Laboratory of Sericulture and Entomoresources, Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya 464-8601, Japan.
Biochem Biophys Res Commun. 2013 Jun 28;436(2):331-7. doi: 10.1016/j.bbrc.2013.05.103. Epub 2013 Jun 4.
Ld652Y cells from the gypsy moth, Lymantria dispar, are extremely sensitive to various apoptotic stimuli, whereas BM-N cells from the silkworm, Bombyx mori, are relatively resistant to apoptotic stimuli. We previously cloned and characterized a B. mori homologue (bm-dronc) of Drosophila melanogaster dronc. In the present study, we cloned and characterized an L. dispar homologue of dronc (ld-dronc) comparatively with Bm-Dronc. The open reading frame of ld-dronc consisted of 1329bp that was predicted to encode a 443 amino-acid polypeptide with a molecular mass of 50,706Da and 54-57% amino acid sequence identity with Dronc homologues from other lepidopteran insects identified to date. Ld-Dronc had a long prodomain, large p20 domain, and small p10 domain, and a catalytic site composed of (308)QTCRG(312), which was distinct from the sites QACRG in Bm-Dronc and QMCRG in Dronc homologues of several other lepidopteran insects. Transiently expressed Ld-Dronc underwent proteolytic processing in the lepidopteran cell lines L. dispar Ld652Y, Spodoptera frugiperda Sf9, and B. mori BM-N, and dipteran D. melanogaster S2, but only triggered apoptosis in the lepidopteran cell lines. Endogenous Ld-Dronc underwent processing in Ld652Y cells upon infection with vAcΔp35, but not in mock-infected Ld652Y cells, supporting the involvement of Ld-Dronc in apoptosis induction. In vAcΔp35-infected apoptotic cells, Ld-Dronc underwent proteolytic processing more rapidly and extensively than Bm-Dronc. Similar results were obtained for Ld-Dronc and Bm-Dronc expressed transiently in S2, Ld652Y, Sf9, and BM-N cells. Taken together, these findings suggest that the intrinsic properties of Dronc proteinsare responsible, at least in part, for the differing sensitivity of Ld652Y and BM-N to apoptosis induction upon NPV infection.
舞毒蛾（Lymantria dispar）的 Ld652Y 细胞对各种凋亡刺激极其敏感，而家蚕（Bombyx mori）的 BM-N 细胞对凋亡刺激相对耐受。我们之前克隆并鉴定了家蚕同源物（bm-dronc）的黑腹果蝇（Drosophila melanogaster） dronc。在本研究中，我们比较了舞毒蛾 Ld-Dronc 的克隆和特性，并与 Bm-Dronc 进行了比较。ld-dronc 的开放阅读框由 1329bp 组成，预测编码一个 443 个氨基酸的多肽，分子量为 50706Da，与迄今为止鉴定的其他鳞翅目昆虫的 Dronc 同源物的氨基酸序列具有 54-57%的同一性。Ld-Dronc 具有长的前导序列、大的 p20 结构域和小的 p10 结构域，以及由（308）QTCRG（312）组成的催化位点，与 Bm-Dronc 的 QACRG 位点和其他几种鳞翅目昆虫的 Dronc 同源物的 QMCRG 位点不同。瞬时表达的 Ld-Dronc 在鳞翅目细胞系舞毒蛾 Ld652Y、草地贪夜蛾 Sf9 和家蚕 BM-N 以及双翅目果蝇 D. melanogaster S2 中进行了蛋白水解加工，但仅在鳞翅目细胞系中引发凋亡。在 vAcΔp35 感染后，内源性 Ld-Dronc 在 Ld652Y 细胞中进行了加工，但在 mock 感染的 Ld652Y 细胞中没有，这支持了 Ld-Dronc 参与凋亡诱导。在 vAcΔp35 感染的凋亡细胞中，Ld-Dronc 的蛋白水解加工比 Bm-Dronc 更快、更广泛。在 S2、Ld652Y、Sf9 和 BM-N 细胞中瞬时表达的 Ld-Dronc 和 Bm-Dronc 也获得了类似的结果。综上所述，这些发现表明，Dronc 蛋白的固有特性至少部分负责舞毒蛾 Ld652Y 和家蚕 BM-N 对 NPV 感染诱导凋亡的不同敏感性。
