Gómez Isabel, Arenas Iván, Benitez Itzel, Miranda-Ríos Juan, Becerril Baltazar, Grande Ricardo, Almagro Juan Carlos, Bravo Alejandra, Soberón Mario
Instituto de Biotecnología, UNAM, Departamento de Microbiología Molecular, Apdo. Postal 510-3, Cuernavaca, Morelos 62250, México.
J Biol Chem. 2006 Nov 10;281(45):34032-9. doi: 10.1074/jbc.M604721200. Epub 2006 Sep 12.
The Bacillus thuringiensis Cry toxins are specific to different insects. In Manduca sexta cadherin (Bt-R1) and aminopeptidase-N (APN) proteins are recognized as Cry1A receptors. Previous work showed that Cry1Ab binds to Bt-R1 promoting the formation of a pre-pore oligomer that binds to APN leading to membrane insertion. In this work we characterized the binding epitopes involved in the sequential interaction of Cry1Ab with Bt-R1 and APN. A Cry1Ab immune M13 phage repertoire was constructed using antibody gene transcripts of bone marrow or spleen from a rabbit immunized with Cry1Ab. We identified antibodies that recognize domain II loop 3 (scFvL3-3) or beta16-beta22 (scFvM22) in domain III. Enzyme-linked immunosorbent assay and toxin overlay binding competition assays in the presence of scFvL3-3, scFvM22, or synthetic peptides showed that domain II loop 3 is an important epitope for interaction with Bt-R1 receptor, whereas domain III beta16 is involved in the interaction with APN. Both scFvL3-3 and scFvM22 lowered the toxicity of Cry1Ab to M. sexta larvae indicating that interaction with both receptors is important for in vivo toxicity. scFvL3-3 and anti-loop2 scFv (scFv73) promoted the formation of the pre-pore oligomer in contrast to scFvM22. In addition, scFvL3-3 and scFv73 preferentially recognized the monomeric toxin rather than the pre-pore suggesting a conformational change in domain II loops upon oligomerization. These results indicate for the first time that both receptor molecules participate in Cry1Ab toxin action in vivo: first the monomeric toxin binds to Bt-R1 through loops 2 and 3 of domain II promoting the formation of the pre-pore inducing some structural changes, then the pre-pore interacts with APN through beta-16 of domain III promoting membrane insertion and cell death.
苏云金芽孢杆菌Cry毒素对不同昆虫具有特异性。在烟草天蛾中，钙黏蛋白（Bt-R1）和氨肽酶N（APN）蛋白被认为是Cry1A受体。先前的研究表明，Cry1Ab与Bt-R1结合，促进形成与APN结合的孔前寡聚体，从而导致膜插入。在这项研究中，我们对Cry1Ab与Bt-R1和APN的顺序相互作用中涉及的结合表位进行了表征。使用用Cry1Ab免疫的兔子的骨髓或脾脏的抗体基因转录本构建了Cry1Ab免疫M13噬菌体文库。我们鉴定出了识别结构域III中结构域II环3（scFvL3-3）或β16-β22（scFvM22）的抗体。在存在scFvL3-3、scFvM22或合成肽的情况下进行的酶联免疫吸附测定和毒素覆盖结合竞争测定表明，结构域II环3是与Bt-R1受体相互作用的重要表位，而结构域IIIβ16参与与APN的相互作用。scFvL3-3和scFvM22均降低了Cry1Ab对烟草天蛾幼虫的毒性，表明与两种受体的相互作用对体内毒性很重要。与scFvM22相反，scFvL3-3和抗环2单链抗体（scFv73）促进了孔前寡聚体的形成。此外，scFvL3-3和scFv73优先识别单体毒素而非孔前体，这表明寡聚化时结构域II环发生了构象变化。这些结果首次表明，两种受体分子都参与了Cry1Ab毒素在体内的作用：首先，单体毒素通过结构域II的环2和环3与Bt-R1结合，促进孔前体的形成，诱导一些结构变化，然后孔前体通过结构域III的β-16与APN相互作用，促进膜插入和细胞死亡。
