Aedes aegypti cadherin serves as a putative receptor of the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis.
Aedes aegypti cadherin serves as a putative receptor of the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis.
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DOI：
10.1042/bj20090730
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发表时间：
2009-11-11
期刊：
影响因子：
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通讯作者：
Gill SS
中科院分区：
文献类型：
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作者：
Chen J;Aimanova KG;Fernandez LE;Bravo A;Soberon M;Gill SS
苏云金芽孢杆菌Cry11Aa israelensis是该菌株中对埃及伊蚊最具活性的毒素。我们以前报道，除了65 kDa GPI（糖基磷脂酰肌醇）锚定的ALP（碱性磷酸酶），毒素还结合250 kDa的膜蛋白。由于这种蛋白质与钙粘蛋白的大小相同，钙粘蛋白在鳞翅目昆虫中是重要的Cry毒素受体，因此我们开发了抗AaeCad抗体。该抗体检测幼虫BBMV（刷状缘膜囊泡）免疫印迹中的250 kDa蛋白。该抗体抑制Cry11Aa毒素与BBMV的结合，并将钙粘蛋白免疫定位于远端和近端盲肠的顶端膜和后中肠上皮细胞。这种定位与Cry11Aa毒素结合并引起致病性的区域一致。因此，从伊蚊幼虫中分离出全长的伊蚊钙粘蛋白cDNA，并在大肠杆菌中表达了覆盖整个蛋白的部分重叠片段。使用毒素覆盖试验，我们发现一个钙粘蛋白片段，其中包含CR7 - 11（钙粘蛋白重复7 - 11），结合Cry11Aa，这种结合主要是通过毒素结构域II环α 8和2。在非变性条件下，钙粘蛋白重复CR8 - 11，但不重复CR7结合Cry11Aa。Cry11Aa以高亲和力结合钙粘蛋白片段，表观Kd为16.7 nM。最后，我们表明，这个Cry11Aa结合位点也可以被Cry11Ba和Cry4Aa竞争，但不Cry4Ba。这些结果表明，伊蚊钙粘蛋白可能是Cry11A的受体，并结合ALP的能力，提出了类似的毒素作用机制，如以前提出的鳞翅目昆虫。
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 α8 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.