Binding Analyses of Bacillus thuringiensis Cry δ-Endotoxins Using Brush Border Membrane Vesicles of Ostrinia nubilalis

Transgenic corn expressing the Bacillus thuringiensis Cry1Ab gene is highly insecticidal to Ostrinia nubilalis (European corn borer) larvae. We ascertained whether Cry1F, Cry9C, or Cry9E recognizes the Cry1Ab binding site on the O. nubilalis brush border by three approaches. An optical biosensor technology based on surface plasmon resonance measured binding of brush border membrane vesicles (BBMV) injected over a surface of immobilized Cry toxin. Preincubation with Cry1Ab reduced BBMV binding to immobilized Cry1Ab, whereas preincubation with Cry1F, Cry9C, or Cry9E did not inhibit BBMV binding. BBMV binding to a Cry1F-coated surface was reduced when vesicles were preincubated in Cry1F or Cry1Ab but not Cry9C or Cry9E. A radioligand approach measured (125)I-Cry1Ab toxin binding to BBMV in the presence of homologous (Cry1Ab) and heterologous (Cry1Ac, Cry1F, Cry9C, or Cry9E) toxins. Unlabeled Cry1Ac effectively competed for (125)I-Cry1Ab binding in a manner comparable to Cry1Ab itself. Unlabeled Cry9C and Cry9E toxins did not inhibit (125)I-Cry1Ab binding to BBMV. Cry1F inhibited (125)I-Cry1Ab binding at concentrations greater than 500 nM. Cry1F had low-level affinity for the Cry1Ab binding site. Ligand blot analysis identified Cry1Ab, Cry1Ac, and Cry1F binding proteins in BBMV. The major Cry1Ab signals on ligand blots were at 145 kDa and 154 kDa, but a strong signal was present at 220 kDa and a weak signal was present at 167 kDa. Cry1Ac and Cry1F binding proteins were detected at 220 and 154 kDa. Anti-Manduca sexta aminopeptidase serum recognized proteins of 145, 154, and 167 kDa, and anti-cadherin serum recognized the 220 kDa protein. We speculate that isoforms of aminopeptidase and cadherin in the brush border membrane serve as Cry1Ab, Cry1Ac, and Cry1F binding proteins.

表达苏云金芽孢杆菌（Bacillus thuringiensis）Cry1Ab基因的转基因玉米，对欧洲玉米螟（Ostrinia nubilalis）幼虫具有极高的杀虫活性。本研究通过三种实验策略，探究Cry1F、Cry9C及Cry9E是否可识别欧洲玉米螟中肠刷状缘膜上的Cry1Ab结合位点。第一种方法采用基于表面等离子体共振（surface plasmon resonance, SPR）的光学生物传感器技术：将刷状缘膜囊泡（brush border membrane vesicles, BBMV）注射至固定化Cry毒素包被的表面，检测二者的结合情况。经Cry1Ab预孵育后，BBMV与固定化Cry1Ab的结合量显著降低；而经Cry1F、Cry9C或Cry9E预孵育后，BBMV的结合并未受到抑制。当囊泡经Cry1F或Cry1Ab预孵育时，BBMV与Cry1F包被表面的结合量会下降，但经Cry9C或Cry9E预孵育则无此抑制效果。第二种方法为放射性配体结合实验：在同源（Cry1Ab）及异源（Cry1Ac、Cry1F、Cry9C或Cry9E）毒素存在的条件下，检测碘-125标记的Cry1Ab毒素与BBMV的结合活性。未标记的Cry1Ac可有效竞争碘-125标记的Cry1Ab的结合位点，其竞争效果与Cry1Ab本身相当；未标记的Cry9C与Cry9E则未对该结合产生抑制作用。当浓度高于500纳摩尔（nM）时，Cry1F可抑制碘-125标记的Cry1Ab与BBMV的结合，提示Cry1F与Cry1Ab结合位点仅具有低水平的亲和力。第三种方法为配体印迹（ligand blot）分析：结果显示BBMV中存在可结合Cry1Ab、Cry1Ac及Cry1F的蛋白。配体印迹实验中，Cry1Ab的主要信号条带位于145 kDa和154 kDa处，同时在220 kDa处存在一条强信号条带，167 kDa处存在一条弱信号条带；可结合Cry1Ac与Cry1F的蛋白条带则分别位于220 kDa和154 kDa处。抗烟草天蛾（Manduca sexta）氨肽酶血清可识别145、154及167 kDa的蛋白，而抗钙粘蛋白血清可识别220 kDa的蛋白。据此，本研究推测，刷状缘膜中的氨肽酶与钙粘蛋白的同工型，可作为Cry1Ab、Cry1Ac及Cry1F的结合蛋白。