Analysis of a new Glycosyltransferase in LRP6/Wnt signalling

LRP6/Wnt 信号传导中新型糖基转移酶的分析

• 批准号: 322812524
• 负责人: Dr. Gary Davidson
• 金额: --
• 依托单位: Institut für Toxikologie und Genetik (ITG) (aufgelöst)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/322812524?language=en
• 关键词: Analysis; new; Glycosyltransferase; LRP6; Wnt

Wnt/b-catenin signalling is transduced through Wnt receptor complexes on the cell membrane that contain a Frizzled receptor and the co-receptor LRP6. My previous research has helped demonstrate that LRP6 plays a crucial regulatory role in transducing Wnt signalling. We have recently identified several new modifiers of LRP6 using cell culture based expression screening approaches and this DFG grant proposal requests funding for the further characterisation of one of them, a novel glycosyltransferase of the B3GNT family. To date, no specific glycosyltransferase (GT) has been implicated in regulating Wnt signaling and it is known that LRP6, like most cell surface receptors, undergoes glycosylation for correct transport and functioning. Of the hundreds of diverse glycosyltransferases that operate in the protein glycosylation pathway, this B3GnT8 family member is predicted to function later in the pathway, likely in the trans-Golgi, to elongate polylactosamine chains on tetraantennary N-glycans. We have extensive preliminary data to show that B3GnT8 is a bona-fide regulator of LRP6 and Wnt signalling. Gain and loss of function experiments show it is both sufficient and required for Wnt signalling and epistasis experiments place its functioning at the level of LRP6. We also have initial evidence that it is involved in hexose induced Wnt signalling. We therefore propose to further characterise the role of B3GnT8 with respect to metabolic regulation of Wnt signalling. Our findings indicate that N-glycosylation of LRP6 plays an important regulatory role in Wnt signalling. Specific Aims 1. Identify LRP6 glycosylation sites (mutagenesis and MS analysis) 2. Characterise LRP6 glycan chain modification using a combination of MS analysis and azidosugar metabolic labelling. 3. Explore the link between glucose (hexose) metabolism and LRP6 signalling. 4. Show the in-vivo relevance of LRP6 glycosylation by the novel LRP6 GT using zebrafish embryos. 5. Understand the global role of the GT in cellular signalling by performing RNA seq experiments.

Wnt/b-catenin信号是通过细胞膜上的Wnt受体复合体转导的，该复合体包含Frizzled型受体和辅助受体LRP6。我之前的研究帮助证明了LRP6在Wnt信号转导中发挥着关键的调节作用。我们最近使用基于细胞培养的表达筛选方法确定了LRP6的几个新的修饰物，这项DFG拨款提案要求为其中之一的进一步表征提供资金，这是B3GNT家族的一个新的糖基转移酶。到目前为止，还没有特定的糖基转移酶(GT)参与调节Wnt信号，我们知道LRP6和大多数细胞表面受体一样，经历糖基化来正确地运输和发挥功能。在蛋白质糖基化途径中的数百种不同的糖基转移酶中，B3GnT8家族成员预计将在该途径的后期发挥作用，很可能是在反式高尔基体中，以延长四天线N-糖链上的聚乳糖胺链。我们有大量的初步数据表明，B3GnT8是LRP6和Wnt信号的真正调节者。功能得失实验表明，它对于WNT信令和上位性实验都是充分和必要的，并将其功能置于LRP6的水平。我们也有初步证据表明，它参与了己糖诱导的Wnt信号传递。因此，我们建议进一步研究B3GnT8在Wnt信号代谢调节中的作用。我们的发现表明，LRP6的N-糖基化在Wnt信号转导中起着重要的调节作用。具体目的1.鉴定LRP6糖基化位点(突变和MS分析)2.结合MS分析和叠氮糖代谢标记鉴定LRP6糖链修饰。3.探索葡萄糖(己糖)代谢与LRP6信号之间的联系。4.用斑马鱼胚胎研究新型LRP6 GT与LRP6糖基化的体内相关性。5.通过进行RNA序列实验，了解GT在细胞信号传递中的全球作用。