Department of Genetics, University of Valencia, Dr. Moliner 50, 46100 Burjassot, Valencia, Spain.
Insect Biochem Mol Biol. 2013 Oct;43(10):924-35. doi: 10.1016/j.ibmb.2013.07.009. Epub 2013 Aug 8.
Aminopeptidase N (APN) isoforms from Lepidoptera are known for their involvement in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis. These enzymes belong to a protein family with at least eight different members that are expressed simultaneously in the midgut of lepidopteran larvae. Here, we focus on the characterization of the APNs from Ostrinia nubilalis (OnAPNs) to identify potential Cry receptors. We expressed OnAPNs in insect cells using a baculovirus system and analyzed their enzymatic activity by probing substrate specificity and inhibitor susceptibility. The interaction with Cry1Ab and Cry1Fa proteins (both found in transgenic insect-resistant maize) was evaluated by ligand blot assays and immunocytochemistry. Ligand blots of brush border membrane proteins showed that both Cry proteins bound mainly to a 150 kDa-band, in which OnAPNs were greatly represented. Binding analysis of Cry proteins to the cell-expressed OnAPNs showed that OnAPN1 interacted with both Cry1Ab and Cry1Fa, whereas OnAPN3a and OnAPN8 only bound to Cry1Fa. Two isoforms, OnAPN2 and OnAPN3b, did not interact with any of these two proteins. This work provides the first evidence of a differential role of OnAPN isoforms in the mode of action of Cry proteins in O. nubilalis.
鳞翅目昆虫的氨肽酶 N（APN）同工酶因其参与苏云金芽孢杆菌杀虫晶体蛋白的作用模式而闻名。这些酶属于一个蛋白质家族，至少有 8 个不同的成员同时在鳞翅目幼虫的中肠中表达。在这里，我们专注于鉴定潜在 Cry 受体的棉铃虫（Ostrinia nubilalis）的 APNs 的特性。我们使用杆状病毒系统在昆虫细胞中表达 OnAPNs，并通过探测底物特异性和抑制剂敏感性来分析它们的酶活性。通过配体印迹分析和免疫细胞化学评估与 Cry1Ab 和 Cry1Fa 蛋白（均存在于转基因抗虫玉米中）的相互作用。刷状缘膜蛋白的配体印迹显示，这两种 Cry 蛋白主要与 150 kDa 带结合，其中 OnAPNs 大量存在。Cry 蛋白与细胞表达的 OnAPNs 的结合分析表明，OnAPN1 与 Cry1Ab 和 Cry1Fa 都相互作用，而 OnAPN3a 和 OnAPN8 仅与 Cry1Fa 结合。两种同工酶 OnAPN2 和 OnAPN3b 与这两种蛋白都没有相互作用。这项工作首次提供了证据表明，OnAPN 同工酶在 Cry 蛋白对棉铃虫的作用模式中具有不同的作用。
