Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle
Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle
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日本甲虫 BBMV 中苏云金芽孢杆菌 Cry8Da 毒素的分子内蛋白水解切口和结合
DOI：
10.1016/j.jip.2010.07.002
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
H. Bando and S.Asano
中科院分区：
文献类型：
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作者：
T. Yamaguchi;K. Sahara;H. Bando and S.Asano
苏云金芽孢杆菌(Bt)Cry8D杀虫蛋白在Cry8家族蛋白中具有独特的杀虫活性。根据与其他Bt Cry蛋白的序列同源性，特别是那些对甲虫有活性的蛋白，如具有3D结构的Cry3Aa，预测Cry8D蛋白的结构是典型的三结构域Cry蛋白类型。此外，据推测，敏感昆虫肠液中Cry8D的激活过程与Cry3A相似(Yamaguchi等人，2008年)。本研究对昆虫肠液中Cry8Da的激活过程进行了研究。日本甲虫肠道汁蛋白酶消化130 kDa的Cry8Da蛋白质，产生64 kDa的蛋白质。该64 kDa蛋白对成虫和幼虫均有活性，被认为是一种活性毒素。对该64 kDa蛋白的N-末端测序表明，从M1到F63的63个氨基酸残基组成的Cry8Da前导序列被移除。与在Cry3Aa的情况下一样，这些酶进一步将64 kDa的蛋白质消化成两个8 kDa和54 kDa的片段。对这些小片段的N-末端氨基酸分析表明，这些酶消化了Alpha Helix(简称Alpha)3和Alpha 4之间的环。这意味着8 kDa的片段由结构域I的Alpha1-3组成，54 kDa的片段包含剩余的结构域I和完整的结构域II和结构域III。尺寸排除层析和阴离子交换层析不能分离这些、54和8 kDa蛋白，表明54 kDa和8 kDa的片段仍在形成与64 kDa蛋白大小和离子电荷相当的毒素复合体。测序和层析结果表明，肠液蛋白水解酶只是在Alpha 3和Alpha 4之间划出了环，这一划伤过程似乎是Cry8Da毒素与受体结合所必需的。BBMV结合分析表明，Cry8Da毒素只有在切断环后才能与成虫和幼虫的BBMV制剂结合。只有54 kDa的片段与BBMV制剂结合，而不结合64 kDa的蛋白。配基印迹杂交显示，该酶激活的Cry8Da毒素，推测为54 kDa的片段，结合到特定的BBMV蛋白上，其中一个或多个将是受体(S)。日本甲虫BBMV的这些Cry8Da结合蛋白的大小和结合亲和力在幼虫和成虫之间不同。
Bacillus thuringiensis (Bt) Cry8D insecticidal proteins are unique among Cry8 family proteins in terms of its insecticidal activity against adult Scarab beetles, such as Japanese beetle (Popillia japonica Newman). From the sequence homology with other Bt Cry proteins especially those active against beetles, such as Cry3Aa whose 3D structure is available, the structure of the Cry8D protein has been predicted to be a typical three-domain Cry protein type. In addition, the activation process of Cry8D in gut juice of susceptible insects is presumed to be similar to that of Cry3A (Yamaguchi et al., 2008). In this study, the activation process of Cry8Da in insect gut juice was closely examined. Japanese beetle gut juice proteases digested the 130kDa Cry8Da protein to produce a 64kDa protein. This 64kDa protein was active against both adult and larval Japanese beetle and considered to be an activated toxin. N-terminal sequencing of this 64kDa protein revealed that the Cry8Da leader sequence consisting of 63 amino acid residues from M1to F63was removed. As in the case of Cry3Aa, the proteases further digested the 64kDa protein to two 8kDa and 54kDa fragments. N-terminal amino acid analysis of these smaller fragments indicated that the proteases digested the loop between Alpha Helix (Alpha for short) 3 and Alpha 4. This means that the 8kDa fragment consists of Alpha 1–3 of Domain I and that the 54kDa fragment contains the remaining Domain I and full Domain II and Domain III. Size exclusion chromatography and anion exchange chromatography could not separate these 64, 54 and 8kDa proteins suggesting that the 54kDa and 8kDa fragments are still forming the toxin complex equivalent to the 64kDa protein by size and ionic charge. The sequencing and chromatography results suggest that the gut juice proteases merely nicked the loop between Alpha 3 and Alpha 4. This nicking process appeared to be essential for receptor binding of the Cry8Da toxin. BBMV binding assay revealed that the Cry8Da toxin bound to BBMV preparations from both adult and larval Japanese beetle only after the loop was nicked. Only the 54kDa fragment bound to the BBMV preparations but not the 64kDa protein. Ligand blot showed that the protease activated Cry8Da toxin, presumably the 54kDa fragment, bound to specific BBMV proteins, one or more of those would be receptor(s). The sizes and binding affinities of these Cry8Da-bound proteins of Japanese beetle BBMV differed between larvae and adults.