Effects on larvicidal activity of single proline substitutions in α3 or α4 of the Bacillus thuringiensis Cry4B toxin
Effects on larvicidal activity of single proline substitutions in α3 or α4 of the Bacillus thuringiensis Cry4B toxin
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DOI：
10.1080/15216549800201872
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发表时间：
1998-04
期刊：
影响因子：
4.6
通讯作者：
P. Uawithya;T. Tuntitippawan;G. Katzenmeier;C. Angsuthanasombat;S. Panyim
中科院分区：
文献类型：
--
作者：
P. Uawithya;T. Tuntitippawan;G. Katzenmeier;C. Angsuthanasombat;S. Panyim
通过定点突变采用脯氨酸取代，研究了α-螺旋3和4在双翅目活性苏云金芽孢杆菌Cry 4 B δ-内毒素毒性中的可能作用。与野生型Cry 4 B类似，突变型毒素在大肠杆菌中作为细胞质内含物过表达，并且在溶解和胰蛋白酶活化后结构稳定。螺旋4中心的谷氨酰胺149被脯氨酸取代（Q149 P）导致对埃及伊蚊蚊幼虫的毒性几乎完全丧失。然而，在螺旋3的中心附近（V119 P）和螺旋4的N末端（Q140 P）处的单个脯氨酸替换并不降低杀幼虫活性。E.表达野生型毒素的大肠杆菌细胞通过与无毒突变体（Q149 P）预孵育2小时而显著减少，从而表明初级结合步骤不受螺旋4中脯氨酸取代的影响。因此，结果揭示了Cry 4 B毒素的螺旋4在毒性中的关键作用，可能是在膜插入和孔形成中，而不是在受体识别中。
The possible role of α‐helices 3 and 4 in toxicity of the dipteran‐active Bacillus thuringiensis Cry4B δ‐endotoxin was investigated by employing proline substitutions via site‐directed mutagenesis. Similar to the wild‐type Cry4B, the mutant toxins were over‐expressed in Escherichia coli as cytoplasmic inclusions and were structurally stable upon solubilization and trypsin activation. The substitution of glutamine 149 by proline in the center of helix 4 (Q149P) resulted in a nearly complete loss of toxicity against Aedes aegypti mosquito‐larvae. However, single proline replacements near the center of helix 3 (V119P) and at the N‐terminus of helix 4 (Q140P) did not decrease larvicidal activity. The toxicity of E. coli cells expressing the wild‐type toxin was significantly reduced by two‐hour preincubation with the non‐toxic mutant (Q149P), thus indicating that the primary binding step was not affected by the proline substitution in helix 4. The results therefore reveal a crucial role for helix 4 of the Cry4B toxin in toxicity, possibly in membrane insertion and pore formation rather than in receptor recognition.