Synergistic and inhibitory effects of aminopeptidase peptides on Bacillus thuringiensis Cry11Ba toxicity in the mosquito Anopheles gambiae.
Synergistic and inhibitory effects of aminopeptidase peptides on Bacillus thuringiensis Cry11Ba toxicity in the mosquito Anopheles gambiae.
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DOI：
10.1021/bi1009908
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发表时间：
2010-09
期刊：
影响因子：
2.9
通讯作者：
Rui Zhang;G. Hua;J. Urbauer;M. Adang
中科院分区：
文献类型：
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作者：
Rui Zhang;G. Hua;J. Urbauer;M. Adang
Cry11Ba 由苏云金芽孢杆菌亚种产生。 jegathesan 是一种针对冈比亚按蚊幼虫的活性毒素。一种名为 AgAPN2 的 106 kDa 氨肽酶 N (APN) 先前被鉴定为冈比亚曲霉中的 Cry11Ba 受体。在大肠杆菌中表达的 AgAPN2 的 70 kDa 片段以高亲和力 (K(d) = 6.4 nM) 结合 Cry11Ba，并在生物测定中抑制 Cry11Ba 活性达 98% [Zhang et al. 2017]。 (2008) 生物化学 47, 11263-11272]。为了确定涉及毒性的区域，我们将 70 kDa APN 片段分别截断为 28 kDa ta 和 tb 的肽，并测试它们介导毒性和结合 Cry11Ba 的能力。 AgAPN2ta 将 Cry11Ba 毒性降低了 85%，而 AgAPN2tb 对 Cry11Ba 毒性表现出显着增强作用。纯化的肽显示出结构折叠的证据，并以高亲和力结合 Cry11Ba 上的相同位点。抑制性 AgAPN2ta 阻断 Cry11Ba 与冈比亚刷状缘膜囊泡 (BBMV) 的结合，而增强毒性的 AgAPN2tb 则增加 Cry11Ba 与 BBMV 的结合。 AgAPN2ta N 末端 ((336)S-P(420)) 的缺失显着降低了 AgAPN2ta 与 Cry11Ba 的结合及其抑制作用。 AgAPN2tb 中心区域 ((676)I-W(760)) 的删除消除了其增加的毒素结合和毒性增强作用，而不影响 Cry11Ba 结合。提出了 AgAPN2tb 作用的“桥”模型，其中肽结合 Cry11Ba 并将其引导至幼虫中肠上的位点。
Cry11Ba produced by Bacillus thuringiensis subsp. jegathesan is an active toxin for larvae of the mosquito Anopheles gambiae. A 106-kDa aminopeptidase N (APN), called AgAPN2, was previously identified as a Cry11Ba receptor in A. gambiae. A 70-kDa fragment of AgAPN2 expressed in Escherichia coli binds Cry11Ba with high affinity (K(d) = 6.4 nM) and inhibits Cry11Ba activity by 98% in bioassays [Zhang et al. (2008) Biochemistry 47, 11263-11272]. To identify regions involved in toxicity, we truncated the 70-kDa APN fragment into peptides of 28- and 30-kDa ta and tb, respectively, and tested their abilities to mediate toxicity and bind Cry11Ba. While AgAPN2ta reduced Cry11Ba toxicity by 85%, AgAPN2tb showed a significant enhancement effect on Cry11Ba toxicity. The purified peptides showed evidence of structural folding and bound the same site(s) on Cry11Ba with high affinity. The inhibitory AgAPN2ta blocked Cry11Ba binding to brush border membrane vesicles (BBMV) of A. gambiae whereas the toxicity enhancing AgAPN2tb increased Cry11Ba binding on BBMV. A deletion at the N-terminus ((336)S-P(420)) of AgAPN2ta significantly reduced AgAPN2ta binding to Cry11Ba and its inhibitory effect. Deletion of the central region ((676)I-W(760)) of AgAPN2tb eliminated its increased toxin binding and toxicity enhancement effect without affecting Cry11Ba binding. A "bridge" model is proposed for AgAPN2tb action whereby the peptide binds Cry11Ba and vectors it to sites on the larval midgut.