Institute of Integrative Biology, University of Liverpool, Liverpool, United Kingdom.
PLoS One. 2011;6(6):e21316. doi: 10.1371/journal.pone.0021316. Epub 2011 Jun 27.
Most species of invertebrate animals cannot synthesise sterols de novo and many that feed on plants dealkylate phytosterols (mostly C(29) and C(28)) yielding cholesterol (C(27)). The final step of this dealkylation pathway involves desmosterol reductase (DHCR24)-catalysed reduction of desmosterol to cholesterol. We now report the molecular characterisation in the silkworm, Bombyx mori, of such a desmosterol reductase involved in production of cholesterol from phytosterol, rather than in de novo synthesis of cholesterol. Phylogenomic analysis of putative desmosterol reductases revealed the occurrence of various clades that allowed for the identification of a strong reductase candidate gene in Bombyx mori (BGIBMGA 005735). Following PCR-based cloning of the cDNA (1.6 kb) and its heterologous expression in Saccharomyces cerevisae, the recombinant protein catalysed reduction of desmosterol to cholesterol in an NADH- and FAD-dependent reaction.Conceptual translation of the cDNA, that encodes a 58.9 kDa protein, and database searching, revealed that the enzyme belongs to an FAD-dependent oxidoreductase family. Western blotting revealed reductase protein expression exclusively in the microsomal subcellular fraction and primarily in the gut. The protein is peripherally associated with microsomal membranes. 2D-native gel and PAGE analysis revealed that the reductase is part of a large complex with molecular weight approximately 250 kDa. The protein occurs in midgut microsomes at a fairly constant level throughout development in the last two instars, but is drastically reduced during the wandering stage in preparation for metamorphosis. Putative Broad Complex transcription factor-binding sites detectable upstream of the DHCR24 gene may play a role in this down-regulation.
大多数无脊椎动物物种不能从头合成甾醇，许多以植物为食的动物会去烷基化植物甾醇（主要是 C(29)和 C(28)），生成胆固醇（C(27)）。这种脱烷基化途径的最后一步涉及到甾醇还原酶（DHCR24）催化的去甲胆固醇还原为胆固醇。我们现在报告了在丝蚕（Bombyx mori）中这种参与从植物甾醇生产胆固醇而不是从头合成胆固醇的去甲胆固醇还原酶的分子特征。对推定的去甲胆固醇还原酶的系统发生基因组分析揭示了各种进化枝的存在，从而可以鉴定出丝蚕中一种强还原酶候选基因（BGIBMGA 005735）。通过基于 PCR 的 cDNA（1.6 kb）克隆及其在酿酒酵母中的异源表达，重组蛋白在 NADH 和 FAD 依赖性反应中催化去甲胆固醇还原为胆固醇。cDNA 的概念翻译，该 cDNA 编码一个 58.9 kDa 的蛋白质，以及数据库搜索，表明该酶属于 FAD 依赖性氧化还原酶家族。Western blot 分析显示还原酶蛋白仅在微粒体亚细胞部分表达，主要在肠道中表达。该蛋白与微粒体膜外周相关。2D-天然凝胶和 PAGE 分析表明，该还原酶是一个分子量约为 250 kDa 的大型复合物的一部分。该蛋白在最后两个龄期的整个发育过程中在中肠微粒体中保持相当稳定的水平，但在变态前的游荡阶段急剧减少。DHCR24 基因上游可检测到的 Broad Complex 转录因子结合位点可能在这种下调中发挥作用。
