Analysis of a new Glycosyltransferase in LRP6/Wnt signalling

Wnt/β-连环蛋白信号传导通过细胞膜上的Wnt受体复合物转导，该复合物含有卷曲受体和共受体LRP 6。我以前的研究已经证明LRP 6在Wnt信号转导中起着至关重要的调节作用。我们最近已经确定了几个新的修饰LRP 6使用细胞培养为基础的表达筛选方法和DFG赠款提案要求资助其中之一，一种新的糖基转移酶的B3 GNT家族的进一步表征。迄今为止，没有特异性糖基转移酶（GT）参与调节Wnt信号传导，并且已知LRP 6与大多数细胞表面受体一样，经历糖基化以正确转运和发挥功能。在蛋白质糖基化途径中操作的数百种不同的糖基转移酶中，该B3 GnT 8家族成员被预测在该途径的后期（可能在反式高尔基体中）起作用，以延长四触角N-聚糖上的聚乳糖胺链。我们有大量的初步数据表明，B3 GnT 8是LRP 6和Wnt信号传导的真正调节剂。功能获得和丧失实验表明它对于Wnt信号传导是足够的和必需的，并且上位性实验将其功能置于LRP 6水平。我们也有初步的证据表明，它参与己糖诱导的Wnt信号转导。因此，我们建议进一步研究B3 GnT 8在Wnt信号传导的代谢调节方面的作用。我们的研究结果表明，N-糖基化LRP 6在Wnt信号转导中起着重要的调节作用。具体目标1.鉴定LRP 6糖基化位点（诱变和MS分析）2.使用MS分析和叠氮糖代谢标记的组合表征LRP 6聚糖链修饰。3.探索葡萄糖（己糖）代谢和LRP 6信号之间的联系。4.使用斑马鱼胚胎显示通过新型LRP 6 GT的LRP 6糖基化的体内相关性。5.通过RNA测序实验了解GT在细胞信号传导中的整体作用。

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.