Cloning and characterization of the Cry1Ac-binding alkaline phosphatase (HvALP) from Heliothis virescens
Cloning and characterization of the Cry1Ac-binding alkaline phosphatase (HvALP) from Heliothis virescens
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DOI：
10.1016/j.ibmb.2009.01.006
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发表时间：
2009-04-01
影响因子：
3.8
通讯作者：
Jurat-Fuentes, Juan L.
中科院分区：
文献类型：
--
作者：
Perera, Omaththage P.;Willis, Jonathan D.;Jurat-Fuentes, Juan L.
已报道昆虫中肠中的膜结合碱性磷酸酶（mALP，EC 3.1.3.1）是来自细菌苏云金芽孢杆菌的Cry毒素的功能性受体。我们以前报道的鉴定HvALP在Heliothis virescens幼虫中肠作为Cry 1Ac结合蛋白，在Cry 1Ac抗性昆虫中下调。为了进一步鉴定HvALP，我们使用抗mALP血清将mALP蛋白定位于前肠和中肠组织，然后从H.绿僵菌幼虫中肠所有5个克隆均显示出高水平的序列同一性（高于90%），表明它们可能代表等位基因变体，并且在序列比对中与其他鳞翅目mALP分组。所有这些克隆的ALP被预测含有糖基磷脂酰肌醇（GPI）锚，并被命名为HvmALP 1 -5。我们在异源系统中表达了两种最多样化的HvmALP，以测试Cry 1Ac的结合和HvALP交叉反应抗血清的识别。我们的数据强调了糖基化对Cry 1Ac与HvALP结合的重要性，并表明，根据糖基化，所有鉴定的HvmALP可能与HvALP同义，HvALP是在H.中肠上皮(C)2009爱思唯尔有限公司保留所有权利。
Membrane-bound alkaline phosphatases (mALPs, EC 3.1.3.1) in the insect midgut have been reported as functional receptors for Cry toxins from the bacterium Bacillus thuringiensis. We previously reported the identification of HvALP in the midgut of Heliothis virescens larvae as a Cry1Ac-binding protein that is down-regulated in Cry1Ac-resistant insects. To further characterize HvALP, we localized mALP protein to foregut and midgut tissues using anti-mALP serum and then cloned five mALPs from H. virescens larval midgut. All five clones displayed high levels of sequence identity (above 90%), suggesting that they may represent allelic variants, and grouped with other lepidopteran mALPs in sequence alignments. All these cloned ALPs were predicted to contain a glycosylphosphatidylinositol (GPI) anchor and were named HvmALP1-5. We expressed two of the most diverse HvmALPs in a heterologous system to test binding of Cry1Ac and recognition by HvALP cross-reacting antiserum. Our data highlight the importance of glycosylation for Cry1Ac binding to HvALP and suggest that, depending on glycosylation, all the identified HvmALPs may be synonymous with HvALP, the Cry1Ac-binding phosphatase identified in H. virescens midgut epithelium. (C) 2009 Elsevier Ltd. All rights reserved.