O-GlcNAc修饰调节SPINDLY蛋白功能的机制研究

O-linked β-N-acetylglucosamine (O-GlcNAc) modification is a widespread dynamic and reversible posttranslational modification, which plays important roles in multiple developmental and growth processes. SPINDLY acts as a repressor in gibberellin response pathway but a positive regulator in cytokinin response, and takes parts in almost all development and growth processes, such as root, shoot, leaf growth, embryo development and abiotic stress responses. Because of high similarity with animal O-GlcNAc transferase (OGT), SPY has long been hypothesized as an OGT. However, SPY was recently report as a novel O-fucosyltransferase, which attaches monofucose to specific serine and threonine residues. SPY is localized in both the nucleus and the cytosol, and this nucleus/cytosol localization of SPY affects its bio-function. However, the regulation of SPY sub-cellular localization is still unclear. We identified an O-GlcNAc modification on SPY protein in our previous study, which supports a hypothesis that a similar post-translational regulation on OGT exists in plant as that in mammal, where self-O-GlcNAc modification regulates subcellular localization and catalytic activity of OGT. This project will identify the exact O-GlcNAc modified site by immuno-precipitation following by Electron Transfer Dissociation (ETD) mass spectrometry analysis, find whether plant OGT, SEC, catalyzes this modification, and analyze the effect on SPY subcellular localization and functional activity by site-directed mutagenesis. This work will elucidate the mechanism by which O-GlcNAc modification regulate plant O-fucosyltransferase, SPY, and the crosstalk between two O-linked glycan modifications in plant development and growth regulation.

氧连氮乙酰葡萄糖胺(O-GlcNAc)修饰是一种重要的动态可逆的蛋白质翻译后修饰。SPINDLY(SPY)在赤霉素、细胞分裂素等激素信号转导以及生长发育的各个阶段发挥着重要作用。由于结构的相似性，长期以来SPY被认为是O-GlcNAc转移酶(OGT)，但最近被鉴定为氧连单岩藻糖转移酶。SPY的核质定位对其功能具有重要影响，但具体调节机制不详。本实验室鉴定到SPY被O-GlcNAc修饰，暗示植物中可能存在类似动物O-GlcNAc修饰调控OGT核质定位的机制。本项目希望利用免疫沉淀结合电子转移解离质谱鉴定SPY蛋白中O-GlcNAc修饰的精确位置，寻找催化该修饰的OGT酶，并构建修饰位点突变的转基因植株，观察该修饰对SPY的亚细胞定位以及生物功能的影响，揭示O-GlcNAc修饰对氧连岩藻糖修饰的调节机制，为理解O-GlcNAc修饰对植物生长发育的调控机制以及氧连糖基化之间交叉作用提供新的认识。

SPINDLY（SPY）蛋白是拟南芥中的氧连岩藻糖基转移酶，在开花、根系和花器官发育、昼夜节律调节、激素信号转导等多个生物学过程中发挥着重要作用。本实验室的前期工作鉴定到SPY蛋白具有氧连氮乙酰葡萄糖胺修饰(O-GlcNAc)。本项目使用特异抗体CTD110.6确认了这一修饰，并利用免疫沉淀结合质谱技术将修饰精确定位到SPY蛋白C末端的两个关键氨基酸Ser878和Thr880。进一步使用酵母双杂交、双分子荧光互补及免疫共沉淀技术证明了SPY与其O-GlcNAc修饰转移酶SEC具有直接的相互作用。虽然直接共表达SEC并未对SPY蛋白的亚细胞定位及蛋白大小未有显著影响,但是，对关键位点进行的点突变实验表明，第878位丝氨酸突变为丙氨酸（S878A）后影响SPY蛋白的核质分配，突变版本的SPY蛋白的核定位减少。SPY-S878A转基因植株的对于赤霉素及其合成抑制剂处理超敏感，SPY的O-GlcNAc修饰可能影响SPY蛋白在赤霉素信号通路中的作用。SPY蛋白上存在氧连岩藻糖修饰（O-FUC）修饰，而且酵母双杂交显示SPY能够与SPY蛋白自身相互作用，暗示SPY蛋白可能对自身进行氧连岩藻糖修饰。SPY蛋白还鉴定到多个磷酸化修饰位点，SPY多个丝氨酸、苏氨酸位点上存在多种修饰，这些修饰之间可能存在竞争或者协同作用精细调控蛋白功能。此外，本项目还鉴定到多个SPY蛋白的相互作用蛋白，为后续解析SPY蛋白的功能提供了思路和实验依据。本项目初步分析发现SPY参与植物的根系生长如主根伸长和根毛发育以及植物的低磷响应。综上所述，本项目研究SPY蛋白的 O-GlcNAc修饰对其核质定位及功能的调节作用，拓展了植物中O-GlcNAc修饰对蛋白质调控机制的认识。本研究也初步揭示O-GlcNAc修饰对O-FUC修饰的调节，以及两种糖基化修饰如何协同精细调控植物生长发育的分子机制，具有重要的科学意义。研究O-GlcNAc修饰的SPY蛋白如何参与植物低磷响应，可以为调控根系形态和提高磷吸收和磷效率提供技术支持，具有重要的应用前景。