灵芝细胞中磷脂酸互作蛋白鉴定

Identification of phosphatidic acid interacting proteins in Ganoderma lingzhi

摘要:

灵芝是名贵药用真菌，三萜是灵芝的关键药效成分。前期研究发现，磷脂酶D (Phospholipase D，PLD) 产生的磷脂酸 (Phosphatidic acid，PA) 可调控三萜合成，为进一步阐明PA调控灵芝三萜合成的分子机制，研究采用PA-beads富集结合LC-MS/MS技术，鉴定灵芝细胞中PA互作蛋白，结果共鉴定到了19个PA互作蛋白，主要包括细胞色素P450单加氧酶 (GL22084)、特异性蛋白激酶MAPK (GL23765)、过氧化氢酶和细胞表面疏水性蛋白等。通过基因克隆、原核表达载体构建、蛋白诱导表达和分离纯化，获得了融合GST标签的GL22084和GL23765蛋白，采用GST-pull down实验，验证了灵芝GL22084和GL23765蛋白与PA互作。研究结果揭示了灵芝细胞中PA互作蛋白，为后续解析PLD介导的PA信号分子调控灵芝三萜合成的分子机理奠定了基础；同时，鉴定到的PA互作蛋白也为其他物种的PLD/PA信号通路相关研究提供借鉴。

Abstract:

Ganoderma lingzhi is widely recognized as a medicinal basidiomycetes. Triterpene acids (TAs) are the key bioactive medicinal components of G. lingzhi. Our previous studies have shown that phospholipid acid (PA) produced by phospholipase D (PLD) plays a regulatory role in TA synthesis. In order to further elucidate the molecular mechanism how PA regulates TA synthesis in G. lingzhi, PA beads enrichment combined with LC-MS/MS technology was used to identify PA interacting proteins in G. lingzhi. A total of 19 PA interacting proteins were identified, including cytochrome P450 monooxygenase (GL22084), specific protein kinase MAPK (GL23765), catalase and cell surface hydrophobicity-associated protein. GST tagged GL22084 and GL23765 proteins were obtained through gene cloning, heterologous expression, and purification. The interactions between GL22084/GL23765 and PA were verified by GST pull down assay. The identification of PA interacting proteins provides a basis for further understanding the molecular mechanism how PLD-mediated PA signaling molecules regulates the TA synthesis in G. lingzhi. Moreover, the PA interacting proteins identified in this study can also provide clues for the research of PLD/PA signaling pathway in other species.