State Key Laboratory for Biology of Plant Disease and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
J Insect Physiol. 2010 Jun;56(6):666-72. doi: 10.1016/j.jinsphys.2010.02.003. Epub 2010 Feb 24.
Midgut membrane-bound alkaline phosphatases (mALP) tethered to the brush border membrane surface by a glycosylphosphatidylinositol (GPI) anchor have been proposed as crucial for Cry1Ac intoxication. In the present work, two full-length cDNAs-encoding alkaline phosphatases in the midgut of Helicoverpa armigera larvae were cloned and named HaALP1 (GenBank accession no. EU729322) and HaALP2 (GenBank accession no. EU729323), respectively. These two clones displayed high identity (above 94%) at the amino acid sequence, indicating that they may represent allelic variants, and were predicted to contain a GPI anchor. Protein sequence alignment revealed that HaALPs were grouped with mALP from the Heliothis virescens midgut. The HaALP1 and HaALP2 ( approximately 68kDa) proteins were heterologously expressed in Sf9 cells using a baculovirus expression system and purified to homogeneity. Ligand blot and dot blot analysis revealed that the Cry1Ac bound to both denatured and native purified HaALPs. Data from lectin blots, competition assays with soybean agglutinin (SBA) lectin and GalNAc binding inhibition assays were indicative of the presence of GalNAc on HaALPs and binding of Cry1Ac toxin to this residue. This observation was further confirmed through N-glycosidase digestion of HaALPs, which resulted in reduced Cry1Ac binding. Our data represent the first report on HaALPs and their putative role as receptors for Cry1Ac toxin in H. armigera.
中肠膜结合的碱性磷酸酶（mALP）通过糖基磷脂酰肌醇（GPI）锚定连接到刷状缘膜表面，被认为是 Cry1Ac 中毒的关键。在本工作中，克隆了棉铃虫幼虫中肠的两个全长 cDNA 编码的碱性磷酸酶，分别命名为 HaALP1（GenBank 登录号 EU729322）和 HaALP2（GenBank 登录号 EU729323）。这两个克隆在氨基酸序列上具有高度的同源性（超过 94%），表明它们可能代表等位基因变异体，并预测含有 GPI 锚。蛋白序列比对表明，HaALPs 与 Heliothis virescens 中肠的 mALP 归为一组。使用杆状病毒表达系统在 Sf9 细胞中异源表达 HaALP1 和 HaALP2（约 68kDa）蛋白，并纯化至均一性。配体印迹和斑点印迹分析表明，Cry1Ac 结合变性和天然纯化的 HaALPs。凝集素印迹、大豆凝集素（SBA）凝集素竞争实验和 GalNAc 结合抑制实验的数据表明 HaALPs 上存在 GalNAc 残基，并与 Cry1Ac 毒素结合。通过 HaALPs 的 N-糖苷酶消化进一步证实了这一观察结果，结果导致 Cry1Ac 结合减少。我们的数据代表了 HaALPs 及其在棉铃虫中作为 Cry1Ac 毒素受体的假定作用的首次报道。
