College of Natural Resources and Life Science, Dong-A University, Busan 604-714, Republic of Korea.
Comp Biochem Physiol B Biochem Mol Biol. 2010 Mar;155(3):272-80. doi: 10.1016/j.cbpb.2009.11.011. Epub 2009 Nov 27.
We cloned and characterized two peroxiredoxins (Prxs), BiPrx1 (a 1-Cys Prx) and BiTPx1 (a 2-Cys Prx) from the bumblebee Bombus ignitus. The BiPrx1 gene consists of 5 exons, encoding 220 amino acid residues with one conserved cysteine residue. The BiTPx1 gene consists of three exons, encoding 195 amino acid residues with 2 conserved cysteine residues. Recombinant BiPrx1 (27 kDa) and BiTPx1 (25 kDa), expressed in baculovirus-infected insect Sf9 cells, reduced H2O2 in the presence of electrons donated by dithiothreitol. Unlike BiTPx1, however, BiPrx1 did not show reduction activity when thioredoxin was used as the electron donor. Both BiPrx1 and BiTPx1 protected super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Tissue distribution analyses showed the presence of BiPrx1 and BiTPx1 in the fat body, midgut, muscle and epidermis, but not in the hemolymph, suggesting that BiPrx1 and BiTPx1 are not secretable. When H2O2 was injected into B. ignitus bees, BiPrx1 and BiTPx1 transcripts were acutely up-regulated in the fat body tissues. We also demonstrated the regulation of BiPrx1 and BiTPx1 expression via reduction of transcript levels in the fat body with RNA interference (RNAi). Under H2O2 overload, the RNAi-induced BiPrx1 knock-down B. ignitus worker bees showed up-regulated expression of BiTPx1. Reciprocally, BiTPx1 RNAi knockdowns showed up-regulated BiPrx1 expression in the fat body. These results indicate that the loss of expression of BiPrx1 or BiTPx1 is compensated by the up-regulation of expression of the other peroxidase in response to H2O2 overload.
我们从大黄蜂 Bombus ignitus 中克隆并鉴定了两种过氧化物酶（Prx），BiPrx1（1-Cys Prx）和 BiTPx1（2-Cys Prx）。BiPrx1 基因由 5 个外显子组成，编码 220 个氨基酸残基，含有一个保守的半胱氨酸残基。BiTPx1 基因由 3 个外显子组成，编码 195 个氨基酸残基，含有 2 个保守的半胱氨酸残基。在杆状病毒感染的昆虫 Sf9 细胞中表达的重组 BiPrx1（27 kDa）和 BiTPx1（25 kDa），在二硫苏糖醇提供电子的情况下，可还原 H2O2。然而，与 BiTPx1 不同的是，当使用硫氧还蛋白作为电子供体时，BiPrx1 没有显示还原活性。BiPrx1 和 BiTPx1 均能在体外保护超螺旋 DNA 免受金属催化氧化（MCO）的损伤。组织分布分析表明，BiPrx1 和 BiTPx1 存在于脂肪体、中肠、肌肉和表皮中，但不存在于血淋巴中，表明 BiPrx1 和 BiTPx1 不可分泌。当将 H2O2 注射到 B. ignitus 蜜蜂中时，脂肪体组织中 BiPrx1 和 BiTPx1 的转录物被急性上调。我们还通过 RNA 干扰（RNAi）降低脂肪体中转录物水平来证明 BiPrx1 和 BiTPx1 表达的调节。在 H2O2 过载的情况下，BiPrx1 敲低的诱导的 RNAi B. ignitus 工蜂表现出 BiTPx1 的上调表达。相反，BiTPx1 RNAi 敲低导致脂肪体中 BiPrx1 的表达上调。这些结果表明，在 H2O2 过载时，一种过氧化物酶表达的丧失由另一种过氧化物酶表达的上调来补偿。
