Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity
Anopheles gambiae cadherin AgCad1 binds the Cry4Ba toxin of Bacillus thuringiensis israelensis and a fragment of AgCad1 synergizes toxicity
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DOI：
10.1021/bi7023578
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发表时间：
2008-05-06
期刊：
影响因子：
2.9
通讯作者：
Adang, Michael J.
中科院分区：
文献类型：
--
作者：
Hua, Gang;Zhang, Rui;Adang, Michael J.
从冈比亚按蚊幼虫中克隆了一个中肠钙粘蛋白AgCad1基因，并对其作为苏云金芽孢杆菌以色列株Cry4BA毒素受体的作用进行了分析。AgCad1钙粘蛋白编码一个1735个残基的蛋白质，由11个钙粘素重复序列组成的胞外区(CR)和膜-近端胞外区(MPED)组成。用聚合酶链式反应(PCR)检测到幼虫中肠AgCad1mRNA的表达。切片幼虫免疫组织化学显示，AgCad1蛋白主要定位于中肠后段的微绒毛。用同样的技术确定了Cry4BA结合的定位，并在中肠后部确定了毒素结合的微绒毛。AgCad1蛋白存在于幼虫的刷状缘膜组分中，Cry4BA毒素在这些组分的斑点上结合了相同大小的蛋白质。AgCad1蛋白在果蝇Schneider 2(S2)细胞中瞬时表达。I-125-Cry4BA毒素以竞争性方式与S2细胞的AgCad1结合。从S2细胞中提取出200 kDa的AgCad1和29 kDa的AgCad1片段。在大肠杆菌中表达了含有细胞近端AgCad1区的多肽(CR11-MPED)。虽然Cry4BA与CR11-MPED的结合有限，但该多肽对Cry4BA对幼虫的毒性有协同作用。位于幼虫刷状缘处的sAgCad1是Cry4BA毒素的结合蛋白。根据结合结果和Cry4BA毒性的CR11-MPED协同作用，AgCad1可能是一种Cry4BA受体。
A midgut cadherin AgCad1 cDNA was cloned from Anopheles gambiae larvae and analyzed for its possible role as a receptor for the Cry4Ba toxin of Bacillus thuringiensis strain israelensis. The AgCad1 cadherin encodes a putative 1735-residue protein organized into an extracellular region of 11 cadherin repeats (CR) and a membrane-proximal extracellular domain (MPED). AgCad1 mRNA was detected in midgut of larvae by polymerase chain reaction (PCR). The AgCad1 protein was localized, by immunochemistry of sectioned larvae, predominately to the microvilli in posterior midgut. The localization of Cry4Ba binding was determined by the same technique, and toxin bound microvilli in posterior midgut. The AgCad1 protein was present in brush border membrane fractions prepared from larvae, and Cry4Ba toxin bound the same-sized protein on blots of those fractions. The AgCad1 protein was expressed transiently in Drosophila melanogaster Schneider 2 (S2) cells. I-125-Cry4Ba toxin bound AgCad1 from S2 cells in a competitive manner. Cry4Ba bound to beads extracted 200 kDa AgCad1 and a 29 kDa fragment of AgCad1 from S2 cells. A peptide containing the AgCad1 region proximal to the cell (CR11-MPED) was expressed in Escherichia coli. Although Cry4Ba showed limited binding to CR11-MPED, the peptide synergized the toxicity of Cry4Ba to larvae. sAgCad1 in the larval brush border is a binding protein for Cry4Ba toxin. On the basis of binding results and CR11-MPED synergism of Cry4Ba toxicity, AgCad1 is probably a Cry4Ba receptor.