School of Bioengineering, Dalian University of Technology, No. 2, Linggong Road, Dalian, 116024, China.
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2 West Yuanmingyuan Road, Beijing, 100193, China.
Insect Biochem Mol Biol. 2022 Jun;145:103783. doi: 10.1016/j.ibmb.2022.103783. Epub 2022 May 5.
The biogenesis of chitin, a major structural polysaccharide found in the cuticle and peritrophic matrix, is crucial for insect growth and development. Chitin synthase, a membrane-integral β-glycosyltransferase, has been identified as the core of the chitin biogenesis machinery. However, a yet unknown number of auxiliary proteins appear to assist in chitin biosynthesis, whose precise function remains elusive. Here, we identified a sarco/endoplasmic reticulum Ca-ATPase (SERCA), in the fruit fly Drosophila melanogaster, as a chitin biogenesis-associated protein. The physical interaction between DmSERCA and epidermal chitin synthase (Krotzkopf verkehrt, Kkv) was demonstrated and analyzed using split-ubiquitin membrane yeast two-hybrid, bimolecular fluorescent complementation, pull-down, and immunoprecipitation assays. The interaction involves N-terminal regions (aa 48-81 and aa 247-33) and C-terminal regions (aa 743-783 and aa 824-859) of DmSERCA and two N-terminal regions (aa 121-179 and aa 369-539) of Kkv, all of which are predicted be transmembrane helices. While tissue-specific knock-down of DmSERCA in the epidermis caused larval and pupal lethality, the knock-down of DmSERCA in wings resulted in smaller and crinkled wings, a significant decrease in chitin deposition, and the loss of chitin lamellar structure. Although DmSERCA is well-known for its role in muscular contraction, this study reveals a novel role in chitin synthesis, contributing to our knowledge on the machinery of chitin biogenesis.
几丁质的生物发生是昆虫生长和发育的关键，它是一种存在于表皮和围食膜中的主要结构多糖。几丁质合酶，一种膜整合的β-糖苷基转移酶，已被确定为几丁质生物发生机制的核心。然而，似乎还有许多未知数量的辅助蛋白参与几丁质的生物合成，其确切功能仍难以捉摸。在这里，我们在果蝇 Drosophila melanogaster 中鉴定出一种肌浆/内质网 Ca-ATP 酶（SERCA）是一种与几丁质生物发生相关的蛋白质。通过分裂泛素膜酵母双杂交、双分子荧光互补、下拉和免疫沉淀实验证实并分析了 DmSERCA 与表皮几丁质合酶（Krotzkopf verkehrt，Kkv）之间的物理相互作用。这种相互作用涉及 DmSERCA 的 N 端区域（aa 48-81 和 aa 247-33）和 C 端区域（aa 743-783 和 aa 824-859）以及 Kkv 的两个 N 端区域（aa 121-179 和 aa 369-539），所有这些区域都预测为跨膜螺旋。虽然在表皮中特异性敲低 DmSERCA 会导致幼虫和蛹期致死，但在翅膀中敲低 DmSERCA 会导致翅膀变小、起皱，几丁质沉积显著减少，几丁质层状结构丧失。尽管 DmSERCA 以其在肌肉收缩中的作用而闻名，但本研究揭示了它在几丁质合成中的新作用，为我们对几丁质生物发生机制的认识做出了贡献。
