Chen Jianwu, Aimanova Karlygash G, Fernandez Luisa E, Bravo Alejandra, Soberon Mario, Gill Sarjeet S
Department of Cell Biology and Neuroscience, University of California Riverside, Riverside, CA 92521, USA.
Biochem J. 2009 Nov 11;424(2):191-200. doi: 10.1042/BJ20090730.
Cry11Aa of Bacillus thuringiensis subsp. israelensis is the most active toxin to Aedes aegypti in this strain. We previously reported that, in addition to a 65 kDa GPI (glycosylphosphatidylinositol)-anchored ALP (alkaline phosphatase), the toxin also binds a 250 kDa membrane protein. Since this protein is the same size as cadherin, which in lepidopteran insects is an important Cry toxin receptor, we developed an anti-AaeCad antibody. This antibody detects a 250 kDa protein in immunoblots of larval BBMVs (brush border membrane vesicles). The antibody inhibits Cry11Aa toxin binding to BBMVs and immunolocalizes the cadherin protein to apical membranes of distal and proximal caecae and posterior midgut epithelial cells. This localization is consistent with areas to which Cry11Aa toxin binds and causes pathogenicity. Therefore, the full-length Aedes cadherin cDNA was isolated from Aedes larvae and partial overlapping fragments that covered the entire protein were expressed in Escherichia coli. Using toxin overlay assays, we showed that one cadherin fragment, which contains CR7-11 (cadherin repeats 7-11), bound Cry11Aa and this binding was primarily through toxin domain II loops alpha8 and 2. Cadherin repeats CR8-11 but not CR7 bound Cry11Aa under non-denaturing conditions. Cry11Aa bound the cadherin fragment with high affinity with an apparent Kd of 16.7 nM. Finally we showed that this Cry11Aa-binding site could also be competed by Cry11Ba and Cry4Aa but not Cry4Ba. These results indicate that Aedes cadherin is possibly a receptor for Cry11A and, together with its ability to bind an ALP, suggest a similar mechanism of toxin action as previously proposed for lepidopteran insects.
苏云金芽孢杆菌以色列亚种的Cry11Aa是该菌株中对埃及伊蚊最具活性的毒素。我们之前报道过，除了一种65 kDa的糖基磷脂酰肌醇（GPI）锚定碱性磷酸酶（ALP）外，该毒素还能结合一种250 kDa的膜蛋白。由于这种蛋白与钙黏蛋白大小相同，而在鳞翅目昆虫中钙黏蛋白是一种重要的Cry毒素受体，我们制备了一种抗埃及伊蚊钙黏蛋白（AaeCad）抗体。该抗体在幼虫刷状缘膜囊泡（BBMVs）的免疫印迹中可检测到一种250 kDa的蛋白。该抗体可抑制Cry11Aa毒素与BBMVs的结合，并将钙黏蛋白定位于远端和近端盲肠以及中肠后部上皮细胞的顶端膜。这种定位与Cry11Aa毒素结合并导致致病性的区域一致。因此，从埃及伊蚊幼虫中分离出全长的埃及伊蚊钙黏蛋白cDNA，并在大肠杆菌中表达覆盖整个蛋白的部分重叠片段。通过毒素覆盖分析，我们发现一个包含CR7 - 11（钙黏蛋白重复序列7 - 11）的钙黏蛋白片段能结合Cry11Aa，且这种结合主要通过毒素结构域II的α8和2环。在非变性条件下，钙黏蛋白重复序列CR8 - 11能结合Cry11Aa，但CR7不能。Cry11Aa以16.7 nM的表观解离常数（Kd）高亲和力结合钙黏蛋白片段。最后我们发现，该Cry-11Aa结合位点也能被Cry11Ba和Cry4Aa竞争，但不能被Cry4Ba竞争。这些结果表明，埃及伊蚊钙黏蛋白可能是Cry11A的受体，并且与其结合ALP的能力一起，提示了一种与之前针对鳞翅目昆虫提出的类似的毒素作用机制。
