Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos 62250, México.
J Biol Chem. 2010 Apr 23;285(17):12497-503. doi: 10.1074/jbc.M109.085266. Epub 2010 Feb 22.
Cry toxins produced by Bacillus thuringiensis have been recognized as pore-forming toxins whose primary action is to lyse midgut epithelial cells in their target insect. In the case of the Cry1A toxins, a prepore oligomeric intermediate is formed after interaction with cadherin receptor. The Cry1A oligomer then interacts with glycosylphosphatidylinositol-anchored receptors. Two Manduca sexta glycosylphosphatidylinositol-anchored proteins, aminopeptidase (APN) and alkaline phosphatase (ALP), have been shown to bind Cry1Ab, although their role in toxicity remains to be determined. Detection of Cry1Ab binding proteins by ligand blot assay revealed that ALP is preferentially expressed earlier during insect development, because it was found in the first larval instars, whereas APN is induced later after the third larval instar. The binding of Cry1Ab oligomer to pure preparations of APN and ALP showed that this toxin structure interacts with both receptors with high affinity (apparent K(d) = 0.6 nM), whereas the monomer showed weaker binding (apparent K(d) = 101.6 and 267.3 nM for APN and ALP, respectively). Several Cry1Ab nontoxic mutants located in the exposed loop 2 of domain II or in beta-16 of domain III were affected in binding to APN and ALP, depending on their oligomeric state. In particular monomers of the nontoxic domain III, the L511A mutant did not bind ALP but retained APN binding, suggesting that initial interaction with ALP is critical for toxicity. Our data suggest that APN and ALP fulfill two roles. First APN and ALP are initial receptors promoting the localization of toxin monomers in the midgut microvilli before interaction with cadherin. Then APN and ALP function as secondary receptors mediating oligomer insertion into the membrane. However, the expression pattern of these receptors and the phenotype of L511A mutant suggest that ALP may have a predominant role in toxin action because Cry toxins are highly effective against the neonate larvae that is the target for pest control programs.
苏云金芽孢杆菌产生的 Cry 毒素已被认为是形成孔的毒素，其主要作用是裂解靶昆虫的中肠上皮细胞。在 Cry1A 毒素的情况下，与钙粘蛋白受体相互作用后形成预孔寡聚中间体。然后，Cry1A 寡聚体与糖基磷脂酰肌醇锚定受体相互作用。已经表明，两种曼陀罗糖基磷脂酰肌醇锚定蛋白，氨肽酶（APN）和碱性磷酸酶（ALP）与 Cry1Ab 结合，尽管它们在毒性中的作用仍有待确定。通过配体印迹分析检测到 Cry1Ab 结合蛋白表明，ALP 在昆虫发育过程中较早表达，因为它在第一龄幼虫中发现，而 APN 在第三龄幼虫后诱导。Cry1Ab 寡聚体与纯 APN 和 ALP 制剂的结合表明，这种毒素结构与两个受体具有高亲和力（表观 K(d) = 0.6 nM）相互作用，而单体的结合较弱（APN 和 ALP 的表观 K(d)分别为 101.6 和 267.3 nM）。位于结构域 II 的暴露环 2或结构域 III 的β-16 中的几个 Cry1Ab 无毒突变体，取决于其寡聚状态，在与 APN 和 ALP 的结合中受到影响。特别是无毒的结构域 III 单体，L511A 突变体不与 ALP 结合，但保留与 APN 的结合，表明与 ALP 的初始相互作用对于毒性至关重要。我们的数据表明，APN 和 ALP 发挥两个作用。首先，APN 和 ALP 是最初的受体，在与钙粘蛋白相互作用之前，促进毒素单体在中肠微绒毛中的定位。然后，APN 和 ALP 作为二级受体起作用，介导寡聚体插入膜中。然而，这些受体的表达模式和 L511A 突变体的表型表明，ALP 可能在毒素作用中起主要作用，因为 Cry 毒素对控制害虫计划的目标新孵化幼虫具有高度有效性。
