但是没有什么是清楚的螺旋插入膜。我们确定了插入顶端细胞膜的结构域1的部分。约24 kDa的肽被认为是插入的，并显示其对应于从α-螺旋2到α-螺旋7的延伸。这是第一个关于结构域1插入区的具体建议 ...更多信息 X-100或CHAPS，因此，它可能局限于非筏区。P252被认为是一种具有同源四聚体的糖蛋白。糖侧链可能是三触角的N-连接的含二等分GlcNAc的糖侧链和O-连接的含GalNAc的糖侧链。P252与Cry 1Aa、Cry 1Ab和Cry 1Ac的kd值分别为10 nM、70 nM和20 nM，是文献中最强的结合位点之一。异源结合实验表明，Cry 1Aa的P252位点与Cry 1Ac的P252位点有50%的同源性，Cry 1Ac的P252位点与Cry 1Aa的P252位点有80%的同源性。P252与Cry 1Ab的kd值为70 nM，该值仍然足够低，可以认为结合是生理事件。但有趣的是，Cry 1Ab与Cry 1Aa和Cry 1Ab的结合位点各为50%，BSA也显示出抑制结合的作用。在三种Cry 1A毒素中，P96仅与Cry 1Ac结合，并且结合被GalNAc抑制。对底物特异性进行了考察，初步认为P96为APN。该反应需要二价金属阳离子，最适pH为6-8。在较高的底物浓度下，反应速度加快。少