Involvement of two amino acid residues in the loop region of Bacillus thuringiensis Cry1Ab toxin in toxicity and binding to Lymantria dispar.
Involvement of two amino acid residues in the loop region of Bacillus thuringiensis Cry1Ab toxin in toxicity and binding to Lymantria dispar.
复制标题
DOI：
10.1006/bbrc.1996.1770
复制
发表时间：
1996-12
影响因子：
3.1
通讯作者：
M. K. Lee;T. You;A. Curtiss;D. Dean
中科院分区：
文献类型：
--
作者：
M. K. Lee;T. You;A. Curtiss;D. Dean
Cry1Ab2毒素II结构域环区282位和283位的两个氨基酸Gly和Ser被Cry1Ab9-033毒素的Ala和Leu取代。与Cry1Ab9-033相比，Cry1Ab2的毒性增加了10倍，与Lymantria dispar的结合亲和力增加了9倍。然而，这些毒素对雌雄蜘蛛和夜蛾的毒性和结合亲和力相似。异源竞争实验和刷状边界膜泡（BBMV）配体印迹实验表明，Cry1Ab2和Cry1Ab9-033毒素识别相同的210 kda L dispar BBMV蛋白。在这两种毒素的解离结合试验中没有可测量的差异。用L - dispar肠道酶和BBMV蛋白酶消化这些毒素的稳定性没有差异。通过定点诱变，将Cry1Ab9-033中的Ala和Leu残基替换为Gly和Ser，产生突变体Cry1Ab α 8。该毒素的毒性完全恢复，并且对L dispar具有结合亲和力。这些数据表明，环区残基Gly和Ser可能直接参与了L dispar的受体结合和毒性。
Two amino acids, Gly and Ser, at positions 282 and 283 in the loop region of domain II of Cry1Ab2 toxin are substituted with Ala and Leu in the Cry1Ab9-033 toxin. Cry1Ab2 exhibited about a 10-fold increase in toxicity and a 9-fold increase in binding affinity to Lymantria dispar compared to Cry1Ab9-033. However, these toxins showed similar toxicity and binding affinity to Manduca sexta and Spodoptera exigua. Heterologous competition assays and brush border membrane vesicle (BBMV) ligand blotting experiments demonstrated that Cry1Ab2 and Cry1Ab9-033 toxins recognized the same 210-kDa L dispar BBMV protein. No measurable differences in dissociation binding assays were observed between these two toxins. Digestion of these toxins with L dispar gut enzymes and BBMV proteases indicated no differences in stability. Ala and Leu residues in Cry1Ab9-033 were substituted with Gly and Ser by site-directed mutagenesis to produce mutant Cry1Ab alpha 8. This toxin exhibited full recovery of toxicity and binding affinity for L dispar. These data suggested that the residues Gly and Ser in the loop region might be directly involved in receptor binding and toxicity in L dispar.