Spruce budworm elastase precipitates Bacillus thuringiensis delta-endotoxin by specifically recognizing the C-terminal region.
Spruce budworm elastase precipitates Bacillus thuringiensis delta-endotoxin by specifically recognizing the C-terminal region.
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DOI：
10.1016/s0965-1748(98)00090-3
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发表时间：
1998-12
影响因子：
3.8
通讯作者：
R. Milne;T. Wright;H. Kaplan;D. Dean
中科院分区：
文献类型：
--
作者：
R. Milne;T. Wright;H. Kaplan;D. Dean
来自 Choristoneura fumiferana（云杉芽虫）幼虫的肠液蛋白可沉淀某些 δ-内毒素，显示出对 C 端氨基酸序列的独特特异性。使用同源扫描突变体，我们鉴定了 Cry1Aa 毒素的一个连续区域，该区域与 75-kDa 毒素沉淀蛋白 (TPP-75)1 相互作用，导致沉淀。 Cry1Aa 的邻近区域可以转移到 Cry1Ac 并导致相同的沉淀反应。沉淀反应发生迅速，其独特之处在于沉淀蛋白与毒素的比率较低（估计为 0.01），这与摩尔比接近 1 的抗体-抗原反应不同。TPP-75 被描述为一种类似弹性蛋白酶的丝氨酸蛋白酶。我们利用了丝氨酸蛋白酶的这种特性，并使用不可逆抑制剂掺入了放射性标记。放射性标记使我们能够在印迹测定中显示催化抑制的 TPP-75 与毒素的一致性，并跟踪储存期间 TPP-75 的降解。 TPP-75 首次证明肠液蛋白在与易感细胞上的假定受体结合之前可以选择性减弱 δ-内毒素的活性。对于那些不表现出基于受体的耐药性的幼虫，应将 TPP-75 作为一种可能的耐药机制进行评估。
A gut juice protein from Choristoneura fumiferana (spruce budworm) larvae that precipitates certain δ-endotoxins shows a unique specificity for the C-terminal amino acid sequence. Using homolog scanning mutants, we have identified a contiguous region of the Cry1Aa toxin which interacts with the 75-kDa toxin precipitating protein (TPP-75)1resulting in precipitation. The contiguous region from Cry1Aa can be transferred to Cry1Ac and results in an identical precipitation reaction. The precipitation reaction occurs rapidly and is unique in that the ratio of precipitating protein to toxin is low (estimated at 0.01), unlike antibody–antigen reactions which exhibit mole ratios close to 1. TPP-75 has been characterized as an elastase-like serine protease. We have taken advantage of this serine protease character and incorporated a radiolabel using an irreversible inhibitor. The radiolabel has allowed us to show the coincidence of the catalytically-inhibited TPP-75 with the toxin in a blotting assay and to follow the degradation of TPP-75 during storage. TPP-75 represents the first evidence that gut juice proteins may selectively attenuate the activity of δ-endotoxins, prior to binding to putative receptors on susceptible cells. TPP-75 should be evaluated as a possible resistance mechanism for those larvae that do not exhibit a receptor-based resistance.